WO2017042814A1 - Use of perforin positive immature dendritic cells in disease treatment - Google Patents

Use of perforin positive immature dendritic cells in disease treatment Download PDF

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WO2017042814A1
WO2017042814A1 PCT/IL2016/051001 IL2016051001W WO2017042814A1 WO 2017042814 A1 WO2017042814 A1 WO 2017042814A1 IL 2016051001 W IL2016051001 W IL 2016051001W WO 2017042814 A1 WO2017042814 A1 WO 2017042814A1
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imdcs
perf
cells
effective amount
therapeutically effective
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PCT/IL2016/051001
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French (fr)
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Yair Reisner
Yael ZLOTNIKOV KLIONSKY
Bar NATHANSOHN-LEVI
Liran YARIMI
Sivan KAGAN
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Yeda Research And Development Co. Ltd.
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Definitions

  • the present invention in some embodiments thereof, relates to perforin positive immature dendritic cells (Perf+ imDCs) and, more particularly, but not exclusively, to the use of same for the treatment of inflammatory conditions.
  • Perf+ imDCs perforin positive immature dendritic cells
  • T regulatory cells T regulatory cells
  • Bregs B regulatory cells
  • DCs dendritic cells
  • mDCs mature DCs
  • imDCs immature DCs
  • Perf-DC are able to selectively kill cognate T cell receptor (TCR) transgenic CD8 + T cells that recognize their peptide-MHC, through a unique perforin/granzyme A-based killing mechanism, regulated through TLR7 and TREM-1 signaling [Zangi L.
  • Perf-DC comprise about 2-4 % of the CD 11c positive cells within the lymph nodes and spleen, and that the abundance of these cells is markedly enhanced upon in-vivo administration of GM-CSF [Zangi L. et al. (2012), supra].
  • Perforin-positive myeloid DCs have also been reported within the human classical DC population [Stary G. et al., J. Exp. Med (2007) 204(6): 1441-51]. Taken together, these initial findings, based exclusively on ex-vivo studies, indicated a potential tolerogenic role for Perf-DCs.
  • U.S. Patent Application No. 20160058792 discloses methods and compositions for producing tolerogenic or immunosuppressive dendritic cells, the methods comprising contacting dendritic cells with an agent that stimulates the IL 27/ectonucleotidase CD39 axis signaling.
  • the cells taught by U.S. 20160058792 can be used for treating an autoimmune disease or disorder.
  • U.S. Patent Application No. 20160095882 discloses tolerogenic dendritic cells for treating a myocardial infarction and a method for preparing the same.
  • dendritic cells are obtained by culturing immature dendritic cells in a medium including TNF-a, IL-4 and GM-CSF, and protein extracted from a region of a myocardial infarction.
  • a method of treating an inflammation in a subject in need thereof comprising administering to the subject a therapeutically effective amount of perforin+ immature DCs (Perf imDCs), thereby treating the inflammation in the subject.
  • Perf imDCs perforin+ immature DCs
  • a therapeutically effective amount of Perff imDCs for use in treating an inflammation in a subject in need thereof.
  • an isolated population of cells comprising Perf+ imDCs generated according to the method of some embodiments of the invention, wherein at least 50 % of the population of cells comprises the Perff imDCs.
  • an isolated population of cells comprising at least 50 % Perf+ imDCs, wherein the Perf+ imDCs maintain an immature phenotype for at least 12 hours when administered to a recipient.
  • a pharmaceutical composition comprising the isolated population of cells of some embodiments of the invention and a pharmaceutically active carrier.
  • an article of manufacture comprising the isolated population of cells of some embodiments of the invention being packaged in a packaging material and identified in print, in or on the packaging material for use in the treatment of an inflammation.
  • the method or therapeutically effective amount of Perf+ imDCs for use further comprises contacting the Perf+ imDCs with a factor capable of inhibiting the Perf+ imDCs from maturing prior to or concomitantly with administration of the Perf imDCs to the subject in need thereof.
  • the method further comprises administering to the subject a therapeutically effective amount of a factor capable of inhibiting the Perf+ imDCs from maturing.
  • the therapeutically effective amount of Perff imDCs for use further comprises the use of a therapeutically effective amount of a factor capable of inhibiting the Perf+ imDCs from maturing.
  • the Perf+ imDCs are inhibited from maturing, by a method comprising: (a) obtaining Perf+ imDCs; and (b) contacting the Perf+ imDCs with a factor capable of inhibiting the Perf+ imDCs from maturing.
  • the method further comprises selecting cells that exhibit the Perff imDCs phenotype.
  • the Perff imDCs are obtained by a method comprising: (a) obtaining CD34+ cells; (b) contacting the CD34+ cells with a factor capable of differentiating the CD34+ cells into early myeloid cells; and (c) contacting the early myeloid cells with a factor capable of differentiating the early myeloid cells into perforin+ immature dendritic cells.
  • the method further comprises obtaining hematopoietic progenitor cells prior to step (a).
  • the factor capable of differentiating the CD34+ cells into early myeloid cells comprises at least one of a stem cell factor (SCF), a thrombopoietin (TPO), a Flt3-ligand (Flt3L), an interleukin-3 (IL-3) and an interleukin-6 (IL-6).
  • SCF stem cell factor
  • TPO thrombopoietin
  • Flt3L Flt3-ligand
  • IL-3 interleukin-3
  • IL-6 interleukin-6
  • contacting the CD34+ cells with a factor capable of differentiating the CD34+ cells into early myeloid cells is effected for 5-20 days.
  • the factor capable of differentiating the early myeloid cells into perforin+ dendritic cells comprises a granulocyte-macrophage colony- stimulating factor (GM-CSF). According to some embodiments of the invention, the factor capable of differentiating the early myeloid cells into perforin+ dendritic cells further comprises an interleukin-4 (IL-4).
  • GM-CSF granulocyte-macrophage colony- stimulating factor
  • IL-4 interleukin-4
  • contacting the early myeloid cells with a factor capable of differentiating the early myeloid cells into perforin+ dendritic cells is effected for 5-20 days.
  • the factor capable of inhibiting the Perf imDCs from maturing comprises an anti-inflammatory agent or an immunosuppressive agent.
  • the anti-inflammatory agent or an immunosuppressive agent is an mTOR inhibitor.
  • the anti-inflammatory agent or an immunosuppressive agent inhibits granulocyte-mediated inflammation.
  • the immunosuppressive agent comprises rapamycin.
  • the anti-inflammatory agent comprises aspirin or a heme oxygenase- 1 (HO-1).
  • contacting the early myeloid cells with a factor capable of differentiating the early myeloid cells into perforin+ dendritic cells is effected concomitantly with the contacting the Perff imDCs with a factor capable of inhibiting the Perff imDCs from maturing.
  • the Perff imDCs comprise cells having the signature perforin+, CDl lc + ,MHC-II + , CD80 and CD86.
  • At least 50 % of the Perf+ imDCs comprise the signature.
  • the Perff imDCs are loaded with an antigen.
  • the method is effected ex vivo.
  • the article further comprises a factor capable of inhibiting the Perff imDCs from maturing.
  • the inflammation is associated with a chronic inflammatory disease. According to some embodiments of the invention, the inflammation is associated with an acute inflammatory disease.
  • the inflammation is associated with a disease selected from the group consisting of a metabolic disease, an autoimmune disease, an infectious disease, a hypersensitivity disease, a transplantation related disease and an injury.
  • the inflammation is associated with a disease selected from the group consisting of multiple sclerosis, metabolic syndrome, diabetes, rheumatoid arthritis, lupus and Crohn's.
  • the therapeutic effective amount of the Perf imDCs is capable of inhibiting an activity or proliferation of a CD4 + T cell and/or a CD8 + T cell.
  • the Perff imDCs are syngeneic with the subject.
  • the Perf+ imDCs are non- syngeneic with the subject.
  • the subject is a human subject.
  • all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.
  • methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control.
  • the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
  • FIGs. 1A-E Characterization of donor type origin in immune cells of WT-PKO and DTA-PKO chimera.
  • Figure la FACS analysis of CDl lc + cells from spleen of DTA- PKO chimeric mice and WT-PKO controls. The CDl lc+ cells could be divided into CDl lc high (cDCs) and CDl lc int subpopulations.
  • Figure lb cDC levels in the spleen of DTA-PKO and WT-PK chimera.
  • Figure lc The cells defined in ( Figure la) were further stained for CD45.1 to identify cell origin from CD1 lc-DTA mice or littermate controls, or from CD45.2 expressed by PKO donors.
  • NK cells pre-gated on CD3 CD19 " , 2.2 + 0.6 vs. 2.1 + 0.2 %), pDCs (pre-gated on CD3 ⁇ CD19Terl l9 ⁇ , 0.27+0.07 vs. 0.29 + 0.02 %), T cells (CD4 + T cells: 13.6 + 1.5 vs. 14.1 + 2.1 and CD8 + T cells: 8.6 + 0.7 vs. 7.9 + 0.4 %) and neutrophils (pre- gated on CD3 " CD19 " MHC-irNKl. r, 1.125 + 0.1 vs. 1.1 + 0.16 %) from DTA-PKO chimeric mice and WT-PKO controls 2 months post- transplant. These percentages indicate the frequencies of each sub-population out of the entire lymphogate in the organ (spleen or BM).
  • FIGs. 2A-F DTA-PKO mice develop a condition resembling metabolic syndrome at 6 months post-transplant.
  • Figure 2a Body weight of chimeric mice 6 months after transplant. Data pooled from three independent experiments; N > 18.
  • Figure 2b Kinetics of weight gain in DTA-PKO (grey curve) and WT-PKO (black curve) mice.
  • Figure 2c Blood chemistry profile of the chimeric mice (Avg + S.D, N > 6, *p ⁇ 0.05, ***p ⁇ 0.001).
  • Figure 2d Serum leptin and
  • Figure 2e TNF-a levels in the DTA-PKO and control WT- PKO chimera (Avg + S.D, N > 5).
  • Figure 2f Percent body fat measured using Echo-MRI (white bars - DTA-PKO, black bars - WT-PKO).
  • FIGs. 3A-E Typical features of T2D and adipose tissue expansion in DTA-PKO mice. Glucose homeostasis determined using (Figure 3a) glucose-tolerance test, and (Figure 3b) insulin-tolerance test in DTA-PKO (red) or WT-PKO (black) chimeric mice, demonstrating impaired glucose tolerance and reduced insulin sensitivity. Mice (n > 6 per group) were injected i.p.
  • FIGs. 4A-F Elevated T cell levels within adipose tissue (AT) of DTA-PKO mice 6 months post-transplant; systemic CD8 + and CD4 + depletion prevents weight gain and increase in leptin levels.
  • Stromal vascular fractions (SVF) cells isolated from visceral AT (VAT) of DTA-PKO (white bars) and WT-PKO (black bars) mice maintained on a normal diet for 6 months were subjected to FACS analysis.
  • Figure 4a Dot plots showing CD4 and CD8 T cells after gating out CD1 lb cells.
  • FIGs. 5A-B A modified TCR repertoire in adipose tissues from DTA-PKO mice compared with WT-PKO
  • Figure 5a Lorenz representation of the TCRP repertoire skewing for T cells from spleen, VAT and subcutaneous AT (SC-AT) of DTA-PKO (blue) and WT-PKO (red) mice.
  • SC-AT subcutaneous AT
  • clonotypes were ordered by frequency. The cumulative frequency was then calculated at each rank (normalized to sample size). The curves represent the mean for each experimental group.
  • Redundancy analysis is an extension of principal component analysis (PCA) that explicitly models response variables (most abundant CDR3 AA sequences, here) as a function of explanatory variables (sample type: mouse (DTA-PKO or WT-PKO) and tissue (spleen, VAT, SC-AT), in this case).
  • PCA principal component analysis
  • This analysis separates between repertoires of DTA-PKO (blue) vs. WT-PKO (red) along RDA1, and between most spleen samples (triangles) vs. adipose tissue samples (circles: SC; squares: VAT) along RDA2.
  • FIGs. 6A-L DTA-PKO chimeras are more prone to high-fat diet (HFD) induced inflammation.
  • Figures 6a-b Two months after chimerism induction, DTA-PKO (gray) and WT-PKO (black) chimeras were maintained on HFD and monitored for weight change over 90 days. Percent of body fat (Figure 6c), liver weight ( Figure 6d) and epididymal fat pad weight ( Figure 6e) in DTA-PKO (white) and WT-PKO chimera (black) maintained for 6 weeks on HFD.
  • FIGs. 7A-E Perforin deficiency within the DC population results in enhanced susceptibility to EAE.
  • Figure 7a Disease progression of DTA-PKO (red) and WT-PKO (black) mice immunized with MOG and the average day of EAE onset (Avg + SEM of two experiments with a total of 18 mice per group).
  • Figure 7b Spinal cord sections from the mice described in Figure 7a, stained with hematoxylin and eosin (H&E); arrows indicate inflammatory infiltrates.
  • Figures 7c-d MOG-specific proliferation of splenocytes from DTA-PKO (red), and WT-PKO (black) mice 30 days after immunization.
  • T cells were isolated from 3-5 pooled spinal cords from the DTA-PKO or WT-PKO mice, 14 days after immunization with MOG, stimulated with soluble CD3 overnight, and then stained for IL-17a and IFN- ⁇ (Avg + SD *P ⁇ 0.05, N > 3).
  • FIGs. 8A-C Perforin expression in CDl lc + DCs.
  • Figure 8a Immuno staining of perforin in CDl lc + DCs isolated by magnetic beads from the spleen of WT or PKO mice, as described in the 'general materials and experimental procedures' section hereinbelow. Due to some non-specific staining experienced with isotype control antibodies (marked by arrow), cells were initially treated with isotype control (Green) and subsequently with perforin- specific antibody (Red). Nuclei are stained by Hoechst in Blue. Inset in the left image shows staining of a Perf-DC under higher magnification.
  • FIG. 9 Immunostaining of perforin in CDl lc "1" DCs isolated by magnetic beads from the spleen of WT mice together with CD4 and CD8 staining (green), as described in the 'general materials and experimental procedures' section hereinbelow. Due to some non-specific staining experienced with isotype control antibodies), cells were initially treated with isotype control (red) and subsequently with perforin- specific antibody (blue). Nuclei are stained by Hoechst Yellow. Dotted circle marks the boundaries of perf-DC cell on the upper image and as can be seen in the lower image it is typically negative for CD4 or CD8 staining (green).
  • FIGs. 10A-D Characterization of CDl lc high and CDl lc int and their perforin expression in WT-PKO and DTA-PKO chimera.
  • Figure 10a FACS analysis of CDl lc + cells from spleen of DTA-PKO chimeric mice and WT-PKO controls.
  • the CDl lc "1" cells could be divided into four sub-populations: CDl lc ⁇ CDl lb ⁇ (I), CDl lc high CDl lb low (II), CDl lc int CDl lb high (III), CDl lc int CDl lb int (IV).
  • FIG. 10b Expression levels of F4/80 were determined in four populations of splenic CDl lc "1" cells by FACS.
  • Figure 10c Proliferation of CFSE labeled Balb/c T cells stimulated for 3 days against C57BL/6 CDl lc high and CDl lc int cells, and measured by FACS analysis for CFSE dilution as an indicator of cell division. The bars indicate mean CFSE value + SD.
  • FIGs. 11A-D Control DTA-WT mice do not exhibit metabolic abnormalities.
  • DTA-PKO red
  • WT-PKO black
  • DTA-WT gray
  • chimeric mice were compared for different metabolic parameters, including:
  • Figure 11a Body weight of chimeric mice 6 months after transplant.
  • Figure 1 lb Percent body fat measured using Echo-MRI.
  • Figure 11c Serum leptin levels (Avg + S.D, N > 5, *p ⁇ 0.05, ***p ⁇ 0.001).
  • FIG. 12 Sequential gating strategy for analysis of T cells in SVF.
  • Collagenase digested stromal vascular fractions (SVF) were stained and analyzed by FACS. T cells were first gated based on their forward and side scatter (Rl). Nonviable cells were excluded using 7AAD staining (R2). Further gating on CD45 + CDl lb " cells (R3) enabled focusing on lymphocytes, gating out CDl lb + macrophages. Moreover, CD4 + and CD8 + T cells within gate R3 were analyzed.
  • FIGs. 13A-B FACS determination of myeloid cell subpopulations in SVF from
  • Macrophages were defined based on CDl lb and F4/80 expression, DCs were defined as CDl lc high and MHC-II high , and neutrophils were CDl lc " F4/80 " CDl lb + Ly6G + ( Figure 13b) Box plots summarizing the number of cells per gram fat (Center lines show Mean; Box limits indicate the 25 th and 75 th percentile, whiskers indicate the minimal and maximal values, ** p ⁇ 0.01).
  • FIG. 14 DTA-PKO chimeras develop inflammation in the hypothalamus 9 months after transplantation.
  • Gene expression level of pro-inflammatory IL-6 and IL- ⁇ genes determined by RT-PCR in hypothalamus of DTA-PKO (red) and WT-PKO (black) mice (N > 3).
  • FIGs. 15A-E Frequency and total cell numbers of different SVF cell subpopulations isolated from VAT of DTA-PKO and WT-PKO mice maintained on HFD for 8 weeks.
  • Cells were identified by FACS analysis of SVF fractions from 6-8 mice; percent gated cell frequencies are indicated for T cells ( Figure 15 a) and macrophages ( Figure 15d).
  • Absolute numbers (in thousands) of CD4 and CD8 T cells and macrophages per gram of fat tissue Figure 15b, 15c and 15e), respectively, are shown (all data are presented as Avg + SD *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001).
  • DTA-PKO red
  • WT- PKO Black.
  • FIG. 16A-B Donor type origin of B cells (Figure 16a) and neutrophils, ( Figure 16b) from spleen of DTA-PKO (gray filled histogram) and WT-PKO (unfilled histogram) chimera before and after onset of pathology.
  • Cells were stained for CD45.1 to identify cell origin from CDl lc-DTA or littermate controls, or from CD45.2 expressed by PKO donors. Numbers indicate percent of CD45.1 cells in the various populations (Ave + SD).
  • FIGs. 17A-C Donor type origin of NK cells and DC from spleen of DTA-PKO (gray filled histogram) and WT-PKO (unfilled histogram) chimera after onset of pathology is not altered compared to data shown in Figures la-e for cells harvested prior to pathology onset.
  • Figure 17a NK cells from spleen were pre-gated for Lin- (Terl l9 ⁇ CD3 ⁇ CD19 ⁇ Gr ) and further defined by various markers including NKl .l, NKp46 and CD49b (DX5).
  • cDCs were defined by staining for MHC-II and CDl lc.
  • FIG. 17b Cells from chimera fed with a regular diet were stained for CD45.1 6 months after transplantation to identify cell origin from CDl lc-DTA or littermate control hosts, versus CD45.2 expressed by PKO donors.
  • Figure 17c Cells from chimera fed with HFD were stained for CD45.1 2 months after transplantation to identify cell origin from CD1 lc-DTA or littermate controls, or from CD45.2 expressed by PKO donors.
  • FIG. 18 Donor type origin of VAT cells from DTA-PKO and WT-PKO chimeric mice fed with normal diet at early (2 months) or late (7 months) time points post- transplant, or with HFD (8 weeks after HFD).
  • Different leukocyte types were defined as indicated in Figures l la-d and Figure 12 and were further stained for CD45.1 to identify cell origin from CDl lc-DTA or littermate controls, or from CD45.2 expressed by PKO donors.
  • FIGs. 19A-B Aspirin-treated imDCs do not undergo maturation in response to induction with LPS. imDCs were generated using the 20 days protocol as previously described [Zangi L. et al. (2012), supra]. One group of cells was additionally treated with 2.5 mM aspirin during the last 10 days of culture, while the second group was not additionally treated. To induce maturation, cells were treated with 1 ⁇ g/ml LPS for 24 hours. Cells from the two groups were stained for MHC II, CD80 and CD86 before (black) and after (red) LPS treatment and were further analyzed by FACS.
  • FIG. 20 Aspirin-treated perff imDCs maintain the ability to kill cognate CD8 + T cells in MLR. FACS evaluation of total numbers of live OT-I CD8 + T cells following 5 hours of incubation with Aspirin-treated imDCs loaded with SIINFEKL peptide (SEQ ID NO: 29), at indicated ratios. Average + SD (N > 4).
  • FIGs. 21A-D MHC-dependent perforin/Granzyme A mediated killing of cognate CD8 + T cells in MLR is associated with triggering of TLR7.
  • Figure 21a FACS evaluation of total numbers of live OT-I CD8 + T cells in the absence (white) or presence of untreated imDCs (patterned) or treated with SIINFEKL (SEQ ID NO: 29) (black) or SIYRYYGL (SEQ ID NO: 30) (grey), at indicated cell ratios (Black).
  • Figure 21b Killing ability of imDCs from C57BL/6 WT or TLR7 _/" mice loaded with SIINFEKL (SEQ ID NO: 29).
  • the present invention in some embodiments thereof, relates to perforin positive immature dendritic cells (Perf+ imDCs) and, more particularly, but not exclusively, to the use of same for the treatment of inflammatory conditions.
  • Perf+ imDCs perforin positive immature dendritic cells
  • Perf+ DCs are able to selectively kill cognate TCR transgenic CD8 + T cells that recognize their peptide-MHC through a unique perforin/granzyme A-based killing mechanism, regulated through TLR7 and TREM-1 signaling [Zangi L. et al., Blood. (2012) 120(8): 1647-57]. While reducing the present invention to practice, the present inventors have uncovered that Perforin positive immature dendritic cells (Perf+ imDCs) represent an important regulatory population of cells that control inflammatory/autoimmune processes.
  • the present inventors further illustrated the regulatory role of Perf+ imDCs in a defined model of autoimmunity, namely Myelin Oligodendrocyte Glycoprotein (MOG) peptide-induced experimental autoimmune encephalomyelitis (EAE).
  • MOG Myelin Oligodendrocyte Glycoprotein
  • EAE experimental autoimmune encephalomyelitis
  • a method of treating an inflammation in a subject in need thereof comprising administering to the subject a therapeutically effective amount of perforin+ immature DCs (Perf+ imDCs), thereby treating the inflammation in the subject.
  • Perf+ imDCs perforin+ immature DCs
  • a therapeutically effective amount of Perf+ imDCs for use in treating an inflammation in a subject in need thereof.
  • DCs refers to antigen-presenting immune cells that process antigenic material and present it to other cells of the immune system e.g. to T cells and B cells (e.g. by presentation on the DCs cell surface via MHC class I and/or MHC class II molecules).
  • the term DCs includes immature and mature dendritic cells.
  • DCs can be typically characterized by expression of cell surface markers, e.g., CDl lc, MHC class II (e.g. HLA DR), CD80 and CD86.
  • DCs can also express any of CD40, CDl lb, CD304 (BDCA4), CD103 and/or CD Id.
  • DCs can also be identified by the absence of T cell, B cell, NK cell, monocyte and/or neutrophil specific lineage markers such as, but not limited to, CD3, T cell receptor (TCR), CD14, CD19, B cell receptor (BCR) and/or CD56.
  • TCR T cell receptor
  • BCR B cell receptor
  • CD56 CD56
  • dendritic cells can be characterized functionally by their capacity to stimulate allo- responses and mixed lymphocyte reactions (MLR).
  • imDCs refers to dendritic cells which are found at an initial maturation state and are capable of capturing and processing an antigen.
  • imDCs can be characterized by expression of CDl lc, CD80, and CD86 as well as by low levels of MHC class II (e.g. HLA DR, as compared to mature dendritic cells).
  • imDCs can also be identified by the presence of CD40, CDl lb, as well as by the absence of T cell, B cell, NK cell, monocyte and/or neutrophil specific lineage markers such as, but not limited to, CD3, T cell receptor (TCR), CD 14, CD 19, B cell receptor (BCR) and/or CD56.
  • immature DCs are typically distributed within all tissues, particularly those that interface with the environment (e.g. skin, mucosal surfaces) and in lymphoid organs, and function to capture and process antigens.
  • Immature DCs are recruited to sites of inflammation in peripheral tissues following pathogen invasion. Internalization of foreign antigens can subsequently trigger their maturation and migration from peripheral tissues to lymphoid organs where the antigens are presented to, for example, antigen- specific immune cells (e.g. T cells and B cells).
  • antigen- specific immune cells e.g. T cells and B cells.
  • the process of DC maturation involves down-regulation of antigen internalization, an increase in the surface expression of MHC molecules and co-stimulatory molecules, morphological changes (e.g. formation of dendrites) and secretion of chemokines, cytokines and proteases.
  • mature dendritic cells refers to DCs which express higher levels of CDl lc, MHC class II, CD80 and CD86 (as compared to imDCs).
  • Mature DCs can also express CD40.
  • Mature DCs release cytokines, including but not limited to, IL- 12, IL- la, IL- ⁇ , IL- 15, IL- 18, IFN-a, IFN- ⁇ , IFN- ⁇ , IL-4, IL- 10, IL-6, IL- 17, IL- 16, TNF-a, and MIF, and can activate naive lymphocytes (e.g. naive T cell).
  • Perforin+ immature DCs or “Perf+ imDCs” refers to a subgroup of imDCs which express perforin and/or granzyme A. Perf+ imDCs are found at an initial maturation state and are capable of capturing and processing an antigen. According to one embodiment, the Perf+ imDCs are characterized by expression of perforin, CDl lc, MHC class II (e.g. HLA DR), CD80 and CD86. According to one embodiment, the Perf+ imDCs may also express any of CD1 lb, CD40 and/or granzyme A.
  • Perf imDCs can also be identified by the absence of T cell, B cell, NK cell, monocyte and/or neutrophil specific lineage markers such as, but not limited to, CD3, T cell receptor (TCR), CD14, CD19, B cell receptor (BCR) and/or CD56.
  • TCR T cell receptor
  • BCR B cell receptor
  • a cell is considered a Perf+ imDC when a single cell of cells has the signature: perforin, CDl lc, MHC class II (e.g. HLA DR), CD80 and CD86.
  • a cell is considered a Perf+ imDC when a single cell of cells has the signature: perforin, granzyme A, CDl lc, MHC class II (e.g. HLA DR), CD80 and CD86.
  • a cell is considered a Perf+ imDC when a single cell of cells has the signature: perforin, CDl lc, MHC class II (e.g. HLA DR), CD80, CD86 and CD40.
  • a cell is considered a Perf+ imDC when a single cell of cells has the signature: perforin, CDl lc, MHC class II (e.g. HLA DR), CD80, CD86 and CD l ib.
  • a cell is considered a Perf+ imDC when a single cell of cells has the signature: perforin, granzyme A, CDl lc, MHC class II (e.g. HLA DR), CD40, CD80 and CD86.
  • a cell is considered a Perf+ imDC when a single cell of cells has the signature: perforin, CDl lc, MHC class II (e.g. HLA DR), CD80, CD86 and CD3 " and/or TCR " .
  • MHC class II e.g. HLA DR
  • CD80 CD86 and CD3 " and/or TCR " .
  • a cell is considered a Perf+ imDC when a single cell of cells has the signature: perforin, CDl lc, MHC class II (e.g. HLA DR), CD80, CD86 and CD 19 “ and/or BCR " .
  • MHC class II e.g. HLA DR
  • CD80 CD86 and CD 19 " and/or BCR " .
  • a cell is considered a Perf+ imDC when a single cell of cells has the signature: perforin, CDl lc, MHC class II (e.g. HLA DR), CD80, CD86 and CD56 " .
  • any method known in the art can be used to identify the cell surface markers, e.g. using FACS analysis or using magnetic cell isolation and cell separation (e.g. by Miltenyi Biotec). Furthermore, any method known in the art can be used for identification of cellular components (e.g. perforin or granzyme A), e.g. using ELISA, PCR or by western blot analysis.
  • FACS analysis e.g. using FACS analysis or using magnetic cell isolation and cell separation (e.g. by Miltenyi Biotec).
  • magnetic cell isolation and cell separation e.g. by Miltenyi Biotec
  • any method known in the art can be used for identification of cellular components (e.g. perforin or granzyme A), e.g. using ELISA, PCR or by western blot analysis.
  • the Perf imDCs comprise immune-regulatory properties (e.g. tolerogenic activity).
  • the Perff imDCs inhibit expansion of antigen specific immune cells by killing cognate T cells (e.g. cognate CD8 + T cells which recognize an antigen presented in the context of a MHC molecule on the surface of the Perf+ imDC) through a unique perforin/granzyme A-based killing mechanism (e.g. regulated through TLR7 and TREM-1 signaling).
  • tolerogenic activity of imDCs refers to the ability of dendritic cells' to suppress immune responses such as suppressing T cell-mediated immune responses (e.g. killing effector T cells).
  • Perf+ imDCs is especially beneficial in situations in which there is a need to down-regulate T cells involved in an inflammatory response, such as an autoimmune disease or metabolic syndrome (as discussed in further detail below).
  • Perf+ imDCs are obtained directly from a cell donor (e.g. from a subject, as discussed in detail hereinbelow).
  • Perf+ DC make about 2-4 % of the CD 11c positive cells in a human body, thus, it is possible to obtain these cells directly from a cell donor by obtaining any biological sample comprising Perf imDCs (e.g. blood, bone marrow, lymph node and fluid, tonsils, adipose tissue, etc.) from the donor and selecting therefrom dendritic cells which express perforin, CDl lc,MHC class II, CD80 and CD86 (e.g.
  • the dendritic cells may further express any of granzyme A, CD l ib and/or CD40.
  • the dendritic cells lack expression of T cell, B cell or NK cell markers, e.g. CD3, CD19, CD56, respectively.
  • the dendritic cells may further lack expression of T cell, B cell, NK cell, monocyte and/or neutrophil specific lineage markers such as, but not limited to, CD3, TCR, CD14, CD19, BCR and/or CD56.
  • the cell donor is administered a factor for inducing mobilization of hematopoietic stem cells (HSCs) from the bone marrow into the bloodstream, such as e.g. GM-CSF and/or Flt3L (e.g. prior to withdrawal of a blood sample).
  • HSCs hematopoietic stem cells
  • GM-CSF hematopoietic stem cells
  • Flt3L hematopoietic stem cells
  • Perf+ imDCs are obtained from a commercial supplier (i.e. as an off the shelf product), as for example, from HemaCare Corporation, from the ATCC, or from Astarte Biologies.
  • Perff imDCs may be difficult to maintain due to their rapid maturation into mature DCs after stimulation with an antigen
  • the present invention contemplates generation of Perf+ imDCs using ex vivo or in vitro protocols.
  • a method of obtaining Perf+ imDCs comprising: (a) obtaining CD34+ cells; (b) contacting the CD34+ cells with a factor capable of differentiating the CD34+ cells into early myeloid cells; and (c) contacting the early myeloid cells with a factor capable of differentiating the early myeloid cells into Perf+ imDCs.
  • the method further comprises obtaining hematopoietic progenitor cells prior to step (a).
  • CD34+ cells may be obtained from a commercial supplier (i.e. as an off the shelf product), as for example, from Stemcell Technologies, from the ATCC, or from Astarte Biologies.
  • Commercially obtained CD34+ cells may include cells obtained from any stem/progenitor cell source, e.g. cord blood CD34+ cells, bone marrow derived CD34+ cells, cell lines, etc.
  • CD34+ cells may also be obtained from a cell donor.
  • This method is typically effected by first promoting mobilization of hematopoietic progenitor cells (comprising CD34 + cells), from bone marrow into peripheral blood in a cell donor (e.g. subject).
  • Such methods comprise administering to the cell donor a mobilization factor, such as granulocyte colony stimulating factor (G-CSF), about 7-21 days prior to cell collection (e.g., 21 days, 14 days, 12 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day).
  • G-CSF granulocyte colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • the dose of the mobilization factor can be effected in a single administration or over a course of several days (e.g. 2, 3, 4, 5, 7 or 10 days).
  • Such methods of promoting mobilization of hematopoietic progenitor cells typically result in sufficient numbers of CD34+ cells in the peripheral blood for subsequent collection.
  • Collecting the mobilized hematopoietic progenitor cells (comprising CD34 + cells) from the peripheral blood of the subject may be effected using various techniques.
  • the collecting step may comprise standard blood retrieval procedures or apheresis.
  • CD34 + cells may be obtained directly from the bone marrow of a subject, e.g. by lumbar puncture.
  • the enriching step typically involves a step in which CD34 + cells are separated from CD34-negative (CD34 ) cells to provide an enriched population of CD34 + cells (e.g. enriched by about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 50-fold, 100-fold, 1000-fold more of CD34 + cells compared to the amount or number of CD34 + cells in the original population of cells, i.e., the population of cells before enrichment).
  • the enriching step optionally comprises the use CD34 " or CD34 + specific antibodies or antibody fragments. Selection may be effected using, but not limited to, magnetic cell isolation and cell separation (e.g. by Miltenyi Biotec). See, for example, PCT publication no. WO 2013126590.
  • the CD34+ cells are then contacted with a factor capable of differentiating the
  • CD34+ cells into early myeloid cells i.e. the immature state of myeloid cells.
  • exemplary factors capable of differentiating CD34+ cells into early myeloid cells include, but are not limited to, stem cell factor (SCF), thrombopoietin (TPO), Flt3-ligand (Flt3L), interleukin- 3 (IL-3) and/or interleukin-6 (IL-6).
  • SCF stem cell factor
  • TPO thrombopoietin
  • Flt3-ligand Flt3-ligand
  • IL-6 interleukin-6
  • differentiating CD34+ cells into early myeloid cells is effected in the presence of SCF, TPO, Flt3L, IL-3 and IL-6.
  • Contacting the CD34+ cells with the factor or combination of factors capable of differentiating the CD34+ cells into early myeloid cells is typically effected for about 2-40 days (e.g. 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 25 days, 30 days or 40 days).
  • contacting the CD34+ cells with the factor or combination of factors capable of differentiating the CD34+ cells into early myeloid cells is effected for 10 days.
  • Early myeloid cells can be selected by the surface expression of CDl la, CDl lb, CDl lc, CD13, CD33, CD34 and CD117. Any method known in the art can be used to identify the cell surface markers, e.g. using FACS analysis or using magnetic cell isolation and cell separation (e.g. by Miltenyi Biotec).
  • the early myeloid cells are contacted with a factor capable of differentiating the early myeloid cells into perf imDCs.
  • factors capable of differentiating the early myeloid cells into perff imDCs include, but are not limited to, granulocyte-macrophage colony- stimulating factor (GM-CSF), Flt-3L, and macrophage colony- stimulating factor (M-CSF).
  • GM-CSF granulocyte-macrophage colony- stimulating factor
  • M-CSF macrophage colony- stimulating factor
  • interleukin-4 (IL-4) can be added to the GM-CSF for differentiation of the early myeloid cells into perff imDCs.
  • Contacting the early myeloid cells with the factor or combination of factors capable of differentiating the early myeloid cells into perff imDCs is typically effected for about 2-40 days (e.g. 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 25 days, 30 days or 40 days).
  • contacting the early myeloid cells with the factor or combination of factors capable of differentiating the early myeloid cells into perf imDCs is effected for 10 days.
  • Perff imDCs can be selected by the expression of perforin, CDl lc,MHC class II, CD80 and CD86. Perff imDCs can be further selected by the additional expression of granzyme A, CD l ib and/or CD40, as well as by the lack expression of T cell, B cell, NK cell, monocyte and/or neutrophil specific lineage markers such as, but not limited to, CD3, TCR, CD14, CD19, BCR and/or CD56, as discussed above. Any method known in the art can be used to identify the cell surface markers, e.g. using FACS analysis or using magnetic cell isolation and cell separation (e.g. by Miltenyi Biotec). Expression of perforin can be carried out using an ELISA, western blot analysis or PCR.
  • generating Perf+ imDCs can be carried out by first obtaining bone marrow cells from a cell donor.
  • the bone marrow cell suspension is enriched for CD34+ cells using e.g. an anti-CD34 antibody, followed by positive selection e.g. on a MACS separation column (e.g. Miltenyi Biotec) or using a FACSAria Sorter.
  • a MACS separation column e.g. Miltenyi Biotec
  • FACSAria Sorter e.g. Miltenyi Biotec
  • at least about 10 5 -10 7 cells/ml e.g. 10 6 cells/ml
  • IMDM Iscove's Modified Dulbecco's Medium
  • SCF stem cell factor
  • TPO thrombopoietin
  • Flt3-ligand Flt3L, e.g. at a dose of about 1-500 ng/ml e.g. 50 ng/ml
  • IL-3 interleukin-3
  • IL-6 interleukin-6
  • Perf+ imDCs are collected at the end of this culture period.
  • the Perf+ imDCs are contacted with a factor which inhibits maturation.
  • Perf+ imDCs are contacted with a factor capable of inhibiting the Perff imDCs from maturing.
  • factors capable of inhibiting the Perf+ imDCs from maturing include e.g. anti-inflammatory agents and/or immunosuppressive agents. Without being bound by theory, these factors inhibit an immune response through different mechanisms e.g. inhibition of NF-kB or inhibition of T-cell mitosis.
  • the anti-inflammatory agent or immunosuppressive agent is an mTOR inhibitor.
  • An exemplary mTOR inhibitor comprises the immunosuppressive agent rapamycin (Sirolimus).
  • immunosuppressive agents which can be used in accordance with some embodiments of the invention include, but are not limited to, methotrexate, cyclophosphamide, cyclosporine, cyclosporin A, chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine), gold salts, D-penicillamine, leflunomide, azathioprine, anakinra, infliximab (REMICADE), etanercept, TNF.alpha. blockers, a biological agent that targets an inflammatory cytokine, and Non-Steroidal Anti-Inflammatory Drug (NSAIDs).
  • methotrexate cyclophosphamide
  • cyclosporine cyclosporin A
  • chloroquine hydroxychloroquine
  • sulfasalazine sulphasalazopyrine
  • gold salts gold salts
  • D-penicillamine le
  • NSAIDs include, but are not limited to acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, tramadol, rapamycin (sirolimus) and rapamycin analogs (such as CCI-779, RAD001, AP23573). These agents may be administered individually or in combination
  • the anti-inflammatory agent or immunosuppressive agent inhibits granulocyte-mediated inflammation.
  • An exemplary agent which inhibits granulocyte-mediated inflammation comprises the anti-inflammatory agent aspirin.
  • anti-inflammatory agents examples include, but are not limited to, Alclofenac; Alclometasone Dipropionate; Algestone Acetonide; Alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride;
  • Clobetasol Propionate Clobetasone Butyrate; Clopirac; Cloticasone Propionate;
  • Etofenamate Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal;
  • Fluquazone Flurbiprofen; Fluretofen; Fluticasone Propionate; Furaprofen; Furobufen;
  • Halcinonide Halobetasol Propionate; Halopredone Acetate; Ibufenac; Ibuprofen;
  • Meclofenamic Acid Meclorisone Dibutyrate
  • Mefenamic Acid Meclorisone Dibutyrate
  • Mefenamic Acid Meclorisone Dibutyrate
  • Mefenamic Acid Meclorisone Dibutyrate
  • Mefenamic Acid Meclorisone Dibutyrate
  • Mefenamic Acid Meclorisone Dibutyrate
  • Mefenamic Acid Meclorisone Dibutyrate
  • Methylprednisolone Suleptanate Momiflumate; Nabumetone; Naproxen;
  • Piroxicam Olamine Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone;
  • Proxazole Proxazole Citrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate;
  • Talmetacin Talniflumate; Talosalate; Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin
  • the anti-inflammatory agent comprises an acetylsalicylic acid (ASA), e.g., AspirinTM, or a heme oxygenase-1 (HO-1).
  • ASA acetylsalicylic acid
  • HO-1 heme oxygenase-1
  • Perf+ imDCs are cultured for about 5-20 days (e.g. 10 days) with aspirin (e.g. at a dose of about 1-25 mM e.g. 2.5 mM), rapamycin (e.g. at a dose of about 1-100 ng/ml e.g. 10 ng/ml) or with Cobalt Protoporphyrin (CoPP), an inducer of HO-1 (e.g. at a dose of about 1-500 ng/ml e.g. 50 niM).
  • aspirin e.g. at a dose of about 1-25 mM e.g. 2.5 mM
  • rapamycin e.g. at a dose of about 1-100 ng/ml e.g. 10 ng/ml
  • CoPP Cobalt Protoporphyrin
  • an inducer of HO-1 e.g. at a dose of about 1-500 ng/ml e.g. 50 niM
  • Contacting the perf+ imDCs with a factor capable of inhibiting the Perf+ imDCs from maturing is typically effected for about 2-40 days (e.g. 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 25 days, 30 days, 35 days or 40 days).
  • contacting the perf imDCs with a factor capable of inhibiting the Perf+ imDCs from maturing is effected for 10 days.
  • contacting the early myeloid cells with a factor capable of differentiating the early myeloid cells into the perff imDCs is effected concomitantly with the factor capable of inhibiting the Perf+ imDCs from maturing (e.g. rapamycin, aspirin or HO-1).
  • this co- culture is effected for about 2-40 days (e.g. 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 25 days, 30 days, 35 days or 40 days).
  • this co-culture is effected for 10 days.
  • the method is effected ex vivo or in vitro (e.g. in a cell culture plate).
  • the method further comprises selecting cells which exhibit the Perf+ imDCs phenotype (as discussed in detail above).
  • At least about 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 99 % or 100 % of the Perf+ imDCs comprise the cell signature (e.g., perforin, CDl lc, MHC class II, CD80 and CD86).
  • at least about 50 % of the Perf+ imDCs comprise the cell signature.
  • the Perff imDCs maintain an immature phenotype
  • the signature for at least about 12 hours to 60 days (e.g. for at least about 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 25 days, 30 days, 45 days, 60 days or more).
  • the Perff imDCs of the present invention may be naive cells (e.g. non-genetically modified) or genetically modified cells (e.g. cells which have been genetically engineered to express or not express specific genes, markers or peptides, such as an antigen or antigens, or to secrete or not secrete specific cytokines). Any method known in the art may be implemented in genetically engineering the cells, such as by inactivation of the relevant gene/s or by insertion of an antisense RNA interfering with polypeptide expression (see e.g.
  • the Perf imDCs of the present invention may be loaded to present short synthetic peptides, protein extracts or purified proteins (e.g. fused or loaded thereto) to immune cells such as T cells or B cells (e.g. for specific tolerogenic activity).
  • Such short peptides, protein extracts or purified proteins may be viral-, bacterial- , fungal-, tumor-, autoimmune- or allergic- antigen derived peptides or peptides representing any other antigen.
  • Dedicated software can be used to analyze viral, bacterial, fungal, tumor, autoimmune or allergic antigen sequences to identify immunogenic short peptides, i.e., peptides presentable in context of major histocompatibility complex (MHC) class I or MHC class II.
  • MHC major histocompatibility complex
  • perf+ imDCs of some embodiments of the present invention may be pulsed with a peptide.
  • the perf+ imDCs may be transfected with a vector encoding a peptide.
  • Such methods are described e.g. in Inzkirweli et al., Anticancer Research (2007) 27: 2121-2130, and in Lesterhuis WJ et al., Anticancer Research (2010) 30(12):5091-7, incorporated herein by reference.
  • an isolated population of cells comprising Perff imDCs generated according to the method of some embodiments of the invention, wherein at least 50 % of the population of cells comprises the Perff imDCs.
  • isolated population of cells refers to cells which have been isolated from their natural environment (e.g., the human body).
  • At least about 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 99 % or 100 % of the population of cells comprise the Perf+ imDCs.
  • at least about 50 % of the population of cells comprises the Perff imDCs.
  • an isolated population of cells comprising at least 50 % Perf+ imDCs, wherein the Perf+ imDCs maintain an immature phenotype for at least about 12 hours to 60 days (e.g.
  • the Perf+ imDCs of some embodiments of the invention can be used in treating an inflammation in a subject.
  • inflammation refers to the general term for accumulation of fluids, plasma proteins, and white blood cells initiated by physical injury, infection, or an immune response. Inflammation may be associated with several signs e.g. redness, pain, heat, swelling and/or loss of function. Inflammation is an aspect of many diseases and disorders, also referred to inflammatory diseases, including but not limited to diseases related to immune disorders, autoimmune diseases, tissue damage, tissue injury, infectious disease (e.g. viral and bacterial infection), arthritis, collagen diseases, allergy, asthma, pollinosis, cancer and atopy (as described in further detail below).
  • diseases related to immune disorders autoimmune diseases, tissue damage, tissue injury, infectious disease (e.g. viral and bacterial infection), arthritis, collagen diseases, allergy, asthma, pollinosis, cancer and atopy (as described in further detail below).
  • the terms "subject” or “subject in need thereof include mammals, specifically human beings at any age or gender.
  • the subject may be healthy or showing preliminary signs of a pathology, e.g. a pathology associated with an inflammation. This term also encompasses individuals who are at risk to develop the pathology (e.g. inflammation).
  • treating refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a disease or disorder (e.g. an inflammation).
  • Inflammatory diseases include chronic inflammatory diseases and acute inflammatory diseases.
  • hypersensitivity examples include, but are not limited to, Type I hypersensitivity, Type II hypersensitivity, Type III hypersensitivity, Type IV hypersensitivity, immediate hypersensitivity, antibody mediated hypersensitivity, immune complex mediated hypersensitivity, T lymphocyte mediated hypersensitivity and DTH.
  • Type I or immediate hypersensitivity such as asthma.
  • Type II hypersensitivity include, but are not limited to, rheumatoid diseases, rheumatoid autoimmune diseases, rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 Jul;15 (3):791), spondylitis, ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res 2001; 3 (3): 189), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998; 17 (l-2):49), sclerosis, systemic sclerosis (Renaudineau Y.
  • vasculitises necrotizing small vessel vasculitises, microscopic polyangiitis, Churg and Strauss syndrome, glomerulonephritis, pauci-immune focal necrotizing glomerulonephritis, crescentic glomerulonephritis (Noel LH. Ann Med Interne (Paris). 2000 May; 151 (3): 178); antiphospholipid syndrome (Flamholz R. et al., J Clin Apheresis 1999; 14 (4): 171); heart failure, agonist-like beta-adrenoceptor antibodies in heart failure (Wallukat G. et al, Am J Cardiol.
  • Type IV or T cell mediated hypersensitivity include, but are not limited to, rheumatoid diseases, rheumatoid arthritis (Tisch R, McDevitt HO. Proc Natl Acad Sci U S A 1994 Jan 18; 91 (2):437), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Datta SK., Lupus 1998; 7 (9):591), glandular diseases, glandular autoimmune diseases, pancreatic diseases, pancreatic autoimmune diseases, Type 1 diabetes (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647); thyroid diseases, autoimmune thyroid diseases, Graves' disease (Sakata S.
  • delayed type hypersensitivity examples include, but are not limited to, contact dermatitis and drug eruption.
  • T lymphocyte mediating hypersensitivity examples include, but are not limited to, helper T lymphocytes and cytotoxic T lymphocytes.
  • helper T lymphocyte-mediated hypersensitivity examples include, but are not limited to, T h l lymphocyte mediated hypersensitivity and T h 2 lymphocyte mediated hypersensitivity.
  • Autoimmune diseases include, but are not limited to, T h l lymphocyte mediated hypersensitivity and T h 2 lymphocyte mediated hypersensitivity.
  • cardiovascular diseases include, but are not limited to, cardiovascular diseases, rheumatoid diseases, glandular diseases, gastrointestinal diseases, cutaneous diseases, hepatic diseases, neurological diseases, muscular diseases, nephric diseases, diseases related to reproduction, connective tissue diseases and systemic diseases.
  • autoimmune cardiovascular diseases include, but are not limited to atherosclerosis (Matsuura E. et al, Lupus. 1998;7 Suppl 2:S 135), myocardial infarction (Vaarala O. Lupus. 1998;7 Suppl 2:S 132), thrombosis (Tincani A. et al, Lupus 1998;7 Suppl 2:S 107-9), Wegener's granulomatosis, Takayasu's arteritis, Kawasaki syndrome (Praprotnik S. et al, Wien Klin Klin Klin Klin Klinschr 2000 Aug 25;112 (15-16):660), anti-factor VIII autoimmune disease (Lacroix-Desmazes S.
  • autoimmune rheumatoid diseases include, but are not limited to rheumatoid arthritis (Krenn V. et al, Histol Histopathol 2000 Jul;15 (3):791; Tisch R, McDevitt HO. Proc Natl Acad Sci units S A 1994 Jan 18;91 (2):437) and ankylosing spondylitis (Jan Voswinkel et al, Arthritis Res 2001; 3 (3): 189).
  • autoimmune glandular diseases include, but are not limited to, pancreatic disease, Type I diabetes, thyroid disease, Graves' disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto's thyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmune anti-sperm infertility, autoimmune prostatitis and Type I autoimmune polyglandular syndrome.
  • Diseases include, but are not limited to autoimmune diseases of the pancreas, Type 1 diabetes (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647; Zimmet P. Diabetes Res Clin Pract 1996 Oct;34 Suppl:S 125), autoimmune thyroid diseases, Graves' disease (Orgiazzi J.
  • autoimmune gastrointestinal diseases include, but are not limited to, chronic inflammatory intestinal diseases (Garcia Herola A. et al, Gastroenterol Hepatol. 2000 Jan; 23 (1): 16), celiac disease (Landau YE. and Shoenfeld Y. Harefuah 2000 Jan 16; 138 (2): 122), colitis, ileitis and Crohn' s disease.
  • autoimmune cutaneous diseases include, but are not limited to, autoimmune bullous skin diseases, such as, but are not limited to, pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.
  • autoimmune hepatic diseases include, but are not limited to, hepatitis, autoimmune chronic active hepatitis (Franco A. et al , Clin Immunol Immunopathol 1990 Mar; 54 (3):382), primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 Nov; 91 (5):551 ; Strassburg CP. et al, Eur J Gastroenterol Hepatol. 1999 Jun; 11 (6):595) and autoimmune hepatitis (Manns MP. J Hepatol 2000 Aug; 33 (2):326).
  • autoimmune neurological diseases include, but are not limited to, multiple sclerosis (Cross AH. et al , J Neuroimmunol 2001 Jan 1 ; 112 (1-2): 1), Alzheimer' s disease (Oron L. et al, J Neural Transm Suppl. 1997;49:77), myasthenia gravis (Infante AJ. And Kraig E, Int Rev Immunol 1999; 18 (l-2):83; Oshima M. et al, Eur J Immunol 1990 Dec;20 (12):2563), neuropathies, motor neuropathies (Kornberg AJ. J Clin Neurosci.
  • autoimmune muscular diseases include, but are not limited to, myositis, autoimmune myositis and primary Sjogren's syndrome (Feist E. et al., Int Arch Allergy Immunol 2000 Sep; 123 (1):92) and smooth muscle autoimmune disease (Zauli D. et al, Biomed Pharmacother 1999 Jun;53 (5-6):234).
  • autoimmune nephric diseases include, but are not limited to, nephritis and autoimmune interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug; 1 (2): 140).
  • autoimmune diseases related to reproduction include, but are not limited to, repeated fetal loss (Tincani A. et al, Lupus 1998; 7 Suppl 2:S 107-9).
  • autoimmune connective tissue diseases include, but are not limited to, ear diseases, autoimmune ear diseases (Yoo TJ. et al, Cell Immunol 1994 Aug; 157 (1):249) and autoimmune diseases of the inner ear (Gloddek B. et al., Ann N Y Acad Sci 1997 Dec 29; 830:266).
  • autoimmune systemic diseases include, but are not limited to, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998; 17 (l-2):49) and systemic sclerosis (Renaudineau Y. et al., Clin Diagn Lab Immunol. 1999 Mar;6 (2): 156); Chan OT. et al, Immunol Rev 1999 Jun; 169: 107).
  • infectious diseases include, but are not limited to, chronic infectious diseases, subacute infectious diseases, acute infectious diseases, viral diseases, bacterial diseases, protozoan diseases, parasitic diseases, fungal diseases, mycoplasma diseases and prion diseases.
  • graft rejection diseases include, but are not limited to, chronic infectious diseases, subacute infectious diseases, acute infectious diseases, viral diseases, bacterial diseases, protozoan diseases, parasitic diseases, fungal diseases, mycoplasma diseases and prion diseases.
  • diseases associated with transplantation of a graft include, but are not limited to, graft rejection, chronic graft rejection, subacute graft rejection, hyper-acute graft rejection, acute graft rejection and graft versus host disease (GVHD).
  • graft rejection chronic graft rejection
  • subacute graft rejection hyper-acute graft rejection
  • acute graft rejection graft versus host disease
  • allergic diseases include, but are not limited to, asthma, hives, urticaria, pollen allergy, dust mite allergy, venom allergy, cosmetics allergy, latex allergy, chemical allergy, drug allergy, insect bite allergy, animal dander allergy, stinging plant allergy, poison ivy allergy and food allergy.
  • asthma hives
  • urticaria pollen allergy
  • dust mite allergy dust mite allergy
  • venom allergy cosmetics allergy
  • latex allergy chemical allergy
  • drug allergy insect bite allergy
  • animal dander allergy stinging plant allergy
  • poison ivy allergy and food allergy.
  • the methods of the invention may be used to treat any injury or damage to a cell, tissue (e.g. soft tissue) or organ which involves an inflammation, including, but not limited to, acute, chronic, ischemic, or traumatic (e.g. such as that associated with a surgery or accident) injury to the skeletal muscle, heart (e.g. cardiac muscle or cardiovascular cell), kidney, liver, intestine, brain, lung, pancreas, vascular, dermal tissue, scalp, or eye as well as ischemia-reperfusion injury (IRI).
  • a cell e.g. soft tissue
  • organ which involves an inflammation
  • acute, chronic, ischemic, or traumatic (e.g. such as that associated with a surgery or accident) injury to the skeletal muscle, heart (e.g. cardiac muscle or cardiovascular cell), kidney, liver, intestine, brain, lung, pancreas, vascular, dermal tissue, scalp, or eye as well as ischemia-reperfusion injury (IRI).
  • IRI ischemia-reperfusion injury
  • the inflammatory condition is associated with an autoimmune disease (e.g. multiple sclerosis).
  • an autoimmune disease e.g. multiple sclerosis.
  • the inflammatory condition is associated with diabetes, metabolic syndrome, and related diseases and conditions.
  • Diabetes is typically characterized by elevated levels of plasma glucose (hyperglycemia) in the fasting state.
  • type 1 diabetes in which patients produce little or no insulin
  • type 2 diabetes in which patients produce "insulin resistance" such that the effect of insulin in stimulating glucose and lipid metabolism in the main insulin-sensitive tissues, namely, muscle, liver, and adipose tissues, is diminished.
  • Abnormal glucose homeostasis is associated both directly and indirectly with obesity, hypertension, and alterations in lipid, lipoprotein, and apolipoprotein metabolism.
  • Patients with diabetes are at increased risk of cardiovascular complications, e.g., atherosclerosis, coronary heart disease, stroke, peripheral vascular disease, hypertension, nephropathy, neuropathy and retinopathy.
  • Persistent or uncontrolled hyperglycemia that occurs in diabetes is also associated with increased morbidity and premature mortality.
  • Metabolic Syndrome is typically characterized by insulin resistance, along with abdominal obesity, hyperinsulinemia, high blood pressure, low HDL levels, high VLDL triglyceride and small dense LDL particles and elevated glucose levels. Subjects having metabolic syndrome, whether or not they develop overt diabetes mellitus, are at increased risk of developing cardiovascular complications (as discussed above).
  • the inflammation is not associated with a cancer.
  • the Perf+ imDC of some embodiments of the invention can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
  • a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • active ingredient refers to the Perf+ imDC accountable for the biological effect.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • neurosurgical strategies e.g., intracerebral injection or intracerebroventricular infusion
  • molecular manipulation of the agent e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB
  • pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers)
  • the transitory disruption of the integrity of the BBB by hyperosmotic disruption resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide).
  • each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.
  • tissue refers to part of an organism consisting of cells designed to perform a function or functions. Examples include, but are not limited to, brain tissue, retina, skin tissue, hepatic tissue, pancreatic tissue, bone, cartilage, connective tissue, blood tissue, muscle tissue, cardiac tissue brain tissue, vascular tissue, renal tissue, pulmonary tissue, gonadal tissue, hematopoietic tissue.
  • the route of administration includes, for example, an injection, ingestion, transfusion, implantation or transplantation.
  • the compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
  • the pharmaceutical composition of the present invention is administered to a patient by intradermal or subcutaneous injection.
  • the pharmaceutical composition of the present invention is administered by i.v. injection.
  • the pharmaceutical composition may be injected directly into a site of inflammation.
  • compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • compositions of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (Perf imDCs) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., inflammation) or prolong the survival of the subject being treated.
  • a therapeutically effective amount means an amount of active ingredients (Perf imDCs) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., inflammation) or prolong the survival of the subject being treated.
  • compositions of the present invention can be administered at a dosage of 5 x 10 4 /kg body weight to 1 x 108 /kg body weight, including all integer values within those ranges.
  • the Perf+ imDCs described herein may be administered at a dosage of about 5 x 10 4 /kg body weight to 1 x 108 /kg body weight, 5 x 10 4 /kg body weight to 1 x 10 7 /kg body weight, 5 x 10 4 /kg body weight to 1 x 10 6 /kg body weight, 5 x 10 4 /kg body weight to 1 x 10 5 /kg body weight, 5 x 10 5 /kg body weight to 1 x
  • the Perf+ imDCs described herein may be administered at a dosage of about 1 x 10 4 /kg body weight, 2.5 x 10 4 /kg body weight, 5 x 10 4 /kg body weight, 1 x 10 5 /kg body weight, 2.5 x 10 5 /kg body weight, 5 x 10 5 /kg body weight, 1 x 10 6 /kg body weight, 2.5 x 10 6 /kg body weight, 5 x 10 6 /kg body weight, 1 x 10 7 /kg body weight, 2.5 x 10 7 /kg body weight, 5 x 10 7 /kg body weight, 1 x 10 8 /kg body weight, 2.5 x 10 8 /kg body weight, 5 x 108 /kg body weight or 1 x 109 /kg body weight.
  • the cell compositions of some embodiments of the invention may also be administered multiple times at these dosages.
  • the cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).
  • the optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.
  • the effect of the active ingredients (e.g., the Perf+ imDCs of some embodiments of the invention) on the pathology can be evaluated by monitoring the level of inflammatory markers, e.g., hormones, glucose, peptides, carbohydrates, etc. in a biological sample of the treated subject using well known methods (e.g. standard blood tests, ELISA, FACS, PCR, etc).
  • active ingredients e.g., the Perf+ imDCs of some embodiments of the invention
  • the level of inflammatory markers e.g., hormones, glucose, peptides, carbohydrates, etc.
  • well known methods e.g. standard blood tests, ELISA, FACS, PCR, etc.
  • the therapeutically effective amount the Perf+ imDCs is an amount capable of inhibiting an activity or proliferation of a CD4 + T cell and/or a CD8 + T cell.
  • Such determinations are well known to one of skill in the art, and may be carried out, for example, by obtaining a blood sample from a subject and determining the activity and/or proliferating levels of CD4 + T cell and/or a CD8 + T cell in the lymphocyte fraction of the blood sample.
  • T cell evaluation may comprise any of the following: quantitative details of specific types of T cells as well as evaluation of T cell characteristics of these cells may be carried out, for example, by complete blood count (CBC) and differential blood count, flow cytometry (FACS), TREC testing and other measures to characterize recent thymic emigrants, and assessment of TCR diversity; functional testing including, for example, lymphocyte proliferation to mitogens, antigens, and/or allogeneic cells; cytokine production; T cell-mediated cytotoxicity and Treg activity, as taught by Rosenzweig and Fleisher, J Allergy Clin Immunol. (2013) 131(2): 622-3.el-4.
  • the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
  • a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).
  • Dosage amount and interval may be adjusted individually to provide the active ingredient at a sufficient amount to induce or suppress the biological effect (minimal effective concentration, MEC).
  • MEC minimum effective concentration
  • the MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • the amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • the Perf imDCs of the invention can be provided to the subject with a factor capable of inhibiting the Perf+ imDCs from entering maturation.
  • factors are described in detail herein above, and include e.g. rapamycin, aspirin or HO-1.
  • the factor capable of inhibiting the Perf+ imDCs from entering maturation is administered to the subject prior to administration of the Perf+ imDCs, e.g. 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days or more prior to administration of the Perf+ imDCs.
  • the factor capable of inhibiting the Perf+ imDCs from entering maturation is administered to the subject in conjunction to administration of the Perf+ imDCs, e.g. at the same time, or within 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours.
  • the factor capable of inhibiting the Perf+ imDCs from entering maturation is administered to the subject following administration of the Perff imDCs, e.g. 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days or more following administration of the Perf+ imDCs.
  • Such administrations may be effected in a single dose or in several doses. Such a determination is well within the capability of one of skill in the art.
  • the Perff imDCs of the invention can be provided to the subject in conjunction with other drug(s) designed for treating the pathology [combination therapy, (e.g., before, simultaneously or following)].
  • the Perff imDCs of some embodiments of the invention are administered to a patient in conjunction with any number of relevant treatment modalities, including but not limited to, treatment with agents such as antiviral agents (e.g. Ganciclovir, Valaciclovir, Acyclovir, Valganciclovir, Foscarnet, Cidofovir, Maribavir, Leflunomide); agents for the treatment of multiple sclerosis (e.g. natalizumab); agents for the treatment of metabolic syndrome or diabetes (e.g. insulin), or anti-inflammatory therapies (e.g. NSAIDs (Non- Steroidal Antiinflammatory Drugs), corticosteroids (such as prednisone) and anti-histamines).
  • agents such as antiviral agents (e.g. Ganciclovir, Valaciclovir, Acyclovir, Valganciclovir, Foscarnet, Cidofovir, Maribavir, Leflunomide); agents for the treatment of multiple sclerosis (e.g. natalizuma
  • compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration.
  • compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
  • agents described hereinabove may be included in a therapeutic kit/article of manufacture preferably along with appropriate instructions for use and labels indicating FDA approval for use in treatment of inflammatory conditions (e.g. metabolic syndrome or autoimmune disease).
  • inflammatory conditions e.g. metabolic syndrome or autoimmune disease.
  • kit/article of manufacture comprising Perf imDCs.
  • kit/article of manufacture may further comprise another active ingredient to improve therapeutic efficacy.
  • kits/article of manufacture comprising Perf+ imDCs and a factor capable of inhibiting the Perff imDCs from entering maturation (e.g. rapamycin, aspirin or HO-1).
  • a factor capable of inhibiting the Perff imDCs from entering maturation e.g. rapamycin, aspirin or HO-1.
  • kits/article of manufacture comprising Perff imDCs and a drug designed for treating the pathology (e.g. inflammation associated with an autoimmune disease, metabolic syndrome, infection, etc.), such a drug may include for example, an antiviral agent, an agent for the treatment of multiple sclerosis, an agent for the treatment of metabolic syndrome or diabetes, or an anti-inflammatory agent (as discussed in detail hereinabove).
  • a drug designed for treating the pathology e.g. inflammation associated with an autoimmune disease, metabolic syndrome, infection, etc.
  • a drug may include for example, an antiviral agent, an agent for the treatment of multiple sclerosis, an agent for the treatment of metabolic syndrome or diabetes, or an anti-inflammatory agent (as discussed in detail hereinabove).
  • the Perf+ imDCs can be packaged in one container while the factor capable of inhibiting the Perff imDCs from entering maturation may be packaged in a second container both for therapeutic treatment.
  • the Perf imDCs and the factor capable of inhibiting the Perf+ imDCs from entering maturation are in a co-formulation.
  • the Perf+ imDCs can be packaged in one container while the anti-inflammatory agent may be packaged in a second container both for therapeutic treatment.
  • the Perf+ imDCs and the anti-inflammatory agent are in a co-formulation.
  • the kit/article of manufacture may also include appropriate buffers and preservatives for improving the shelf-life of the kit.
  • the method may be effected using Perf+ imDCs which are syngeneic or non-syngeneic with the subject.
  • syngeneic refers to cells which are derived from an individual who is essentially genetically identical with the subject. Typically, essentially fully inbred mammals, mammalian clones, or homozygotic twin mammals are syngeneic.
  • syngeneic cells include cells derived from the subject (also referred to in the art as “autologous”), a clone of the subject, or a homozygotic twin of the subject.
  • non-syngeneic refers to cells which are derived from an individual who is allogeneic or xenogeneic with the subject's lymphocytes (also referred to in the art as “non-autologous”).
  • allogeneic refers to cells which are derived from a donor who is of the same species as the subject, but which is substantially non-clonal with the subject. Typically, outbred, non-zygotic twin mammals of the same species are allogeneic with each other. It will be appreciated that an allogeneic donor may be HLA identical or HLA non-identical with respect to the subject.
  • xenogeneic refers to cells which substantially express antigens of a different species relative to the species of a substantial proportion of the lymphocytes of the subject. Typically, outbred mammals of different species are xenogeneic with each other.
  • xenogeneic cells are derived from a variety of species such as, but not limited to, bovines (e.g., cow), equids (e.g., horse), porcines (e.g. pig), ovids (e.g., goat, sheep), felines (e.g., Felis domestica), canines (e.g., Canis domestica), rodents (e.g., mouse, rat, rabbit, guinea pig, gerbil, hamster) or primates (e.g., chimpanzee, rhesus monkey, macaque monkey, marmoset).
  • Cells of xenogeneic origin e.g. porcine origin
  • human-derived cells are preferably obtained from substantially pathogen-free sources.
  • the cells of the present invention may be obtained from a prenatal organism, postnatal organism, an adult or a cadaver donor. Moreover, depending on the application needed, the cells may be naive or genetically modified. Such determinations are well within the ability of one of ordinary skill in the art
  • the Perf+ imDCs are syngeneic with the subject (e.g. obtained from the subject).
  • the Perf imDCs are non- syngeneic with the subject.
  • both the subject and the donor of the Perf+ DCs are humans.
  • the subject may be further administered an immunosuppressive regimen in order to reduce rejection of the transplanted Perf+ DCs.
  • an immunosuppressive regimen may be determined by one of ordinary skill in the art and takes into account the age and disease severity of the subject. Thus, for example, an elderly subject (e.g. one who is over 60 years of age) may be treated with a mild immunosuppressive regimen.
  • immunosuppressive regimens include administration of immunosuppressive drugs and/or immunosuppressive irradiation.
  • the immunosuppressive regimen consists of administering at least one immunosuppressant agent to the subject.
  • immunosuppressive agents are described in detail hereinabove. Any of the immunosuppressive agents described herein may be administered individually or in combination.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
  • the phrases "ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
  • the term "method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • any Sequence Identification Number can refer to either a DNA sequence or a RNA sequence, depending on the context where that SEQ ID NO is mentioned, even if that SEQ ID NO is expressed only in a DNA sequence format or a RNA sequence format.
  • RNA sequence format e.g. , reciting U for uracil
  • it can refer to either the sequence of a RNA molecule comprising a dsRNA, or the sequence of a DNA molecule that corresponds to the RNA sequence shown. In any event, both DNA and RNA molecules having the sequences disclosed with any substitutes are envisioned.
  • mice used were 6-12 week old females or males (in the metabolic studies) of the following strains: C57BL/6, perforin KO (PKO) (H-2 db /I-A b ) 1 ,CDl lc-eYFP 2 (Weizmann Institute Animal Breeding Center, Israel).
  • CDllc-Cre mice [previously disclosed in Caton M.L. et al., J. Exp. Med. (2007) 204, 1653-1664]
  • R26-DTA mice backcrossed for ten generations onto C57BL/6) [previously disclosed in Brockschnieder D. et al., Genesis (2006) 44, 322-327].
  • R26-DTA mice were crossed with CDl lc-Cre transgenic mice to generate CD l lc-Cre:DTA mice.
  • Mixed [50 % DTA-50 % PKO > WT], [50 % WT-50 % PKO > WT], BM chimeras were generated by exposure of C57BL/6 WT mice to a single dose of 950 rad total body irradiation. The following day, the mice received i.v. 5 x 10 6 mixed BM cells, as indicated. The mice were allowed to rest for 6 weeks before use. Animals were maintained under conditions approved by the Institutional Animal Care and Use Committee at the Weizmann Institute of Science.
  • mice were maintained on high-fat diet (HFD, D 12492, 60 Kcal % fat, Research Diets), the control group was fed either a standard chow diet or low fat sucrose-matched diet (NCD, D12450J, 10 Kcal % fat, Research Diets).
  • HFD high-fat diet
  • NCD low fat sucrose-matched diet
  • mice DTA-PKO and WT-PKO chimeric mice were weighed regularly. For glucose tolerance tests, fasted (16 hours) mice were given 1 g glucose per kg body weight; for insulin tolerance test, 0.75 U per kg body weight human insulin was used (NovoNordisk, Denmark). Glucose, triglycerides, total cholesterol and high density lipoprotein (HDL) levels were measured in mouse serum by SpotChem EZ Chemistry Analyzer (Arkray, Japan). Serum cytokine concentrations were detected using ELISA kits (TNF-a, IL-6 and IL- ⁇ from eBioscience, leptin from ENZO). Body composition analysis was performed using EchoMRI Analyzer (EchoMRI LLC, USA)
  • Flow cytometry was performed using a FACS-CantoII (BD Biosciences) and the analysis was done by FlowJo (Tree Star, Inc.) software.
  • Single cell suspensions were stained with anti-CD8a, CD4, CD3, MHC-II (IA/IE), CD45, CDl lc, CDl lb, Ly6G, Gr-1, B220, CD19, F4/80, CD45.1, CD25, Foxp-3, TNF-a, IL-17a, or IFN- ⁇ conjugated to FITC, PE, PerCp, APC, APC-Cy7, PE-Cy7, Pacific Blue, or Brilliant Violet 711 (Biolegend).
  • mice were systemically perfused with PBS, visceral AT (VAT) and inguinal SC pads were removed (avoiding lymph nodes), mashed into small pieces (approximately 1-2 mm) and digested with collagenase II (Sigma, 1 mg/ml in DMEM for 20 minutes at 37 °C, with vigorous agitation). The digests were then pelleted and filtered through 100 ⁇ filters. The cells were washed with PBS, incubated for 5 minutes in erythrocyte-lysing buffer (ACK), and finally resuspended in PBS supplemented with 2 % FBS. The nonspecific staining was blocked by antibody against FC receptor (Biolegend), and then the isolated cells were labeled with either monoclonal antibody or isotype control antibody (Biolegend). 7AAD was used to exclude dead cells.
  • FC receptor Biolegend
  • Tissue samples from spinal cord were fixed in 4 % PFA, processed and embedded in paraffin. Sections of 6 ⁇ were stained with hematoxylin and eosin (H&E). Tissue samples from visceral adipose tissue were fixed in 4 % PFA, processed and embedded in paraffin. Sections of 5 ⁇ were stained with hematoxylin and eosin (H&E). Fat cell area on H&E stained slides was measured using ImageJ software, quantifying two different tissue sections per mouse (at least 3 mice with at least 100 fat cells in each image).
  • MAC-2 antibody (Cedarlane); MAC-2 positive CLS per field were counted manually in a blinded manner and used as a measure of adipose tissue CLS content.
  • splenic DCs from WT and PKO mice were isolated by two stage magnetic sorting (first negative depletion of CD3, Terl 19, Gr-1, B220 positive cells and then positive selection of CDl lc + cells) and were spun onto glass slides and fixed with 4 % PFA. The cells were then blocked first with 5 % horse serum and 0.2 % Triton for 5 minutes, and subsequently with 100 % horse serum for 1 hour.
  • Isotype blocking was done with rat IgG2a overnight in 25 % horse serum and 0.1 % Triton at 4 °C followed by secondary anti-RatCy2 or anti-Rat DyLight594 (Jackson) antibody to visualize the non-specific binding.
  • Specific rat anti -perforin Ab (Clone CB5.4, Abeam) was applied overnight in 25 % horse serum and 0.1 % Triton at 4 °C followed by secondary anti-rat DyLight 594 or anti-rat AMCA (Jackson) antibody.
  • the nuclei were stained with Hoechst blue or yellow stain.
  • Cytotoxic T-lymphocytes (CTL) from C57BL/6 WT and PKO were prepared as previously described [Reich-Zeliger S.
  • CD8-FITC (53-6.7) were used (Biolegend).
  • Anti-CD4 (hybridoma GK1.5) or CD8 (hybridoma 53-6.72) (BioXcell) depleting antibodies were given i.p. weekly at a dose of 3 ⁇ g/gram body weight to chimeric mice starting 4 months post-transplant before the appearance of metabolic syndrome symptoms.
  • the control mice received weekly i.p. injections of PBS.
  • Each ⁇ primer was linked to a restriction-site sequence for the ACUI restriction enzyme (New England BioLabs). PCR reactions were performed in duplicate, and PCR products were then pooled and cleaned using the QIAquick PCR Purification Kit (Qiagen), followed by enzymatic digestion, in accordance with the ACUI protocol (New England BioLabs).
  • the ACUI enzyme was used to cleave the 14 bp downstream of its binding site, enabling positioning of the Illumina sequencing primer in close proximity to the junction region and ensuring sequencing of the entire variable CDR3 region. Digestion produced a 2-bp overhang for the ligation of the Illumina 5' adapter, which was linked to a 3-bp barcode sequence at its 3' end.
  • the Smith-Waterman alignment algorithm was used to assign to each sequencing read its variable ( ⁇ ) and joining ( ⁇ ) gene, using germ-line ⁇ / ⁇ gene segment sequences downloaded from the IMGT database. Reads that were not assigned to either a ⁇ or ⁇ , and other erroneous reads were discarded. The library- derived reads were then clustered using a version of the quality threshold clustering algorithm to correct for nucleotide copying errors (up to two errors for each read). The clustering procedure identified unique CDR3 ⁇ clonotypes, defined as the most prevalent read found in each cluster.
  • the clonotype sequences were then translated, and those clonotypes that lacked a stop codon in-frame with the V/D/J sequences were considered for further analysis.
  • This analysis computed statistics of V/D/J use, statistical properties of the number of deletions and insertions of nucleotides at both VD and DJ junctions, as well as distributions of CDR3 lengths. The analysis was done using the R statistical software package (R Development Core Team; www(dot)r-project(dot)org). Frequencies of ⁇ and ⁇ segment use were measured for all samples in each treatment group. Correlation coefficients were calculated, based on the sample mean of combined ⁇ and ⁇ use in each group, using MATLAB (Mathworks). Hierarchical clustering was performed, based on combined ⁇ and ⁇ use, in all groups (clustergram; MATLAB).
  • RNA from fat pads was extracted with the RNeasy lipid tissue mini kit (Qiagen, Germantown, MD) and analyzed with Nanodrop ⁇ .
  • RNA 100 ng was reverse- transcribed with qScript cDNA Synthesis Kit (Quanta Biosciences). SYBR Green system (Roche) was used for real-time PCR amplification. Data were normalized based on TBP expression as housekeeping gene, using appropriate primers (see Table 1, below).
  • Table 1 Primers used for RT-PCR
  • Chronic EAE was induced in C57BL/6 chimeric mice by injecting a peptide consisting of amino acids 35-55 of myelin oligodendrocyte glycoprotein (MOG), synthesis by Genscript (Piscataway, NJ, USA). Mice were injected subcutaneously at the flank, with 200 ⁇ emulsion containing 200 ⁇ g of the encephalitogenic peptide in incomplete Freund's adjuvant enriched with 5 mg/ml heat-inactivated Mycobacterium tuberculosis (Sigma, St. Louis, MO, USA). Mice were examined daily.
  • MOG myelin oligodendrocyte glycoprotein
  • EAE was scored as follows: 0 - no disease, 1 - limp tail, 2 - hind limb paralysis, 3 - paralysis of all limbs, 4 - moribund condition, and 5 - death, as previously described [Aharoni R. et al. Exp. Neurol. (2013) 240, 130-144].
  • Isolated lymph node cells from chimeric mice 14 or 30 days after EAE induction were isolated and stained with 5 ⁇ CFSE (CellTrace CFSE Cell Proliferation Kit, Invitrogen) for 15 minutes. After staining, 0.5 x 10 6 cells were incubated in full RPMI medium supplemented with 10 % FCS for 4 days in the presence of either MOG, PPD (adjuvant related peptide) or MBP (unrelated encephalitogenic peptide) peptides. The cells were then harvested and analyzed by flow cytometry for CFSE staining in conjunction with either CD4 or CD8 staining to examine the proliferation status based on CFSE dilution.
  • CFSE CellTrace CFSE Cell Proliferation Kit, Invitrogen
  • Perf imDCs were generated using the 20 days protocol as previously described [Zangi L. et al., Blood. (2012) 120(8): 1647-57].
  • bone marrow cells were obtained from tibiae and femurs of C57BL/6, CB6/F1, FVB, gld-/-, PKO, TLR7-/- and Dap 12-/- mice.
  • the cell suspension was enriched for the SCA1+ population using anti-SCAl-PE antibody (clone E13-161.7, Miltenyi Biotec), followed by incubation with anti-PE MicroBeads and positive selection on a MACS separation column (Miltenyi Biotec).
  • the cells were then stained for lineage positive markers with the following biotinylated Abs: CD3e (clone 145-2C11), CDl lb (clone Ml/70), CD45R/B220 (clone RA3-6B2), Ly6G/ Ly-6C (clone RB6-8C5), and TER-119 (clone TER-119) (BD Pharmingen) followed by staining with streptavidin-APC (Jackson Immunoresearch Laboratories). SCA-1+ Lin-, C- Kit+ cells were then sorted using FACSAria Sorter.
  • IMDM Iscove's Modified Dulbecco's Medium
  • FCS 10 % FCS
  • 2 mM L-glutamine 100 U/ml penicillin and 0.1 mg/ml streptomycin
  • Cultures were supplemented every second day with 50 ng/ml stem cell factor (SCF), 1 ng/ml thrombopoietin (TPO), 50 ng/ml Flt3-ligand (Flt3L), 10 ng/ml interleukin- 3 (IL-3), and 10 ng/ml interleukin-6 (IL-6) (Peprotech).
  • SCF stem cell factor
  • TPO 1 ng/ml thrombopoietin
  • Flt3-ligand Flt3-ligand
  • IL-3 10 ng/ml interleukin- 3
  • IL-6 10 ng/ml interleukin-6
  • differentiated cells (early myeloid cells) were cultured for an additional 10 days in RPMI 1640 complete tissue culture medium (CTMC), 20 ng/ml granulocyte-macrophage colony- stimulating factor (GM-CSF) (Peprotech). imDCs were collected after 20 days.
  • CMC complete tissue culture medium
  • GM-CSF granulocyte-macrophage colony- stimulating factor
  • Perf imDCs were generated using the 20 days protocol as described above.
  • One group of cells was additionally treated with 2.5 mM aspirin during the last 10 days of culture.
  • a second group of cells was additionally treated with 10 ng/ml Rapamycin during the last 10 days of culture.
  • a third group of cells was additionally treated with 50 mM CoPP / SnPP (an inducer and an inhibitor of HO-1, respectively) during the last 10 days of culture.
  • a fourth group was not additionally treated. To induce maturation, cells were treated with 1 ⁇ g/ml LPS for 24 hours.
  • Aspirin-treated Perf+ imDCs were loaded with SIINFEKL peptide (SEQ ID NO: 29) or with SIYRYYGL peptide (SEQ ID NO: 30). These cells were tested for their ability to kill cognate OT-I CD8 + T cells in short-term mixed lymphocyte reaction (MLR).
  • MLR short-term mixed lymphocyte reaction
  • results are expressed as means + SD or S.E.M, as indicated in each figure legend.
  • the statistical significance of differences between two groups was determined using Student's t-tests; values of p ⁇ 0.05 were considered significant.
  • Perf-DC also referred to herein as Perforin positive immature DCs
  • WT wild type C57BL/6 mice
  • Figure 8a This rare subpopulation of perf-DCs is markedly enhanced upon in vivo administration of GM-CSF [Zangi et al. (2012), supra], and to a lesser extent, upon treatment with Flt3L ( Figure 8b and 8c). Further immuno staining analysis indicated that this subpopulation does not express CD4 or CD8 consistent with double negative (DN) DCs ( Figure 9).
  • mice were generated which selectively lack perforin expression in CDl lc hlgh DC. Chronic selective ablation of these cells can be attained in transgenic mice expressing the diphtheria toxin (DTx) A subunit (DTA) under control of a CDl lc promoter (CDl lc Cre :R26-STOP-DTA mice as previously taught [Birnberg T. et al., Immunity (2008) 29, 986-997]. Of note, most macrophages, NK cells and pDC, expressing low to intermediate levels of CDl lc are spared in these mice [Birnberg et al., (2008), supra].
  • DTx diphtheria toxin
  • DTA diphtheria toxin
  • CDl lc Cre CDl lc Cre :R26-STOP-DTA mice
  • BM bone marrow
  • DTA-PKO chimeric mice spontaneously gained significantly more weight compared to their WT-PKO counterparts, starting at approximately 5 months post-transplant ( Figures 2a-b).
  • WT-DTA mixed BM from WT and DTA donors
  • Figures l la-d The increased weight gain observed in DTA-PKO mice prompted the present inventors to test whether this phenomenon was accompanied by metabolic alterations.
  • DTA-PKO mice manifested by elevated serum cholesterol and triglyceride levels in comparison to those exhibited by the controls ( Figure 2c).
  • percent of total body fat was significantly elevated in DTA-PKO mice compared to both control groups ( Figure 2f).
  • DTA-PKO mice, but not the control chimeras displayed highly elevated levels of TNF-a (which has been linked to obesity-associated inflammation and insulin resistance) and leptin, the proinflammatory adipokine associated with obesity, overeating, hypertension, cardiovascular diseases and metabolic syndrome ( Figures 2d-e).
  • DTA-PKO chimeric mice exhibited a decreased ability to handle glucose challenge (assessed by glucose-tolerance test; GTT) ( Figure 3a), as well as reduced insulin sensitivity (determined by insulin-tolerance test; ITT) ( Figure 3b).
  • GTT glucose-tolerance test
  • ITT insulin-tolerance test
  • adipose tissue (AT) expansion occurs by enlargement of the fat pad mass through enhanced recruitment of adipocyte precursor cells that differentiate into small adipocytes, along with the recruitment of other stromal cell types. Subsequently, vascularization, minimal induction of ECM and minimal inflammation occur.
  • pathological expansion of AT is characterized by rapid growth of the fat pad through enlargement of existing fat cells, a high degree of macrophage infiltration, limited vessel development, and massive fibrosis. Such pathological expansion is associated with chronic inflammation, which ultimately results in the development of systemic insulin resistance.
  • the observed metabolic syndrome in DTA-PKO chimeras indicated that pathological AT expansion might also occur in these mice.
  • adipocytes in the AT tissue of DTA-PKO chimera were significantly larger, less organized and more loosely packed compared to adipocytes in WT-PKO control chimeras.
  • the visceral AT of DTA-PKO chimeras contained significantly more 'crownlike' structures (CLS), which are formed when macrophages within inflamed AT cluster around dead adipocytes ( Figures 3c-e).
  • CD4 and CD8 T cells are required for the development of metabolic syndrome in
  • the present inventors initially analyzed immune cell populations in collagenase-digested stromal vascular fractions (SVF) from epididymal adipose tissue, as previously described [Brake D.K. et al., Am. J. Physiol. Cell. Physiol. (2006) 291, C 1232-9]. The gating strategy for the identification and quantification of these cell subpopulations is shown in Figure 12.
  • SVF collagenase-digested stromal vascular fractions
  • the present inventors further investigated the potential roles of CD4 + and CD8 + T cells, by ablating these cell subpopulations prior to onset of disease in the DTA-PKO chimera.
  • a developing inflammatory response was detected within the AT tissue based on cell composition (i.e. expansion of CD4 + and CD8 + T cells) and serum markers (Leptin, TNF-a, ILl- ⁇ ) starting at 5 months post-transplant, weekly i.p. injections were administered of anti-CD4 antibodies, anti-CD8 antibodies or both, starting at 1 month prior to development of the earliest inflammatory signs (4 month post BM transplant).
  • TCR repertoire from AT tissue of DTA-PKO and WT-PKO chimeras at 6 months post-transplant was analyzed by high-throughput TCR sequencing.
  • the TCRP CDR3 region of T cells was sequenced and was compared the repertoire found in spleen, visceral AT (VAT), and subcutaneous AT (SC-AT) of DTA-PKO and WT-PKO chimeras.
  • the level of skewing in the frequencies of observed TCR sequences was evaluated.
  • a more diverse repertoire consists of sequences that are found at similar frequencies, whereas in a skewed repertoire, a small number of sequences are dominant and are found at higher frequencies than the others.
  • the level of skewing of a repertoire can be evaluated by the deviation of its Lorentz curve from the diagonal; such deviation is larger for more skewed repertoires.
  • the present inventors started by comparing the repertoires of T cells that are resident within AT to the repertoire found in the spleen of WT-PKO animals. Consistent with previous observations of a restricted TCR repertoire within adipose tissues [Yang H. et al., J. Immunol.
  • the present inventors observed a higher level of skewing in the repertoires of T cells derived from VAT and SC-AT compared with splenic T cells of WT- PKO mice ( Figure 5a).
  • specific ⁇ segments were identified that are enriched in the repertoires derived from AT compared with the splenic repertoire.
  • the present inventors compared the TCR repertoires between WT-PKO and DTA-PKO. As shown in Figure 5 a, while there was no difference in the spleens of these chimera, the TCR repertoire observed in the AT tissues derived from DTA-PKO mice was less skewed compared to WT-PKO mice (i.e. the Lorentz curve was closer to the diagonal).
  • the TCR repertoire in AT of the WT-PKO chimera is dominated by expansion of a small number of clonotypes, whereas the DTA-PKO AT repertoire is less skewed and contains a greater number of distinct T cell clones found at intermediate levels.
  • RDA redundancy analysis
  • TCR sequences were selected with the highest contribution to the separation between the experimental groups, resulting in a list of signature sequences that are found at high levels in one group but not in the other. This analysis was able to separate between repertoires of DTA-PKO vs. WT-PKO along the first RDA axis (RDA1), and between most spleen samples vs. VAT samples along RDA2 ( Figure 5b).
  • RDA1 first RDA axis
  • Figure 5b the present inventors identified CDR3 sequences that were most significantly enriched in repertoires of DTA-PKO mice compared with WT-PKO, by RDA analysis.
  • TCR sequences that are consistently up-regulated across DTA-PKO animals, suggesting their potential role in the observed phenotype.
  • many of these TCRs are encoded by different nucleotide sequences in different animals (data not shown), which is an indication of clonal selection driven by TCR specificity.
  • These TCR sequences which exist in the DTA-PKO animals but not in the WT-PKO, may be mechanistically related to the metabolic phenotype.
  • PKO-DTA chimeras exhibit enhanced development of obesity in response to a high fat diet
  • mice lacking Perf-DC respond to excess adiposity used a classical model of obesity, in which mice are chronically fed a high-fat diet (HFD).
  • HFD high-fat diet
  • normal WT C57BL/6 mice develop glucose intolerance and insulin resistance by the 12 th week, with elevated levels of pro-inflammatory cytokines and adipokines.
  • DTA-PKO mice have a tendency to spontaneously develop these symptoms, the present inventors chose to follow the dynamics of metabolic and functional changes at early time points.
  • DTA-PKO mice gained weight much earlier than WT-PKO chimeras when fed HFD, with higher percentage of body fat, increased liver weight and epididymal fat pad weight ( Figures 6c- e). Furthermore, the levels of leptin were significantly higher as early as 1 month after initiation of HFD. At 1.5 months from initiation of feeding, levels of insulin and both IL- 1 ⁇ and TNF-a were elevated in DTA-PKO mice ( Figures 6f-h and 61).
  • T cell subsets from spleen of DTA-PKO or WT-PKO mice fed either NCD for 7 months, or HFD for 8 weeks were defined by various markers including CD3, CD8, CD4, CD44, CD62L, CD25 and Foxp3, and analyzed by FACS.
  • T cell subsets from chimera were also stained for CD45.1 to identify cell origin from CD1 lc-DTA or littermate control hosts, versus CD45.2 expressed by PKO donors.
  • the present inventors showed that selective deletion of perforin in the rare subpopulation of Perf-DC leads to a distinct metabolic phenotype, and that this phenotype can be prevented by T cell depletion in-vivo. Collectively, this strongly indicates a role for Perf-DC in the control of otherwise deleterious T cell-mediated inflammatory processes.
  • the present inventors extended the study to evaluate whether Perf-DC may also exhibit a regulatory role in a well-defined mouse model for autoimmunity, in which the pathological antigen is known.
  • DTA-PKO chimera are more susceptible to development of EAE, a T-cell mediated disorder widely used as a model of multiple sclerosis [Aharoni, (2013), supra].
  • EAE a T-cell mediated disorder widely used as a model of multiple sclerosis
  • MOG-induced EAE results in a chronic persistent disease course.
  • Aspirin the most common analgesic and anti-inflammatory substance, was shown to also have a broad spectrum of pharmacological actions, including the inhibition of NF- KB and other molecular pathways of inflammation.
  • the cellular targets of Aspirin in the immune system are poorly understood.
  • Aspirin added to DCs cultures was shown to inhibit DCs maturation in a dose-dependent manner, promote relative increase in the numbers of CDl lc + DCs and inhibit the DCs stimulatory activity on allogeneic T cells [Hackstein H. et al, J Immunol (2001) 166:7053-7062].
  • HO- 1 is an intracellular enzyme that degrades heme and inhibits immune responses and inflammation in-vivo.
  • DCs express HO-1 and that this expression is downregulated with maturation led to the assumption that the induction of the expression of HO-1 in DCs may inhibit their maturation.
  • CoPP Cobalt Protoporphyrin
  • the present inventors modified the 20 days Perf+ imDCs generation protocol by supplementing the cultures with 10 ng/ml Rapamycin from day 10 of the culture, 2.5 mM Aspirin from day 10 of the culture or 50 mM CoPP / SnPP (an inducer and an inhibitor of HO-1, respectively) for 2 hours.
  • the treated cells were collected and checked for their phenotype before and after treatment with 1 ⁇ g/ml LPS for 24 hours.
  • treated cells were checked for their ability to kill cognate OT-I CD8 + T cells in short-term MLR.
  • FIG 20 shows that Aspirin-treated Perf+ imDCs maintain their potent killing capability of OT-I CD8 + T cells and in comparable levels to the killing exhibited by WT Perf+ imDCs (as seen in Figures 21a-d). Taken together, these results highlight the great potential of Aspirin as a maturation inhibitor of imDCs.
  • Perf-DC i.e. Perf+ imDCs
  • MOG peptide a factor capable of inhibiting the Perf+ imDCs from maturing (e.g. aspirin, rapamycin, HO-1).
  • the present inventors are testing the efficacy of these Perf-DCs in creating a tolerogenic state by deleting cognate reactive T-cells (e.g. CD8+ T cells) and the potential of the Perf+ imDCs to ameliorate clinical symptoms of EAE which appear after injection of the MOG peptide.
  • the present inventors are further analyzing the length of time by which these cells maintain their immature phenotype following administration to a recipient.

Abstract

A method of treating an inflammation in a subject in need thereof is disclosed. The method comprises administering to the subject a therapeutically effective amount of perforin+ immature DCs (Perf+ im DCs), thereby treating the inflammation in the subject. Methods of generating Perf+ im DCs, wherein the Perf+ im DCs are inhibited from maturing are also disclosed. Isolated population of cells, pharmaceutical compositions and articles of manufacture are also disclosed.

Description

USE OF PERFORIN POSITIVE IMMATURE DENDRITIC CELLS IN DISEASE
TREATMENT FIELD AND BACKGROUND OF THE INVENTION
The present invention, in some embodiments thereof, relates to perforin positive immature dendritic cells (Perf+ imDCs) and, more particularly, but not exclusively, to the use of same for the treatment of inflammatory conditions.
Studies of cellular mechanisms involved in maintenance of peripheral immune tolerance have largely focused on T regulatory cells (Tregs), but other key cellular mediators such as B regulatory cells (Bregs), myeloid suppressor cells and various types of dendritic cells (DCs) have also been implicated. DCs have a tolerogenic capacity in their immature state [Tiao M.M. et al., Ann. Surg. (2005) 241, 497]. However, this rather simplistic dichotomy between mature DCs (mDCs) and immature DCs (imDCs) was challenged by the demonstration that fully mature DCs can also induce tolerance under appropriate conditions [Yu P. et al., Immunology (2009) 127, 500-511].
Zangi et al. recently described the generation of a highly defined population of DCs from Lin-Scal+cKit+ (LSK) hematopoietic progenitors [Zangi L. et al., Blood. (2012) 120(8): 1647-57]. These DCs were shown to express perforin and granzyme A in discrete granules, and hence termed 'Perf-DC [Zangi L. et al. (2012), supra]. Perf-DC are able to selectively kill cognate T cell receptor (TCR) transgenic CD8+ T cells that recognize their peptide-MHC, through a unique perforin/granzyme A-based killing mechanism, regulated through TLR7 and TREM-1 signaling [Zangi L. et al. (2012), supra]. Zangi et al. further demonstrated by immune-histological staining that Perf-DC comprise about 2-4 % of the CD 11c positive cells within the lymph nodes and spleen, and that the abundance of these cells is markedly enhanced upon in-vivo administration of GM-CSF [Zangi L. et al. (2012), supra].
Perforin-positive myeloid DCs have also been reported within the human classical DC population [Stary G. et al., J. Exp. Med (2007) 204(6): 1441-51]. Taken together, these initial findings, based exclusively on ex-vivo studies, indicated a potential tolerogenic role for Perf-DCs.
Additional background art includes:
U.S. Patent Application No. 20160058792 discloses methods and compositions for producing tolerogenic or immunosuppressive dendritic cells, the methods comprising contacting dendritic cells with an agent that stimulates the IL 27/ectonucleotidase CD39 axis signaling. The cells taught by U.S. 20160058792 can be used for treating an autoimmune disease or disorder.
U.S. Patent Application No. 20160095882 discloses tolerogenic dendritic cells for treating a myocardial infarction and a method for preparing the same. According to U.S. 20160095882, dendritic cells are obtained by culturing immature dendritic cells in a medium including TNF-a, IL-4 and GM-CSF, and protein extracted from a region of a myocardial infarction. SUMMARY OF THE INVENTION
According to an aspect of some embodiments of the present invention there is provided a method of treating an inflammation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of perforin+ immature DCs (Perf imDCs), thereby treating the inflammation in the subject.
According to an aspect of some embodiments of the present invention there is provided a therapeutically effective amount of Perff imDCs for use in treating an inflammation in a subject in need thereof.
According to an aspect of some embodiments of the present invention there is provided an isolated population of cells comprising Perf+ imDCs generated according to the method of some embodiments of the invention, wherein at least 50 % of the population of cells comprises the Perff imDCs.
According to an aspect of some embodiments of the present invention there is provided an isolated population of cells comprising at least 50 % Perf+ imDCs, wherein the Perf+ imDCs maintain an immature phenotype for at least 12 hours when administered to a recipient.
According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising the isolated population of cells of some embodiments of the invention and a pharmaceutically active carrier.
According to an aspect of some embodiments of the present invention there is provided an article of manufacture comprising the isolated population of cells of some embodiments of the invention being packaged in a packaging material and identified in print, in or on the packaging material for use in the treatment of an inflammation. According to some embodiments of the invention, the method or therapeutically effective amount of Perf+ imDCs for use further comprises contacting the Perf+ imDCs with a factor capable of inhibiting the Perf+ imDCs from maturing prior to or concomitantly with administration of the Perf imDCs to the subject in need thereof.
According to some embodiments of the invention, the method further comprises administering to the subject a therapeutically effective amount of a factor capable of inhibiting the Perf+ imDCs from maturing.
According to some embodiments of the invention, the therapeutically effective amount of Perff imDCs for use further comprises the use of a therapeutically effective amount of a factor capable of inhibiting the Perf+ imDCs from maturing.
According to some embodiments of the invention, the Perf+ imDCs are inhibited from maturing, by a method comprising: (a) obtaining Perf+ imDCs; and (b) contacting the Perf+ imDCs with a factor capable of inhibiting the Perf+ imDCs from maturing.
According to some embodiments of the invention, the method further comprises selecting cells that exhibit the Perff imDCs phenotype.
According to some embodiments of the invention, the Perff imDCs are obtained by a method comprising: (a) obtaining CD34+ cells; (b) contacting the CD34+ cells with a factor capable of differentiating the CD34+ cells into early myeloid cells; and (c) contacting the early myeloid cells with a factor capable of differentiating the early myeloid cells into perforin+ immature dendritic cells.
According to some embodiments of the invention, the method further comprises obtaining hematopoietic progenitor cells prior to step (a).
According to some embodiments of the invention, the factor capable of differentiating the CD34+ cells into early myeloid cells comprises at least one of a stem cell factor (SCF), a thrombopoietin (TPO), a Flt3-ligand (Flt3L), an interleukin-3 (IL-3) and an interleukin-6 (IL-6).
According to some embodiments of the invention, contacting the CD34+ cells with a factor capable of differentiating the CD34+ cells into early myeloid cells is effected for 5-20 days.
According to some embodiments of the invention, the factor capable of differentiating the early myeloid cells into perforin+ dendritic cells comprises a granulocyte-macrophage colony- stimulating factor (GM-CSF). According to some embodiments of the invention, the factor capable of differentiating the early myeloid cells into perforin+ dendritic cells further comprises an interleukin-4 (IL-4).
According to some embodiments of the invention, contacting the early myeloid cells with a factor capable of differentiating the early myeloid cells into perforin+ dendritic cells is effected for 5-20 days.
According to some embodiments of the invention, the factor capable of inhibiting the Perf imDCs from maturing comprises an anti-inflammatory agent or an immunosuppressive agent.
According to some embodiments of the invention, the anti-inflammatory agent or an immunosuppressive agent is an mTOR inhibitor.
According to some embodiments of the invention, the anti-inflammatory agent or an immunosuppressive agent inhibits granulocyte-mediated inflammation.
According to some embodiments of the invention, the immunosuppressive agent comprises rapamycin.
According to some embodiments of the invention, the anti-inflammatory agent comprises aspirin or a heme oxygenase- 1 (HO-1).
According to some embodiments of the invention, contacting the early myeloid cells with a factor capable of differentiating the early myeloid cells into perforin+ dendritic cells is effected concomitantly with the contacting the Perff imDCs with a factor capable of inhibiting the Perff imDCs from maturing.
According to some embodiments of the invention, the Perff imDCs comprise cells having the signature perforin+, CDl lc+,MHC-II+, CD80 and CD86.
According to some embodiments of the invention, at least 50 % of the Perf+ imDCs comprise the signature.
According to some embodiments of the invention, the Perff imDCs are loaded with an antigen.
According to some embodiments of the invention, the method is effected ex vivo. According to some embodiments of the invention, the article further comprises a factor capable of inhibiting the Perff imDCs from maturing.
According to some embodiments of the invention, the inflammation is associated with a chronic inflammatory disease. According to some embodiments of the invention, the inflammation is associated with an acute inflammatory disease.
According to some embodiments of the invention, the inflammation is associated with a disease selected from the group consisting of a metabolic disease, an autoimmune disease, an infectious disease, a hypersensitivity disease, a transplantation related disease and an injury.
According to some embodiments of the invention, the inflammation is associated with a disease selected from the group consisting of multiple sclerosis, metabolic syndrome, diabetes, rheumatoid arthritis, lupus and Crohn's.
According to some embodiments of the invention, the therapeutic effective amount of the Perf imDCs is capable of inhibiting an activity or proliferation of a CD4+ T cell and/or a CD8+ T cell.
According to some embodiments of the invention, the Perff imDCs are syngeneic with the subject.
According to some embodiments of the invention, the Perf+ imDCs are non- syngeneic with the subject.
According to some embodiments of the invention, the subject is a human subject. Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced. In the drawings:
FIGs. 1A-E: Characterization of donor type origin in immune cells of WT-PKO and DTA-PKO chimera. (Figure la) FACS analysis of CDl lc+ cells from spleen of DTA- PKO chimeric mice and WT-PKO controls. The CDl lc+ cells could be divided into CDl lchigh (cDCs) and CDl lcint subpopulations. (Figure lb) cDC levels in the spleen of DTA-PKO and WT-PK chimera. (Figure lc) The cells defined in (Figure la) were further stained for CD45.1 to identify cell origin from CD1 lc-DTA mice or littermate controls, or from CD45.2 expressed by PKO donors. Numbers indicate percent of CD45.1 cells in the various populations (Ave + SD, N > 5). (Figures ld-e) Donor origin as defined by FACS analysis of CD45.1 and CD45.2 of DN CDl lc+ cells (pre-gated on CDl lchighMHC-IIhigh 0.83 + 0.07 % in WT-PKO vs. 0.91 + 0.04 % in DTA-PKO. BM pre-DCs (pre-gated on CD3"CD19 erl l9"GRrMHC-ir,CDl lc+, 0.26 + 0.06 vs. 0.25 + 0.03 %), B cells (61.4 + 1.2 vs. 60.1 + 2.2 %), NK cells (pre-gated on CD3 CD19", 2.2 + 0.6 vs. 2.1 + 0.2 %), pDCs (pre-gated on CD3~CD19Terl l9~, 0.27+0.07 vs. 0.29 + 0.02 %), T cells (CD4+ T cells: 13.6 + 1.5 vs. 14.1 + 2.1 and CD8+ T cells: 8.6 + 0.7 vs. 7.9 + 0.4 %) and neutrophils (pre- gated on CD3"CD19"MHC-irNKl. r, 1.125 + 0.1 vs. 1.1 + 0.16 %) from DTA-PKO chimeric mice and WT-PKO controls 2 months post- transplant. These percentages indicate the frequencies of each sub-population out of the entire lymphogate in the organ (spleen or BM).
FIGs. 2A-F: DTA-PKO mice develop a condition resembling metabolic syndrome at 6 months post-transplant. (Figure 2a) Body weight of chimeric mice 6 months after transplant. Data pooled from three independent experiments; N > 18. (Figure 2b) Kinetics of weight gain in DTA-PKO (grey curve) and WT-PKO (black curve) mice. (Figure 2c) Blood chemistry profile of the chimeric mice (Avg + S.D, N > 6, *p < 0.05, ***p < 0.001). (Figure 2d) Serum leptin and (Figure 2e) TNF-a levels in the DTA-PKO and control WT- PKO chimera (Avg + S.D, N > 5). (Figure 2f) Percent body fat measured using Echo-MRI (white bars - DTA-PKO, black bars - WT-PKO).
FIGs. 3A-E: Typical features of T2D and adipose tissue expansion in DTA-PKO mice. Glucose homeostasis determined using (Figure 3a) glucose-tolerance test, and (Figure 3b) insulin-tolerance test in DTA-PKO (red) or WT-PKO (black) chimeric mice, demonstrating impaired glucose tolerance and reduced insulin sensitivity. Mice (n > 6 per group) were injected i.p. with 2 gr glucose/kg body weight (BW) (Figure 3a), or with 0.75 U insulin/kg BW (Figure 3b) and plasma glucose was measured at the indicated time points (Avg + SEM; statistical analysis was performed using GraphPad Prism software by repeated-measurements two-way ANOVA, followed by Bonferroni post-hoc analysis *, p < 0.05 p < 0.001). (Figure 3c) Average (Avg) size of adipocytes in DTA-PKO vs. WT-PKO mice. The surface area of at least 100 adipocytes per image was counted, and a total of five images per epididymal fat pad were analyzed by ImageJ software. Number of mice per group > 3. (Figure 3d) Number of CLS per surface unit. (Figure 3e) Representative hematoxylin and eosin (H&E) staining of epididymal fat pad from each group.
FIGs. 4A-F: Elevated T cell levels within adipose tissue (AT) of DTA-PKO mice 6 months post-transplant; systemic CD8+ and CD4+ depletion prevents weight gain and increase in leptin levels. Stromal vascular fractions (SVF) cells isolated from visceral AT (VAT) of DTA-PKO (white bars) and WT-PKO (black bars) mice maintained on a normal diet for 6 months were subjected to FACS analysis. (Figure 4a) Dot plots showing CD4 and CD8 T cells after gating out CD1 lb cells. Absolute numbers of CD4 and CD8 T cells (Figure 4b), Tregs (Figure 4c), and B cells (Figure 4d) were determined per gram of fat tissue (Avg + SD *P < 0.05, N > 5). (Figure 4e) DTA-PKO and WT-PKO mice were treated by weekly i.p. injections of anti-CD4 antibodies (triangle), anti-CD8 antibodies (square) or both antibodies (circle). The control animals were treated with weekly injections of PBS (diamond). The weight changes were monitored weekly, and calculated as the percent of initial weight. (Figure 4f) Leptin levels were measured by ELISA.
FIGs. 5A-B: A modified TCR repertoire in adipose tissues from DTA-PKO mice compared with WT-PKO (Figure 5a) Lorenz representation of the TCRP repertoire skewing for T cells from spleen, VAT and subcutaneous AT (SC-AT) of DTA-PKO (blue) and WT-PKO (red) mice. For each mouse, clonotypes were ordered by frequency. The cumulative frequency was then calculated at each rank (normalized to sample size). The curves represent the mean for each experimental group. (Figure 5b) Redundancy analysis (RDA) is an extension of principal component analysis (PCA) that explicitly models response variables (most abundant CDR3 AA sequences, here) as a function of explanatory variables (sample type: mouse (DTA-PKO or WT-PKO) and tissue (spleen, VAT, SC-AT), in this case). This analysis separates between repertoires of DTA-PKO (blue) vs. WT-PKO (red) along RDA1, and between most spleen samples (triangles) vs. adipose tissue samples (circles: SC; squares: VAT) along RDA2.
FIGs. 6A-L: DTA-PKO chimeras are more prone to high-fat diet (HFD) induced inflammation. (Figures 6a-b) Two months after chimerism induction, DTA-PKO (gray) and WT-PKO (black) chimeras were maintained on HFD and monitored for weight change over 90 days. Percent of body fat (Figure 6c), liver weight (Figure 6d) and epididymal fat pad weight (Figure 6e) in DTA-PKO (white) and WT-PKO chimera (black) maintained for 6 weeks on HFD. (Figures 6f-h) Levels of Leptin, IL-Ιβ and TNF-a were tested in sera of DTA-PKO (white) and WT-PKO chimera (black) maintained on high and low fat. Blood was drawn at different time points, as indicated (Avg + SD *p < 0.05, ***p < 0.001, N > 5). (Figure 6i) Serum cholesterol and triglyceride (TG) levels of chimeric mice fed HFD (Avg + SD, N > 5). (Figure 6j) Gene expression profile of M1/M2 genes determined by RT-PCR in VAT of DTA-PKO (white) and WT-PKO (black) chimeras, maintained on HFD for 2 months. (Figure 6k) Liver triglyceride levels in DTA-PKO (white) and WT-PKO chimera maintained for 6 weeks on HFD (Avg + SD, N > 4). (Figure 61) Serum insulin levels in DTA-PKO (white) and WT-PKO chimeras maintained for 6 weeks on HFD (Avg + SD, N > 4).
FIGs. 7A-E: Perforin deficiency within the DC population results in enhanced susceptibility to EAE. (Figure 7a) Disease progression of DTA-PKO (red) and WT-PKO (black) mice immunized with MOG and the average day of EAE onset (Avg + SEM of two experiments with a total of 18 mice per group). (Figure 7b) Spinal cord sections from the mice described in Figure 7a, stained with hematoxylin and eosin (H&E); arrows indicate inflammatory infiltrates. (Figures 7c-d) MOG-specific proliferation of splenocytes from DTA-PKO (red), and WT-PKO (black) mice 30 days after immunization. Proliferation was assessed by CFSE dilution together with CD4 and CD8 antibody staining by FACS (***, P < 0.001 Avg + SD 5-mice per group) (Figure 7e) Analysis of spinal cord T cells. T cells were isolated from 3-5 pooled spinal cords from the DTA-PKO or WT-PKO mice, 14 days after immunization with MOG, stimulated with soluble CD3 overnight, and then stained for IL-17a and IFN-γ (Avg + SD *P < 0.05, N > 3).
FIGs. 8A-C: Perforin expression in CDl lc+ DCs. (Figure 8a) Immuno staining of perforin in CDl lc+ DCs isolated by magnetic beads from the spleen of WT or PKO mice, as described in the 'general materials and experimental procedures' section hereinbelow. Due to some non-specific staining experienced with isotype control antibodies (marked by arrow), cells were initially treated with isotype control (Green) and subsequently with perforin- specific antibody (Red). Nuclei are stained by Hoechst in Blue. Inset in the left image shows staining of a Perf-DC under higher magnification. Typical immunostaining (Figure 8b) and quantification (Figure 8c) of perforin"1" DCs in the spleen of CDl lc-eYFP mice 14 days after infusion of B-16 melanoma cells secreting GM-CSF or Flt3-L as compared to non-secreting cells.
FIG. 9: Immunostaining of perforin in CDl lc"1" DCs isolated by magnetic beads from the spleen of WT mice together with CD4 and CD8 staining (green), as described in the 'general materials and experimental procedures' section hereinbelow. Due to some non-specific staining experienced with isotype control antibodies), cells were initially treated with isotype control (red) and subsequently with perforin- specific antibody (blue). Nuclei are stained by Hoechst Yellow. Dotted circle marks the boundaries of perf-DC cell on the upper image and as can be seen in the lower image it is typically negative for CD4 or CD8 staining (green).
FIGs. 10A-D: Characterization of CDl lchigh and CDl lcint and their perforin expression in WT-PKO and DTA-PKO chimera. (Figure 10a) FACS analysis of CDl lc+ cells from spleen of DTA-PKO chimeric mice and WT-PKO controls. The CDl lc"1" cells could be divided into four sub-populations: CDl lc^CDl lb^ (I), CDl lchighCDl lblow (II), CDl lcintCDl lbhigh (III), CDl lcintCDl lbint (IV). (Figure 10b) Expression levels of F4/80 were determined in four populations of splenic CDl lc"1" cells by FACS. (Figure 10c) Proliferation of CFSE labeled Balb/c T cells stimulated for 3 days against C57BL/6 CDl lchigh and CDl lcint cells, and measured by FACS analysis for CFSE dilution as an indicator of cell division. The bars indicate mean CFSE value + SD. (Figure lOd) Differential expression of perforin as determined by RT-PCR (Avg + SD; n=3) in DC sub- populations I-IV isolated from DTA-PKO (red) or WT-PKO (black) spleen cells by FACS.
FIGs. 11A-D: Control DTA-WT mice do not exhibit metabolic abnormalities. DTA-PKO (red), WT-PKO (black) or DTA-WT (gray) chimeric mice were compared for different metabolic parameters, including: (Figure 11a) Body weight of chimeric mice 6 months after transplant. (Figure 1 lb) Percent body fat measured using Echo-MRI. (Figure 11c) Serum leptin levels (Avg + S.D, N > 5, *p < 0.05, ***p < 0.001). (Figure l id) Glucose homeostasis as determined by glucose-tolerance test, demonstrating impaired glucose tolerance. Mice (n=5 per group) were injected i.p. with 2 gr glucose/kg body weight.
FIG. 12: Sequential gating strategy for analysis of T cells in SVF. Collagenase digested stromal vascular fractions (SVF) were stained and analyzed by FACS. T cells were first gated based on their forward and side scatter (Rl). Nonviable cells were excluded using 7AAD staining (R2). Further gating on CD45+CDl lb" cells (R3) enabled focusing on lymphocytes, gating out CDl lb+ macrophages. Moreover, CD4+ and CD8+ T cells within gate R3 were analyzed.
FIGs. 13A-B: FACS determination of myeloid cell subpopulations in SVF from
VAT of DTA-PKO and WT-PKO chimera. Collagenase digested SVF from VAT of DTA- PKO (red bars) and WT-PKO (black bars) mice was stained and analyzed by FACS. (Figure 13 a) Leukocyte gates were defined based on forward and side scatter. Non- viable and non-lymphoid cells were excluded using 7AAD and CD45 staining, respectively. Macrophages were defined based on CDl lb and F4/80 expression, DCs were defined as CDl lchigh and MHC-IIhigh, and neutrophils were CDl lc"F4/80"CDl lb+Ly6G+ (Figure 13b) Box plots summarizing the number of cells per gram fat (Center lines show Mean; Box limits indicate the 25th and 75th percentile, whiskers indicate the minimal and maximal values, ** p<0.01).
FIG. 14: DTA-PKO chimeras develop inflammation in the hypothalamus 9 months after transplantation. Gene expression level of pro-inflammatory IL-6 and IL-Ιβ genes determined by RT-PCR in hypothalamus of DTA-PKO (red) and WT-PKO (black) mice (N > 3). * p < 0.05; ** p < 0.01.
FIGs. 15A-E: Frequency and total cell numbers of different SVF cell subpopulations isolated from VAT of DTA-PKO and WT-PKO mice maintained on HFD for 8 weeks. Cells were identified by FACS analysis of SVF fractions from 6-8 mice; percent gated cell frequencies are indicated for T cells (Figure 15 a) and macrophages (Figure 15d). Absolute numbers (in thousands) of CD4 and CD8 T cells and macrophages per gram of fat tissue (Figure 15b, 15c and 15e), respectively, are shown (all data are presented as Avg + SD *P < 0.05, **P < 0.01, ***P < 0.001). DTA-PKO (red) and WT- PKO (Black). FIGs. 16A-B: Donor type origin of B cells (Figure 16a) and neutrophils, (Figure 16b) from spleen of DTA-PKO (gray filled histogram) and WT-PKO (unfilled histogram) chimera before and after onset of pathology. Cells were stained for CD45.1 to identify cell origin from CDl lc-DTA or littermate controls, or from CD45.2 expressed by PKO donors. Numbers indicate percent of CD45.1 cells in the various populations (Ave + SD).
FIGs. 17A-C: Donor type origin of NK cells and DC from spleen of DTA-PKO (gray filled histogram) and WT-PKO (unfilled histogram) chimera after onset of pathology is not altered compared to data shown in Figures la-e for cells harvested prior to pathology onset. (Figure 17a) NK cells from spleen were pre-gated for Lin- (Terl l9~ CD3~CD19~Gr ) and further defined by various markers including NKl .l, NKp46 and CD49b (DX5). cDCs were defined by staining for MHC-II and CDl lc. (Figure 17b) Cells from chimera fed with a regular diet were stained for CD45.1 6 months after transplantation to identify cell origin from CDl lc-DTA or littermate control hosts, versus CD45.2 expressed by PKO donors. (Figure 17c) Cells from chimera fed with HFD were stained for CD45.1 2 months after transplantation to identify cell origin from CD1 lc-DTA or littermate controls, or from CD45.2 expressed by PKO donors. Representative FACS data WT-PKO (black), DTA-PKO (gray), N > 4 mice from a single experiment on normal chow diet (NCD) and HFD. Numbers indicate percent of CD45.1 cells in the various populations (Ave + SD).
FIG. 18: Donor type origin of VAT cells from DTA-PKO and WT-PKO chimeric mice fed with normal diet at early (2 months) or late (7 months) time points post- transplant, or with HFD (8 weeks after HFD). Different leukocyte types were defined as indicated in Figures l la-d and Figure 12 and were further stained for CD45.1 to identify cell origin from CDl lc-DTA or littermate controls, or from CD45.2 expressed by PKO donors.
FIGs. 19A-B: Aspirin-treated imDCs do not undergo maturation in response to induction with LPS. imDCs were generated using the 20 days protocol as previously described [Zangi L. et al. (2012), supra]. One group of cells was additionally treated with 2.5 mM aspirin during the last 10 days of culture, while the second group was not additionally treated. To induce maturation, cells were treated with 1 μg/ml LPS for 24 hours. Cells from the two groups were stained for MHC II, CD80 and CD86 before (black) and after (red) LPS treatment and were further analyzed by FACS. Of note, while untreated perf+ imDCs upregulate levels of MHC-II, CD80 and CD86 in response to LPS, Aspirin-treated perf imDCs did not change the expression of these 3 markers, indicating a maturation-resistant status.
FIG. 20: Aspirin-treated perff imDCs maintain the ability to kill cognate CD8+ T cells in MLR. FACS evaluation of total numbers of live OT-I CD8+ T cells following 5 hours of incubation with Aspirin-treated imDCs loaded with SIINFEKL peptide (SEQ ID NO: 29), at indicated ratios. Average + SD (N > 4).
FIGs. 21A-D: MHC-dependent perforin/Granzyme A mediated killing of cognate CD8+ T cells in MLR is associated with triggering of TLR7. (Figure 21a) FACS evaluation of total numbers of live OT-I CD8+ T cells in the absence (white) or presence of untreated imDCs (patterned) or treated with SIINFEKL (SEQ ID NO: 29) (black) or SIYRYYGL (SEQ ID NO: 30) (grey), at indicated cell ratios (Black). (Figure 21b) Killing ability of imDCs from C57BL/6 WT or TLR7_/" mice loaded with SIINFEKL (SEQ ID NO: 29). (Figure 21c) Killing ability of imDCs from C57BL/6 WT or Dapl2_/" mice loaded with SIINFEKL (SEQ ID NO: 29) (Figure 21d) Killing ability of imDCs treated with RNAse. Average + SD (N > 3).
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
The present invention, in some embodiments thereof, relates to perforin positive immature dendritic cells (Perf+ imDCs) and, more particularly, but not exclusively, to the use of same for the treatment of inflammatory conditions.
The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
Previous work illustrated that Perf+ DCs are able to selectively kill cognate TCR transgenic CD8+ T cells that recognize their peptide-MHC through a unique perforin/granzyme A-based killing mechanism, regulated through TLR7 and TREM-1 signaling [Zangi L. et al., Blood. (2012) 120(8): 1647-57]. While reducing the present invention to practice, the present inventors have uncovered that Perforin positive immature dendritic cells (Perf+ imDCs) represent an important regulatory population of cells that control inflammatory/autoimmune processes.
As is illustrated hereinbelow and in the Examples section which follows, the present inventors generated chimeric mice in which perforin expression was selectively impaired in CDl lc+ DC (see Example 1 of the Example section which follows), the present inventors found that within 5 months these mice developed many features of metabolic syndrome (see Example 2 of the Examples section which follows). These results point to Perf imDCs as a regulatory cell population which control inflammatory processes in the adipose tissue (AT). Importantly, in these chimeric mice, induction of metabolic syndrome occurred spontaneously in mice fed with a regular diet (see Example 2 of the Examples section which follows) as well as in mice fed with a high fat diet (see Example 5 of the Examples section which follows).
The present inventors further illustrated the regulatory role of Perf+ imDCs in a defined model of autoimmunity, namely Myelin Oligodendrocyte Glycoprotein (MOG) peptide-induced experimental autoimmune encephalomyelitis (EAE). Specifically, chimeric mice lacking expression of Perf+ imDCs were substantially more prone to induction of EAE, exhibiting significantly elevated levels of MOG-specific autoimmune T cell clones compared to their WT counterparts (see Example 6 of the Examples section which follows). Taken together, these results point to the immune-regulatory role of Perf+ imDCs in inflammatory conditions and to the use thereof for the treatment of inflammation.
However, adoptive transfer of Perff imDCs can pose a drawback as immature dendritic cells typically undergo rapid maturation following encounter with an antigen. Thus, following implantation, Perf+ imDCs typically undergo rapid maturation and lose their tolerogenic activity. The present inventors uncovered that treating Perff imDCs with a factor which inhibits maturation (e.g. Aspirin, Rapamycin and Heme Oxygenase- 1 (HO- 1)) prior to implantation, results in immature dendritic cells which stably maintain their immature phenotype while preserving their killing-mediated tolerogenic capacity (see Example 7 of the Examples section which follows). Thus, according to one aspect of the present invention there is provided a method of treating an inflammation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of perforin+ immature DCs (Perf+ imDCs), thereby treating the inflammation in the subject.
According to another aspect of the present invention there is provided a therapeutically effective amount of Perf+ imDCs for use in treating an inflammation in a subject in need thereof.
As used herein, the term "dendritic cells" or "DCs" refers to antigen-presenting immune cells that process antigenic material and present it to other cells of the immune system e.g. to T cells and B cells (e.g. by presentation on the DCs cell surface via MHC class I and/or MHC class II molecules). The term DCs includes immature and mature dendritic cells. DCs can be typically characterized by expression of cell surface markers, e.g., CDl lc, MHC class II (e.g. HLA DR), CD80 and CD86. According to one embodiment, DCs can also express any of CD40, CDl lb, CD304 (BDCA4), CD103 and/or CD Id. DCs can also be identified by the absence of T cell, B cell, NK cell, monocyte and/or neutrophil specific lineage markers such as, but not limited to, CD3, T cell receptor (TCR), CD14, CD19, B cell receptor (BCR) and/or CD56. In addition, dendritic cells can be characterized functionally by their capacity to stimulate allo- responses and mixed lymphocyte reactions (MLR).
The term "immature dendritic cells" or "imDCs" refers to dendritic cells which are found at an initial maturation state and are capable of capturing and processing an antigen. Typically, imDCs can be characterized by expression of CDl lc, CD80, and CD86 as well as by low levels of MHC class II (e.g. HLA DR, as compared to mature dendritic cells). imDCs can also be identified by the presence of CD40, CDl lb, as well as by the absence of T cell, B cell, NK cell, monocyte and/or neutrophil specific lineage markers such as, but not limited to, CD3, T cell receptor (TCR), CD 14, CD 19, B cell receptor (BCR) and/or CD56.
Without being bound by theory, immature DCs are typically distributed within all tissues, particularly those that interface with the environment (e.g. skin, mucosal surfaces) and in lymphoid organs, and function to capture and process antigens. Immature DCs are recruited to sites of inflammation in peripheral tissues following pathogen invasion. Internalization of foreign antigens can subsequently trigger their maturation and migration from peripheral tissues to lymphoid organs where the antigens are presented to, for example, antigen- specific immune cells (e.g. T cells and B cells). In general, the process of DC maturation, involves down-regulation of antigen internalization, an increase in the surface expression of MHC molecules and co-stimulatory molecules, morphological changes (e.g. formation of dendrites) and secretion of chemokines, cytokines and proteases.
Accordingly, the term "mature dendritic cells" refers to DCs which express higher levels of CDl lc, MHC class II, CD80 and CD86 (as compared to imDCs). Mature DCs can also express CD40. Mature DCs release cytokines, including but not limited to, IL- 12, IL- la, IL- Ιβ, IL- 15, IL- 18, IFN-a, IFN-β, IFN-γ, IL-4, IL- 10, IL-6, IL- 17, IL- 16, TNF-a, and MIF, and can activate naive lymphocytes (e.g. naive T cell).
As used herein, the term "perforin+ immature DCs" or "Perf+ imDCs" refers to a subgroup of imDCs which express perforin and/or granzyme A. Perf+ imDCs are found at an initial maturation state and are capable of capturing and processing an antigen. According to one embodiment, the Perf+ imDCs are characterized by expression of perforin, CDl lc, MHC class II (e.g. HLA DR), CD80 and CD86. According to one embodiment, the Perf+ imDCs may also express any of CD1 lb, CD40 and/or granzyme A. According to one embodiment, Perf imDCs can also be identified by the absence of T cell, B cell, NK cell, monocyte and/or neutrophil specific lineage markers such as, but not limited to, CD3, T cell receptor (TCR), CD14, CD19, B cell receptor (BCR) and/or CD56.
According to one embodiment, a cell is considered a Perf+ imDC when a single cell of cells has the signature: perforin, CDl lc, MHC class II (e.g. HLA DR), CD80 and CD86.
According to one embodiment, a cell is considered a Perf+ imDC when a single cell of cells has the signature: perforin, granzyme A, CDl lc, MHC class II (e.g. HLA DR), CD80 and CD86.
According to one embodiment, a cell is considered a Perf+ imDC when a single cell of cells has the signature: perforin, CDl lc, MHC class II (e.g. HLA DR), CD80, CD86 and CD40.
According to one embodiment, a cell is considered a Perf+ imDC when a single cell of cells has the signature: perforin, CDl lc, MHC class II (e.g. HLA DR), CD80, CD86 and CD l ib. According to one embodiment, a cell is considered a Perf+ imDC when a single cell of cells has the signature: perforin, granzyme A, CDl lc, MHC class II (e.g. HLA DR), CD40, CD80 and CD86.
According to one embodiment, a cell is considered a Perf+ imDC when a single cell of cells has the signature: perforin, CDl lc, MHC class II (e.g. HLA DR), CD80, CD86 and CD3" and/or TCR".
According to one embodiment, a cell is considered a Perf+ imDC when a single cell of cells has the signature: perforin, CDl lc, MHC class II (e.g. HLA DR), CD80, CD86 and CD 19" and/or BCR".
According to one embodiment, a cell is considered a Perf+ imDC when a single cell of cells has the signature: perforin, CDl lc, MHC class II (e.g. HLA DR), CD80, CD86 and CD56".
Any method known in the art can be used to identify the cell surface markers, e.g. using FACS analysis or using magnetic cell isolation and cell separation (e.g. by Miltenyi Biotec). Furthermore, any method known in the art can be used for identification of cellular components (e.g. perforin or granzyme A), e.g. using ELISA, PCR or by western blot analysis.
According to some embodiments of the invention, the Perf imDCs comprise immune-regulatory properties (e.g. tolerogenic activity). Typically, the Perff imDCs inhibit expansion of antigen specific immune cells by killing cognate T cells (e.g. cognate CD8+ T cells which recognize an antigen presented in the context of a MHC molecule on the surface of the Perf+ imDC) through a unique perforin/granzyme A-based killing mechanism (e.g. regulated through TLR7 and TREM-1 signaling).
The term "tolerogenic activity" of imDCs refers to the ability of dendritic cells' to suppress immune responses such as suppressing T cell-mediated immune responses (e.g. killing effector T cells).
The use of Perf+ imDCs is especially beneficial in situations in which there is a need to down-regulate T cells involved in an inflammatory response, such as an autoimmune disease or metabolic syndrome (as discussed in further detail below).
Any method used for obtaining or generating of Perf+ imDCs can be used in accordance with the present teachings. According to one embodiment, Perf+ imDCs are obtained directly from a cell donor (e.g. from a subject, as discussed in detail hereinbelow). Perf+ DC make about 2-4 % of the CD 11c positive cells in a human body, thus, it is possible to obtain these cells directly from a cell donor by obtaining any biological sample comprising Perf imDCs (e.g. blood, bone marrow, lymph node and fluid, tonsils, adipose tissue, etc.) from the donor and selecting therefrom dendritic cells which express perforin, CDl lc,MHC class II, CD80 and CD86 (e.g. using aphaeresis, magnetic beads or FACS). According to one embodiment, the dendritic cells may further express any of granzyme A, CD l ib and/or CD40. According to one embodiment, the dendritic cells lack expression of T cell, B cell or NK cell markers, e.g. CD3, CD19, CD56, respectively. According to one embodiment, the dendritic cells may further lack expression of T cell, B cell, NK cell, monocyte and/or neutrophil specific lineage markers such as, but not limited to, CD3, TCR, CD14, CD19, BCR and/or CD56. According to a specific embodiment, the cell donor is administered a factor for inducing mobilization of hematopoietic stem cells (HSCs) from the bone marrow into the bloodstream, such as e.g. GM-CSF and/or Flt3L (e.g. prior to withdrawal of a blood sample). It will be appreciated that any method of obtaining a biological sample (e.g. blood sample, bone marrow sample, lymph node sample, tonsils, adipose tissue, etc.) can be used, as for example, using standard blood retrieval procedures, apheresis, biopsy, lumbar puncture, liposuction or surgery.
According to one embodiment, Perf+ imDCs are obtained from a commercial supplier (i.e. as an off the shelf product), as for example, from HemaCare Corporation, from the ATCC, or from Astarte Biologies.
As Perff imDCs may be difficult to maintain due to their rapid maturation into mature DCs after stimulation with an antigen, the present invention contemplates generation of Perf+ imDCs using ex vivo or in vitro protocols.
Methods of producing Perff imDCs are well known to one of skill in the art, such as disclosed in Zangi L. et al., Blood. (2012) 120(8): 1647-57, incorporated herein by reference.
According to one embodiment, there is provided a method of obtaining Perf+ imDCs, the method comprising: (a) obtaining CD34+ cells; (b) contacting the CD34+ cells with a factor capable of differentiating the CD34+ cells into early myeloid cells; and (c) contacting the early myeloid cells with a factor capable of differentiating the early myeloid cells into Perf+ imDCs.
According to one embodiment, the method further comprises obtaining hematopoietic progenitor cells prior to step (a).
Methods of obtaining CD34+ cells are well known to one of skill in the art. For example, CD34+ cells may be obtained from a commercial supplier (i.e. as an off the shelf product), as for example, from Stemcell Technologies, from the ATCC, or from Astarte Biologies. Commercially obtained CD34+ cells may include cells obtained from any stem/progenitor cell source, e.g. cord blood CD34+ cells, bone marrow derived CD34+ cells, cell lines, etc.
CD34+ cells may also be obtained from a cell donor. This method is typically effected by first promoting mobilization of hematopoietic progenitor cells (comprising CD34+ cells), from bone marrow into peripheral blood in a cell donor (e.g. subject). Such methods comprise administering to the cell donor a mobilization factor, such as granulocyte colony stimulating factor (G-CSF), about 7-21 days prior to cell collection (e.g., 21 days, 14 days, 12 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day). Typically, G-CSF is administered at a dose of about 10-100 μg/kg (e.g. 100 μg/kg, 75 μg/kg, 50 μg/kg, 40 μg/kg, 30 μg/kg, 25 μg/kg or 10 μg/kg), the dose of the mobilization factor (e.g. GM-CSF) can be effected in a single administration or over a course of several days (e.g. 2, 3, 4, 5, 7 or 10 days). Such methods of promoting mobilization of hematopoietic progenitor cells typically result in sufficient numbers of CD34+ cells in the peripheral blood for subsequent collection. Collecting the mobilized hematopoietic progenitor cells (comprising CD34+ cells) from the peripheral blood of the subject may be effected using various techniques. For example, the collecting step may comprise standard blood retrieval procedures or apheresis. Alternatively, CD34+ cells may be obtained directly from the bone marrow of a subject, e.g. by lumbar puncture.
Following collection, the biological sample is typically enriched for CD34+ cells. The enriching step typically involves a step in which CD34+ cells are separated from CD34-negative (CD34 ) cells to provide an enriched population of CD34+ cells (e.g. enriched by about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 50-fold, 100-fold, 1000-fold more of CD34+ cells compared to the amount or number of CD34+ cells in the original population of cells, i.e., the population of cells before enrichment). The enriching step optionally comprises the use CD34" or CD34+ specific antibodies or antibody fragments. Selection may be effected using, but not limited to, magnetic cell isolation and cell separation (e.g. by Miltenyi Biotec). See, for example, PCT publication no. WO 2013126590.
The CD34+ cells are then contacted with a factor capable of differentiating the
CD34+ cells into early myeloid cells (i.e. the immature state of myeloid cells). Exemplary factors capable of differentiating CD34+ cells into early myeloid cells include, but are not limited to, stem cell factor (SCF), thrombopoietin (TPO), Flt3-ligand (Flt3L), interleukin- 3 (IL-3) and/or interleukin-6 (IL-6). According to a specific embodiment, differentiating CD34+ cells into early myeloid cells is effected in the presence of SCF, TPO, Flt3L, IL-3 and IL-6.
Contacting the CD34+ cells with the factor or combination of factors capable of differentiating the CD34+ cells into early myeloid cells is typically effected for about 2-40 days (e.g. 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 25 days, 30 days or 40 days). According to a specific embodiment, contacting the CD34+ cells with the factor or combination of factors capable of differentiating the CD34+ cells into early myeloid cells is effected for 10 days.
Early myeloid cells can be selected by the surface expression of CDl la, CDl lb, CDl lc, CD13, CD33, CD34 and CD117. Any method known in the art can be used to identify the cell surface markers, e.g. using FACS analysis or using magnetic cell isolation and cell separation (e.g. by Miltenyi Biotec).
In order to generate perf+ imDCs from the early myeloid cells, the early myeloid cells are contacted with a factor capable of differentiating the early myeloid cells into perf imDCs. Exemplary factors capable of differentiating the early myeloid cells into perff imDCs include, but are not limited to, granulocyte-macrophage colony- stimulating factor (GM-CSF), Flt-3L, and macrophage colony- stimulating factor (M-CSF). According to one embodiment, interleukin-4 (IL-4) can be added to the GM-CSF for differentiation of the early myeloid cells into perff imDCs.
Contacting the early myeloid cells with the factor or combination of factors capable of differentiating the early myeloid cells into perff imDCs is typically effected for about 2-40 days (e.g. 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 25 days, 30 days or 40 days). According to a specific embodiment, contacting the early myeloid cells with the factor or combination of factors capable of differentiating the early myeloid cells into perf imDCs is effected for 10 days.
Perff imDCs can be selected by the expression of perforin, CDl lc,MHC class II, CD80 and CD86. Perff imDCs can be further selected by the additional expression of granzyme A, CD l ib and/or CD40, as well as by the lack expression of T cell, B cell, NK cell, monocyte and/or neutrophil specific lineage markers such as, but not limited to, CD3, TCR, CD14, CD19, BCR and/or CD56, as discussed above. Any method known in the art can be used to identify the cell surface markers, e.g. using FACS analysis or using magnetic cell isolation and cell separation (e.g. by Miltenyi Biotec). Expression of perforin can be carried out using an ELISA, western blot analysis or PCR.
According to one embodiment, generating Perf+ imDCs can be carried out by first obtaining bone marrow cells from a cell donor. The bone marrow cell suspension is enriched for CD34+ cells using e.g. an anti-CD34 antibody, followed by positive selection e.g. on a MACS separation column (e.g. Miltenyi Biotec) or using a FACSAria Sorter. Next, at least about 105-107 cells/ml (e.g. 106 cells/ml) are cultured for 5-20 days (e.g. 10 days) in cell culture medium (e.g. Iscove's Modified Dulbecco's Medium (IMDM)) containing, for example, FCS (e.g. at a dose of about 1-20 % e.g. 10 %), L-glutamine (e.g. at a dose of about 1-10 mM e.g. 2 mM), penicillin (e.g. at a dose of about 10-1000 U/ml e.g. 100 U/ml) and streptomycin (e.g. at a dose of about 0.01-10 mg/ml e.g. 0.1 mg/ml). Cultures are supplemented every second day with stem cell factor (SCF, e.g. at a dose of about 1-500 ng/ml e.g. 50 ng/ml), thrombopoietin (TPO, e.g. at a dose of about 0.01-10 ng/ml e.g. 1 ng/ml), Flt3-ligand (Flt3L, e.g. at a dose of about 1-500 ng/ml e.g. 50 ng/ml), interleukin-3 (IL-3, e.g. at a dose of about 1-100 ng/ml e.g. 10 ng/ml), and interleukin-6 (IL-6, e.g. at a dose of about 1-100 ng/ml e.g. 10 ng/ml). At the end of the culture period, differentiated cells (i.e. early myeloid cells) are cultured for an additional 5-20 days (e.g. 10 days) in culture medium (e.g. RPMI 1640 complete tissue culture medium e.g. CTMC) along with granulocyte-macrophage colony- stimulating factor (GM-CSF, at a dose of about 1-100 ng/ml e.g. 20 ng/ml). Perf+ imDCs are collected at the end of this culture period. According to one embodiment, in order to preserve the tolerogenic activity of the Perf imDCs for adoptive transfer into a subject (as discussed in detail below), the Perf+ imDCs are contacted with a factor which inhibits maturation.
Thus, according to one embodiment, Perf+ imDCs are contacted with a factor capable of inhibiting the Perff imDCs from maturing. Exemplary factors capable of inhibiting the Perf+ imDCs from maturing include e.g. anti-inflammatory agents and/or immunosuppressive agents. Without being bound by theory, these factors inhibit an immune response through different mechanisms e.g. inhibition of NF-kB or inhibition of T-cell mitosis.
According to one embodiment, the anti-inflammatory agent or immunosuppressive agent is an mTOR inhibitor. An exemplary mTOR inhibitor comprises the immunosuppressive agent rapamycin (Sirolimus).
Examples of immunosuppressive agents which can be used in accordance with some embodiments of the invention include, but are not limited to, methotrexate, cyclophosphamide, cyclosporine, cyclosporin A, chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine), gold salts, D-penicillamine, leflunomide, azathioprine, anakinra, infliximab (REMICADE), etanercept, TNF.alpha. blockers, a biological agent that targets an inflammatory cytokine, and Non-Steroidal Anti-Inflammatory Drug (NSAIDs). Examples of NSAIDs include, but are not limited to acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, tramadol, rapamycin (sirolimus) and rapamycin analogs (such as CCI-779, RAD001, AP23573). These agents may be administered individually or in combination
According to one embodiment, the anti-inflammatory agent or immunosuppressive agent inhibits granulocyte-mediated inflammation. An exemplary agent which inhibits granulocyte-mediated inflammation comprises the anti-inflammatory agent aspirin.
Examples of anti-inflammatory agents which can be used in accordance with some embodiments of the invention include, but are not limited to, Alclofenac; Alclometasone Dipropionate; Algestone Acetonide; Alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride;
Bromelains; Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen;
Clobetasol Propionate; Clobetasone Butyrate; Clopirac; Cloticasone Propionate;
Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide; Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium; Diflorasone
Diacetate; Diflumidone Sodium; Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide;
Drocinonide; Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac;
Etofenamate; Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal;
Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid; Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; Fluocortin Butyl; Fluorometholone Acetate;
Fluquazone; Flurbiprofen; Fluretofen; Fluticasone Propionate; Furaprofen; Furobufen;
Halcinonide; Halobetasol Propionate; Halopredone Acetate; Ibufenac; Ibuprofen;
Ibuprofen Aluminum; Ibuprofen Piconol; Ilonidap; Indomethacin; Indomethacin Sodium;
Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lomoxicam; Loteprednol Etabonate; Meclofenamate Sodium;
Meclofenamic Acid; Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine;
Meseclazone; Methylprednisolone Suleptanate; Momiflumate; Nabumetone; Naproxen;
Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein; Orpanoxin;
Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride; Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone; Piroxicam; Piroxicam Cinnamate;
Piroxicam Olamine; Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone;
Proxazole; Proxazole Citrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate;
Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam; Sulindac; Suprofen;
Talmetacin; Talniflumate; Talosalate; Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin
Sodium; Triclonide; Triflumidate; Zidometacin; Zomepirac Sodium.
According to a specific embodiment, the anti-inflammatory agent comprises an acetylsalicylic acid (ASA), e.g., Aspirin™, or a heme oxygenase-1 (HO-1).
According to one embodiment, Perf+ imDCs are cultured for about 5-20 days (e.g. 10 days) with aspirin (e.g. at a dose of about 1-25 mM e.g. 2.5 mM), rapamycin (e.g. at a dose of about 1-100 ng/ml e.g. 10 ng/ml) or with Cobalt Protoporphyrin (CoPP), an inducer of HO-1 (e.g. at a dose of about 1-500 ng/ml e.g. 50 niM). Perf+ imDCs are collected at the end of this culture period.
Contacting the perf+ imDCs with a factor capable of inhibiting the Perf+ imDCs from maturing is typically effected for about 2-40 days (e.g. 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 25 days, 30 days, 35 days or 40 days). According to a specific embodiment, contacting the perf imDCs with a factor capable of inhibiting the Perf+ imDCs from maturing is effected for 10 days.
According to one embodiment, contacting the early myeloid cells with a factor capable of differentiating the early myeloid cells into the perff imDCs (e.g. GM-CSF) is effected concomitantly with the factor capable of inhibiting the Perf+ imDCs from maturing (e.g. rapamycin, aspirin or HO-1). According to a specific embodiment, this co- culture is effected for about 2-40 days (e.g. 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 25 days, 30 days, 35 days or 40 days). According to a specific embodiment, this co-culture is effected for 10 days.
According to one embodiment, the method is effected ex vivo or in vitro (e.g. in a cell culture plate).
According to one embodiment, the method further comprises selecting cells which exhibit the Perf+ imDCs phenotype (as discussed in detail above).
According to one embodiment, at least about 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 99 % or 100 % of the Perf+ imDCs comprise the cell signature (e.g., perforin, CDl lc, MHC class II, CD80 and CD86). According to a specific embodiment, at least about 50 % of the Perf+ imDCs comprise the cell signature.
According to one embodiment, the Perff imDCs maintain an immature phenotype
(e.g. upon stimulation with an antigen, such as following administration to a subject) and the signature for at least about 12 hours to 60 days (e.g. for at least about 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 25 days, 30 days, 45 days, 60 days or more).
According to some embodiments, the Perff imDCs of the present invention may be naive cells (e.g. non-genetically modified) or genetically modified cells (e.g. cells which have been genetically engineered to express or not express specific genes, markers or peptides, such as an antigen or antigens, or to secrete or not secrete specific cytokines). Any method known in the art may be implemented in genetically engineering the cells, such as by inactivation of the relevant gene/s or by insertion of an antisense RNA interfering with polypeptide expression (see e.g. WO/2000/039294, which is hereby incorporated by reference), or using an exogenous polynucleotide sequence designed and constructed to express at least a functional portion of a protein (see e.g. Suhoski MM et al, Mol Ther. (2007) 15(5): 981-988).
According to some embodiments, the Perf imDCs of the present invention may be loaded to present short synthetic peptides, protein extracts or purified proteins (e.g. fused or loaded thereto) to immune cells such as T cells or B cells (e.g. for specific tolerogenic activity). Such short peptides, protein extracts or purified proteins may be viral-, bacterial- , fungal-, tumor-, autoimmune- or allergic- antigen derived peptides or peptides representing any other antigen. Dedicated software can be used to analyze viral, bacterial, fungal, tumor, autoimmune or allergic antigen sequences to identify immunogenic short peptides, i.e., peptides presentable in context of major histocompatibility complex (MHC) class I or MHC class II.
Methods of loading DCs are well known in the art. For example, perf+ imDCs of some embodiments of the present invention may be pulsed with a peptide. Alternatively, the perf+ imDCs may be transfected with a vector encoding a peptide. Such methods are described e.g. in Inzkirweli et al., Anticancer Research (2007) 27: 2121-2130, and in Lesterhuis WJ et al., Anticancer Research (2010) 30(12):5091-7, incorporated herein by reference.
According to one aspect of the invention, there is provided an isolated population of cells comprising Perff imDCs generated according to the method of some embodiments of the invention, wherein at least 50 % of the population of cells comprises the Perff imDCs.
The phrase "isolated population of cells" as used herein refers to cells which have been isolated from their natural environment (e.g., the human body).
According to one embodiment, at least about 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 99 % or 100 % of the population of cells comprise the Perf+ imDCs. According to a specific embodiment, at least about 50 % of the population of cells comprises the Perff imDCs. According to one aspect of the invention, there is provided an isolated population of cells comprising at least 50 % Perf+ imDCs, wherein the Perf+ imDCs maintain an immature phenotype for at least about 12 hours to 60 days (e.g. for at least about 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 25 days, 30 days, 45 days, 60 days or more) when administered to a mouse.
As mentioned, the Perf+ imDCs of some embodiments of the invention can be used in treating an inflammation in a subject.
The term "inflammation" as used herein refers to the general term for accumulation of fluids, plasma proteins, and white blood cells initiated by physical injury, infection, or an immune response. Inflammation may be associated with several signs e.g. redness, pain, heat, swelling and/or loss of function. Inflammation is an aspect of many diseases and disorders, also referred to inflammatory diseases, including but not limited to diseases related to immune disorders, autoimmune diseases, tissue damage, tissue injury, infectious disease (e.g. viral and bacterial infection), arthritis, collagen diseases, allergy, asthma, pollinosis, cancer and atopy (as described in further detail below).
As used herein, the terms "subject" or "subject in need thereof include mammals, specifically human beings at any age or gender. The subject may be healthy or showing preliminary signs of a pathology, e.g. a pathology associated with an inflammation. This term also encompasses individuals who are at risk to develop the pathology (e.g. inflammation).
As used herein the term "treating" refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a disease or disorder (e.g. an inflammation).
A number of diseases and conditions, which involve an inflammatory response, can be treated using the methodology described hereinabove. Examples of such diseases and conditions are summarized infra.
Inflammatory diseases - Include, but are not limited to, chronic inflammatory diseases and acute inflammatory diseases.
Inflammatory diseases associated with hypersensitivity
Examples of hypersensitivity include, but are not limited to, Type I hypersensitivity, Type II hypersensitivity, Type III hypersensitivity, Type IV hypersensitivity, immediate hypersensitivity, antibody mediated hypersensitivity, immune complex mediated hypersensitivity, T lymphocyte mediated hypersensitivity and DTH.
Type I or immediate hypersensitivity, such as asthma.
Type II hypersensitivity include, but are not limited to, rheumatoid diseases, rheumatoid autoimmune diseases, rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 Jul;15 (3):791), spondylitis, ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res 2001; 3 (3): 189), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998; 17 (l-2):49), sclerosis, systemic sclerosis (Renaudineau Y. et al., Clin Diagn Lab Immunol. 1999 Mar;6 (2): 156); Chan OT. et al., Immunol Rev 1999 Jun;169: 107), glandular diseases, glandular autoimmune diseases, pancreatic autoimmune diseases, diabetes, Type I diabetes (Zimmet P. Diabetes Res Clin Pract 1996 Oct;34 Suppl:S 125), thyroid diseases, autoimmune thyroid diseases, Graves' disease (Orgiazzi J. Endocrinol Metab Clin North Am 2000 Jun;29 (2):339), thyroiditis, spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol 2000 Dec 15;165 (12):7262), Hashimoto's thyroiditis (Toyoda N. et al, Nippon Rinsho 1999 Aug;57 (8): 1810), myxedema, idiopathic myxedema (Mitsuma T. Nippon Rinsho. 1999 Aug;57 (8): 1759); autoimmune reproductive diseases, ovarian diseases, ovarian autoimmunity (Garza KM. et al, J Reprod Immunol 1998 Feb;37 (2):87), autoimmune anti-sperm infertility (Diekman AB. et al., Am J Reprod Immunol. 2000 Mar;43 (3): 134), repeated fetal loss (Tincani A. et al., Lupus 1998;7 Suppl 2:S 107-9), neurodegenerative diseases, neurological diseases, neurological autoimmune diseases, multiple sclerosis (Cross AH. et al., J Neuroimmunol 2001 Jan 1 ; 112 (1-2): 1), Alzheimer's disease (Oron L. et al., J Neural Transm Suppl. 1997;49:77), myasthenia gravis (Infante AJ. And Kraig E, Int Rev Immunol 1999; 18 (l-2):83), motor neuropathies (Kornberg AJ. J Clin Neurosci. 2000 May;7 (3): 191), Guillain-Barre syndrome, neuropathies and autoimmune neuropathies (Kusunoki S. Am J Med Sci. 2000 Apr;319 (4):234), myasthenic diseases, Lambert-Eaton myasthenic syndrome (Takamori M. Am J Med Sci. 2000 Apr;319 (4):204), paraneoplastic neurological diseases, cerebellar atrophy, paraneoplastic cerebellar atrophy, non-paraneoplastic stiff man syndrome, cerebellar atrophies, progressive cerebellar atrophies, encephalitis, Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de la Tourette syndrome, polyendocrinopathies, autoimmune polyendocrinopathies (Antoine JC. and Honnorat J. Rev Neurol (Paris) 2000 Jan; 156 (1):23); neuropathies, dysimmune neuropathies (Nobile-Orazio E. et al., Electroencephalogr Clin Neurophysiol Suppl 1999;50:4-19); neuromyotonia, acquired neuromyotonia, arthrogryposis multiplex congenita (Vincent A. et al., Ann N Y Acad Sci. 1998 May 13;841:482), cardiovascular diseases, cardiovascular autoimmune diseases, atherosclerosis (Matsuura E. et al., Lupus. 1998;7 Suppl 2:S 135), myocardial infarction (Vaarala O. Lupus. 1998;7 Suppl 2:S 132), thrombosis (Tincani A. et al, Lupus 1998;7 Suppl 2:S 107-9), granulomatosis, Wegener's granulomatosis, arteritis, Takayasu's arteritis and Kawasaki syndrome (Praprotnik S. et al, Wien Klin Wochenschr 2000 Aug 25;112 (15-16):660); anti-factor VIII autoimmune disease (Lacroix-Desmazes S. et al, Semin Thromb Hemost.2000;26 (2): 157); vasculitises, necrotizing small vessel vasculitises, microscopic polyangiitis, Churg and Strauss syndrome, glomerulonephritis, pauci-immune focal necrotizing glomerulonephritis, crescentic glomerulonephritis (Noel LH. Ann Med Interne (Paris). 2000 May; 151 (3): 178); antiphospholipid syndrome (Flamholz R. et al., J Clin Apheresis 1999; 14 (4): 171); heart failure, agonist-like beta-adrenoceptor antibodies in heart failure (Wallukat G. et al, Am J Cardiol. 1999 Jun 17;83 (12A):75H), thrombocytopenic purpura (Moccia F. Ann Ital Med Int. 1999 Apr- Jun; 14 (2): 114); hemolytic anemia, autoimmune hemolytic anemia (Efremov DG. et al., Leuk Lymphoma 1998 Jan;28 (3-4):285), gastrointestinal diseases, autoimmune diseases of the gastrointestinal tract, intestinal diseases, chronic inflammatory intestinal disease (Garcia Herola A. et al., Gastroenterol Hepatol. 2000 Jan;23 ( 1): 16), celiac disease (Landau YE. and Shoenfeld Y. Harefuah 2000 Jan 16; 138 (2): 122), autoimmune diseases of the musculature, myositis, autoimmune myositis, Sjogren's syndrome (Feist E. et al., Int Arch Allergy Immunol 2000 Sep;123 (1):92); smooth muscle autoimmune disease (Zauli D. et al., Biomed Pharmacother 1999 Jun;53 (5-6):234), hepatic diseases, hepatic autoimmune diseases, autoimmune hepatitis (Manns MP. J Hepatol 2000 Aug; 33 (2):326) and primary biliary cirrhosis (Strassburg CP. et al., Eur J Gastroenterol Hepatol. 1999 Jun; 11 (6):595).
Type IV or T cell mediated hypersensitivity, include, but are not limited to, rheumatoid diseases, rheumatoid arthritis (Tisch R, McDevitt HO. Proc Natl Acad Sci U S A 1994 Jan 18; 91 (2):437), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Datta SK., Lupus 1998; 7 (9):591), glandular diseases, glandular autoimmune diseases, pancreatic diseases, pancreatic autoimmune diseases, Type 1 diabetes (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647); thyroid diseases, autoimmune thyroid diseases, Graves' disease (Sakata S. et al, Mol Cell Endocrinol 1993 Mar;92 (1):77); ovarian diseases (Garza KM. et al, J Reprod Immunol 1998 Feb;37 (2):87), prostatitis, autoimmune prostatitis (Alexander RB. et al, Urology 1997 Dec;50 (6):893), polyglandular syndrome, autoimmune polyglandular syndrome, Type I autoimmune polyglandular syndrome (Hara T. et al, Blood. 1991 Mar 1;77 (5): 1127), neurological diseases, autoimmune neurological diseases, multiple sclerosis, neuritis, optic neuritis (Soderstrom M. et al, J Neurol Neurosurg Psychiatry 1994 May;57 (5):544), myasthenia gravis (Oshima M. et al, Eur J Immunol 1990 Dec;20 (12):2563), stiff-man syndrome (Hiemstra HS. et al, Proc Natl Acad Sci U S A 2001 Mar 27;98 (7):3988), cardiovascular diseases, cardiac autoimmunity in Chagas' disease (Cunha-Neto E. et al, J Clin Invest 1996 Oct 15;98 (8): 1709), autoimmune thrombocytopenic purpura (Semple JW. et al, Blood 1996 May 15;87 (10):4245), anti-helper T lymphocyte autoimmunity (Caporossi AP. et al, Viral Immunol 1998; 11 (1):9), hemolytic anemia (Sallah S. et al, Ann Hematol 1997 Mar;74 (3): 139), hepatic diseases, hepatic autoimmune diseases, hepatitis, chronic active hepatitis (Franco A. et al, Clin Immunol Immunopathol 1990 Mar;54 (3):382), biliary cirrhosis, primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 Nov; 91 (5):551), nephric diseases, nephric autoimmune diseases, nephritis, interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug; 1 (2): 140), connective tissue diseases, ear diseases, autoimmune connective tissue diseases, autoimmune ear disease (Yoo TJ. et al, Cell Immunol 1994 Aug; 157 (1):249), disease of the inner ear (Gloddek B. et al, Ann N Y Acad Sci 1997 Dec 29; 830:266), skin diseases, cutaneous diseases, dermal diseases, bullous skin diseases, pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.
Examples of delayed type hypersensitivity include, but are not limited to, contact dermatitis and drug eruption.
Examples of types of T lymphocyte mediating hypersensitivity include, but are not limited to, helper T lymphocytes and cytotoxic T lymphocytes.
Examples of helper T lymphocyte-mediated hypersensitivity include, but are not limited to, Thl lymphocyte mediated hypersensitivity and Th2 lymphocyte mediated hypersensitivity. Autoimmune diseases
Include, but are not limited to, cardiovascular diseases, rheumatoid diseases, glandular diseases, gastrointestinal diseases, cutaneous diseases, hepatic diseases, neurological diseases, muscular diseases, nephric diseases, diseases related to reproduction, connective tissue diseases and systemic diseases.
Examples of autoimmune cardiovascular diseases include, but are not limited to atherosclerosis (Matsuura E. et al, Lupus. 1998;7 Suppl 2:S 135), myocardial infarction (Vaarala O. Lupus. 1998;7 Suppl 2:S 132), thrombosis (Tincani A. et al, Lupus 1998;7 Suppl 2:S 107-9), Wegener's granulomatosis, Takayasu's arteritis, Kawasaki syndrome (Praprotnik S. et al, Wien Klin Wochenschr 2000 Aug 25;112 (15-16):660), anti-factor VIII autoimmune disease (Lacroix-Desmazes S. et al, Semin Thromb Hemost.2000;26 (2): 157), necrotizing small vessel vasculitis, microscopic polyangiitis, Churg and Strauss syndrome, pauci-immune focal necrotizing and crescentic glomerulonephritis (Noel LH. Ann Med Interne (Paris). 2000 May; 151 (3): 178), antiphospholipid syndrome (Flamholz R. et al, J Clin Apheresis 1999; 14 (4): 171), antibody-induced heart failure (Wallukat G. et al, Am J Cardiol. 1999 Jun 17;83 (12A):75H), thrombocytopenic purpura (Moccia F. Ann Ital Med Int. 1999 Apr- Jun; 14 (2): 114; Semple JW. et al, Blood 1996 May 15;87 (10):4245), autoimmune hemolytic anemia (Efremov DG. et al, Leuk Lymphoma 1998 Jan;28 (3-4):285; Sallah S. et al, Ann Hematol 1997 Mar;74 (3): 139), cardiac autoimmunity in Chagas' disease (Cunha-Neto E. et al, J Clin Invest 1996 Oct 15;98 (8): 1709) and anti-helper T lymphocyte autoimmunity (Caporossi AP. et al, Viral Immunol 1998;11 (1):9).
Examples of autoimmune rheumatoid diseases include, but are not limited to rheumatoid arthritis (Krenn V. et al, Histol Histopathol 2000 Jul;15 (3):791; Tisch R, McDevitt HO. Proc Natl Acad Sci units S A 1994 Jan 18;91 (2):437) and ankylosing spondylitis (Jan Voswinkel et al, Arthritis Res 2001; 3 (3): 189).
Examples of autoimmune glandular diseases include, but are not limited to, pancreatic disease, Type I diabetes, thyroid disease, Graves' disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto's thyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmune anti-sperm infertility, autoimmune prostatitis and Type I autoimmune polyglandular syndrome. Diseases include, but are not limited to autoimmune diseases of the pancreas, Type 1 diabetes (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647; Zimmet P. Diabetes Res Clin Pract 1996 Oct;34 Suppl:S 125), autoimmune thyroid diseases, Graves' disease (Orgiazzi J. Endocrinol Metab Clin North Am 2000 Jun;29 (2):339; Sakata S. et al, Mol Cell Endocrinol 1993 Mar;92 (1):77), spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol 2000 Dec 15; 165 (12):7262), Hashimoto' s thyroiditis (Toyoda N. et al, Nippon Rinsho 1999 Aug;57 (8): 1810), idiopathic myxedema (Mitsuma T. Nippon Rinsho. 1999 Aug;57 (8): 1759), ovarian autoimmunity (Garza KM. et al, J Reprod Immunol 1998 Feb;37 (2):87), autoimmune anti-sperm infertility (Diekman AB. et al., Am J Reprod Immunol. 2000 Mar;43 (3): 134), autoimmune prostatitis (Alexander RB. et al, Urology 1997 Dec;50 (6):893) and Type I autoimmune polyglandular syndrome (Hara T. et al, Blood. 1991 Mar 1 ;77 (5): 1127).
Examples of autoimmune gastrointestinal diseases include, but are not limited to, chronic inflammatory intestinal diseases (Garcia Herola A. et al, Gastroenterol Hepatol. 2000 Jan; 23 (1): 16), celiac disease (Landau YE. and Shoenfeld Y. Harefuah 2000 Jan 16; 138 (2): 122), colitis, ileitis and Crohn' s disease.
Examples of autoimmune cutaneous diseases include, but are not limited to, autoimmune bullous skin diseases, such as, but are not limited to, pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.
Examples of autoimmune hepatic diseases include, but are not limited to, hepatitis, autoimmune chronic active hepatitis (Franco A. et al , Clin Immunol Immunopathol 1990 Mar; 54 (3):382), primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 Nov; 91 (5):551 ; Strassburg CP. et al, Eur J Gastroenterol Hepatol. 1999 Jun; 11 (6):595) and autoimmune hepatitis (Manns MP. J Hepatol 2000 Aug; 33 (2):326).
Examples of autoimmune neurological diseases include, but are not limited to, multiple sclerosis (Cross AH. et al , J Neuroimmunol 2001 Jan 1 ; 112 (1-2): 1), Alzheimer' s disease (Oron L. et al, J Neural Transm Suppl. 1997;49:77), myasthenia gravis (Infante AJ. And Kraig E, Int Rev Immunol 1999; 18 (l-2):83; Oshima M. et al, Eur J Immunol 1990 Dec;20 (12):2563), neuropathies, motor neuropathies (Kornberg AJ. J Clin Neurosci. 2000 May;7 (3): 191); Guillain-Barre syndrome and autoimmune neuropathies (Kusunoki S. Am J Med Sci. 2000 Apr; 319 (4):234), myasthenia, Lambert- Eaton myasthenic syndrome (Takamori M. Am J Med Sci. 2000 Apr;319 (4):204); paraneoplastic neurological diseases, cerebellar atrophy, paraneoplastic cerebellar atrophy and stiff-man syndrome (Hiemstra HS. et al., Proc Natl Acad Sci units S A 2001 Mar 27;98 (7):3988); non-paraneoplastic stiff man syndrome, progressive cerebellar atrophies, encephalitis, Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de la Tourette syndrome and autoimmune polyendocrinopathies (Antoine JC. and Honnorat J. Rev Neurol (Paris) 2000 Jan;156 (1):23); dysimmune neuropathies (Nobile- Orazio E. et al., Electroencephalogr Clin Neurophysiol Suppl 1999;50:4-19); acquired neuromyotonia, arthrogryposis multiplex congenita (Vincent A. et al., Ann N Y Acad Sci. 1998 May 13;841:482), neuritis, optic neuritis (Soderstrom M. et al., J Neurol Neurosurg Psychiatry 1994 May;57 (5):544) and neurodegenerative diseases.
Examples of autoimmune muscular diseases include, but are not limited to, myositis, autoimmune myositis and primary Sjogren's syndrome (Feist E. et al., Int Arch Allergy Immunol 2000 Sep; 123 (1):92) and smooth muscle autoimmune disease (Zauli D. et al, Biomed Pharmacother 1999 Jun;53 (5-6):234).
Examples of autoimmune nephric diseases include, but are not limited to, nephritis and autoimmune interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug; 1 (2): 140).
Examples of autoimmune diseases related to reproduction include, but are not limited to, repeated fetal loss (Tincani A. et al, Lupus 1998; 7 Suppl 2:S 107-9).
Examples of autoimmune connective tissue diseases include, but are not limited to, ear diseases, autoimmune ear diseases (Yoo TJ. et al, Cell Immunol 1994 Aug; 157 (1):249) and autoimmune diseases of the inner ear (Gloddek B. et al., Ann N Y Acad Sci 1997 Dec 29; 830:266).
Examples of autoimmune systemic diseases include, but are not limited to, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998; 17 (l-2):49) and systemic sclerosis (Renaudineau Y. et al., Clin Diagn Lab Immunol. 1999 Mar;6 (2): 156); Chan OT. et al, Immunol Rev 1999 Jun; 169: 107).
Infectious diseases
Examples of infectious diseases include, but are not limited to, chronic infectious diseases, subacute infectious diseases, acute infectious diseases, viral diseases, bacterial diseases, protozoan diseases, parasitic diseases, fungal diseases, mycoplasma diseases and prion diseases. Graft rejection diseases
Examples of diseases associated with transplantation of a graft include, but are not limited to, graft rejection, chronic graft rejection, subacute graft rejection, hyper-acute graft rejection, acute graft rejection and graft versus host disease (GVHD).
Allergic diseases
Examples of allergic diseases include, but are not limited to, asthma, hives, urticaria, pollen allergy, dust mite allergy, venom allergy, cosmetics allergy, latex allergy, chemical allergy, drug allergy, insect bite allergy, animal dander allergy, stinging plant allergy, poison ivy allergy and food allergy. Injuries
The methods of the invention may be used to treat any injury or damage to a cell, tissue (e.g. soft tissue) or organ which involves an inflammation, including, but not limited to, acute, chronic, ischemic, or traumatic (e.g. such as that associated with a surgery or accident) injury to the skeletal muscle, heart (e.g. cardiac muscle or cardiovascular cell), kidney, liver, intestine, brain, lung, pancreas, vascular, dermal tissue, scalp, or eye as well as ischemia-reperfusion injury (IRI).
In a specific embodiment, the inflammatory condition is associated with an autoimmune disease (e.g. multiple sclerosis).
In a specific embodiment, the inflammatory condition is associated with diabetes, metabolic syndrome, and related diseases and conditions.
Diabetes is typically characterized by elevated levels of plasma glucose (hyperglycemia) in the fasting state. There are two generally recognized forms of diabetes: type 1 diabetes (in which patients produce little or no insulin) and type 2 diabetes (in which patients produce "insulin resistance" such that the effect of insulin in stimulating glucose and lipid metabolism in the main insulin-sensitive tissues, namely, muscle, liver, and adipose tissues, is diminished).
Abnormal glucose homeostasis is associated both directly and indirectly with obesity, hypertension, and alterations in lipid, lipoprotein, and apolipoprotein metabolism. Patients with diabetes are at increased risk of cardiovascular complications, e.g., atherosclerosis, coronary heart disease, stroke, peripheral vascular disease, hypertension, nephropathy, neuropathy and retinopathy. Persistent or uncontrolled hyperglycemia that occurs in diabetes is also associated with increased morbidity and premature mortality.
Metabolic Syndrome is typically characterized by insulin resistance, along with abdominal obesity, hyperinsulinemia, high blood pressure, low HDL levels, high VLDL triglyceride and small dense LDL particles and elevated glucose levels. Subjects having metabolic syndrome, whether or not they develop overt diabetes mellitus, are at increased risk of developing cardiovascular complications (as discussed above).
According to some embodiments, the inflammation is not associated with a cancer.
The Perf+ imDC of some embodiments of the invention can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
Herein the term "active ingredient" refers to the Perf+ imDC accountable for the biological effect.
Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.
Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.
Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.
Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide). However, each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.
Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.
The term "tissue" refers to part of an organism consisting of cells designed to perform a function or functions. Examples include, but are not limited to, brain tissue, retina, skin tissue, hepatic tissue, pancreatic tissue, bone, cartilage, connective tissue, blood tissue, muscle tissue, cardiac tissue brain tissue, vascular tissue, renal tissue, pulmonary tissue, gonadal tissue, hematopoietic tissue.
According to one embodiment, the route of administration includes, for example, an injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the pharmaceutical composition of the present invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the pharmaceutical composition of the present invention is administered by i.v. injection. The pharmaceutical composition may be injected directly into a site of inflammation.
Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (Perf imDCs) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., inflammation) or prolong the survival of the subject being treated.
Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
When "therapeutic amount" is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, disease state, e.g. extent of inflammation, and the condition of the patient (subject). It can be generally be stated that a pharmaceutical composition comprising the Perff imDCs described herein may be administered at a dosage of 5 x 10 4 /kg body weight to 1 x 108 /kg body weight, including all integer values within those ranges.
According to one embodiment, the Perf+ imDCs described herein may be administered at a dosage of about 5 x 10 4 /kg body weight to 1 x 108 /kg body weight, 5 x 104/kg body weight to 1 x 107/kg body weight, 5 x 104/kg body weight to 1 x 106/kg body weight, 5 x 104/kg body weight to 1 x 105/kg body weight, 5 x 105/kg body weight to 1 x
10 8 /kg body weight, 5 x 105 /kg body weight to 1 x 107 /kg body weight, 5 x 105 /kg body weight to 1 x 106/kg body weight, 5 x 106/kg body weight to 1 x 108/kg body weight, 5 x
10 6 /kg body weight to 1 x 107 /kg body weight or 5 x 107 /kg body weight to 1 x 108 /kg body weight.
According to one embodiment, the Perf+ imDCs described herein may be administered at a dosage of about 1 x 104/kg body weight, 2.5 x 104/kg body weight, 5 x 104/kg body weight, 1 x 105/kg body weight, 2.5 x 105/kg body weight, 5 x 105/kg body weight, 1 x 106/kg body weight, 2.5 x 106/kg body weight, 5 x 106/kg body weight, 1 x 107/kg body weight, 2.5 x 107/kg body weight, 5 x 107/kg body weight, 1 x 108/kg body weight, 2.5 x 10 8 /kg body weight, 5 x 108 /kg body weight or 1 x 109 /kg body weight.
The cell compositions of some embodiments of the invention may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.
For example, the effect of the active ingredients (e.g., the Perf+ imDCs of some embodiments of the invention) on the pathology can be evaluated by monitoring the level of inflammatory markers, e.g., hormones, glucose, peptides, carbohydrates, etc. in a biological sample of the treated subject using well known methods (e.g. standard blood tests, ELISA, FACS, PCR, etc).
According to one embodiment, the therapeutically effective amount the Perf+ imDCs is an amount capable of inhibiting an activity or proliferation of a CD4+ T cell and/or a CD8+ T cell.
Such determinations are well known to one of skill in the art, and may be carried out, for example, by obtaining a blood sample from a subject and determining the activity and/or proliferating levels of CD4+ T cell and/or a CD8+ T cell in the lymphocyte fraction of the blood sample. For example, T cell evaluation may comprise any of the following: quantitative details of specific types of T cells as well as evaluation of T cell characteristics of these cells may be carried out, for example, by complete blood count (CBC) and differential blood count, flow cytometry (FACS), TREC testing and other measures to characterize recent thymic emigrants, and assessment of TCR diversity; functional testing including, for example, lymphocyte proliferation to mitogens, antigens, and/or allogeneic cells; cytokine production; T cell-mediated cytotoxicity and Treg activity, as taught by Rosenzweig and Fleisher, J Allergy Clin Immunol. (2013) 131(2): 622-3.el-4.
For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
Animal models for inflammation are described for example in Webb DR, Biochem Pharmacol. (2014) 87(1): 121-30.
Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).
Dosage amount and interval may be adjusted individually to provide the active ingredient at a sufficient amount to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. According to some embodiments of the invention, the Perf imDCs of the invention can be provided to the subject with a factor capable of inhibiting the Perf+ imDCs from entering maturation. Such factors are described in detail herein above, and include e.g. rapamycin, aspirin or HO-1.
According to one embodiment, the factor capable of inhibiting the Perf+ imDCs from entering maturation is administered to the subject prior to administration of the Perf+ imDCs, e.g. 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days or more prior to administration of the Perf+ imDCs.
According to one embodiment, the factor capable of inhibiting the Perf+ imDCs from entering maturation is administered to the subject in conjunction to administration of the Perf+ imDCs, e.g. at the same time, or within 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours.
According to one embodiment, the factor capable of inhibiting the Perf+ imDCs from entering maturation is administered to the subject following administration of the Perff imDCs, e.g. 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days or more following administration of the Perf+ imDCs.
Such administrations may be effected in a single dose or in several doses. Such a determination is well within the capability of one of skill in the art.
According to some embodiments of the invention, the Perff imDCs of the invention can be provided to the subject in conjunction with other drug(s) designed for treating the pathology [combination therapy, (e.g., before, simultaneously or following)].
In certain embodiments of the present invention, the Perff imDCs of some embodiments of the invention are administered to a patient in conjunction with any number of relevant treatment modalities, including but not limited to, treatment with agents such as antiviral agents (e.g. Ganciclovir, Valaciclovir, Acyclovir, Valganciclovir, Foscarnet, Cidofovir, Maribavir, Leflunomide); agents for the treatment of multiple sclerosis (e.g. natalizumab); agents for the treatment of metabolic syndrome or diabetes (e.g. insulin), or anti-inflammatory therapies (e.g. NSAIDs (Non- Steroidal Antiinflammatory Drugs), corticosteroids (such as prednisone) and anti-histamines).
Further exemplary anti-inflammatory agents which may be used according to the present teachings are described in detail hereinabove. Any of the anti-inflammatory agents described herein may be administered individually or in combination. Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
The agents described hereinabove may be included in a therapeutic kit/article of manufacture preferably along with appropriate instructions for use and labels indicating FDA approval for use in treatment of inflammatory conditions (e.g. metabolic syndrome or autoimmune disease).
According to one embodiment of the present invention, there is provided a kit/article of manufacture comprising Perf imDCs.
It will be appreciated that the kit/article of manufacture may further comprise another active ingredient to improve therapeutic efficacy.
According to one embodiment of the present invention, there is provided a kit/article of manufacture comprising Perf+ imDCs and a factor capable of inhibiting the Perff imDCs from entering maturation (e.g. rapamycin, aspirin or HO-1).
According to one embodiment of the present invention, there is provided a kit/article of manufacture comprising Perff imDCs and a drug designed for treating the pathology (e.g. inflammation associated with an autoimmune disease, metabolic syndrome, infection, etc.), such a drug may include for example, an antiviral agent, an agent for the treatment of multiple sclerosis, an agent for the treatment of metabolic syndrome or diabetes, or an anti-inflammatory agent (as discussed in detail hereinabove).
Thus, for example, the Perf+ imDCs can be packaged in one container while the factor capable of inhibiting the Perff imDCs from entering maturation may be packaged in a second container both for therapeutic treatment. According to another embodiment, the Perf imDCs and the factor capable of inhibiting the Perf+ imDCs from entering maturation are in a co-formulation. Likewise, the Perf+ imDCs can be packaged in one container while the anti-inflammatory agent may be packaged in a second container both for therapeutic treatment. According to another embodiment, the Perf+ imDCs and the anti-inflammatory agent are in a co-formulation.
The kit/article of manufacture may also include appropriate buffers and preservatives for improving the shelf-life of the kit.
Depending on the application, the method may be effected using Perf+ imDCs which are syngeneic or non-syngeneic with the subject.
As used herein, the term "syngeneic" refers to cells which are derived from an individual who is essentially genetically identical with the subject. Typically, essentially fully inbred mammals, mammalian clones, or homozygotic twin mammals are syngeneic.
Examples of syngeneic cells include cells derived from the subject (also referred to in the art as "autologous"), a clone of the subject, or a homozygotic twin of the subject.
As used herein, the term "non-syngeneic" refers to cells which are derived from an individual who is allogeneic or xenogeneic with the subject's lymphocytes (also referred to in the art as "non-autologous").
As used herein, the term "allogeneic" refers to cells which are derived from a donor who is of the same species as the subject, but which is substantially non-clonal with the subject. Typically, outbred, non-zygotic twin mammals of the same species are allogeneic with each other. It will be appreciated that an allogeneic donor may be HLA identical or HLA non-identical with respect to the subject.
As used herein, the term "xenogeneic" refers to cells which substantially express antigens of a different species relative to the species of a substantial proportion of the lymphocytes of the subject. Typically, outbred mammals of different species are xenogeneic with each other.
The present invention envisages that xenogeneic cells are derived from a variety of species such as, but not limited to, bovines (e.g., cow), equids (e.g., horse), porcines (e.g. pig), ovids (e.g., goat, sheep), felines (e.g., Felis domestica), canines (e.g., Canis domestica), rodents (e.g., mouse, rat, rabbit, guinea pig, gerbil, hamster) or primates (e.g., chimpanzee, rhesus monkey, macaque monkey, marmoset). Cells of xenogeneic origin (e.g. porcine origin) are preferably obtained from a source which is known to be free of zoonoses, such as porcine endogenous retroviruses. Similarly, human-derived cells are preferably obtained from substantially pathogen-free sources.
Depending on the application and available sources, the cells of the present invention may be obtained from a prenatal organism, postnatal organism, an adult or a cadaver donor. Moreover, depending on the application needed, the cells may be naive or genetically modified. Such determinations are well within the ability of one of ordinary skill in the art
According to an embodiment of the present invention, the Perf+ imDCs are syngeneic with the subject (e.g. obtained from the subject).
According to an embodiment of the present invention, the Perf imDCs are non- syngeneic with the subject.
According to an embodiment of the present invention, both the subject and the donor of the Perf+ DCs are humans.
In cases where non-syngeneic Perf+ DCs are used, the subject may be further administered an immunosuppressive regimen in order to reduce rejection of the transplanted Perf+ DCs. The type of regimen may be determined by one of ordinary skill in the art and takes into account the age and disease severity of the subject. Thus, for example, an elderly subject (e.g. one who is over 60 years of age) may be treated with a mild immunosuppressive regimen.
Examples of suitable types of immunosuppressive regimens include administration of immunosuppressive drugs and/or immunosuppressive irradiation.
Ample guidance for selecting and administering suitable immunosuppressive regimens is provided in the literature of the art (for example, refer to: Kirkpatrick CH. and Rowlands DT Jr., 1992. JAMA. 268, 2952; Higgins RM. et al., 1996. Lancet 348, 1208; Suthanthiran M. and Strom TB., 1996. New Engl. J. Med. 331, 365; Midthun DE. et al., 1997. Mayo Clin Proc. 72, 175; Morrison VA. et al., 1994. Am J Med. 97, 14; Hanto DW, 1995. Annu Rev Med. 46, 381; Senderowicz AM. et al., 1997. Ann Intern Med. 126, 882; Vincenti F. et al., 1998. New Engl. J. Med. 338, 161; Dantal J. et al. 1998. Lancet 351, 623). According to a specific embodiment, the immunosuppressive regimen consists of administering at least one immunosuppressant agent to the subject.
Examples of immunosuppressive agents are described in detail hereinabove. Any of the immunosuppressive agents described herein may be administered individually or in combination.
As used herein the term "about" refers to ± 10 %.
The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".
The term "consisting of means "including and limited to".
The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.
Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween. As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
It is understood that any Sequence Identification Number (SEQ ID NO) disclosed in the instant application can refer to either a DNA sequence or a RNA sequence, depending on the context where that SEQ ID NO is mentioned, even if that SEQ ID NO is expressed only in a DNA sequence format or a RNA sequence format. Similarly, though some sequences are expressed in a RNA sequence format (e.g. , reciting U for uracil), depending on the actual type of molecule being described, it can refer to either the sequence of a RNA molecule comprising a dsRNA, or the sequence of a DNA molecule that corresponds to the RNA sequence shown. In any event, both DNA and RNA molecules having the sequences disclosed with any substitutes are envisioned.
EXAMPLES
Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non-limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
GENERAL MATERIALS AND EXPERIMENTAL PROCEDURES
Animals
Mice used were 6-12 week old females or males (in the metabolic studies) of the following strains: C57BL/6, perforin KO (PKO) (H-2db/I-Ab)1,CDl lc-eYFP2 (Weizmann Institute Animal Breeding Center, Israel). CDllc-Cre mice [previously disclosed in Caton M.L. et al., J. Exp. Med. (2007) 204, 1653-1664], R26-DTA mice (backcrossed for ten generations onto C57BL/6) [previously disclosed in Brockschnieder D. et al., Genesis (2006) 44, 322-327]. R26-DTA mice were crossed with CDl lc-Cre transgenic mice to generate CD l lc-Cre:DTA mice. Mixed [50 % DTA-50 % PKO > WT], [50 % WT-50 % PKO > WT], BM chimeras were generated by exposure of C57BL/6 WT mice to a single dose of 950 rad total body irradiation. The following day, the mice received i.v. 5 x 106 mixed BM cells, as indicated. The mice were allowed to rest for 6 weeks before use. Animals were maintained under conditions approved by the Institutional Animal Care and Use Committee at the Weizmann Institute of Science.
Diets
For excessive caloric intake experiments, mice were maintained on high-fat diet (HFD, D 12492, 60 Kcal % fat, Research Diets), the control group was fed either a standard chow diet or low fat sucrose-matched diet (NCD, D12450J, 10 Kcal % fat, Research Diets).
Metabolic studies
DTA-PKO and WT-PKO chimeric mice were weighed regularly. For glucose tolerance tests, fasted (16 hours) mice were given 1 g glucose per kg body weight; for insulin tolerance test, 0.75 U per kg body weight human insulin was used (NovoNordisk, Denmark). Glucose, triglycerides, total cholesterol and high density lipoprotein (HDL) levels were measured in mouse serum by SpotChem EZ Chemistry Analyzer (Arkray, Japan). Serum cytokine concentrations were detected using ELISA kits (TNF-a, IL-6 and IL-Ιβ from eBioscience, leptin from ENZO). Body composition analysis was performed using EchoMRI Analyzer (EchoMRI LLC, USA)
Cytofluorometric analysis
Flow cytometry was performed using a FACS-CantoII (BD Biosciences) and the analysis was done by FlowJo (Tree Star, Inc.) software. Single cell suspensions were stained with anti-CD8a, CD4, CD3, MHC-II (IA/IE), CD45, CDl lc, CDl lb, Ly6G, Gr-1, B220, CD19, F4/80, CD45.1, CD25, Foxp-3, TNF-a, IL-17a, or IFN-γ conjugated to FITC, PE, PerCp, APC, APC-Cy7, PE-Cy7, Pacific Blue, or Brilliant Violet 711 (Biolegend). Fat-associated stromal vascular fractions (SVF) cell isolation
Anesthetized mice (Ketamine-Xylazine) were systemically perfused with PBS, visceral AT (VAT) and inguinal SC pads were removed (avoiding lymph nodes), mashed into small pieces (approximately 1-2 mm) and digested with collagenase II (Sigma, 1 mg/ml in DMEM for 20 minutes at 37 °C, with vigorous agitation). The digests were then pelleted and filtered through 100 μηι filters. The cells were washed with PBS, incubated for 5 minutes in erythrocyte-lysing buffer (ACK), and finally resuspended in PBS supplemented with 2 % FBS. The nonspecific staining was blocked by antibody against FC receptor (Biolegend), and then the isolated cells were labeled with either monoclonal antibody or isotype control antibody (Biolegend). 7AAD was used to exclude dead cells.
Histology and immunohistochemistry
Tissue samples from spinal cord were fixed in 4 % PFA, processed and embedded in paraffin. Sections of 6 μηι were stained with hematoxylin and eosin (H&E). Tissue samples from visceral adipose tissue were fixed in 4 % PFA, processed and embedded in paraffin. Sections of 5 μηι were stained with hematoxylin and eosin (H&E). Fat cell area on H&E stained slides was measured using ImageJ software, quantifying two different tissue sections per mouse (at least 3 mice with at least 100 fat cells in each image). In addition, sections were stained with MAC-2 antibody (Cedarlane); MAC-2 positive CLS per field were counted manually in a blinded manner and used as a measure of adipose tissue CLS content. For staining of perforin in Perf-DC, splenic DCs from WT and PKO mice were isolated by two stage magnetic sorting (first negative depletion of CD3, Terl 19, Gr-1, B220 positive cells and then positive selection of CDl lc+ cells) and were spun onto glass slides and fixed with 4 % PFA. The cells were then blocked first with 5 % horse serum and 0.2 % Triton for 5 minutes, and subsequently with 100 % horse serum for 1 hour. Isotype blocking was done with rat IgG2a overnight in 25 % horse serum and 0.1 % Triton at 4 °C followed by secondary anti-RatCy2 or anti-Rat DyLight594 (Jackson) antibody to visualize the non-specific binding. Specific rat anti -perforin Ab (Clone CB5.4, Abeam) was applied overnight in 25 % horse serum and 0.1 % Triton at 4 °C followed by secondary anti-rat DyLight 594 or anti-rat AMCA (Jackson) antibody. The nuclei were stained with Hoechst blue or yellow stain. Cytotoxic T-lymphocytes (CTL) from C57BL/6 WT and PKO were prepared as previously described [Reich-Zeliger S. et al., The Journal of Immunology (2004) 173, 6660] and used as positive and negative controls, respectively, for perforin staining. 14 perforin positive cells from total of 375 DCs were counted in 6 fields of WT sample, 11 out of 431 cells in 6 fields of WT-PKO and 1 out of 1053 in 5 fields of DTA-PKO. The calculation was done using ImageJ software.
For CD4 and CD8 staining, anti-mouse CD4-FITC (clone GK1.5) and anti-mouse
CD8-FITC (53-6.7) were used (Biolegend).
Depletion of CD4 and CD8 T cell populations
Anti-CD4 (hybridoma GK1.5) or CD8 (hybridoma 53-6.72) (BioXcell) depleting antibodies were given i.p. weekly at a dose of 3 μg/gram body weight to chimeric mice starting 4 months post-transplant before the appearance of metabolic syndrome symptoms. The control mice received weekly i.p. injections of PBS.
cDNA Library Preparation for TCRfi Sequencing
Total RNA was extracted from spleen, visceral adipose tissue and subcutaneous adipose tissue of DTA-PKO and WT-PKO mice. RNA was extracted using an RNeasy Mini Kit (Qiagen) and reverse-transcribed with Superscript II reverse transcriptase (Invitrogen) using a primer specific for the TCRP constant region linked to the Solexa 3' adapter (CP-3'adp). cDNA was then used as a template for high fidelity PCR amplification (Phusion; Finnzymes) using a pool of 23 νβ-specific primers, divided into five primer groups, to minimize potential for cross-hybridization. Each νβ primer was linked to a restriction-site sequence for the ACUI restriction enzyme (New England BioLabs). PCR reactions were performed in duplicate, and PCR products were then pooled and cleaned using the QIAquick PCR Purification Kit (Qiagen), followed by enzymatic digestion, in accordance with the ACUI protocol (New England BioLabs). The ACUI enzyme was used to cleave the 14 bp downstream of its binding site, enabling positioning of the Illumina sequencing primer in close proximity to the junction region and ensuring sequencing of the entire variable CDR3 region. Digestion produced a 2-bp overhang for the ligation of the Illumina 5' adapter, which was linked to a 3-bp barcode sequence at its 3' end. Overnight ligation was performed using T4 ligase (Fermentas) at 16 °C in accordance with the manufacturer's protocol. A second round of PCR amplification was performed (24 cycles), using primers for the 5' and 3' Illumina adapters. Final PCR products were run on an agarose gel (2 %), and purified using the Wizard SV Gel and PCR Clean-Up System (Promega). Final library concentrations were measured using a NanoDrop spectrophotometer. The libraries were sequenced using an Illumina Genome Analyzer II.
Computational Analysis of TCRfi Sequencing Data
For preprocessing of the data, the Smith-Waterman alignment algorithm was used to assign to each sequencing read its variable (νβ) and joining (Ιβ) gene, using germ-line νβ/Ιβ gene segment sequences downloaded from the IMGT database. Reads that were not assigned to either a νβ or Ιβ, and other erroneous reads were discarded. The library- derived reads were then clustered using a version of the quality threshold clustering algorithm to correct for nucleotide copying errors (up to two errors for each read). The clustering procedure identified unique CDR3β clonotypes, defined as the most prevalent read found in each cluster. The clonotype sequences were then translated, and those clonotypes that lacked a stop codon in-frame with the V/D/J sequences were considered for further analysis. This analysis computed statistics of V/D/J use, statistical properties of the number of deletions and insertions of nucleotides at both VD and DJ junctions, as well as distributions of CDR3 lengths. The analysis was done using the R statistical software package (R Development Core Team; www(dot)r-project(dot)org). Frequencies of νβ and Ιβ segment use were measured for all samples in each treatment group. Correlation coefficients were calculated, based on the sample mean of combined νβ and Ιβ use in each group, using MATLAB (Mathworks). Hierarchical clustering was performed, based on combined νβ and Ιβ use, in all groups (clustergram; MATLAB).
RNA Extraction and Quantitative RT-PCRfor adipose tissue (AT)
Total RNA from fat pads was extracted with the RNeasy lipid tissue mini kit (Qiagen, Germantown, MD) and analyzed with Nanodrop©. RNA (100 ng) was reverse- transcribed with qScript cDNA Synthesis Kit (Quanta Biosciences). SYBR Green system (Roche) was used for real-time PCR amplification. Data were normalized based on TBP expression as housekeeping gene, using appropriate primers (see Table 1, below). Table 1: Primers used for RT-PCR
Figure imgf000052_0001
EAE induction and evaluation
Chronic EAE was induced in C57BL/6 chimeric mice by injecting a peptide consisting of amino acids 35-55 of myelin oligodendrocyte glycoprotein (MOG), synthesis by Genscript (Piscataway, NJ, USA). Mice were injected subcutaneously at the flank, with 200 μΐ emulsion containing 200 μg of the encephalitogenic peptide in incomplete Freund's adjuvant enriched with 5 mg/ml heat-inactivated Mycobacterium tuberculosis (Sigma, St. Louis, MO, USA). Mice were examined daily. EAE was scored as follows: 0 - no disease, 1 - limp tail, 2 - hind limb paralysis, 3 - paralysis of all limbs, 4 - moribund condition, and 5 - death, as previously described [Aharoni R. et al. Exp. Neurol. (2013) 240, 130-144].
CFSE proliferation assay of CD4 and CD8 T cells
Isolated lymph node cells from chimeric mice 14 or 30 days after EAE induction were isolated and stained with 5 μΜ CFSE (CellTrace CFSE Cell Proliferation Kit, Invitrogen) for 15 minutes. After staining, 0.5 x 106 cells were incubated in full RPMI medium supplemented with 10 % FCS for 4 days in the presence of either MOG, PPD (adjuvant related peptide) or MBP (unrelated encephalitogenic peptide) peptides. The cells were then harvested and analyzed by flow cytometry for CFSE staining in conjunction with either CD4 or CD8 staining to examine the proliferation status based on CFSE dilution.
Generation of Perforin+ Immature DCs (Perf+ imDCs)
Perf imDCs were generated using the 20 days protocol as previously described [Zangi L. et al., Blood. (2012) 120(8): 1647-57]. In short, bone marrow cells were obtained from tibiae and femurs of C57BL/6, CB6/F1, FVB, gld-/-, PKO, TLR7-/- and Dap 12-/- mice. The cell suspension was enriched for the SCA1+ population using anti-SCAl-PE antibody (clone E13-161.7, Miltenyi Biotec), followed by incubation with anti-PE MicroBeads and positive selection on a MACS separation column (Miltenyi Biotec). The cells were then stained for lineage positive markers with the following biotinylated Abs: CD3e (clone 145-2C11), CDl lb (clone Ml/70), CD45R/B220 (clone RA3-6B2), Ly6G/ Ly-6C (clone RB6-8C5), and TER-119 (clone TER-119) (BD Pharmingen) followed by staining with streptavidin-APC (Jackson Immunoresearch Laboratories). SCA-1+ Lin-, C- Kit+ cells were then sorted using FACSAria Sorter. Then 105 cells/ml were cultured for 10 days in 24-well plates in Iscove's Modified Dulbecco's Medium (IMDM) containing 10 % FCS, 2 mM L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin (Biological Industries). Cultures were supplemented every second day with 50 ng/ml stem cell factor (SCF), 1 ng/ml thrombopoietin (TPO), 50 ng/ml Flt3-ligand (Flt3L), 10 ng/ml interleukin- 3 (IL-3), and 10 ng/ml interleukin-6 (IL-6) (Peprotech). After 10 days, differentiated cells (early myeloid cells) were cultured for an additional 10 days in RPMI 1640 complete tissue culture medium (CTMC), 20 ng/ml granulocyte-macrophage colony- stimulating factor (GM-CSF) (Peprotech). imDCs were collected after 20 days.
Inhibition of maturation of perforin + immature DCs
Perf imDCs were generated using the 20 days protocol as described above. One group of cells was additionally treated with 2.5 mM aspirin during the last 10 days of culture. A second group of cells was additionally treated with 10 ng/ml Rapamycin during the last 10 days of culture. A third group of cells was additionally treated with 50 mM CoPP / SnPP (an inducer and an inhibitor of HO-1, respectively) during the last 10 days of culture. A fourth group was not additionally treated. To induce maturation, cells were treated with 1 μg/ml LPS for 24 hours.
Cell Killing Assay
Aspirin-treated Perf+ imDCs were loaded with SIINFEKL peptide (SEQ ID NO: 29) or with SIYRYYGL peptide (SEQ ID NO: 30). These cells were tested for their ability to kill cognate OT-I CD8+ T cells in short-term mixed lymphocyte reaction (MLR).
Statistical analysis
The results are expressed as means + SD or S.E.M, as indicated in each figure legend. The statistical significance of differences between two groups was determined using Student's t-tests; values of p < 0.05 were considered significant.
EXAMPLE 1
Characterization of Perf-DC in radiation chimeras
As previously described [Zangi L. et al. (2012), supra] Perf-DC (also referred to herein as Perforin positive immature DCs) represent a minor subpopulation (approximately 4 %) within the CDl lc+ population in the spleen of wild type (WT) C57BL/6 mice (Figure 8a). This rare subpopulation of perf-DCs is markedly enhanced upon in vivo administration of GM-CSF [Zangi et al. (2012), supra], and to a lesser extent, upon treatment with Flt3L (Figure 8b and 8c). Further immuno staining analysis indicated that this subpopulation does not express CD4 or CD8 consistent with double negative (DN) DCs (Figure 9). These results are in line with sequencing data from Immgen (immgen(dot)org) suggesting that while Zbtb46, a recently introduced marker for classical DC (cDC), is expressed in all three subtypes of CDl lchlgh cells, perforin is expressed largely in the DN DCs.
Furthermore, as shown in Figures lOa-d perforin is expressed in CDl lchlghMHC- jjhi h ηο^ m CDl lcint macrophages identified by their F4/80 expression and by their inability to stimulate effectively T cells.
To evaluate the functional role of Perf-DC in the steady state in-vivo, mice were generated which selectively lack perforin expression in CDl lchlgh DC. Chronic selective ablation of these cells can be attained in transgenic mice expressing the diphtheria toxin (DTx) A subunit (DTA) under control of a CDl lc promoter (CDl lcCre:R26-STOP-DTA mice as previously taught [Birnberg T. et al., Immunity (2008) 29, 986-997]. Of note, most macrophages, NK cells and pDC, expressing low to intermediate levels of CDl lc are spared in these mice [Birnberg et al., (2008), supra]. To induce a restricted perforin deficiency within the CDl lchlgh DC population, the present inventors generated bone marrow (BM) chimeras using a 1: 1 mixture of perforin KO BM as previously taught [Kagi D. et al., Nature (1994) 369, 31-37] and from BM of mice expressing DTA in CDl lchigh cells (CDl lc-DTA mice) [Birnberg et al., (2008), supra]. Based on the reported specifics of the CDl lc-DTA mice [Birnberg et al., (2008), supra], in the resulting chimeras (DTA- PKO), cells other than CDl lchlgh DC will be derived from both types of donor BM (and hence 50 % will express perforin). In contrast, due to the DTA-mediated ablation of perforin-proficient cells, the CDl lchlgh population will be exclusively derived from the PKO donor, and therefore lack perforin. These chimeras should hence allow determining whether the absence of Perf-DC leads to a particular autoimmune phenotype, similarly to that found in mice lacking other immune regulatory cells [Sakaguchi S. et al. Cell (2000) 101, 455-458]. In parallel the present inventors generated control BM chimeras, by transplantation of a 1: 1 mixture of BM from PKO mice and BM from CDl 1 -DTA negative littermates (WT-PKO mice). Histological evaluation using ImageJ software of cDCs isolated from spleens of DTA-PKO chimeras showed indeed absence of perforin expressing DCs (0.09 + 0.19 %), while in WT-PKO chimera the level of Perf-DC was reduced to 2.4 + 1.9 % from 4.1 + 1 % found in WT mice (data not shown).
Confirming this ablation strategy, cDCs CDl lchigh MHC-IIhigh cells (Figure la), although reaching the same steady state levels as in the WT-PKO chimera (Figure lb), were exclusively derived from the CD45.2+ PKO donor (Figure lc). Likewise, BM pre- DC (Figure Id), as well as double negative (DN) DC (Figure Id) largely originated from the CD45.2+ PKO donor. In contrast, WT-PKO chimera exhibited CDl lchigh cells that were equally derived from both BM donors (Figure Id). Notably, T cells, B cells, neutrophils, pDCs, NK cells and macrophages in the spleen of DTA-PKO chimeras were equally derived from both BM donors (Figure le and Table 2, hereinbelow).
Taken together, these results establish that DTA-PKO chimeras, in which all CDl lchlgh DC were derived from PKO donors and thus do not express perforin offer a robust model for studying the role of Perf-DC in the steady state EXAMPLE 2
Perf-DC deficient chimeras develop metabolic syndrome
Following the generation of DTA-PKO chimeras, these mice were followed up for a prolonged period of time to assess the development of a potential pathological phenotype that might be associated with the lack of Perf-DC. Interestingly, DTA-PKO chimeric mice spontaneously gained significantly more weight compared to their WT-PKO counterparts, starting at approximately 5 months post-transplant (Figures 2a-b). A similar difference was found when comparing the animals to an additional control group in which recipient mice received mixed BM from WT and DTA donors (WT-DTA) (Figures l la-d). The increased weight gain observed in DTA-PKO mice prompted the present inventors to test whether this phenomenon was accompanied by metabolic alterations. Indeed, dyslipidemia was detected in DTA-PKO mice, manifested by elevated serum cholesterol and triglyceride levels in comparison to those exhibited by the controls (Figure 2c). In addition, the percent of total body fat, as measured by body composition MRI, was significantly elevated in DTA-PKO mice compared to both control groups (Figure 2f). DTA-PKO mice, but not the control chimeras, displayed highly elevated levels of TNF-a (which has been linked to obesity-associated inflammation and insulin resistance) and leptin, the proinflammatory adipokine associated with obesity, overeating, hypertension, cardiovascular diseases and metabolic syndrome (Figures 2d-e). Furthermore, DTA-PKO chimeric mice exhibited a decreased ability to handle glucose challenge (assessed by glucose-tolerance test; GTT) (Figure 3a), as well as reduced insulin sensitivity (determined by insulin-tolerance test; ITT) (Figure 3b). Taken together, these findings support a tendency of DTA-PKO chimeric mice to develop type 2 diabetes and a general condition resembling metabolic syndrome.
In healthy individuals, adipose tissue (AT) expansion occurs by enlargement of the fat pad mass through enhanced recruitment of adipocyte precursor cells that differentiate into small adipocytes, along with the recruitment of other stromal cell types. Subsequently, vascularization, minimal induction of ECM and minimal inflammation occur. In contrast, pathological expansion of AT is characterized by rapid growth of the fat pad through enlargement of existing fat cells, a high degree of macrophage infiltration, limited vessel development, and massive fibrosis. Such pathological expansion is associated with chronic inflammation, which ultimately results in the development of systemic insulin resistance. The observed metabolic syndrome in DTA-PKO chimeras indicated that pathological AT expansion might also occur in these mice. Indeed, as can be seen in Figures 3c-e, adipocytes in the AT tissue of DTA-PKO chimera were significantly larger, less organized and more loosely packed compared to adipocytes in WT-PKO control chimeras. Moreover, the visceral AT of DTA-PKO chimeras contained significantly more 'crownlike' structures (CLS), which are formed when macrophages within inflamed AT cluster around dead adipocytes (Figures 3c-e).
EXAMPLE 3
CD4 and CD8 T cells are required for the development of metabolic syndrome in
DTA-PKO chimera
In general, it is difficult to discern cause and effect when studying inflammatory processes in the AT. However, the present observation that mice in which all CDl lchlgh DC are perforin-deficient, develop a profound spontaneous metabolic phenotype, strongly indicated a regulatory role for Perf-DC.
To define candidate effector cells that might be controlled by Perf-DC, the present inventors initially analyzed immune cell populations in collagenase-digested stromal vascular fractions (SVF) from epididymal adipose tissue, as previously described [Brake D.K. et al., Am. J. Physiol. Cell. Physiol. (2006) 291, C 1232-9]. The gating strategy for the identification and quantification of these cell subpopulations is shown in Figure 12.
Interestingly, WT-PKO and DTA-PKO chimeras exhibited similar distributions of four subpopulations of CD1 lchighCDl lbhigh (I), CD1 lchighCDl lbint (II), CD1 lcintCDl lbhigh (III), CDl lcintCDl lbint (IV) cells in the AT, similar to the ones described above for the spleen. While these subpopulations residing in the AT might differ in function and origin from the splenic cells bearing the same phenotypes, no significant difference in the frequencies of the CDl lchigh DC was found between the two types of chimera (Figure 13a). However, as can be seen in Figures 4a and 4b, AT of DTA-PKO mice displayed significantly more CD4+ and CD8+ T cells compared to WT-PKO chimera at 6 months post-transplant, the time of disease onset, while no significant differences were found in neutrophil or macrophage levels (Figure 13b), nor in B or Treg cells (Figures 4c and 4d).
Since a major difference in immune cell composition was observed mainly in the T cell compartment, the present inventors further investigated the potential roles of CD4+ and CD8+ T cells, by ablating these cell subpopulations prior to onset of disease in the DTA-PKO chimera. As a developing inflammatory response was detected within the AT tissue based on cell composition (i.e. expansion of CD4+ and CD8+ T cells) and serum markers (Leptin, TNF-a, ILl-β) starting at 5 months post-transplant, weekly i.p. injections were administered of anti-CD4 antibodies, anti-CD8 antibodies or both, starting at 1 month prior to development of the earliest inflammatory signs (4 month post BM transplant). This treatment resulted in over 95 % depletion of the target population(s) in the spleen, lymph nodes (LNs) and peripheral blood, with no changes in other cell populations (data not shown). More importantly though, the present inventors found that DTA-PKO mice treated with anti-CD4 antibodies, anti-CD8 antibodies, or a combination of them, did not gain weight, and their leptin levels remained low, similarly to the WT- PKO controls (Figure 4e). Likewise, serum leptin levels were found to be elevated only in the group of DTA-PKO chimera that did not receive antibody treatment (Figure 4f). This observed role of both CD4 and CD8 T cells is consistent with potential triggering of the AT pathology by effector CD8 T cells requiring initial help from CD4 T cells.
Considering that alterations in central regulatory functions are likely involved in the metabolic changes observed and recent data that demonstrate changes in inflammatory status in the hypothalamus that may be important in the regulation of body weight, the expression of IL-6 and IL-Ιβ was evaluated in the hypothalamus of DTA-PKO and control WT-PKO chimeric mice before (4 months post transplant) and after disease onset (9 months). As can be seen in Figure 14, significantly elevated levels of these cytokines were indeed detected in DTA-PKO chimera, albeit only after disease onset, thereby indicating that these changes might be induced, as a result of the inflammatory process in AT and not vice versa.
EXAMPLE 4
A modified T cell repertoire within the adipose tissue of DTA-PKO chimeras
One potential explanation for the observed phenotype in DTA-PKO chimeras is that autoimmune T cells might be poorly controlled in the absence of regulatory Perf-DC. To address this possibility, the TCR repertoire from AT tissue of DTA-PKO and WT-PKO chimeras at 6 months post-transplant was analyzed by high-throughput TCR sequencing. The TCRP CDR3 region of T cells was sequenced and was compared the repertoire found in spleen, visceral AT (VAT), and subcutaneous AT (SC-AT) of DTA-PKO and WT-PKO chimeras.
As a measure of the composition of the TCR repertoire, the level of skewing in the frequencies of observed TCR sequences was evaluated. A more diverse repertoire consists of sequences that are found at similar frequencies, whereas in a skewed repertoire, a small number of sequences are dominant and are found at higher frequencies than the others. The level of skewing of a repertoire can be evaluated by the deviation of its Lorentz curve from the diagonal; such deviation is larger for more skewed repertoires. The present inventors started by comparing the repertoires of T cells that are resident within AT to the repertoire found in the spleen of WT-PKO animals. Consistent with previous observations of a restricted TCR repertoire within adipose tissues [Yang H. et al., J. Immunol. (2010) 185, 1836-1845], the present inventors observed a higher level of skewing in the repertoires of T cells derived from VAT and SC-AT compared with splenic T cells of WT- PKO mice (Figure 5a). In addition, specific νβ segments were identified that are enriched in the repertoires derived from AT compared with the splenic repertoire. Next, the present inventors compared the TCR repertoires between WT-PKO and DTA-PKO. As shown in Figure 5 a, while there was no difference in the spleens of these chimera, the TCR repertoire observed in the AT tissues derived from DTA-PKO mice was less skewed compared to WT-PKO mice (i.e. the Lorentz curve was closer to the diagonal). Thus, the TCR repertoire in AT of the WT-PKO chimera is dominated by expansion of a small number of clonotypes, whereas the DTA-PKO AT repertoire is less skewed and contains a greater number of distinct T cell clones found at intermediate levels. To identify signature TCR sequences that distinguish the two types of chimera, the present inventors used an ordination method, similar to principle component analysis, termed redundancy analysis (RDA) [Sadys M. et al., Int. J. Biometeorol (2014) 59(l):25-36]. Briefly, this method attempts to explain the variance in the data, constrained by the grouping of samples, by fitting a linear model that maximizes the variance between groups. Using RDA, TCR sequences were selected with the highest contribution to the separation between the experimental groups, resulting in a list of signature sequences that are found at high levels in one group but not in the other. This analysis was able to separate between repertoires of DTA-PKO vs. WT-PKO along the first RDA axis (RDA1), and between most spleen samples vs. VAT samples along RDA2 (Figure 5b). Next, the present inventors identified CDR3 sequences that were most significantly enriched in repertoires of DTA-PKO mice compared with WT-PKO, by RDA analysis. Thus, the present inventors were able to detect several TCRP sequences that are consistently up-regulated across DTA-PKO animals, suggesting their potential role in the observed phenotype. Of note, many of these TCRs are encoded by different nucleotide sequences in different animals (data not shown), which is an indication of clonal selection driven by TCR specificity. These TCR sequences, which exist in the DTA-PKO animals but not in the WT-PKO, may be mechanistically related to the metabolic phenotype. EXAMPLE 5
PKO-DTA chimeras exhibit enhanced development of obesity in response to a high fat diet
To learn how mice lacking Perf-DC respond to excess adiposity, the present inventors used a classical model of obesity, in which mice are chronically fed a high-fat diet (HFD). When fed HFD, normal WT C57BL/6 mice develop glucose intolerance and insulin resistance by the 12th week, with elevated levels of pro-inflammatory cytokines and adipokines. Bearing in mind that DTA-PKO mice have a tendency to spontaneously develop these symptoms, the present inventors chose to follow the dynamics of metabolic and functional changes at early time points. As can be seen in Figures 6a-b, DTA-PKO mice gained weight much earlier than WT-PKO chimeras when fed HFD, with higher percentage of body fat, increased liver weight and epididymal fat pad weight (Figures 6c- e). Furthermore, the levels of leptin were significantly higher as early as 1 month after initiation of HFD. At 1.5 months from initiation of feeding, levels of insulin and both IL- 1β and TNF-a were elevated in DTA-PKO mice (Figures 6f-h and 61). Plasma levels of both cholesterol and triglycerides were elevated in DTA-PKO mice relative to control WT-PKO animals, starting at 1 month from initiation of HFD (Figure 6f and 6i), and liver triglyceride (TG) were also markedly higher (Figures 6i-k). Taken together, these results suggest that the increased susceptibility to diet-induced obesity could offer an additional, more rapid model for investigating the impact of Perf-DC on different immune cell subpopulations.
As in the normal-fat diet, the present inventors also found that larger numbers of both CD8+ and CD4+ T cells infiltrated the VAT in DTA-PKO mice fed with HFD compared to the levels found in controls (Figures 15a-d). Importantly, while there was no significant elevation of macrophages within the VAT of the DTA-PKO chimera (Figure 15e), the present inventors found, by RT-PCR, a significant shift towards a gene expression profile typical of inflammatory Ml macrophages (Figure 6j), which is consistent with increased inflammatory properties of macrophages in the AT of obese mice.
It could be suggested that activation of T cells and NK cells upon onset of inflammation or administration of HFD, might induce up-regulation of CD1 lc and thereby of diphtheria toxin (DT) at a sufficient level to cause the deletion of these populations. Clearly, such deletion would not be apparent in analysis of cell populations in the naive mice, shown in Figures la-e prior to onset of disease. To address this possibility, the present inventors re-evaluated potential changes in the level of PKO-derived cells, within various leukocyte populations in the BM, spleen and VAT before (2 months post BMT) and after (7 months after BMT) the onset of the metabolic phenotype. Likewise, such potential changes were analyzed in chimeric mice after 8 weeks of HFD, by which time the full-blown syndrome develops in DTA-PKO mice.
As can be seen in Figures 16a-b, 17a-c and 18 and Table 2, below, the relative abundance of the various cell types found in naive mice remains unaltered after the inflammatory onset. Table 2: T cell analysis of spleen cells from DTA-PKO versus WT-PKO mice fed
NCD or HFD (Ave ± SD)
Figure imgf000062_0001
No. T cell subsets from spleen of DTA-PKO or WT-PKO mice fed either NCD for 7 months, or HFD for 8 weeks were defined by various markers including CD3, CD8, CD4, CD44, CD62L, CD25 and Foxp3, and analyzed by FACS. T cell subsets from chimera were also stained for CD45.1 to identify cell origin from CD1 lc-DTA or littermate control hosts, versus CD45.2 expressed by PKO donors.
EXAMPLE 6
Perf-DC exhibit a major regulatory role in EAE
The present inventors showed that selective deletion of perforin in the rare subpopulation of Perf-DC leads to a distinct metabolic phenotype, and that this phenotype can be prevented by T cell depletion in-vivo. Collectively, this strongly indicates a role for Perf-DC in the control of otherwise deleterious T cell-mediated inflammatory processes. To further investigate the regulatory role of perf-DC, the present inventors extended the study to evaluate whether Perf-DC may also exhibit a regulatory role in a well-defined mouse model for autoimmunity, in which the pathological antigen is known. To that end, the present inventors examined whether DTA-PKO chimera are more susceptible to development of EAE, a T-cell mediated disorder widely used as a model of multiple sclerosis [Aharoni, (2013), supra]. In this mouse model, MOG-induced EAE results in a chronic persistent disease course.
As can be seen in Figures 7a-b, MOG administration in DTA-PKO mice resulted in a greater clinical severity of the disease with earlier onset and higher disease score. Moreover, in accordance with the clinical course of EAE, lymph node cells from DTA- PKO mice showed more antigen- specific proliferation of CD4 T cells, both during the peak of the disease at day 14, and during the chronic stage at day 30. This specificity is clearly illustrated when comparing the marked enhancement of anti-MOG CD4 T cells to that exhibited by T cells directed against PPD (adjuvant related peptide) or MBP (unrelated encephalitogenic peptide) (Figure 7c) or of CD8 T cells directed against MOG (Figure 7d). Importantly, MOG specific IFN-γ and IL-17a positive T cells were also found in the central nervous system 14 days after immunization (Figure 7e). Taken together, these results demonstrate the immune regulatory role of Perf-DC, and the ability of these cells to inhibit detrimental expansion of antigen specific autoimmune clones in the context of EAE.
EXAMPLE 7
Aspirin inhibits imDCs maturation while maintaining their killing-mediated tolerogenic capacity
The elusive nature of the immature developmental stage of DCs, together with the fact that the presently described 20 days Perf+ imDCs generation protocol allows a narrow time window of activity, led the present inventors to test alternative modes of generating Perf imDCs resistant to maturation-inducing factors. Clearly, this could be of great importance when performing adoptive transfer of Perff imDCs, considering that following implantation they typically undergo rapid maturation and lose their tolerogenic activity.
It has been previously demonstrated that maturation-resistant imDCs can be generated in-vitro through exposure to various anti-inflammatory and immunosuppressive agents [Morelli, A. E. and Thomson, A. W. Nature Reviews Immunology (2007) 7, 610- 621]. Thus, to further develop optimal tolerizing imDCs, the present inventors focused on several potentially promising agents, including Rapamycin, Aspirin and Heme Oxygenase- 1 (HO-1). Rapamycin is a well-established immunosuppressive drug that operates through the inhibition of mTOR and used to prevent rejection in organ transplantation. In the context of DCs, Rapamycin was shown to inhibit DCs maturation and their T cell alio stimulatory activity both in-vivo and in-vitro [Hackstein H. et al., Blood. (2003) 101:4457-4463].
Aspirin, the most common analgesic and anti-inflammatory substance, was shown to also have a broad spectrum of pharmacological actions, including the inhibition of NF- KB and other molecular pathways of inflammation. The cellular targets of Aspirin in the immune system are poorly understood. Aspirin added to DCs cultures was shown to inhibit DCs maturation in a dose-dependent manner, promote relative increase in the numbers of CDl lc+ DCs and inhibit the DCs stimulatory activity on allogeneic T cells [Hackstein H. et al, J Immunol (2001) 166:7053-7062].
HO- 1 is an intracellular enzyme that degrades heme and inhibits immune responses and inflammation in-vivo. The fact that DCs express HO-1 and that this expression is downregulated with maturation led to the assumption that the induction of the expression of HO-1 in DCs may inhibit their maturation. Indeed, induction of HO-1 expression in DCs by Cobalt Protoporphyrin (CoPP) rendered DCs refractory to LPS-induced maturation and decreased their capacity to stimulate T cells in MLRs [Chauveau C. et al., Blood (2005) 106: 1694-1702].
Thus, the present inventors modified the 20 days Perf+ imDCs generation protocol by supplementing the cultures with 10 ng/ml Rapamycin from day 10 of the culture, 2.5 mM Aspirin from day 10 of the culture or 50 mM CoPP / SnPP (an inducer and an inhibitor of HO-1, respectively) for 2 hours. At the end of the culture, the treated cells were collected and checked for their phenotype before and after treatment with 1 μg/ml LPS for 24 hours. In addition, treated cells were checked for their ability to kill cognate OT-I CD8+ T cells in short-term MLR.
As illustrated in Figures 19a-b, while untreated Perf+ imDCs showed upregulated levels of MHC-II, CD80 and CD86 in response to LPS, Aspirin-treated Perf imDCs levels of these three markers remained unchanged. Since MHC-II, CD80 and CD86 are indicative markers which are upregulated upon DCs maturation, it can be concluded that treatment of Perf+ imDCs with aspirin prevents their conversion into maturity and enables their use in cell therapy. Next, the present inventors wanted to confirm whether Aspirin-treated Perf+ imDCs loaded with SIINFEKL peptide (SEQ ID NO: 29) maintain the capacity to kill cognate OT-I CD8+ T cells in short-term MLR. Figure 20 shows that Aspirin-treated Perf+ imDCs maintain their potent killing capability of OT-I CD8+ T cells and in comparable levels to the killing exhibited by WT Perf+ imDCs (as seen in Figures 21a-d). Taken together, these results highlight the great potential of Aspirin as a maturation inhibitor of imDCs.
EXAMPLE 8
Perf-DC loaded with MOG peptides are effective in the treatment of EAE
The present inventors showed that selective deletion of Perf-DC score in a well- defined EAE mouse model for multiple sclerosis leads to a greater clinical severity of the disease with earlier onset and higher disease. In this mouse model, MOG-induced EAE results in a chronic persistent disease course (Figures 7a-b).
Next, the present inventors are administering Perf-DC (i.e. Perf+ imDCs) loaded with a MOG peptide to EAE mice (e.g. by injection). These cells are further cultured with or without a factor capable of inhibiting the Perf+ imDCs from maturing (e.g. aspirin, rapamycin, HO-1).
The present inventors are testing the efficacy of these Perf-DCs in creating a tolerogenic state by deleting cognate reactive T-cells (e.g. CD8+ T cells) and the potential of the Perf+ imDCs to ameliorate clinical symptoms of EAE which appear after injection of the MOG peptide. The present inventors are further analyzing the length of time by which these cells maintain their immature phenotype following administration to a recipient.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

WHAT IS CLAIMED IS:
1. A method of treating an inflammation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of perforin+ immature DCs (Perf imDCs), thereby treating the inflammation in the subject.
2. A therapeutically effective amount of Perf+ imDCs for use in treating an inflammation in a subject in need thereof.
3. The method of claim 1, or therapeutically effective amount of Perf+ imDCs for use of claim 2, further comprising contacting said Perf+ imDCs with a factor capable of inhibiting said Perf+ imDCs from maturing prior to or concomitantly with administration of said Perf+ imDCs to said subject in need thereof.
4. The method of claim 1 or 3, further comprising administering to the subject a therapeutically effective amount of a factor capable of inhibiting said Perf+ imDCs from maturing.
5. The therapeutically effective amount of Perff imDCs for use of claim 2 or 3, further comprising the use of a therapeutically effective amount of a factor capable of inhibiting said Perff imDCs from maturing.
6. A method of generating Perff imDCs, wherein the Perff imDCs are inhibited from maturing, the method comprising:
(a) obtaining Perf+ imDCs; and
(b) contacting said Perf+ imDCs with a factor capable of inhibiting said Perf+ imDCs from maturing.
7. The method of claim 6, further comprising selecting cells which exhibit said Perff imDCs phenotype.
8. The method of claim 6 or 7, wherein said Perf imDCs are obtained by a method comprising:
(a) obtaining CD34+ cells;
(b) contacting said CD34+ cells with a factor capable of differentiating said CD34+ cells into early myeloid cells; and
(c) contacting said early myeloid cells with a factor capable of differentiating said early myeloid cells into perforin+ immature dendritic cells.
9. The method of claim 8, further comprising obtaining hematopoietic progenitor cells prior to step (a).
10. The method of claim 8, wherein said factor capable of differentiating said CD34+ cells into early myeloid cells comprises at least one of a stem cell factor (SCF), a thrombopoietin (TPO), a Flt3-ligand (Flt3L), an interleukin-3 (IL-3) and an interleukin-6 (IL-6).
11. The method of claim 8 or 10, wherein said contacting said CD34+ cells with a factor capable of differentiating said CD34+ cells into early myeloid cells is effected for 5-20 days.
12. The method of claim 8, wherein said factor capable of differentiating said early myeloid cells into perforin+ dendritic cells comprises a granulocyte-macrophage colony-stimulating factor (GM-CSF).
13. The method of claim 12, wherein said factor capable of differentiating said early myeloid cells into perforin+ dendritic cells further comprises an interleukin-4 (IL-4).
14. The method of any one of claims 8, 12 or 13, wherein said contacting said early myeloid cells with a factor capable of differentiating said early myeloid cells into perforin+ dendritic cells is effected for 5-20 days.
15. The method of any one of claims 3-4 or 6-14, or therapeutically effective amount of Perf+ imDCs for use of claim 3 or 5, wherein said factor capable of inhibiting said Perf+ imDCs from maturing comprises an anti-inflammatory agent or an immunosuppressive agent.
16. The method of claim 15, or therapeutically effective amount of Perf+ imDCs for use of claim 15, wherein said an anti-inflammatory agent or an immunosuppressive agent is an mTOR inhibitor.
17. The method of claim 15, or therapeutically effective amount of Perf+ imDCs for use of claim 15, wherein said an anti-inflammatory agent or an immunosuppressive agent inhibits granulocyte-mediated inflammation.
18. The method of claim 15 or 16, or therapeutically effective amount of Perf+ imDCs for use of claim 15 or 16, wherein said immunosuppressive agent comprises rapamycin.
19. The method of claim 13, or therapeutically effective amount of Perf imDCs for use of claim 15, wherein said anti-inflammatory agent comprises aspirin or a heme oxygenase- 1 (HO-1).
20. The method of any one of claims 8-17, wherein said contacting said early myeloid cells with a factor capable of differentiating said early myeloid cells into perforin+ dendritic cells is effected concomitantly with said contacting said Perf+ imDCs with a factor capable of inhibiting said Perf+ imDCs from maturing.
21. The method of any one of claims 1, 3, 4, 6-20, or therapeutically effective amount of Perf+ imDCs for use of any one of claims 2, 3 or 5, wherein said Perf imDCs comprise cells having the signature perforin+, CDl lc+,MHC-II+, CD80 and CD86.
22. The method of claim 21, or therapeutically effective amount of Perf+ imDCs for use of claim 21, wherein at least 50 % of said Perf+ imDCs comprise said signature.
23. The method of any one of claims 6-22, wherein the Perf+ imDCs are loaded with an antigen.
24. The method of any one of claims 3 or 6-23, wherein the method is effected ex vivo.
25. An isolated population of cells comprising Perf+ imDCs generated according to the method of any one of claims 6-24, wherein at least 50 % of said population of cells comprises said Perf imDCs.
26. An isolated population of cells comprising at least 50 % Perf+ imDCs, wherein said Perf+ imDCs maintain an immature phenotype for at least 12 hours when administered to a recipient.
27. A pharmaceutical composition comprising the isolated population of cells of claim 25 or 26 and a pharmaceutically active carrier.
28. An article of manufacture comprising the isolated population of cells of claim 25 or 26 being packaged in a packaging material and identified in print, in or on said packaging material for use in the treatment of an inflammation.
29. The article of claim 28, further comprising a factor capable of inhibiting said Perff imDCs from maturing.
30. The method of claim 1, 3 or 4, or therapeutically effective amount of Perf+ imDCs for use of claims 2, 3 or 5, being effected using the isolated population of cells of claim 25 or 26.
31. The method of any one of claims 1, 3, 4 or 30, therapeutically effective amount of Perf+ imDCs for use of any one of claims 2, 3, 5 or 30, or article of claim 28, wherein said inflammation is associated with a chronic inflammatory disease.
32. The method of any one of claims 1, 3, 4 or 30, therapeutically effective amount of Perf+ imDCs for use of any one of claims 2, 3, 5 or 30, or article of claim 28, wherein said inflammation is associated with an acute inflammatory disease.
33. The method of any one of claims 1, 3, 4, 30, 31 or 32, therapeutically effective amount of Perf+ imDCs for use of any one of claims 2, 3, 5, 30, 31 or 32, or article of claim 28, wherein said inflammation is associated with a disease selected from the group consisting of a metabolic disease, an autoimmune disease, an infectious disease, a hypersensitivity disease, a transplantation related disease and an injury.
34. The method of any one of claims 1, 3, 4, 30, 31 or 32, therapeutically effective amount of Perf+ imDCs for use of any one of claims 2, 3, 5, 30, 31 or 32, or article of claim 28, wherein said inflammation is associated with a disease selected from the group consisting of multiple sclerosis, metabolic syndrome, diabetes, rheumatoid arthritis, lupus and Crohn's.
35. The method of any one of claims 1, 3, 4, 30, 31, 32, 33 or 34, or therapeutically effective amount of Perf+ imDCs for use of any one of claims 2, 3, 5, 30, 31, 32, 33 or 34, wherein said therapeutic effective amount of said Perf+ imDCs is capable of inhibiting an activity or proliferation of a CD4+ T cell and/or a CD8+ T cell.
36. The method of any one of claims 1, 3, 4, 30, 31, 32, 33, 34 or 35, or therapeutically effective amount of Perf+ imDCs for use of any one of claims 2, 3, 5, 30, 31, 32, 33, 34 or 35, wherein said Perf+ imDCs are syngeneic with the subject.
37. The method of any one of claims 1, 3, 4, 30, 31, 32, 33, 34 or 35, or therapeutically effective amount of Perf+ imDCs for use of any one of claims 2, 3, 5, 30, 31, 32, 33, 34 or 35, wherein said Perf+ imDCs are non-syngeneic with the subject.
38. The method of any one of claims 1, 3, 4, 30, 31, 32, 33, 34, 35, 36 or 37, or therapeutically effective amount of Perf+ imDCs for use of any one of claims 2, 3, 5, 30, 31, 32, 33, 34, 35, 36 or 37, wherein said subject is a human subject.
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