WO2000017657A9 - A flow cytometric, whole blood dendritic cell immune function assay - Google Patents
A flow cytometric, whole blood dendritic cell immune function assayInfo
- Publication number
- WO2000017657A9 WO2000017657A9 PCT/US1999/021731 US9921731W WO0017657A9 WO 2000017657 A9 WO2000017657 A9 WO 2000017657A9 US 9921731 W US9921731 W US 9921731W WO 0017657 A9 WO0017657 A9 WO 0017657A9
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- specific
- dendritic cell
- antibody
- sample
- antibodies
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5002—Partitioning blood components
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
- G01N33/56972—White blood cells
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/962—Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
- Y10T436/25375—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
Definitions
- This invention relates to assays of blood cell function, and in particular to assays of dendritic cell function in whole blood.
- Dendritic cells first identified a quarter century ago by a characteristic "dendritic" morphology observable in peripheral lymphoid tissues, Steinman et al . , J. Exp . Med. 137:1142-1162 (1973), are now known to be a morphologically-diverse and widely- distributed cell population. Today, these. diverse cells are collectively distinguished by a common function: dendritic cells are the most potent antigen- presenting cells (APCs) of the mammalian immune system, and alone among the various antigen-presenting cells appear capable of triggering a primary T lymphocyte response.
- APCs antigen- presenting cells
- WO 97/24438 describes compositions and methods for co-culturing dendritic cells with T lymphocytes and protein antigen in vitro, thus driving the ex vivo antigen-specific activation of T cells.
- the activated T cells are then administered autologously to effect an antigen-specific immune response in vivo .
- WO 97/29183 describes a method of activating T cells in vi tro by contacting the T lymphocytes with DC that directly express an antigenic protein from a recombinant construct. Again, the activated T cells are intended for autologous infusion.
- Specific application of DC-driven ex vivo T cell activation to the treatment of prostate cancer is described and claimed in U.S. Patent No. 5,788,963.
- Nemazee U.S. Patent No. 5,698,679, describes and claims immunoglobulin fusion proteins that deliver antigenic peptides to targeted antigen presenting cells (APCs) , including dendritic cells, in vivo.
- APCs targeted antigen presenting cells
- Dendritic cells have also been implicated as important in the pathogenesis and pathophysiology of AIDS.
- One type of DC the Langerhans cells (LC)
- LC Langerhans cells
- DC are believed to act not only during the initial phase of HIV disease, but also during the chronic phase, facilitating infection and depletion of T lymphocytes. Zoeteweij et al . , J Biomed Sci 5(4):253-259 (1998).
- DCs in lymphoid mucosa may represent a key reservoir of viral nucleic acid and virions throughout the course of the disease. Grouard et al . , Curr . Opin . Immunol .
- DCs have been difficult to study, and particularly difficult to study in their native milieu.
- the difficulty stems in part from the rarity of dendritic cells.
- DC are sparse, even in lymphoid tissues, and represent no more than about 0.3% - 0.5% of nucleated cells in human peripheral blood.
- a further difficulty arises from the absence of DC-specific cell surface markers that would readily permit the positive immunoselection of DCs from mixed populations of cells.
- DCs are presently identified by multiple- marker panels, with identification based primarily on the absence of staining with markers for other lineages (i.e., as lin " cells).
- typical DC immunopurification protocols require at least one immunodepletion step, eliminating cells of various nondendritic blood lineages — lymphocyte, monocyte, granulocyte, and NK lineages, e . g. — coupled with at least one immunoenrichment step.
- the immunoenrichment step may, for example, include selection for CD4 + cells (Blood Dendritic Cell Isolation Kit, Miltenyi Biotec #468-01, Auburn, CA) , or, in the alternative or in addition, selection for HLA-DR expression, Ghanekar et al . , J. Immunol . 157:4028-4036 (1996).
- a flow cytometric method for measuring dendritic cell function in whole blood comprising the steps of: (a) contacting a whole blood sample with a dendritic cell activator; (b) contacting the sample with a plurality of dendritic cell-distinguishing antibodies and at least one cytokine-specific antibody; and then (c) flow cytometrically assaying the sample for the binding of cytokine-specific antibody by at least one distinguishable DC subset.
- the dendritic cell activator contacting step is performed in the presence of Brefeldin A, and the antibody contacting step itself comprises the steps, in order, of: (bl) adding a plurality of dendritic cell-distinguishing antibodies to the sample ; (b2) lysing erythrocytes in the sample; (b3) permeabilizing nucleated cells in the sample; and then (b4) adding at least one cytokine-specific antibody to the sample.
- the dendritic cell-distinguishing antibodies may include a plurality of non-DC lineage-specific antibodies.
- each of the non-DC lineage-specific antibodies be conjugated to the identical fluorophore.
- the dendritic cell-distinguishing antibodies further include an antibody specific for HLA-DR.
- subsets of dendritic cells are distinguishably labeled.
- the dendritic cell-distinguishing antibodies include at least one antibody that binds differentially to the surface of the different dendritic cell subsets. Particularly preferred in this embodiment is the use of antibody specific for CDllc or CD123.
- DCs can be characterized by their cytokine expression patterns and by the dynamic regulation of differentiation/activation markers (CMRF- 44, CMRF-56, CD83, CD25) , of co-stimulatory molecules (CD40, CD80, CD86) and of class II major histocompatibility complexes (MHC class II) .
- CMRF- 44, CMRF-56, CD83, CD25 dynamic regulation of differentiation/activation markers
- CD40, CD80, CD86 co-stimulatory molecules
- MHC class II class II major histocompatibility complexes
- the invention provides a flow cytometric method for measuring dendritic cell function in whole blood, comprising: (a) contacting a whole blood sample with a dendritic cell activator; (b) adding to the sample a plurality of dendritic cell- distinguishing antibodies and at least one antibody specific for a dendritic cell surface marker indicative of dendritic cell activation; and then (c) flow cytometrically assaying said sample for the binding of said antibody specific for the dendritic cell surface activation marker by at least one distinguishable DC subset.
- the dendritic cell surface activation marker is usefully selected from the group consisting of differentiation/activation markers (CMRF-44, CMRF-56, CD83, CD25) , of co-stimulatory molecules (CD40, CD80, CD86) and of class II major histocompatibility complexes (MHC class II) .
- CMRF-44 differentiation/activation markers
- CMRF-56 CMRF-56, CD83, CD25
- co-stimulatory molecules CD40, CD80, CD86
- MHC class II class II major histocompatibility complexes
- FIG. 1 is a flow chart schematizing the basic steps in a whole blood flow cytometric assay for dendritic cell function, with LPS exemplified as the dendritic cell activator;
- FIG. 2 presents a series of dot plots generated during the flow cytometric analysis of whole blood activated with LPS in the presence of Brefeldin A.
- CDllc + dendritic cells are painted green and CDllc " dendritic cells are painted red; nondendritic cells appear gray. The colors are arbitrarily chosen, and bear no relationship to the fluorophores used for analysis;
- FIG. 3 presents a series of dot plots generated during the flow cytometric analysis of whole blood activated with PMA+I in the presence of Brefeldin A.
- CDllc* dendritic cells are painted green and CDllc" dendritic cells are painted red; nondendritic cells appear gray. The colors are arbitrarily chosen, and bear no relationship to the fluorophores used for analysis;
- FIG. 3 presents a series of dot plots generated during the flow cytometric analysis of whole blood activated with PMA+I in the presence of Brefeldin A.
- CDllc* dendritic cells are painted green and CDll
- CDllc + dendritic cells are painted green and CDllc " dendritic cells are painted red; nondendritic cells appear gray.
- the colors are arbitrarily chosen, and bear no relationship to the fluorophores used for analysis;
- FIG. 5 presents the differential expression of TNF ⁇ , IL-8, CD80 and CD86 in CDllc + dendritic cells from two donors, each activated alternatively with LPS or PMA+I;
- FIG. 6 presents a series of histograms summarizing the effects of three different dendritic activators on the surface expression of the identified markers on peripheral blood dendritic cells in whole blood;
- FIG. 7 shows a comparison of cytokine expression between monocytes (gray bars) and CDllc* DCs (black bars) in activated whole blood, with FIG. 7A showing LPS + Brefeldin A-stimulated cells, and FIG. 7B showing PMA+I + Brefeldin A-stimulated cells; and
- FIG. 8 shows kinetics of TNF ⁇ , IL-l ⁇ , IL-6 and CD80 in LPS activated CDllc + DCs.
- the time course of LPS incubation was 0 to 8 hours.
- Intracellular cytokine and CD80 expression is measured as PE mean fluorescence intensity (MFI) .
- whole blood is intended a fluid blood sample as drawn from a mammal and substantially unfractionated thereafter. That is, if fractionation is performed subsequent to blood draw, • the fractionation has raised the percentage of dendritic cells to no more than about 5%, preferably no more than about 1 - 4%, most preferably no more than 1%, of total nucleated cells;
- Antibody includes all products, derived or derivable from antibodies or from antibody genes, that are useful as markers in the flow cytometric methods described herein. “Antibody” thus includes, inter alia, natural antibodies, antibody fragments, antibody derivatives, and genetically-engineered antibodies, antibody fragments, and antibody derivatives;
- Dendritic cell-distinguishing antibody includes any antibody that may be used, alone or in combination with other antibodies, to facilitate identification of dendritic cells, and thus includes antibodies that are specific for epitopes displayed by non-DC lineages and further includes antibodies that bind to structures displayed by DC that prove useful for positive immuno-identification;
- Lineage negative also abbreviated “lin "”
- absence is intended a level of surface expression, as measured in an immunoassay, such as a flow cytometric assay, that is not significantly different from background;
- a “dendritic cell activator” is any substance that is capable of inducing or upregulating expression of cytokines, chemokines, or detectable cell surface proteins by dendritic cells; All remaining terms have their usual meaning in the flow cytometric arts, as set forth, inter alia, in Ormerod (ed.), Flow Cvtometrv: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al . (eds.), Flow Cvtometrv Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); and Practical Flow Cytometrv, 3rd ed., Wiley-Liss (1995).
- DC Dendritic cells capture, process and present antigen to naive and memory T cells, and thus play a pivotal role in the mammalian immune response.
- An understanding of DC function is critical to any detailed understanding of mammalian immune function.
- Yet functional studies of dendritic cells have in the past been hampered by the functional diversity of the cells that are collectively so denominated.
- peripheral blood dendritic cells For example, studies of peripheral blood dendritic cells were for two decades conducted without awareness of the fact that peripheral blood dendritic cells fall into two mutually-exclusive subsets distinguishable by cell surface immunophenotype.
- CD19, CD20, CD16, CD56 The subsets are distinguished from one another by their divergent expression of CDllc and CD123: one subset is CDllc + CD123 low , the other CDllc ' CD123 + .
- the two peripheral blood DC subsets that were first identified by fortuitous cell surface distinctions have now been shown to be functionally distinct. It is known, for example, that the CDllc + CD123 low DC subset proves more potent than the CDllc " CD123 + subset in stimulating T cells in a mixed lymphocyte reaction (MLR) . And as shown newly herein, the CDllc + subset alone responds to DC activators with upregulation of cytokine production and increased surface expression of T cell costimulatory molecules.
- MLR mixed lymphocyte reaction
- the present invention permits peripheral blood dendritic cells to be described and distinguished based upon differences in their functional responses to DC activators.
- the invention further permits these functional responses to be measured with minimal experimental intervention, precluding the known phenotypic plasticity of dendritic cells from confounding the results.
- FIG. 1 schematizes the basic method of the present invention.
- a sample of whole blood is first incubated with a DC activator.
- LPS is exemplified in the figure.
- BFA Brefeldin A
- the surface of the cells is stained with fluorophore-conjugated antibodies.
- This surface staining step includes, as a first class of antibodies, a plurality of dendritic cell-distinguishing antibodies.
- a dendritic cell- distinguishing antibody is any antibody that may be used, alone or in combination with other antibodies, to facilitate identification of dendritic cells.
- the antibodies used in this step may include (1) antibodies that preferentially bind non-dendritic cells, and (2) antibodies that bind to dendritic cell surface structures useful in identifying DC.
- a cocktail of lineage-specific antibodies labeled with the identical fluorophore may advantageously be used.
- One such cocktail available commercially is the lin 1 FITC lineage cocktail from Becton Dickinson Immunocytometry Systems (BDIS, San Jose, CA, catalogue number 340546) , which contains a mixture of antibodies specific for CD3, CD14, CD16, CD19, CD20, and CD56, each conjugated to fluorescein isothiocyanate (FITC) .
- the antibodies in the cocktail stain lymphocytes, monocytes, eosinophils, and neutrophils, but not dendritic cells.
- the DC in the labeled sample thus assort into the FITC " or FITC low class.
- the lin 1 cocktail is particularly advantageous in that the concentration of antibodies and degree of conjugation have been titrated to provide equivalent intensity fluorescence signals from the cells of the various non- DC lineages that are bound by the antibodies.
- the second category of dendritic cell- distinguishing antibodies there is, as yet, no cell surface marker that alone positively identifies dendritic cells.
- an antibody thereto may be used alone in this stage of the protocol.
- the use of antibodies in the second category of DC-distinguishing antibodies antibodies that affirmatively bind to dendritic cell surface structures — obligates the additional use of DC- distinguishing antibodies from the first category, i.e., those that identify non-dendritic lineages.
- antibodies from the first category of DC-distinguishing antibodies those that preferentially bind non-dendritic cells - cannot at present be used without at least one antibody from the second category.
- Basophils are lin " CD123 hi ⁇ h CDllc + but HLA-DR " ; when antibodies that preferentially bind non- dendritic cells (category 1) are used in the assay, an anti-HLA-DR antibody must also be used.
- the antibodies in the two categories are preferentially labeled with fluorophores that are flow cytometrically distinguishable .
- the surface staining step may optionally also include, as a second broad class, antibodies that distinguish known dendritic cell subsets.
- antibodies specific for CDllc or CD123 prove particularly useful, as these antigens are known to define mutually exclusive peripheral blood DC subsets.
- the fluorophore used should be flow cytometrically distinguishable.
- PE phycoerythrin
- a typical surface staining scheme would include, e.g., lin 1 FITC, HLA-DR PerCP, and CDllc APC (in this nomenclature, the antibody is identified by its specificity, followed by the fluorophore) .
- the red cells in the sample are lysed and the nucleated cells then permeabilized.
- FACS ® Permeabilizing Solution FACS ® Lysing solution (BDIS catalogue numbers 340457 and 349202, respectively) , according to the manufacturer's instructions.
- the cells are stained intracellularly using fluorophore-conjugated antibodies that are specific for cytokines.
- the fluorophore conjugated to the cytokine-specific antibodies is preferentially distinguishable in a flow cytometric assay from any of those used for surface staining.
- FIG. 1 does not schematize the assay for detecting changes in the surface expression of dendritic cell activation markers, which differs in some respects from that used to detect changes in cytokine expression.
- activation of dendritic cells in whole blood is performed in the absence of secretion inhibitor, such as Brefeldin A. This precludes the concurrent measurement, in any such sample, of intracellular cytokine expression.
- the surface of the cells is stained with fluorophore-conjugated antibodies.
- a plurality of dendritic-cell distinguishing antibodies is used, optionally with antibodies that distinguish known dendritic cell subsets, as above-described.
- a third class of surface-staining antibodies is used. These are antibodies that recognize surface structures, typically proteins, the expression of which is altered by the prior incubation with dendritic cell activator. For example, activation of peripheral blood dendritic cells is known to cause upregulation of the T cell costimulatory molecules CD80 (B7.1), CD86 (B7.2) and HLA-DQ. Olweus et al . , Proc . Natl . Acad. Sci . USA 94:12551-12556 (1997) .
- the surface staining step may include antibodies specific for one or more of these antigens.
- CD83 and CMRF-44 are typically conjugated to a fluorophore that is flow cytometrically distinguishable from the antibodies described above.
- a typical surface staining scheme would include, e.g., lin 1 FITC, HLA-DR PerCP, CDllc APC, and an antibody specific to a DC surface activation antigen labeled with PE.
- the red cells in the sample are lysed and the cells are washed and then analyzed using a flow cytometer, preferably one capable of simultaneous excitation and detection of multiple fluorophores .
- a flow cytometer preferably one capable of simultaneous excitation and detection of multiple fluorophores .
- LPS lipopolysaccharide
- PMA phorbol 12- myristate 13 acetate
- I ionomycin
- PMA + I CD40-crosslinking
- CD40-crosslinking effected changes in surface antigen expression but failed to elicit cytokine production.
- Table 1 lists the cytokines that were assayed in one or more of the experiments, further classified according to the DC activator used in the experiment.
- a plus (“+”) indicates that expression of the respective cytokine was assessed in one or more experiments; a minus (“-”) indicates that expression of the respective cytokine was not assessed.
- Table 1 does not report the level of expression, which follows in Table 2.
- Table 2 presents the functional responses of the CDllc+ and CDllc- dendritic cell subsets, stimulated with either LPS or PMA+I, and assayed in whole blood. Cytokine expression is measured as mean fluorescence intensity (MFI); the change in surface molecule expression is measured as a ratio of mean fluorescence intensities (MFI) of activated versus control sample.
- MFI mean fluorescence intensity
- CDllc + CD123 low DCs showed easily measured changes in cytokine expression when stimulated with LPS or PMA+I.
- CDllc + cells produced high levels of TNF ⁇ and IL-l ⁇ , lower levels of IL-6, IL-1RA and IL-8, and trace levels of IL-12 and IL-la.
- the response to LPS is surprising: the CDllc + CD123 iow DC are CD14 " , and CD14 is the principal LPS receptor. It seems likely that LPS acts additionally through a second receptor, perhaps CDllc itself. Consistent with that hypothesis, the CDllc " DCs, which lack both CD14 and CDllc, fail to respond to LPS stimulation with increased intracellular cytokine expression. Further consistent with this hypothesis, CD14 + CDllc + monocytes respond to LPS stimulation much more potently than do CD14"CDllc + DCs (FIG. 7A) , without showing significantly increased response to PMA+I (FIG. 7B) .
- FIG. 8 further shows the kinetics of the response of CDllc + DCs to LPS.
- TNF ⁇ is produced first, followed by IL-l ⁇ and IL-6.
- CD80 is intensely upregulated after about 4 hours of activation.
- CDllc + cells produced IL-8 and IL-l ⁇ , lower but significant levels of IL-1RA and TNF ⁇ , trace amounts of IL-l ⁇ , and no detectable IL-6.
- CDllc + DCs in whole blood also led to an increased expression of' accessory molecules.
- LPS activation triggered upregulation of CD25, CD40, CD80, CD86, HLA-DR and HLA-DQ.
- PMA+I led to an upregulation of CD86, CD80, HLA-DQ and HLA-DR.
- Minimal increase of CD25 and CD40 were observed.
- Activation via crosslinking of CD40 resulted in increased levels of CD86, CD80, and minimal upregulation of HLA-DR.
- peripheral blood DC subsets may readily be distinguished in whole blood by their differential production of cytokines and/or cell surface proteins in response to DC activators.
- the dendritic cells that were observed to respond to DC activators fall into a subset (CDllc + ) known to be more potent in T cell activation than is the subset (CDllc " ) showing no such response
- the data further demonstrate that the parameters measured in the method of the present invention — cytokine production and upregulation of surface activation antigens — directly correlate with DC function.
- the methods of the present invention eliminate the cell loss attendant upon all DC purification schemes, increasing sensitivity and reducing possible systematic bias. Additionally, the minimal perturbation effected by the methods of the present invention reduces the chance for phenotypic changes resulting from experimental intervention. And as a flow cytometric assay, the methods of the present invention permit DC function to be assessed on a cell-by-cell, rather than bulk, basis, permitting fine discrimination.
- the methods of the present invention permit, for the first time, the ready and rapid assessment of DC function in whole blood.
- the methods of the present invention thus permit the measurement of DC function to be added to the existing roster of immune function assays, and will find utility in clinical situations in which such existing immune function tests are presently used.
- the methods of the present invention may advantageously be used, alone or in conjunction with flow cytometric quantitation of CD4 + T lymphocyte levels, in the clinical staging of AIDS progression.
- the methods of the present invention may also be used, alone or in conjunction with existing assays, in the assessment of immune function in congenital, rather than acquired, immunodeficiency syndromes, and in the assessment of immune competence following therapeutic immunosuppression or immunoablation.
- the methods of the present invention will also profitably find use, alone or in ' conjunction with existing assays, in the clinical assessment of various forms of immune hypersensitivity, allergies, or in the clinical assessment of autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, sarcoidosis, or the like.
- the methods of the present invention also permit the ready evaluation of the effects that agents circulating in the blood may have on DC function.
- the assay permits the assessment of the specific effects on DC function of pharmaceutical agents that either intentionally or fortuitously affect DC function.
- the methods of the present invention allow pharmaceutical agents to be tested for their in vivo effects on DC function, permitting the selection of agents that desirably demonstrate immunomodulatory effects, or the selection of agents that specifically lack such effects.
- the methods of the present invention permit the ready assessment of the intentional or fortuitous effects on DC function of drugs that circulate in the patient's blood, as a complement to existing immune function assays.
- the methods of the present invention may be used to assist the monitoring and titration of immunosuppressive agents.
- the methods prove particularly useful in the monitoring and titration of immunosuppressive agents that abrogate, downmodulate, or otherwise interfere with the function of cytokines, chemokines, or growth factors.
- the methods of the present invention also prove particularly useful in the monitoring of the effects of affirmative cytokine therapy, such as therapies involving the administration of interferons in the treatment of multiple sclerosis, the administration of growth factors after myeloablation, or the like.
- the methods of the present invention may also be used to monitor immunomodulatory side effects of agents given to effect unrelated clinical goals.
- the methods of the present invention are particularly well suited to the experimental and clinical assessment of therapies involving DC cells themselves.
- the methods of the present invention find use in the design, assessment, and monitoring of therapies in which autologous dendritic cells are administered after in vi tro manipulation, therapies in which dendritic cells are targeted for ablation, either in vi tro, to facilitate transplantation, or in vi vo, to effect immunosuppression or induction of tolerance, or therapies in which dendritic cells are targeted to increase global or specific immune function.
- the dendritic cells that are found circulating in the peripheral blood at the moment that blood is drawn — those assayed in the methods of the present invention are drawn from a temporal window in the maturation of one or more cell lineages.
- the cells circulate preferentially during particular phases in the maturational process.
- the methods of the present invention is limited -to particular phases in DC maturation.
- the methods may equally be applied to CD34 + committed DC precursors that circulate spontaneously, or to CD34 + DC precursors that are mobilized by pharmacological intervention or the like.
- IgGl PE cat. no. 340013, 50 ⁇ g/mL
- CD83 PE BDIS research conjugate, PC#1520; 50 ⁇ g/mL, used at 20 ⁇ L per 300 ⁇ L whole blood
- Pharmingen catalogue number 36935X
- the lin 1 FITC lineage cocktail is also available commercially (BDIS, catalogue number 340546) , and contains a titrated mixture of antibodies specific for CD3, CD14, CD16, CD19, CD20, and CD56, all labeled with FITC. In combination, the antibodies stain lymphocytes, monocytes, eosinophils, and neutrophils. The following antibodies were obtained from BDIS, catalogue number 340546) , and contains a titrated mixture of antibodies specific for CD3, CD14, CD16, CD19, CD20, and CD56, all labeled with FITC. In combination, the antibodies stain lymphocytes, monocytes, eosinophils, and neutrophils. The following antibodies were obtained from BDIS, catalogue number 340546) , and contains a titrated mixture of antibodies specific for CD3, CD14, CD16, CD19, CD20, and CD56, all labeled with FITC. In combination, the antibodies stain lymphocytes, monocytes,
- IL-10 PE IgG2a
- IL-12 PE IgGl
- CD86 PE clone IT2.2; Cat #33435X, IgG2b
- FACS® Permeabilizing Solution and FACS® Lysing Solution were obtained as 10X stock solutions from BDIS (catalogue numbers 340457 and 349202, respectively) , and were diluted and used in accordance with the package insert.
- LPS Lipopolysaccharide
- I (Catalogue number 1-0634)
- PMA Phorbol 12-myristate 13 acetate
- CD40 crosslinking was performed using polystyrene beads (0.84 urn, Baxter) coated with CD40 antibody (PharMingen, San Diego) .
- Brefeldin A (catalogue number B-7651) was made 5 mg/mL in DMSO, and stored at -20°C.
- Wash buffer consisted of phosphate-buffered saline ("DPBS") (obtained as a 10X stock solution from GibCoBRL (Grand Island, NY) , then diluted with deionized water to IX), containing 0.5% fetal calf serum (Sigma, St. Louis, MO) (fetal calf serum added after dilution of 10X PBS stock to IX) .
- DPBS phosphate-buffered saline
- Venous blood of normal donors was collected in sodium heparin VACUTAINER® tubes.
- LPS the blood was stimulated with 1 ⁇ g/mL LPS.
- PMA+I whole blood was first diluted 1:1 with RPMI medium (Biowhittaker, Watersville, MD) . PMA was then added at 5 ng/mL and ionomycin at 1 ⁇ g/mL.
- RPMI medium Biowhittaker, Watersville, MD
- ionomycin 1 ⁇ g/mL
- 50 ⁇ L CD40-coated polystyrene beads was added to 1 mL whole blood. All samples were incubated for four hours at 37°C in a humidified incubator with 5% C0 2 .
- BFA Brefeldin A
- PMA+I treated blood samples Prior to staining, PMA+I treated blood samples were reduced to half volume by centrifugation and removal of supernatant.
- the cells were then collected by centrifugation for 10 min at 500 x g, and the pellet gently broken off for further processing. Next, 10 mL FACS® Permeabilizing Solution was added and the cells were incubated for 10 min. The permeabilization reaction was stopped by addition of 40 mL of buffer (DPBS IX, 0.5% fetal calf serum) . The permeabilized cells were pelleted for 10 min at 500 x g and resuspended in the supernatant remaining in the tube after decanting (approximate volume 500 ⁇ L) .
- buffer DPBS IX, 0.5% fetal calf serum
- PMA+I treated blood samples Prior to staining, PMA+I treated blood samples were reduced to half volume by centrifugation and removal of supernatant.
- FIGS. 2A - 2C show the surface immunophenotypic characteristics of peripheral blood DC from a single LPS-activated whole blood sample.
- CDllc + dendritic cells are painted green, CDllc " DC are painted red, and nondendritic cells appear gray.
- the colors are arbitrarily chosen for purposes of display, and bear no relationship to the fluorophores used for analysis.
- FIG. 2A demonstrates that both dendritic cell subsets are lin 1 FITC dim and HLA-DR bri 9 ht , in agreement with O'Doherty et al . , Immunology 82:487-493 (1994); Olweus et al . , Proc . Na tl . Acad. Sci . USA 94(23): 12551-12556 (1997), with FIG. 2B further demonstrating that the two subsets have similar side scatter and forward scatter properties.
- FIG. 2C shows discrimination of the two subsets based on differential levels of
- FIGS. 2D - 2J show the result of assays for expression of IL-IRA (FIG. 2D), TNF ⁇ (FIG. 2E) , IL-6
- FIG. 2F shows results using an isotype-matched PE-conjugated negative control antibody.
- FIGS. 2D - 2J demonstrate that the CDllc " (CD123 + ) subset (red) is unresponsive to LPS stimulation, at least as evidenced by the absence of detectable cytokine production. Although not shown directly on these figures, the cytokine levels measured in the LPS-activated CDllc " DC are indistinguishable from those produced in the absence of activator; as shown in FIG. 4, neither CDllc " nor CDllc + subset produces detectable levels of cytokine in the absence of DC activators.
- FIGS. 3A - 3C show the surface immunophenotypic characteristics of peripheral blood DC from a single whole blood sample activated with PMA+I.
- CDllc + dendritic cells are painted green, CDllc " DC are painted red, and nondendritic cells appear gray. The colors are arbitrarily chosen for purposes of display, and bear no relationship to the fluorophores used for analysis.
- FIG. 3A demonstrates that both dendritic cell subsets are lin 1 FITC dim and HLA-DR bri ⁇ ht , with FIG. 3B further demonstrating that the two subsets have similar side scatter and forward scatter properties.
- FIG. 3C shows discrimination of the two subsets based on differential levels of CDllc expression.
- FIGS. 3D - 31 show the result of assays for expression of TNF ⁇ (FIG. 3D), IL-l ⁇ (FIG. 3E) , IL-l ⁇
- FIG. 31 shows results using an isotype-matched PE-conjugated negative control antibody.
- FIGS. 3D - 31 demonstrate that the CDllc " (CD123 + ) subset (red) is unresponsive to PMA + I stimulation, at least as evidenced by the absence of detectable cytokine production. Although not shown directly on these figures, the cytokine levels measured in the LPS-activated CDllc " DC are indistinguishable from those produced in the absence of activator
- FIG. 3E demonstrates that CDllc + cells produced trace amounts of
- monocytes The cytokine expression of monocytes was evaluated in some of the same samples. Monocytes were identified based on their scatter characteristics, their bright lin 1 FITC, anti-HLA-DR PerCP and CDllc
- APC staining As can be seen, in LPS+BFA stimulated samples, monocytes express cytokines at higher levels than do CDllc + DCs. Upon PMA+I+BFA activation, the cytokine secretion of CDllc + DCs and monocytes is equivalent, but much less compared to LPS activated samples. Intracellular protein secretion is evaluated as PE mean fluorescence intensity (MFI) .
- MFI PE mean fluorescence intensity
- the CDllc " subset demonstrated clear upregulation of CD25 expression upon PMA+I activation; upregulation of CD25 was the only distinct response observed in CDllc " subset.
- the CDllc + subset showed upregulation of CD25, CD40, CD80, CD86, HLA--DR and HLA- DQ upon LPS activation.
- the T cell co-stimulatory molecules, in particular CD80 gave the strongest signal.
- PMA+I led to an upregulation in CDllc + cells of CD86, CD80, HLA-DQ and HLA-DR.
- Minimal increase of CD25 and CD40 were observed.
- Activation via crosslinking of CD40 resulted in increased levels of CD86, CD80, and minimal upregulation of HLA-DR.
- FIG. 5 highlights the differences in the TNF ⁇ , IL-8, CD80 and CD86 responses of the CDllc + peripheral blood DC subset during activation with PMA+I versus LPS.
- Two donors are displayed.
- the expression pattern of cytokines and co-stimulatory molecules varies between different stimuli and is consistent between donors.
- IL-8 and CD86 are the dominant signal in PMA+I stimulation.
- TNF ⁇ and CD80 are produced to a greater extent.
- This Example presents protocols modified slightly from those set forth in Examples 1 and 2; the modified protocols are faster, permit data to be acquired from a greater number of cells, thus improving statistics, and contain fewer aliquoting steps.
- DC Surface Antigen Expression in Whole Blood Response of CDllc + DC to LPS stimulation reported as the mean fluorescence intensity (mean MFI) of surface expression of the co-stimulatory molecule CD80 or the DC activation marker CD83, is measured as follows .
- LPS lipopolysaccharide
- CDllc APC by BDIS, Cat#340544, use 15 ⁇ L/300 ⁇ L whole blood (WB)
- lineage cocktail 1 (lin 1) FITC, by BDIS, Cat#340546, 60 ⁇ L/300 ⁇ L WB
- CD83 PE by BDIS (custom conjugate), PC#1520, 50 ⁇ g/mL, use 20 ⁇ L/300 ⁇ L WB
- FCS file • Acquire the FCS file using anti-human HLA-DR PerCP positive / lin 1 FITC dim events. Use the entire cell suspension for acquisition (about 4,000 -7,000 events).
- LPS lipopolysaccharide
- lineage cocktail 1 (lin 1) FITC, by BDIS, Cat#340546, 120 ⁇ L/300 ⁇ L WB
- Anti-human HLA-DR PerCP by BDIS, Cat#347364, use 60 ⁇ L/300 ⁇ L WB
- Anti-human TNF ⁇ PE by BDIS, Cat#340512, use 20 ⁇ L/test
- FACSTM Permeabilizing Solution lOx by BDIS, Cat#340457, working solution is [1:10] dilution in deionized water
- FACSTM lysing Solution lOx by BDIS, Cat#349202, working solution is a [1:10] dilution in deionized water
- FCS file • Acquire the FCS file using anti-human HLA-DR PerCP positive / lin 1 FITC dim events. Use the entire cell suspension for acquisition (4,000 -7,000 events).
Abstract
Description
Claims
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DE69917051T DE69917051T2 (en) | 1998-09-22 | 1999-09-21 | "FLOW-CYTOMETRIC" WHOLE BLOOD DENDRITZ CELL IMMUNFUNCTION TEST |
JP2000571267A JP4450998B2 (en) | 1998-09-22 | 1999-09-21 | Flow cytometric whole blood dendritic cell immune function assay |
EP99946994A EP1116037B1 (en) | 1998-09-22 | 1999-09-21 | Flow cytometric, whole blood dendritic cell immune function assay |
AT99946994T ATE266204T1 (en) | 1998-09-22 | 1999-09-21 | ßFLOW CYTOMETRIC ß WHOLE BLOOD DENDRITE CELL IMMUNE FUNCTION TEST |
US10/635,972 US7351546B2 (en) | 1998-09-22 | 2003-08-07 | Flow cytometric, whole blood dendritic cell immune function assay |
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