EP1842068A1 - Use of multiparametric flow cytometry for the diagnosis, prognosis, and validation of immunotherapies in autoimmune, hematologic, and lymphoproliferative diseases - Google Patents
Use of multiparametric flow cytometry for the diagnosis, prognosis, and validation of immunotherapies in autoimmune, hematologic, and lymphoproliferative diseasesInfo
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- EP1842068A1 EP1842068A1 EP04745177A EP04745177A EP1842068A1 EP 1842068 A1 EP1842068 A1 EP 1842068A1 EP 04745177 A EP04745177 A EP 04745177A EP 04745177 A EP04745177 A EP 04745177A EP 1842068 A1 EP1842068 A1 EP 1842068A1
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- prognosis
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Classifications
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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
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
- G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
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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
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/42—Low-temperature sample treatment, e.g. cryofixation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N2015/1477—Multiparameters
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
Definitions
- This invention concerns a method which allows to perform flow cytometric analysis using determined antibody combinations; these antibodies are conjugated with different fluorochromes, and the antibody combinations include several panels which allow to perform an analysis of the immunological status of the individual (in case of screening for autoimmune disorders or chronic infections) or a detailed study of neoplastic cell populations (when screening for haematological diseases).
- the increased knowledge on how the immune system works has brought, in recent years, to a better understanding of the pathogenesis of many diseases, and, consequently, to new therapeutic approaches.
- the interactions between the different components of the immune system are indeed very complex, and new technologies have revealed that cellular populations Which apparently seem homogeneous are in fact composed of several subpopulations with different phenotypes and functions.
- the cells of the immune system circulate in the blood and the lymph which can also migrate to tissue to protect the organism efficiently. Lymphocytes specifically recognise and respond to foreign antigens, while phagocytes and granulocytes are classified as "inflammatory" cells, and they play a major role in the innate immune response.
- lymphocytes appear to be macroscopically homogeneous, they can actually be divided in distinct subpopulations: in the first place one must distinguish between B-lymphocytes, which produce antibodies, and T lymphocytes. Based on expression of surface molecules, the latter can be further divided in CD4+ "helper” cells and CD8+ cytotoxic cells. Both CD4+ and CD8+ cells comprise subsets, which have not yet encountered a foreign antigen, so called “na ⁇ ve" cells, and memory cells, which represent the expanded pool of antigen experienced cells. But these subsets are still heterogeneous: indeed some memory cells produce proinflammatory proteins, while other tend to regulate and to switch off the immune response.
- the complexity of the immune system is such that the identification of cellular subsets with unique functional characteristics requires the simultaneous measurement of multiple parameters on each single cell.
- multiparametric flow cytometry it is possible to measure the presence of surface markers, which permit the identification of distinct cellular subsets: for instance, during a viral infection, it is possible to identify virus-specific cells activated by the infection and to follow them during the anti-viral response.
- virus-specific cells activated by the infection and to follow them during the anti-viral response.
- the cells of the immune system become activated and acquire the functions which permit the elimination of the offending agent.
- adhesion molecules which enable the cells of the immune system to interact with the endothelial cells lining the capillaries: this interaction represents the initial step of the migration process by which the cells of the immune system gain access to inflamed tissues.
- the study of the membrane receptors expressed by the different subsets of lymphocytes represents a powerful tool for the evaluation of the "immunological status" of an individual.
- autoimmune diseases are the results of the dysregulation of the complex interactions between cells of the immune system, so these pathologies represent a good target for studies involving the definition of fine cellular subsets.
- Flow cytometry represents the ideal tool for the study of non adherent cells (in suspension), and can be used also for the study of lymphoid tissues, from which single cell suspensions can be easily obtained. Data obtained with flow cytometry - both concerning the distribution of the different cellular subsets and the expression of multiple cellular markers are then evaluated with powerful software.
- Fig. 1 show the multiparametric analysis of CD3+CD8+ lymphocytes obtained from the peripheral blood of a healthy subject.
- Fig.2. show the multiparametric analysis of CD3+CD8+ lymphocytes in an MS patient
- Fig.3. show the analysis of the ability of CD3+CD8+ lymphocytes to produce the inflammatory cytokine interferon gamma (IFN ⁇ ).
- This invention concerns a method which allows to perform flow cytometric analysis using determined antibody combinations.
- These antibodies are conjugated with different fluorochromes, and the antibody combinations include several panels which allow to perform an analysis of the immunological status of the individual (in case of screening for autoimmune disorders or chronic infections) or a detailed study of neoplastic cell populations (when screening for haematological diseases).
- Antibodies are aliquoted in small tubes at predetermined concentrations, and then lyophilised.
- Antibodies which we have tested are: CD3 PE-Cy7, CD8 PE-Cy7, CD8 PE-TxRed, CD45RA PE-TxRed, CD62L PE-TxRed, CD45RO PE-TxRed, CD8 beta PE, CD49dFlTC, NKRP1A PE, CD3 FITC, CD3 PE, CD8 FITC, CD8 PE, CD8 CyChrome, CD8 APC, CD4 FITC, CD4 APC, CD62L PE-Cy5, CD62L APC, CD162 PE, CCR7 PE, CD45RO FITC, CD11a PE, CD11a APC, CD49d PE, CD8 APC-Cy7, CDHa ' FITC, CD45RA PE-Cy5, CD5 PE-Cy5, CD23 PE-TxRed, CD19 PE-Cy7, CD19 APC- Cy7, CD79b APC, CD38 PE, kappa FITC, lambda PE, F
- Antibodies are used are optimal predetermined concentrations, (usually 0,25 ⁇ g/ml). Each new antibody lot is accurately tested in serial dilutions, and antibodies are aliquoted in 1 ,5 ml tubes in different combinations. Tubes are then put in the speedvac, until completely lyophilised (20 minutes). Before use each antibody combinations is reconstituted with saline solution, and added to the tube containing cells to be analyzed. For each antibody combination, 2x10 6 cells are stained. This method can be used for staining of whole blood or of peripheral blood lymphocytes (PBLs) obtained from centrifugation on a density gradient.
- PBLs peripheral blood lymphocytes
- heparinized blood will be diluted with 1 volume of RPMI media and gently layered over the Fycoll-Hypaque (Pharmacia). Following centrifugation at 660g for 30 minutes, cells at the interface of the gradient will be collected and washed three times in medium. After the final wash cells will be resuspended in PBS with 1% human serum. For each labelling cells will be resuspended in a volume of 100 ⁇ l. All antibodies will have been tested at saturating conditions, to exclude differences in staining.
- Samples will then be acquired at the flow cytometer using constant instrument settings, obtained by a careful daily calibration of the instruments with beads and by the use of appropriate compensation controls (beads coated with the antibodies used in the stainings). For each sample 1x106 events will be acquired, in order to guarantee an appropriate representation of all cellular subsets, and allowing significant statistical analysis.
- To study cytokine production we will follow the procedure of intracellular staining, as established in our laboratory. Briefly, cells are plated at a density of 2x10 6 /ml in 96 well plates and triggered with either mitogens (PHA, PMA+ionomycin) or antibodies directed against cell surface molecules (such as CD3), for 6 hours at 37°C, in the presence of monensin (10 mM).
- Flow cytometric analysis permits not only the phenotypic analysis of cellular subpopulations r but also the ability of each subpopulation to produce soluble factors which modulate the immune response (Fig. 3).
- the panel of cytokine produced by each cell type provides important information on the immunological status of the patient, and can be used as a "marker" of the efficacy of immunomodulatory treatments.
- this invention may be used for the diagnosis of hematologic and lymphoproliferative disorders.
- flow cytometry is a powerful tool used for the characterization of neoplastic cells present in these diseases.
- the ability to simultaneously measure multiple markers on each ' single cell enables to carry out a detailed analysis of tumoral cells, and to perform an accurate diagnosis.
- the diagnosis of hematologic tumours requires an accurate characterization of the neoplastic clone, since treatment and prognosis depend closely on the diagnosis.
- a standard panel of antibodies specific for membrane or cytoplasmic markers is used in order to define the lineage and differentiation stage of the neoplastic clone (Tab.1).
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Abstract
This invention concerns a method which allows to perform flow cytometric analysis using determined antibody combinations. These antibodies are conjugated with different fluorochromes, and the antibody combinations include several panels which allow to perform an analysis of the immunological status of the individual (in case of screening for autoimmune disorders or chronic infections) or a detailed study of neoplastic cell populations (when screening for haematological diseases). Antibodies are aliquoted in small tubes at predetermined concentrations, and then lyophilised.
Description
USE OF MULT I PARAMETRIC FLOW CYTOMERY FOR THE DIAGNOSIS , PROGNOSIS , AND VALIDATIO N OF IMMUNOTHERAPIES IN AUTOIMMUNE , HEMATOLOGIC, AND LYMPHOPROLIFERATIVE DISEASE S
Technical field This invention concerns a method which allows to perform flow cytometric analysis using determined antibody combinations; these antibodies are conjugated with different fluorochromes, and the antibody combinations include several panels which allow to perform an analysis of the immunological status of the individual (in case of screening for autoimmune disorders or chronic infections) or a detailed study of neoplastic cell populations (when screening for haematological diseases).
Description of the Related Art
The increased knowledge on how the immune system works has brought, in recent years, to a better understanding of the pathogenesis of many diseases, and, consequently, to new therapeutic approaches. The interactions between the different components of the immune system are indeed very complex, and new technologies have revealed that cellular populations Which apparently seem homogeneous are in fact composed of several subpopulations with different phenotypes and functions.
The cells of the immune system circulate in the blood and the lymph which can also migrate to tissue to protect the organism efficiently. Lymphocytes specifically recognise and respond to foreign antigens, while phagocytes and granulocytes are classified as "inflammatory" cells, and they play a major role in the innate immune response. Although lymphocytes appear to be macroscopically homogeneous, they can actually be divided in distinct subpopulations: in the first place one must distinguish between B-lymphocytes, which produce antibodies, and T lymphocytes. Based on expression of surface molecules, the latter can be further divided in CD4+ "helper" cells and CD8+ cytotoxic cells. Both CD4+ and CD8+ cells comprise subsets, which have not yet encountered a foreign antigen, so called "naϊve" cells, and memory cells, which represent the expanded pool of antigen experienced cells. But these subsets are still heterogeneous: indeed some memory cells produce proinflammatory proteins, while other tend to regulate and to switch off the immune response. Thus, it is clear that the complexity of the immune system is such that the identification of cellular subsets with unique functional characteristics requires the simultaneous measurement of multiple parameters on each single cell. With multiparametric flow cytometry it is possible to measure the presence of surface markers, which permit the identification of distinct cellular subsets: for instance, during a viral infection, it is possible to identify virus-specific cells activated by the infection and to follow them during the anti-viral response. Thus, it is possible to study the initial expansion of these cells, to evaluate the panel of anti-viral proteins produced, and finally to monitor the contraction of the virus specific population and the return to the steady state, with the maintenance of a memory population able to respond more rapidly and vigorously in. the case of re-infection.
During the immune response, be it directed against infectious agents, tumours, or, in the case of autoimmune disorders, against tisues of the host, the cells of the immune system become activated and acquire the functions which permit the elimination of the offending agent. For instance, it is crucial to study the expression of adhesion molecules which enable the cells of the immune system to interact with the endothelial cells lining the capillaries: this interaction represents the initial step of the migration process by which the cells of the immune system gain access to inflamed tissues. The study of the membrane receptors expressed by the different subsets of lymphocytes represents a powerful tool for the evaluation of the "immunological status" of an individual. Notably, autoimmune diseases are the results of the dysregulation of the complex interactions between cells of the immune system, so these pathologies represent a good target for studies involving the definition of fine cellular subsets.
For instance, in the case of Multiple Sclerosis, an autoimmune disorder of the nervous system in which the cells of the immune system erroneously attack the myelin sheath, it would be useful to delineate a strategy by which the different cellular subsets can be uniquely identified andJbllowed in time with the progression of the disease. Flow cytometry is also a fundamental tool for the diagnosis and characterization of hematologic and lymphoproliferative disorders. This technology indeed represents a necessary complement for the traditional diagnostic tools based on the use of the microscope, and it also adds finer discrimination abilities which can not be achieved with other diagnostic tools. Flow cytometry represents the ideal tool for the study of non adherent cells (in suspension), and can be used also for the study of lymphoid tissues, from which single cell suspensions can be easily obtained. Data obtained with flow cytometry - both concerning the distribution of the different cellular subsets and the
expression of multiple cellular markers are then evaluated with powerful software.
The identification of tumoral clones, the study of the expression of tumoral antigens by leukemias, the measurement of cell cycle and the detection of intracellular antigens represent useful applications of flow cytometry in haematology. Generally, in clinical labs where blood samples obtained from patients with hematologic diseases, diagnosis is made using 4 or 5 parameter-flow cytometry (2 or three colours). Given that neoplastic cells may coexpress numerous markers, the possibility to perform multiparametric analyses ' with 7 colours permits a more accurate definition of. neoplastic populations thus allowing a faster and more precise analysis.
Brief description of the drawings
Fig. 1 show the multiparametric analysis of CD3+CD8+ lymphocytes obtained from the peripheral blood of a healthy subject.
Fig.2. show the multiparametric analysis of CD3+CD8+ lymphocytes in an MS patient
Fig.3. show the analysis of the ability of CD3+CD8+ lymphocytes to produce the inflammatory cytokine interferon gamma (IFNγ).
Detailed description of the preferred embodiment
This invention concerns a method which allows to perform flow cytometric analysis using determined antibody combinations. These antibodies are conjugated with different fluorochromes, and the antibody combinations include several panels which allow to perform an analysis of the immunological status of the individual (in case of screening for autoimmune disorders or chronic infections) or a detailed
study of neoplastic cell populations (when screening for haematological diseases). Antibodies are aliquoted in small tubes at predetermined concentrations, and then lyophilised. Antibodies which we have tested are: CD3 PE-Cy7, CD8 PE-Cy7, CD8 PE-TxRed, CD45RA PE-TxRed, CD62L PE-TxRed, CD45RO PE-TxRed, CD8 beta PE, CD49dFlTC, NKRP1A PE, CD3 FITC, CD3 PE, CD8 FITC, CD8 PE, CD8 CyChrome, CD8 APC, CD4 FITC, CD4 APC, CD62L PE-Cy5, CD62L APC, CD162 PE, CCR7 PE, CD45RO FITC, CD11a PE, CD11a APC, CD49d PE, CD8 APC-Cy7, CDHa 'FITC, CD45RA PE-Cy5, CD5 PE-Cy5, CD23 PE-TxRed, CD19 PE-Cy7, CD19 APC- Cy7, CD79b APC, CD38 PE, kappa FITC, lambda PE, FMC7 FITC, CD20 PE-Cy7.
Antibodies are used are optimal predetermined concentrations, (usually 0,25μg/ml). Each new antibody lot is accurately tested in serial dilutions, and antibodies are aliquoted in 1 ,5 ml tubes in different combinations. Tubes are then put in the speedvac, until completely lyophilised (20 minutes). Before use each antibody combinations is reconstituted with saline solution, and added to the tube containing cells to be analyzed. For each antibody combination, 2x106 cells are stained. This method can be used for staining of whole blood or of peripheral blood lymphocytes (PBLs) obtained from centrifugation on a density gradient. Briefly, heparinized blood will be diluted with 1 volume of RPMI media and gently layered over the Fycoll-Hypaque (Pharmacia). Following centrifugation at 660g for 30 minutes, cells at the interface of the gradient will be collected and washed three times in medium. After the final wash cells will be resuspended in PBS with 1% human serum. For each labelling cells will be resuspended in a volume of 100 μl. All antibodies will have been tested at saturating conditions, to exclude differences in staining. Samples will then be acquired at the flow cytometer using constant instrument settings, obtained by a careful daily calibration of the instruments with beads and by the use of
appropriate compensation controls (beads coated with the antibodies used in the stainings). For each sample 1x106 events will be acquired, in order to guarantee an appropriate representation of all cellular subsets, and allowing significant statistical analysis. To study cytokine production we will follow the procedure of intracellular staining, as established in our laboratory. Briefly, cells are plated at a density of 2x106/ml in 96 well plates and triggered with either mitogens (PHA, PMA+ionomycin) or antibodies directed against cell surface molecules (such as CD3), for 6 hours at 37°C, in the presence of monensin (10 mM). At the end of the incubation period, cells will be collected, spun, and staining of extracellular markers will be performed, followed by two washes. Samples will then be fixed with 4% paraformaldehyde in PBS for 10 min at 40C, and permeabilized by washing twice with a solution containing PBS (without Ca++ and Mg++), 1% FBS, and 0.1% saponin at pH 7.4. Cells will be resuspended in permeabilization buffer plus fluorochrome-conjugated anticytokine antibody (1μg per ml) for 30 min at 4°C in the dark. Cells will be washed twice, resuspended in 250 μl of staining buffer (PBS, 1% FBS, 0.01% sodium azide) and analyzed by FACS. This invention has been successfully used in our laboratory for the study of- the distribution of different cell subsets in healthy individuals and in patients with autoimmune diseases. The accurate analysis of the different cellular subsets allows to identify a disequilibrium of the immune system (due for instance to the presence of an autoimmune disease, or to a chronic infection which determines a persistent stimulation of immune cells). Moreover based on our studies, it is possible to say that the results of a detailed phenotypic analysis can also be taken in consideration for the prognosis of the disease. For instance we have shown that multiple sclerosis patients during the acute phase of the disease show a very high proportion of "naϊve" and perforin+ effector T cells, and this percentage varies in the course of the disease (Fig.2)
Flow cytometric analysis permits not only the phenotypic analysis of cellular subpopulationsr but also the ability of each subpopulation to produce soluble factors which modulate the immune response (Fig. 3). The panel of cytokine produced by each cell type provides important information on the immunological status of the patient, and can be used as a "marker" of the efficacy of immunomodulatory treatments. Moreover, this invention may be used for the diagnosis of hematologic and lymphoproliferative disorders. As mentioned above, flow cytometry is a powerful tool used for the characterization of neoplastic cells present in these diseases. The ability to simultaneously measure multiple markers on each' single cell enables to carry out a detailed analysis of tumoral cells, and to perform an accurate diagnosis. The diagnosis of hematologic tumours requires an accurate characterization of the neoplastic clone, since treatment and prognosis depend closely on the diagnosis. Usually a standard panel of antibodies specific for membrane or cytoplasmic markers is used in order to define the lineage and differentiation stage of the neoplastic clone (Tab.1).
Discrimination between B and T lymphocytes CD2 CD3 I CD5 CD7 CD10 I CD19 CD20 CD24 B cells + + T cells + +
Tab.l Commonly used markers for the diasgnosis of hematologic tumours.
In clinical labs these analyses are performed using two or three markers in each staining. With this invention it is possible to analyze simultaneously 7 markers on each single cell, with the following advantages: 1. Less sample needs to be used in each test; 2. Saving of reagents; 3. Accurate definition of the neoplastic cells; 4. Test can be performed in a smaller amount of time.
Again, a set of pre-defined antibody combinations are used in order to accurately define cell populations.
Claims
Claims
1. Method for the use of multiparametric flow cytometry for the prognosis and the validation of immunotherapies of autoimmune and haematological diseases. This method is characterized by the ability to use different fluorochrome-conjugated antibody combinations which enable to perform an analysis of the individual's immunological status; the antibodies are aliquoted in predetermined concentrations and lyophilized. At the moment of use, each antibody combination is reconstituted by the addition of saline solution, and added to the tube containing the cells to be analyzed. For each antibody combination 2x106 cells are stained. Each antibody is tested and used in saturating concentration so as to avoid differences in sample staining. Samples are then analyzed at the flow cytometer using constant instrument setting obtained with a careful daily calibration with beads. For each sample 1x106 events are acquired in order to achieve an appropriate representation of all cellular subsets and to allow significant statistical analysis.
2.Method for the use of multiparametric flow cytometry for the prognosis and the validation of immunotherapies of autoimmune and haematological diseases, as described above, and characterized by the fact that the antibodies whose efficacy has been tested are: CD3 PE-Cy7, CD8 PE-Cy7, CD8 PE-TxRed, CD45RA PE-TxRed, CD62L, PE-TxRed, CD45RO PE-TxRed, CD8 beta PE, CD49dFITC, NKRP1A PE, CD3 FITC, CD3 PE, CD8 FITC, CD8 PE, CD8 CyChrome, CD8
APC, CD4 FITC, CD4 APC, CD62L PE-Cy5, CD62L APC, CD162 PE, CCR7 PE, CD45RO FITC, CD11a PE, CD11a APC, CD49d PE, CD8 APC-Cy7, CD11a FITC, CD45RA PE-Cy5, CD5 PE-Cy5, CD23 PE- TxRed, CD19 PE-Cy7, CD19 APC-Cy7, CD79b APC, CD38 PE, kappa FITC, lambda PE, FMC7 FITC, CD20 PE-Cy7. Two examples of antibody combinations used are: a) for haematological diseases, Mix 1 = CD19 CD20 CD79b, CD5, CD23, CD38, FMC7, and b) for the study of Multiple Sclerosis Mix 2= CD3 CD8 CCR7 CD45RA CD11a CD62L CD27. 3. Method for the use of multiparametric flow cytometry for the prognosis and the validation of immunotherapies of autoimmune and haematological diseases, where the combination for the study and the diagnosis of lymphoproliferative diseases is: CD19 CD20, CD5, CD23, CD38, FMC7 CD79b. 4.Method for the use of multiparametric flow cytometry for the prognosis and the validation of immunotherapies of autoimmune and haematological diseases, where the combination for the study of Multiple Sclerosis is: CD3 CD8 CCR7 CD45RA CD11a CD62L CD27. δ.Method for the use of multiparametric flow cytometry for the prognosis and the validation of immunotherapies of autoimmune and haematological diseases, as described above, and characterized by the fact that this method can be used both on whole blood and on peripheral blood mononuclear cells purified with a gradient. Briefly, heparinized blood is diluted with 1 volume of RPMI media and gently
layered over the Fycoll-Hypaque (Pharmacia). Following centrifugation at 660g for 30 minutes, cells at the interface of the gradient will be collected and washed three times in medium. After the final wash cells are resuspended in PBS with 1% human serum. For each labelling 5 cells are resuspended in a volume of 100 μl. 6. Method for the use of multiparametric flow cytometry for the prognosis and the validation of immunotherapies of autoimmune and haematological diseases, as described above, and characterized by the fact that for the study of cytokine production the technique of ιo intracellular staiinng is performed, by which cells are plated at a density of 2x106/ml in 96 well plates and triggered with either mitogens (PHA, PMA+ionomycin) or antibodies directed against cell surface molecules (such as CD3), for 6 hours at 37°C, in the presence of monensin (10 mM). At the end of the incubation period, cells will be5 collected, spun, and staining of extracellular markers will be performed, followed by two washes. Samples will then be fixed with 4% paraformaldehyde in PBS for 10 min at 40C, and permeabilized by washing twice with a solution containing PBS (without Ca++ and Mg++), 1% FBS, and 0.1% saponin at pH 7.4. Cells will beo resuspended in permeabilization buffer plus fluorochrome-conjugated anticytokine antibody (1 Dg per ml) for 30 miri at 4°C in the dark. Cells will be washed twice, resuspended in 250 μl of staining buffer (PBS, 1% FBS, 0.01% sodium azide) and analyzed by FACS.
Claims
1. Method for the use of multiparametric flow cytometry for the prognosis and the validation of immunotherapies of autoimmune and haematological diseases. This method is characterized by the ability to use different fluorochrome-conjugated antibody combinations which enable to perform an analysis of the individual's immunological status; the antibodies are aliquoted in predetermined concentrations and lyophilized. At the moment of use, each antibody combination is reconstituted by the addition of saline solution, and added to the tube containing the cells to be analyzed. For each antibody combination 2x106 cells are stained. Each antibody is tested and used in saturating concentration so as to avoid differences in sample staining. Samples are then analyzed at the flow cytometer using constant instrument setting obtained with a careful daily calibration with beads. For each sample 1x106 events are acquired in order to achieve an appropriate representation of all cellular subsets and to allow significant statistical analysis.
2.Method for the use of multiparametric flow cytometry for the prognosis and the validation of immunotherapies of autoimmune and haematological diseases, as described above, and characterized by the fact that the antibodies whose efficacy has been tested are: CD3 PE-Cy7, CD8 PE-Cy7, CD8 PE-TxRed, CD45RA PE-TxRed, CD62L, PE-TxRed, CD45RO PE-TxRed, CD8 beta PE, CD49dFITC, NKRP1A PE, CD3 FITC, CD3 PE, CD8 FITC, CD8 PE, CD8 CyChrome, CD8
APC, CD4 FITC, CD4 APC, CD62L PE-Cy5, CD62L APC, CD162 PE, CCR7 PE, CD45RO FITC, CD11a PE, CD11a APC, CD49d PE, CD8 APC-Cy7, CD11a FITC, CD45RA PE-Cy5, CD5 PE-Cy5, CD23 PE- TxRed, CD19 PE-Cy7, CD19 APC-Cy7, CD79b APC, CD38 PE, kappa FITC, lambda PE, FMC7 FITC, CD20 PE-Cy7. Two examples of antibody combinations used are: a) for haematological diseases, Mix 1 = CD19 CD20 CD79b, CD5, CD23, CD38, FMC7, and b) for the study of Multiple Sclerosis Mix 2= CD3 CD8 CCR7 CD45RA CD11a CD62L CD27. 3.Method for the use of multiparametric flow cytometry for the prognosis and the validation of immunotherapies of autoimmune and haematological diseases, where the combination for the study and the diagnosis of lymphoproliferative diseases is: CD19 CD20, CD5, CD23, CD38, FMC7 CD79b. 4.Method for the use of multiparametric flow cytometry for the prognosis and the validation of immunotherapies of autoimmune and haematological diseases, where the combination for the study of Multiple Sclerosis is: CD3 CD8 CCR7 CD45RA CD11a CD62L CD27. β.Method for the use of multiparametric flow cytometry for the prognosis and the validation of immunotherapies of autoimmune and haematological diseases, as described above, and characterized by the fact that this method can be used both on whole blood and on peripheral blood mononuclear cells purified with a gradient. Briefly, heparinized blood is diluted with 1 volume of RPMI media and gently
layered over the Fycoll-Hypaque (Pharmacia). Following centrifugation at 660g for 30 minutes, cells at the interface of the gradient will be collected and washed three times in medium. After the final wash cells are resuspended in PBS with 1% human serum. For each labelling cells are resuspended in a volume of 100 μl.
6. Method for the use of multiparametric flow cytometry for the prognosis and the validation of immunotherapies of autoimmune and haematological diseases, as described above, and characterized by the fact that for the study of cytokine production the technique of intracellular staiinng is performed, by which cells are plated at a density of 2x106/ml in 96 well plates and triggered with either mitogens (PHA, PMA+ionomycin) or antibodies directed against cell surface molecules (such as CD3), for 6 hours at 37°C, in the presence of monensin (10 m ). At the end of the incubation period, cells will be collected, spun, and staining of extracellular markers will be performed, followed by two washes. Samples will then be fixed with 4% paraformaldehyde in PBS for 10 min at 40C, and permeabilized by washing twice with a solution containing PBS (without Ca++ and Mg++), 1% FBS, and 0.1% saponin at pH 7.4. Cells will be resuspended in permeabilization buffer plus fluorochrome-conjugated anticytokine antibody (1 Dg per ml) for 30 min at 4°C in the dark. Cells will be washed twice, resuspended in 250 μl of staining buffer (PBS, 1% FBS, 0.01% sodium azide) and analyzed by FACS.
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