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WO1998040745A1 - Procede de detection de cellules t specifiques de l'antigene - Google Patents

Procede de detection de cellules t specifiques de l'antigene Download PDF

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Publication number
WO1998040745A1
WO1998040745A1 PCT/EP1998/001479 EP9801479W WO9840745A1 WO 1998040745 A1 WO1998040745 A1 WO 1998040745A1 EP 9801479 W EP9801479 W EP 9801479W WO 9840745 A1 WO9840745 A1 WO 9840745A1
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WO
WIPO (PCT)
Prior art keywords
cells
antigen
specific
cell population
gad
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP1998/001479
Other languages
German (de)
English (en)
Inventor
Josef Endl
Barbara Dreisbusch
Sabine Fessele
Peter Stahl
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Roche Diagnostics GmbH
Original Assignee
Roche Diagnostics GmbH
Boehringer Mannheim GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Roche Diagnostics GmbH, Boehringer Mannheim GmbH filed Critical Roche Diagnostics GmbH
Priority to AU70351/98A priority Critical patent/AU7035198A/en
Priority to EP98916946A priority patent/EP0972199A1/fr
Priority to JP53923898A priority patent/JP2002500627A/ja
Publication of WO1998040745A1 publication Critical patent/WO1998040745A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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

Definitions

  • the invention relates to a method for producing a T cell population enriched in antigen-specific T cells, the use of this T cell population in diagnostics and for the production of a therapeutic agent, and a test kit for a method for detecting the antigen-specific activation of a T cell population.
  • the immune system of the vertebrates is a complex system in which a large number of molecules and cells interact in fine-tuned reactions. Its primary task is to protect the organism from microorganisms such as viruses, bacteria and other parasites. For this purpose, two different systems have emerged in the course of development, which complement each other in a communicative manner to ensure this task.
  • the T-Ly ⁇ nphozyten stimulate the humoral immune response by interacting with the B cells, the precursors of the plasma cells.
  • the immune system must distinguish its own structures, ie cells of its own organism from foreign components and cells that could damage the organism.
  • the body's own structures are also referred to as “self” and foreign structures as “not-self”. Disorders of this "self-recognition mechanism" can lead to diseases such as allergies or autoimmune diseases.
  • IDDM insulin-dependent diabetes mellitus
  • T-lymphocyte-mediated destruction of the insulin-producing beta cells of the pancreas a disease in which an autoimmune defect is suspected to be the cause.
  • IDDM insulin-dependent diabetes mellitus
  • GAD glutamate decarboxylase
  • the GAD antigen contained in the beta cells leads to a proliferative T cell response with the simultaneous onset of insulitis and subsequently by the interaction of various processes for the destruction of the beta cells of the pancreas.
  • the detection of autoreactive T cells that can be stimulated by the GAD antigen provides a method by means of which the emergence or the presence of IDDM can be diagnosed. This detection can be carried out by in vitro stimulation of the proliferation or the expression of activation markers or the cytokine release of peripheral blood lymphocytes with one or more autoantigens in a blood sample.
  • a problem with this detection method is the low frequency of autoantigen-specific T cells in peripheral blood, which are present with a frequency of only 1 in 10 5 to 1 in 10 6 of the total T cells.
  • autostimulation which is referred to as the so-called “autologous mixed ly phocyte reaction”
  • the object of the invention is achieved by a method for producing a cell population enriched in antigen-specific T cells, which is characterized in that naive T cells and a subpopulation of B-, T- from a mononuclear population of cells by specific depletion steps. or / and NK cells which cause non-specific activation or / and proliferation are removed.
  • binding molecules eg antibodies
  • binding molecules eg antibodies
  • unwanted cells can be removed by means of chromatographic separation processes if one uses binding molecules immobilized on a support to which the desired antigen-specific T cells are not bound. In this way, the antigen-specific T cells do not come into contact with molecules that can bind to them, which prevents unspecific auto-stimulation.
  • Suitable surface determinants of the cells to be depleted can be determined and suitable binding molecules directed against them, e.g. Antibodies are used. Monoclonal antibodies or fragments thereof are preferably used as antibodies. However, polyclonal antisera with sufficient specificity can also be used.
  • Antibodies against the surface determinants CD62L (Reinherz, EL et al., 1982, J. Immunol. 184, 1508) or / and CD45RA (Morimoto C. et al., 1985, J. Immunol.) are preferably used to remove the naive T cells 184, 1508).
  • Antibodies against the surface determinant CD39 are preferably used to remove a subpopulation of B, T or / and NK cells (Kansas, G. S. et al., 1991, J. Immunol. 146, 2235).
  • the method of the invention can be adapted so that in each case desired cell populations can be used as the starting material.
  • Peripheral blood lymphocytes are a preferred starting material; peripheral mononuclear cells, for example from blood, are particularly preferred.
  • a cell population enriched with memory T cells and T cells preactivated in vivo is preferably obtained by the method.
  • the invention relates to an enriched cell population that is obtainable by the method outlined above.
  • the content of the cell population is ⁇ 80% of memory T cells and T cells preactivated in vivo. The content is particularly preferably 90 90%.
  • the content of naive T cells and of the disruptive subpopulation of B, T, or / and NK cells is reduced by the method according to the invention.
  • the cell population preferably contains a naive T cell content of 10 10%.
  • the content of the disruptive subpopulation of B, T or / and NK cells in the cell population is preferably ⁇ 20% of the initial value.
  • the invention relates to the use of the cell population obtainable by the described method in diagnostics, preferably in a method for the detection of the T cell reaction against a specific antigen, which is an autoantigen, tumor antigen and / or pathogen or a Fragment can act about it.
  • the antigen is particularly preferably an autoantigen or a fragment thereof which can be used for the detection of autoimmune diseases or a predisposition therefor.
  • An example of this is the detection of IDDM, which via specific autoantigens such as human GAD (Baekkeskov S. et al., 1987, J. Clin. Invest., 79, 926) or IA-2 (Payton, MA, et al., 1995, J. Clin. Invest., 96, 1506).
  • human GAD or a fragment thereof e.g. a partial peptide as described in German Patent Application No. P 4401 629.8.
  • the invention relates to the use of the cell population in a method for monitoring the success of a vaccination, for example in the vaccination against a pathogen, such as tetanus.
  • the invention relates to the use of the cell population in a method for controlling the reduction in antigen-specific T cells.
  • a method can be used for therapy monitoring.
  • therapeutic measures such as anergy induction or depletion, e.g. B. by antibody mediated complement lysis, such a control method can be used.
  • the antigen is used in the process in a suitable form, preferably it is used in a highly purified form.
  • the antigen can also be chemically modified to increase uptake in the antigen-presenting cells
  • the antigen is particularly preferably glycosylated, e.g. B. mannosylated, since this results in an increased uptake via the mannose receptor, which is primarily expressed on dendritic cells (Sallusto, F. et al., 1995, J. Exp. Med., 182, 389).
  • the antigen is particularly preferably used as an immune complex bound to an antibody or to an antibody fragment, since the amount of antigen can be reduced in this way by a factor of up to 10 or the maximum activation can be increased.
  • the antibody can be any suitable polyclonal or monoclonal antibody. Complexes of antigen and antibody fragments can also be used.
  • the antibody is preferably a human antibody.
  • human sera which already contain antibodies which are directed against the relevant antigen such as e.g. B. Autoantibody positive sera from patients with autoimmune diseases or sera from people with antibodies to pathogens.
  • the T cell population according to the invention can also be used to produce a therapeutic agent, e.g. B. an agent for gene therapy.
  • the desired T cell population is obtained on a preparative scale and can then be subjected to the treatment steps customary in the context of gene therapy methods in vitro.
  • the invention relates to a test kit for a method for detecting the antigen-specific reactivity of T cells, comprising a reagent for removing naive T cells, a reagent for removing a subpopulation of B, T or / and NK cells that cause non-specific activation and / or proliferation, and reagents for carrying out an assay for the detection of antigen-specific T cells.
  • the test kit preferably contains antibodies against CD45RA or / and L-selectin (CD62L) as a reagent for removing naive T cells.
  • CD45RA or / and L-selectin CD62L
  • the test kit preferably contains antibodies against CD39 as a reagent.
  • the test kit contains the corresponding autoantigens, tumor antigens and / or pathogens or fragments thereof as reagents for a T cell assay.
  • the reagents are in a suitable form, for example as a solution, suspension or lyophilisate, and they can be reconstituted with suitable buffers or solutions.
  • the test kit particularly preferably contains the human autoantigen GAD or / and fragments thereof. Most preferably, the GAD antigen is complexed with an antibody.
  • the antibody is preferably a human antibody against GAD.
  • suitable antibodies are the MICA3 and / or MICA4 antibodies (Richter, W. et al., 1992, PNAS 89, 8467).
  • the invention relates to a method for the detection of antigen-specific T cells, in particular autoantigen-specific human T cells, wherein a cell population to be tested is brought into contact with an immune complex which binds the relevant antigen to an antibody directed against it, in particular to a human antibody, and then the antigen-specific reactivity of the T cell population is determined.
  • Figure 1 shows the stimulation of the T cell line 40/2 # 38 (ICL 40/2 # 38) by free or by complexed with MICA 4 GAD.
  • Example 1 Obtaining activated or memory T cells by positive enrichment
  • peripheral mononuclear cells PBMNC
  • PBMNC peripheral mononuclear cells
  • the marked cells were marked with magnetizable microbeads and separated from the unmarked cells using a magnet.
  • the non-retained cell fraction negative fraction
  • the immobilized fraction was also collected by washing the column (positive fraction).
  • TT tetanus toxoid
  • the PBMNC obtained by means of Ficoll separation were adjusted to a cell number of approx. 3 ⁇ 10 6 Z / ml in cold (4 ° C.) complete medium.
  • the monoclonal antibodies were added at a concentration of 5-10 ⁇ g / ml. Then was for approx. Incubated for 30 min at 4 ° C.
  • the PBMNC were then washed first in medium (RPMI / 10% human serum), then in PBS / 0.5% RSA.
  • the cell pellet was then resuspended in buffer (max. 10 7 cells in 80 ⁇ l).
  • dialyzed and sterile-filtered microbeads (Miltenyi) were added (20 ⁇ l to 10 7 cells or 80 ⁇ l cell suspension) and incubated for 15 minutes at 6 to 12 ° C. The cells were washed and then resuspended in the same (100 ⁇ l) volume of buffer as before.
  • the separating column was hung in the separator (with negative selection with flow resistor).
  • the separation column was rinsed with 500 ⁇ l buffer and the well-resuspended cell suspension was applied to the column and allowed to run slowly. Then was rinsed with 500 ul buffer. Both fractions were combined to form the negative fraction.
  • the flow resistor was removed and the column was rinsed two to three times with 500 ⁇ l buffer.
  • the column was then removed from the separator, placed on a sterile 15 ml cell culture tube and the labeled cell fraction was pressed out of the column with 1 ml of buffer. This resulted in the positive fraction.
  • the cells were then aspirated through a filter and the built-in radioactivity was determined directly using a beta counter.
  • the evaluation was carried out by determining the stimulation index (SI), which is defined as follows: cpm (counts per minute) in the presence of TT / cpm with medium.
  • SI stimulation index
  • Table 1 shows the results of these tests.
  • 10,000 PBMNCs from the positive fraction (P) or the negative fraction (N) were used together with 100,000 irradiated unseparated feeder cells in the stimulation tests.
  • a positive selection with antibodies directed against CD25, CD71, CD54 or CD80 leads to an increase in the stimulation index compared to non-separated PBMNC.
  • a relative enrichment of the TT-reactive T cells can also be found in the positively selected fraction with the monoclonal antibodies against CD45RO compared to the cell population depleted with these markers.
  • the positive fractions which were obtained by labeling with anti-HLA-DR and anti-HLA-DQ antibodies, are significantly reduced in relation to TT.
  • a surprising result was observed in the separation experiment with anti-CD39 antibodies.
  • Example 1 first shows that the positive selection with anti-CD25 antibodies led to the greatest increase in the stimulation index compared to the unseparated PBMNC. The following experiment was carried out to determine whether this stimulation increase is antigen-specific:
  • PBMNC were labeled with anti-CD25 antibodies.
  • the cells thus loaded with monoclonal antibodies were not further separated, but were used directly in a proliferation assay as described under 1.
  • a control approach with the same number of lines of unmarked PBMNC was used for comparison.
  • Table 2 shows the result of this experiment.
  • Table 2 Modulation of the immune response by anti IL-2 receptor
  • the monoclonal antibody against the IL-2 receptor modulates the in vitro immune response against TT positively.
  • the increase in stimulation after positive selection of the PBMNC with the anti-CD25 antibody is therefore probably not due to an enrichment of the relevant T cell population, but is the result of a costimulation via the antibody directed against the IL-2 receptor.
  • a positive selection could not be selected for an enrichment of the relevant T cell subpopulation, since the antibodies bound to cell receptors either a positive modulation (e.g. by CD25) or a negative modulation (e.g. by HLA-DR or HLA-DQ) of the T cells in the subsequent antigen-specific stimulation.
  • Example 3 Stimulation experiments with PBMNC after removal of naive T cells or removal of the CD39 positive cell population
  • the PBMNC isolated via Ficoll separation were, as described under Example 1, with anti-CD39 antibodies alone or with the antibody mixtures against CD39 / CD62L or CD39 / CD45RA marked.
  • the further treatment of the cells proceeded as described under example 1 for the negative separation.
  • the relevant PBMNC population is thus optimally enriched with the combination of the anti-CD39 / anti-CD45RA antibodies.
  • a possible combination of markers is the anti-CD39 / anti-CD62L antibody pair.
  • Example 4 Cell separation with samples from control persons and patients compared
  • Example 5 Increasing the in vitro stimulation by GAD after immune complex formation with MICA 4
  • GAD-specific T cell lines can be stimulated by PBMNC from partially HLA-identical donors if these PBMNC have been pulsed with 10 ⁇ g / ml GAD.
  • MICA GAD-specific human monoclonal island-cell-specific antibodies
  • the GAD-specific antibody MICA 4 was used for complexation (for the nomenclature and characteristics of the antibodies, see Richter et al., 1992, PNAS, 89, 8467 and published application DE 41 29 849 AI). This recognizes a conformal epitope in the middle of the GAD sequence (amino acids 309- 440).
  • the GAD concentration was varied between 0.1 and 6.3 ⁇ g / ml.
  • MICA 4 was used in eightfold for complexation molar excess added. In previous experiments, this ratio had led to an optimal increase in stimulation
  • Duplicates were prepared from each dilution step, for which 50 ⁇ l free or complexed GAD were combined with 50 ⁇ l PBMNC suspension (2 ⁇ 10 6 Z / ml) in the wells of a 96-well round-bottom plate.
  • the GAD was diluted by a factor of two.
  • a PBMNC concentration of 100,000 cells / well resulted.
  • 50 ⁇ l of T cell suspension (1.6 ⁇ 10 5 Z / ml) were added per well, which resulted in a T cell concentration of 8,000 cells / well.
  • the cells were pulsed with 1 ⁇ Ci [ 3 H] -thymidine per well and, after a further 16 hours of incubation, sucked off through a filter.
  • the built-in radioactivity was determined directly using a beta counter. It is used as a measure of stimulation.
  • PBMNC of a normal person were administered with the following antibodies as described in Example 1 for the negative separation:
  • anti-CD45RA alone
  • anti-CD39 alone
  • anti-CD39 in combination with anti-CD45RA
  • TT tetanus toxoid

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Abstract

L'invention concerne un procédé pour la production d'une population de cellules enrichie en cellules T spécifiques de l'antigène, l'utilisation de cette population de cellules pour le diagnostic et la mise au point d'un agent thérapeutique, ainsi qu'un matériel de diagnostic pour un procédé permettant de détecter la réactivité spécifique de l'antigène d'une population de cellules T.
PCT/EP1998/001479 1997-03-13 1998-03-13 Procede de detection de cellules t specifiques de l'antigene Ceased WO1998040745A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU70351/98A AU7035198A (en) 1997-03-13 1998-03-13 Method of detecting antigen-specific t-cells
EP98916946A EP0972199A1 (fr) 1997-03-13 1998-03-13 Procede de detection de cellules t specifiques de l'antigene
JP53923898A JP2002500627A (ja) 1997-03-13 1998-03-13 単核細胞集団の濃縮後の抗原特異的ｔ細胞の検出方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19710496.7 1997-03-13
DE19710496A DE19710496A1 (de) 1997-03-13 1997-03-13 Verfahren zum Nachweis antigenspezifischer T-Zellen nach Anreicherung von mononukleären Zellpopulationen

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WO1998040745A1 true WO1998040745A1 (fr) 1998-09-17

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PCT/EP1998/001479 Ceased WO1998040745A1 (fr) 1997-03-13 1998-03-13 Procede de detection de cellules t specifiques de l'antigene

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EP (1) EP0972199A1 (fr)
JP (1) JP2002500627A (fr)
AU (1) AU7035198A (fr)
DE (1) DE19710496A1 (fr)
WO (1) WO1998040745A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1218182C (zh) * 1999-05-28 2005-09-07 干细胞技术公司 利用免疫玫瑰花结分离细胞的方法
EP1146119A1 (fr) * 2000-04-12 2001-10-17 Institut National De La Sante Et De La Recherche Medicale (Inserm) Méthodes pour obtenir des lymphocytes-T spécifiques et pour identifier des épitopes inconnus
EP1146120A1 (fr) * 2000-04-12 2001-10-17 Institut National De La Sante Et De La Recherche Medicale (Inserm) Méthodes pour obtenir des lymphocytes-T spécifiques et pour identifier des épitopes inconnus

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992004462A1 (fr) * 1990-09-06 1992-03-19 Immulogic Pharmaceutical Corporation Therapie par cellules t cytotoxiques (ctl) specifiques d'agents pathogenes
WO1996028732A1 (fr) * 1995-03-15 1996-09-19 Miltenyi Biotech, Inc. Isolement de cellules dendritiques hematopoietiques par triage magnetique de cellules a gradient eleve
CA2178984A1 (fr) * 1995-06-16 1996-12-17 Terry Thomas Compositions anticorps pour l'obtention de preparations de cellules hematopoietiques humaines enrichies
WO1997018473A1 (fr) * 1995-11-13 1997-05-22 Coulter Corporation Procede et dispositif d'enrichissement en volume d'une population ou sous-population de cellules
WO1998010284A1 (fr) * 1996-09-06 1998-03-12 Ortho Pharmaceutical Corporation Purification de cellules t specifiques de l'antigene

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992004462A1 (fr) * 1990-09-06 1992-03-19 Immulogic Pharmaceutical Corporation Therapie par cellules t cytotoxiques (ctl) specifiques d'agents pathogenes
WO1996028732A1 (fr) * 1995-03-15 1996-09-19 Miltenyi Biotech, Inc. Isolement de cellules dendritiques hematopoietiques par triage magnetique de cellules a gradient eleve
CA2178984A1 (fr) * 1995-06-16 1996-12-17 Terry Thomas Compositions anticorps pour l'obtention de preparations de cellules hematopoietiques humaines enrichies
WO1997018473A1 (fr) * 1995-11-13 1997-05-22 Coulter Corporation Procede et dispositif d'enrichissement en volume d'une population ou sous-population de cellules
WO1998010284A1 (fr) * 1996-09-06 1998-03-12 Ortho Pharmaceutical Corporation Purification de cellules t specifiques de l'antigene

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AU7035198A (en) 1998-09-29
JP2002500627A (ja) 2002-01-08
DE19710496A1 (de) 1998-09-17
EP0972199A1 (fr) 2000-01-19

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