WO1998040745A1 - Method of detecting antigen-specific t-cells - Google Patents

Abstract

The invention concerns a method of producing a cell population enriched with antigen-specific T-cells, the use of this cell population in diagnosis and for producing a therapeutic agent, and a test kit for a method of detecting the antigen-specific reactivity of a T-cell population.

Description

Method for the detection of antigen-specific T cells

description

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.

A distinction is made between the recognition system of the humoral immune response, which consists of soluble proteins, the antibodies that are produced in plasma cells, and the cellular immune response, in which cells with foreign structures on their surfaces are recognized by T-Lyτnphozyten and from Organism to be removed. In addition, the T-Lyτnphozyten stimulate the humoral immune response by interacting with the B cells, the precursors of the plasma cells.

In these processes, 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.

An example of a disease in which an autoimmune defect is suspected to be the cause is insulin-dependent diabetes mellitus (IDDM). This leads to T-lymphocyte-mediated destruction of the insulin-producing beta cells of the pancreas. Autoimmunity to glutamate decarboxylase (GAD) is believed to mediate the T cell response for other beta cell antigens. 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.

These findings indicate that an anti-GAD immune response plays a critical role in the clinical picture of the IDDM. 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. In addition, autostimulation, which is referred to as the so-called "autologous mixed ly phocyte reaction", can often lead to a high background signal, which has a decisive effect on the sensitivity and reliability of the detection.

It was therefore an object of the present invention to at least partially overcome the disadvantages of the prior art eliminate and in particular increase the sensitivity and reliability of T cell proliferation tests.

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.

Surprisingly, it was found that depletion of naive T cells and a mononuclear subpopulation of Lyτnphozyten, which causes an unspecific activation and / or proliferation of T cells and in particular a subpopulation of B-, T- and NK- (natural killer ) Cells, not only increases the frequency of the antigen-specific T cells, but also the autostimulation reaction can be at least largely prevented.

In contrast, it was found that it was not possible to obtain antigen-specific T cells by means of targeted “positive” enrichment steps, since this always results in a very considerable increase in auto-stimulation. It is surprising and new that only the depletion of naive T cells and a subpopulation of B, T or / and NK cells according to the method of the invention leads to the desired antigen-specific cell population.

The removal of unwanted cells through the combination of depletion steps according to the invention using binding molecules, eg antibodies, which can bind specifically to characteristic surface structures of the unwanted cells has proven to be particularly favorable. It is of great advantage that 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.

It was particularly surprising that the combination according to the invention of a depletion of naive T cells and a subpopulation of B, T or / and NK cells led to a high degree of accumulation of antigen-specific T cells without at the same time a disadvantageous autostimulation occurred.

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. In another aspect, the invention relates to an enriched cell population that is obtainable by the method outlined above. In a preferred embodiment, 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.

In a further aspect, 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). Most preferred is human GAD or a fragment thereof, e.g. a partial peptide as described in German Patent Application No. P 4401 629.8.

In a further aspect, 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. In yet another aspect, the invention relates to the use of the cell population in a method for controlling the reduction in antigen-specific T cells. In general, such a method can be used for therapy monitoring. Especially after 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

(dendritic cells, macrophages). 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.

As an alternative to the use of prefabricated immune complexes, 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. For this purpose, 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.

In a further aspect, 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. To remove the subpopulation of B, T or / and NK cells that cause non-specific activation and / or proliferation, 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. Examples of suitable antibodies are the MICA3 and / or MICA4 antibodies (Richter, W. et al., 1992, PNAS 89, 8467). In yet another aspect, 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.

The following examples and figure serve to explain the invention. 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.

Examples

Example 1: Obtaining activated or memory T cells by positive enrichment

1.1 Introduction

To enrich activated T cells or memory T cells, peripheral mononuclear cells (PBMNC) were labeled with various monoclonal antibodies which were directed against surface antigens which are expressed on activated lymphocytes or memory T cells. In a second step, the marked cells were marked with magnetizable microbeads and separated from the unmarked cells using a magnet. The non-retained cell fraction (negative fraction) was collected. After removing the magnet, the immobilized fraction was also collected by washing the column (positive fraction). In order to measure the effect of cell accumulation, a stimulation test with tetanus toxoid (TT) as an antigen was carried out. .2 Carrying out the experiment

The following antibodies were evaluated

Die mittels Ficollseparation gewonnenen PBMNC wurden in kaltem (4°C) Vollmedium auf eine Zellzahl von ca. 3 x 10⁶ Z/ml eingestellt. Es erfolgte die Zugabe der monoklonalen Antiköper in einer Konzentration von 5 - 10 μg/ml . Anschließend wurde für ca . 30 Min. bei 4 °C inkubiert. Die PBMNC wurden dann zuerst in Medium (RPMI/10 % Humanserum), dann in PBS/0,5 % RSA gewaschen. Das Zellpellet wurde dann in Puffer resuspendiert (max. 10⁷ Zellen in 80 μl) . Anschließend wurden dialysierte und sterilfiltrierte Microbeads (Miltenyi) zugegeben (20 μl auf 10⁷ Zellen bzw. 80 μl Zellsuspension) und 15 Minuten bei 6 bis 12 °C inkubiert. Die Zellen wurden gewaschen und anschließend in gleichem (100 μl) Volumen Puffer wie zuvor resuspendiert.

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 μg / 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 μl). Then dialyzed and sterile-filtered microbeads (Miltenyi) were added (20 μl to 10 ⁷ cells or 80 μl cell suspension) and incubated for 15 minutes at 6 to 12 ° C. The cells were washed and then resuspended in the same (100 μl) volume of buffer as before.

Now the separating column was hung in the separator (with negative selection with flow resistor). The separation column was rinsed with 500 μl 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.

Now the flow resistor was removed and the column was rinsed two to three times with 500 μl 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.

Positive and negative fractions were washed once with full medium, resuspended in full medium and then distributed in 96-well round-bottom plates in different cell concentrations. If very few (below 20,000 PBMNC per well) were used, irradiated so-called feeder cells were added. These were PBMNC from the same blood donor, which were irradiated with 4000 rad to prevent the proliferation of these feeder cells. Mostly triplicates without antigen (only with medium) and with tetanus toxoid (TT) were used as stimulation antigen. It was then incubated for 5 days at 37 ° C./7% CO ₂ . beer. Then there was a pulse with ³ H-Thyτnidin for 16 hours. 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.

Table 1 shows the results of these tests. In this series of experiments, 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.

Table 1: Results with positive markers for the enrichment of activated or memory T cells

Fortsetzung Tabelle 1

Continuation of table 1

The following conclusions can be drawn from this experiment: 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. Here there was a higher stimulation index with the negative fraction compared to the positive fraction. This was mainly due to a significantly reduced spontaneous proliferation with the media control.

Example 2: Investigation of the modulation of the immune response by the presence of monoclonal antibody against IL-2 receptor (CD 25)

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:

As described in Example 1, 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

I.

It can be seen that 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. As a result, 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

An attempt has now been made to enrich the desired antigen-specific T cell population by negative selection. For this purpose, naive and immature T cells were labeled using the markers CD45RA (Morimoto C. et al., 1985. J. Immunol. 184, 1508) and CD62L (Reinherz, EL et al., 1982. J. Immunol. 128, 4639) away. Furthermore, the marker CD39, already identified in Example 1, was used for the negative selection, which recognizes a mononuclear subpopulation of B, T and NK cells, which causes unspecific activation or proliferation.

For this experiment, PBMNC from IDDM patients were used for the first time. TT was still tested as a model antigen, but also the IDDM-relevant antigen GAD.

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.

Table 3 shows the result of this series of tests.

Removal of the CD39 subpopulation alone caused a sharp decrease in proliferation with the media control in all cases, but also in the presence of the antigen TT in patients 27 and 31. No proliferation was observed with GAD, neither with unseparated PBL nor with cells depleted with anti-CD39 antibodies. If depletion was additionally carried out with anti-CD62L antibodies, a significant increase in the stimulation index compared to the two antigens GAD and TT was observed in patients 27 and 31. The combination of anti-CD39 / anti-CD45RA antibodies proved to be the ideal combination for increasing the stimulation index, in particular against the autoantigen GAD. A highly significant increase in in vitro stimulation with GAD and with TT was observed.

The relevant PBMNC population is thus optimally enriched with the combination of the anti-CD39 / anti-CD45RA antibodies. However, 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

In a further series of experiments, the question was clarified whether the depletion of the CD39 and CD45RA subpopulation did not have the undesirable effect that an increase in the in vitro proliferation against the diabetes-relevant autoantigen GAD also occurs in normal people. For this purpose a series of PBMNC was compared. Since the reactivity of T cells to certain antigens strongly depends on the HLA type of the PBMNC donor, only those control persons were used which also expressed the HLA class II susceptibility antigens characteristic of type I diabetics. The individual results are shown in Table 4.

The following results from this series of experiments:

1. After depletion of CD39 and CD45RA subpopulations, the stimulation index increased significantly compared to TT in the vast majority of control persons. On the other hand, the vast majority of SI control persons decrease compared to the autoantigen GAD.

2. In contrast, a clear increase in SI versus GAD was observed in the IDDM patients examined after cell depletion.

3. Lowering the SI compared to GAD in the control group and simultaneously increasing the SI in the patient results in a highly significant difference in the GAD reactivity of these two comparison groups. This method therefore offers itself as an improved detection system for diagnosing the T-cell mediated reactivity against the autoantigen GAD.

Table 3 Comparison of the stimulation of unseparated and separated PBMNC from IDDM patients with GAD and TT as antigen

Table 4 Cell separation with PBMNC of control persons (KP) and patients (patients) in comparison

Example 5: Increasing the in vitro stimulation by GAD after immune complex formation with MICA 4

5.1 Introduction

nnen durch PBMNC von partiell HLA- identischen Spendern stimuliert werden, wenn diese PBMNC mit 10 μg/ml GAD gepulst worden sind. Durch Komplexierung der GAD durch GAD-spezifische humane monoklonale inselzellspezi- fische Antikörper (MICA) sollte die Immunogenität der GAD verstärkt werden. Es wurde erwartet, daß komplexierte GAD bereits in Konzentrationen unter 10 μg/ml stimulatorisch

wirkt .

Es wurde die Proliferation der T-Zell-Linie 40/2#38 (TCL 40/2#38) in Anwesendheit verschiedener Konzentrationen an freier bzw. komplexierter GAD miteinander verglichen. Zur

Komplexierung wurde der GAD-spezifische Antikörper MICA 4 verwendet (zur Nomenklatur und Charakteristik der Antikörper siehe Richter et al . , 1992, PNAS, 89, 8467 sowie Offenle- gungsschrift DE 41 29 849 AI) . Dieser erkennt ein Konforma- tionsepitop in der Mitte der GAD-Sequenz (Aminosäuren 309-

440) . Die GAD-Konzentration wurde zwischen 0,1 und 6,3 μg/ml variiert. Zur Komplexierung wurde jeweils MICA 4 in achtfachem

molaren Überschuß zugegeben. Dieses Verhältnis hatte in Vorversuchen zu einer optimalen Steigerung der Stimulation ge GAD-specific T cell lines

can be stimulated by PBMNC from partially HLA-identical donors if these PBMNC have been pulsed with 10 μg / ml GAD. By complexing the GAD with GAD-specific human monoclonal island-cell-specific antibodies (MICA), the immunogenicity of the GAD should be increased. It was expected that complexed GAD was stimulatory even at concentrations below 10 μg / ml

works.

The proliferation of the T cell line 40/2 # 38 (TCL 40/2 # 38) in the presence of different concentrations of free or complexed GAD was compared. to

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 μg / 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

also 12.5 μg / ml). The two batches were incubated for 2 hours at 37 ° C., 7% CO ₂ and then serially diluted.

Duplicates were prepared from each dilution step, for which 50 μl free or complexed GAD were combined with 50 μl 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. After a three-hour antigen pulse at 37 ° C, 7% CO ₂ , the cells were exposed to 40 Gy radiation. Finally, 50 μl 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.

After 48 hours of incubation at 37 ° C., 7% CO _{2, the} cells were pulsed with 1 μCi [ 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.

5.3 Result

The titration curves determined for free or complexed GAD are shown in Figure 1. While free GAD in a concentration of 0.4 μg / ml does not have a stimulating effect, considerable stimulation can already be observed in the presence of 0.4 μg / ml complexed GAD. Even at the higher GAD concentrations tested, the complexed GAD causes a significantly stronger stimulation than the free GAD.

In order to stimulate the strength, which is achieved in the presence of 6.3 or 3.1 μg / ml of free GAD, a concentration of complexed GAD that is ten times lower is sufficient. By complexing with MICA 4, the GAD concentration required for stimulation is reduced. At a given concentration, complexed GAD stimulates more than free GAD.

Example 6 Comparison of the simulation ability of PBMNC after depletion with anti-CD39 or anti-CD45RA antibodies alone and in combination

PBMNC of a normal person were administered with the following antibodies as described in Example 1 for the negative separation:

a. anti-CD45RA alone b. anti-CD39 alone c. anti-CD39 in combination with anti-CD45RA

The depleted cell populations were then stimulated with tetanus toxoid (TT) as described in Example 1 and the proliferation was measured. The results of this series of tests are summarized in Table 5.

Table 5

The results shown in Table 5 clearly show that depletion using anti-CD39 or anti-CD45RA antibodies alone did not improve the stimulation index (SI). First a combination of anti CD39 and anti-CD45RA antibodies led to an optimized enrichment of the relevant mononuclear cell population and an increase in the SI value by 2.5 to 5 times.

Claims

claims 1. A process for producing a cell population enriched in antigen-specific T cells, characterized in that a) naive T cells and b) a subpopulation of B-, T- and / or NK- from a mononuclear population of cells by specific depletion steps. Cells that cause non-specific activation and / or proliferation are removed. 2. The method according to claim 1, characterized in that one uses antibodies to CD45RA and / or CD62L to remove the naive T cells. 3. The method according to any one of claims 1 to 3, characterized in that one uses antibodies against CD39 to remove the subpopulation of B, T or / and NK cells. 4. The method according to any one of claims 1 to 3, characterized in that a cell population is produced which is enriched in memory T cells and in vivo pre-activated T cells. 5. Enriched cell population obtainable by the method according to one of claims 1 to 4. 6. Cell population according to claim 5, characterized in that it contains a content of ≥ 80% of memory T cells and in vivo preactivated T cells. 7. Cell population according to claim 5 or 6, characterized in that it has a content. Contains ≤ 10% of naive T cells. 5 8. Use of the cell population according to one of claims 5 to 7 in diagnostics. 9. Use according to claim 8 in a method for detecting the T cell reaction against a specific antigen. 0 10. Use according to claim 9, characterized in that the specific antigen is an autoantigen, tumor antigen and / or pathogen. 5 11. Use according to claim 9 for the detection of diabetes, characterized in that the antigen is an autoantigen which comprises human GAD or fragments thereof. 20th 12. Use of the cell population according to one of claims 5 to 7 in a method for monitoring a vaccination success. 25 13. Use of the cell population according to one of claims 5 to 7 in a method for controlling the reduction of antigen-specific T cells. 14. Use according to claim 12, 30 characterized in that the antigen is a tetanus toxoid. 15. Use according to one of claims 9 to 14, characterized in 35 that the antigen is used as an immune complex bound to an antibody. 16. Use according to claim 15, characterized in that the antibody is a human antibody. 17. Use of the cell population according to one of claims 5 to 7 for the production of a therapeutic agent. 18. A test kit for a method for detecting the antigen-specific reactivity of T cells, comprising a) a reagent for removing naive T cells, b) a reagent for removing a subpopulation of B, T or / and NK cells, which causes non-specific activation or / and proliferation, and c) reagents for carrying out an assay for the detection of antigen-specific T cells. 19. Test kit according to claim 18, characterized in that the reagent for removing naive T cells comprises antibodies against CD45RA and / or CD62L. 20. Test kit according to claim 18 or 19, characterized in that the reagent for removing a subpopulation of B, T or / and NK cells comprises antibodies against CD39. 21. Test kit according to one of claims 18 to 20, characterized in that it comprises autoantigens, tumor antigens, and / or pathogens. 22. Test kit according to one of claims 18 to 21, characterized in that it comprises human GAD and / or fragments thereof. 23. Test kit according to claim 22, characterized in that the GAD antigen is present in complex with an antibody. 24. Test kit according to claim 23, characterized in that the antibody is a human antibody against GAD,