WO2004009801A1 - Non-antigen presenting cells for selecting t cells - Google Patents

Abstract

The invention relates to the use of non-antigen presenting cells, which are transfected using CIITA, (CII transactivator), for the ex-vivo selection of T lymphocytes.

Description

 Non-anti-presenting cells for selection of T cells

The present invention relates to the use of non-antigen-presenting cells for the selection of T cells, methods for producing such cells, and preparation and medicament containing such cells, and use of the cells.

CIITA is a transcription factor that activates the MHC II gene locus. By introducing CIITA into any non-APC (antigen presenting cell) that may not belong to the blood system, CIITA and then MHC II, as well as other associated genes of the MHC II locus, are transcribed from this cell. The cells then become artificial APC. You can turn off immune responses because they don't produce costimulatory molecules.

Major histocompatibility complexes (MHC class II) are heterodimeric glycoproteins that are found in the plasma membrane. Their expression on the surface of antigen-presenting cells is essential for the recognition of foreign antigens via the T cell receptor. T cell activation and antigen presentation depend on the amount of MHC-II on individual cells. The regulation and expression of MHC-II genes is important for the control of immune responses.

In humans, the genes coding for the α and β chains of the HLA-DP, HLA-DQ and HLA-DR - class II molecules are grouped on chromosome 6 in the D region of the MHC locus. The genes are subject to a sometimes complex regulatory control. Their expression is generally coordinated and mainly restricted to cells of the immune system, such as B lymphocytes, activated T lymphocytes, macrophages (MQ), dendritic cells (DC) and certain specialized cells such as Kupffer cells and Langerhans cells (Radka, SF, Charron, DJ.6k Brodsky, FM Class II molecules of the major histocompatibility complex considered as differentiation markers.Hum Immunol 16, 390-400. (1986)). In certain class II negative cells, expression can be induced by stimulation with lymphokines such as interferon-γ or interleukin 4. Class II molecules are also expressed transiently during the developmental pathway of many haematopoietic cell types (Radka, SF, Charron, DJ. & Brodsky, FM Class II molecules of the major histocompatibility complex considered as differentiation markers. Hum Immunol 16, 390-400. ( 1986)).

MHC II genes represent a particularly complex type of regulated gene expression. This regulation affects not only the amount of expression of class II molecules but also the very restricted cell type specificity, since most cells in the body are normally MHC II negative. The complexity of regulation includes two different types of control: constitutive expression in cells such as B cells and inducible expression in certain cell types such as monocytes and fibroblasts. Finally, the family of genes includes the α and β chains from three different HLA class II isotypes. A number of protein factors can bind to the promoter of the MHC II genes in vitro and in vivo. CIITA is such a factor. CIITA not only regulates the constitutive expression of MHC II genes in cells such as B lymphocytes, but also controls the inducible expression of the same genes in other cell types. It is therefore a factor that is involved in the general control of the MHC II genes. CIITA is a protein with a sequence of 1130 amino acids in length, whose mRNA is only produced in small quantities. When CIITA cDNA is transfected into MHC class II negative cells, these cells begin producing MHC II molecules (Bradley, MB et al. Correction of defective expression in MHC class II deficiency (bare lymphocyte syndrome) cells by retroviral transduction of CIITA. J Immunol 159, 1086-1095. (1997)), (Mori-Aoki, A. et al. Class II transactivator suppresses transcription of thyroid-specific genes. Biochem Biophys Res Commun 278, 58-62. ( 2000)), (Armstrong, TD, Clements, VK, Martin, BK, Ting, JP & Ostrand-Rosenberg, S. Major histocompatibility complex class II- transfected tumor cells present endogenous antigen and are potent inducers of tumor-specific immunity. Proc Natl Acad Sei USA 94, 6886-6891. (1997)).

The human gene for the MHC class II transactivator is located on chromosome 16 (16P13). The mouse gene for CIITA is located on mouse chromosome 16. The overall intron and exon structure of the mouse MHC II TA gene was determined. It consists of 19 exons, which require a total of 42 kb of the genomic DNA (Reith, W. & Mach, B. The bare lymphocyte syndrome and the regulation of MHC expression. Annu Rev Immunol 19, 331-373 (2001)). The transgenic expression of CIITA can reactivate the expression of all three MHC II isotypes. The same applies to the invariant chain, HLA-DMA and HLA-DMB. The MHC II promoter contains the conserved S, X, X2 and Y sequences. The key transcription factors that control promoter activity are RFX, X2BP and NF-Y. They cooperatively bind to the X, X2 and Y sequences of the promoter and form a very stable nucleoprotein complex. This is called the MHC II enhanceosome. The proteins that bind to the S-Box have not yet been well characterized. CIITA is a non-DNA-binding coactivator that is recruited to the promoter via protein-protein interactions with the DNA-binding components of the enhanceosome (Reith, W. & Mach, B. The bare lymphocyte syndrome and the regulation of MHC expression. Annu Rev Immunol 19, 331-373 (2001)).

The X-Box of the promoter is bound by RFX, the X2-Box by X2BP and the Y-Box by NF-Y. All proteins of the enhanceosome, or at least those that bind to the promoter boxes mentioned, are required for CIITA recruitment. CIITA activates transcription via an N-terminal transcription activation domain (AD). CIITA interacts directly with the RFX-ANK and RFX-5, subunits from RFX, as well as the B and C subunits from NF-Y and with CREB. CIITA is a nuclear protein and contains three regions that appear important for transport to the cell nucleus. The first sits at the C-terminus of CIITA and contains five amino acids that resemble a nuclear localization signal (NLS). If these amino acids are removed, CIITA can no longer be imported directly into the cell nucleus. The second region is a GTP-binding motif. Binding of GTP also appears to be necessary for the transport of CIITA into the cell nucleus. The third region, called LRR, was also recognized by a mutation analysis as important for the direct transport into the cell nucleus. If CIITA does not get into the cell nucleus, the MHC II locus is not read either (Reith, W. & Mach, B. The bare lymphocyte syndrome and the regulation of MHC expression. Annu Rev Immunol 19, 331-373 (2001)) ,

In contrast to the ubiquitous DNA binding factors (also RFX) that form the MHC II enhososome, the expression of CIITA is very strictly regulated. The expression of CIITA dictates whether and to what extent MHC II genes are expressed. The MHC II TA gene is therefore the main regulator of MHC II expression and therefore also has an essential immunomodulatory role. Most cell types do not form CIITA and are consequently MHC II negative. Expression of CIITA and thus of MHC II can be activated by stimulating such cells with interferon-γ (gamma). This has been demonstrated on many established cell lines. These include: fibroblasts, melanoma cells, macrophages, but also some primary cell types, such as mouse embryonic fibroblasts, peritoneal macrophages, microglia and astrocytes. Transfection of MHC II negative cells with CIITA vectors is usually sufficient to induce MHC II expression.

Regulation of CIITA expression

A large (> 12 kb) and complex regulatory region contains various independent promoters that control the transcription of CIITA. 4 promoters pl to pIV were identified in the human genes. Three of these p1, pH, pIII and pIV are also highly conserved in the mouse gene. The use of these promoters leads to the synthesis of distinct CIITA mRNAs (types I, III and IV). These contain alternative first exons that are spliced to a conserved second exon. The analysis of the different MHC II-TA promoters has shown that their different activity is crucial for the complex expression pattern of MHC II genes is. Each of them has a specific physiological relevance. In both humans and mice, pl is very specific for dendritic cells, but pl is not the only promoter active in DCs. Significant transcriptions can also be detected by the plll promoter. The type I transcripts contain an alternative first exon which contains a translation initiation codon which codes for a specific 94 amino acid N-terminal extension of CIITA. pIII is mainly used in B cells. Type III-CIITA mRNA contains an alternative first exon that contains a translation initiation codon and encodes a specific 17 amino acid N-terminal extension. pIV is activated by interferon gamma. Unlike type I and type III, type IV mRNA does not contain a translation initiation codon and therefore does not code for an N-terminal extension. The translation of CIITA is initiated by the first AUG of the second exon (Reith, W. & Mach, B. The bare lymphocyte syndrome and the regulation of MHC expression. Annu Rev Immunol 19, 331-373 (2001)).

A problem on which the invention is based is to provide therapeutically usable products for the reduction of allogeneic immune reactions in bone marrow transplants.

Suppression of graft versus host reactions of bone marrow transplants by ex vivo selection of donor T cells, maintenance of the graft versus leukemia reactions (also mediated by donor T cells) is to be achieved and the generation of donor suppressor T - Cells for the alloantigens to be able to generate a permanent anti-GvHD effect.

The problem is solved by the MHC II-producing non-antigen-producing cell (APC) according to the invention.

The invention relates to the use of non-antigen-presenting cells which are stimulated to produce MHC II for the ex vivo selection of T lymphocytes. This can be achieved by interferon gamma or transfection with CIITA (CII transactivator). The induction can be brought about by interferon gamma or the transactivator CIITA, which in turn can be achieved by introducing the gene for CIITA into the target cell. The MHC II-producing cell according to the invention has the advantage that it cannot stimulate potent immune reactions via CD4 + T cells. The switching off of T cells is improved by small amounts of CD80 (B7-1) and CD86 (B7-2), so that these molecules can also be produced in the MHC II-producing cell according to the invention. This can be achieved by transfection with CD80 and / or CD86. B7-1 / B7-2 have a 20-50-fold higher affinity for CTLA-4 than for CD28. B7 in small amounts therefore preferentially binds to CTLA-4. In contrast to CD28, CTLA-4 has a tolerogenic effect on T cells. This effect is used.

By transfecting the MHCII transactivator (CIITA), fibroblasts that are not professional APC express MHCII.

In the absence of sufficient costimulatory B7 signals, immune responses will be shut off.

Fig. 1: Schematic representation of the planned treatment process

Fig. 2a: The human MHC-II gene locus

Fig. 2b: The murine MHC-II gene locus

Fig. 3: Transcriptional regulation of the murine MHC-II. Cis-acting elements W, X and Y boxes with the transcription factors and the CIITA. All factors together cause the transcription of the MHC-II genes.

Fig. 4: Cloned human CIITA in the pcD N A3.1 vector (see also the accompanying diagrams).

FIG. 5 shows FACS analysis that the primary human fibroblasts express MHCII after infection with hCIITA. The gene was transferred by lentiviral gene transfer. FIG. 6 shows FACS analysis that the HEK293 cells express MHCII after transfection with hCIITA. It is a selected transgenic cell line.

Figure 7 relates to T cell proliferation on artificial APC

Figure 8 shows a T cell proliferation assay with artificial APC and control cells

FIG. 9 shows a scheme for carrying out an incubation of non-APC with allogeneic cells

Fig. 10 shows a plate layout

11 and 12 show different CD4 T cell proliferation of T cells

Fig. 13: Cloned murine CIITA in the pcDNA3.1 vector

14 shows FACS analysis that the L929 cells express MHCII after transfection with mCIITA. It is a selected transgenic line.

Fig. 15: Scheme of the experiments

Fig. 16: Proliferated CD4 / CD4CD25 (A) and CD8 T cells (B) after first contact with L929. The individual L929 are shown. T cells without L929 (T cells only) served as controls for background proliferation. The evaluation was carried out at the FACS.

Fig. 17 shows a scheme for experiments for the incubation of non-APC with allogeneic T cells.

Fig. 18: Proliferated CD4 and CD8 T cells on second contact with allogeneic L929.

The use according to the invention can lead, in particular, to the production of a medicament for suppressing immune reactions during transplantations, in particular bone marrow transplantations. In particular, the non-AP cell can be selected from the group consisting of fibroblasts, epithelial cells, muscle cells, keratinocytes, hepatocytes, parenchymal cells, chondrocytes and / or melanocytes.

Interferon gamma and / or CIITA can cause any cell to produce MHC II. Those that are easy to obtain are the best target cells for the desired treatment.

The invention also relates to a method for producing a composition which contains T cells for a recipient, comprising the following steps:

Removal of T cells from a donor,

- bringing these T cells into contact with non-AP cells incubated with interferon gamma and / or with CIITA and possibly with B7-1 and / or B7-2, the non-AP cells originating from the recipient ,

The T cells preferably originate from a bone marrow donation or a bone marrow donor, in particular an allogeneic bone marrow donation.

The ex vivo selection offers the advantage of rendering alloreactive T cells harmless before contact with the recipient. In this way, only "useful" T cells are infused into the recipient, so that there is no fear of GvHD reactions. A clinically relevant alternative is to remove T cells before infusing the haematopoietic stem cells. However, this method has the disadvantage that the patients usually develop blood cancer again, since leukaemic cells that have survived radiation and chemotherapy proliferate again. The absence of allogeneic T cells also prevents the useful GvL reactions.

Typically, the non-AP cell is selected from the group consisting of fibroblasts, epithelial cells, muscle cells, keratinocytes, hepatocytes, parenchymal cells, chondrocytes, and / or melanocytes. It may be advantageous to separate the haematopoietic stem cells from T cells before using the non-AP cell to be used according to the invention.

According to the invention, the stem cells can be separated from T cells.

Sorting out T lymphocytes from bone marrow donations is clinical practice because of the risk of GvHD. The cultivation and storage of T lymphocytes is also routine.

The composition with T cells obtainable by the method according to the invention is also the subject of the invention.

Only the co-cultivation of non-APC and T cells can cause a selective shutdown of the allogeneic reactions.

A medicament can be produced from the composition according to the invention. This medicament is also the subject of the present invention.

The drug is a selected T cell with reduced GvHD potential. These T cells with reduced risk potential can be infused into a recipient. In a leukemia patient, it also pays off that the GvL potential of the allogeneic T cells is still present.

The modified non-antigen presenting cells produce MHC II by incubation with interferon gamma (INFγ) or transfection with CIITA and they may also be transfected with B7-1 and / or B7-2. Then they are incubated with allogeneic bone marrow transplants. These grafts contain lymphocytes. In a preferred embodiment, the cells not presenting antigen can also be incubated alone with the lymphocytes or T cells (T lymphocytes). Among the lymphocytes, the T cells are of particular interest because they trigger important reactions for the recipient / patient. One of the reactions is wanted and useful, the other is harmful and often leads to the death of the recipient. The useful response is the anti-leukemic GvL, the most harmful to the GvHD. Incubation of the cells to be used according to the invention (modified non-AP) with the transplant and here in particular with the T cells occurring therein switches off the GvHD reaction and only maintains the GvL reaction. For this purpose, the non-antigen presenting cells are selected or treated in such a way that they come into contact with T cells via MHC II, but cannot activate them, but still present the recipient's alloantigens. In addition, such cells that do not belong to the blood system do not present tumor or cancer antigens because they are not affected by cancer. Therefore, they do not stop immune responses to these antigens. If an alloreactive T cell encounters such a cell that does not present an antigen, it is permanently anergic (stopped), or sent to programmed cell death (apoptosis), or differentiated into a regulatory T cell. A regulatory T cell is understood to be a T cell that suppresses immune responses to specific antigens, here alloantigens.

Exemplary embodiments of the invention are described below.

According to the invention, a non-antigen-presenting cell that originates from the recipient of the transplant and is characterized in that this cell is provided with CIITA or is caused to produce CIITA is used. CIITA induces the synthesis of MHC II molecules, which in turn can present CD4 + T cell antigens. Without the simultaneous contact with B7 or large B7 and at the same time large amounts of MHC II on the functionally modified cell according to the invention, the T cell is not activated. The T cells are switched off in this way and / or they are driven to programmed cell death (apoptosis). They can differentiate into regulatory T cells if the T cells recognize a presented antigen with their T cell receptor. The antigens recognized by this method are predominantly transplantation antigens [main histocompatibility complexes (HLA, MHC I, MHC II), rhesus factor, Minor histocompatibility antigens], since these differ between donor and recipient of the transplant.

B.

According to the invention, the cell is transfected with at least one gene for a CIITA. By expressing CIITA, the cells can be made to produce their MHC II molecules.

C.

The cell to be used according to the invention can additionally or alternatively be transfected with CIITA as the protein. With CIITA protein, the cells can be made to produce their MHC II molecules.

D.

The cell to be used according to the invention can also be produced by incubation with INFγ.

E.

The cell according to A and / or D to be used according to the invention can additionally be transfected with B7-1 and / or B7-2 in order to force the negative selection of the T cells.

F.

The cell according to the invention is incubated in vitro with allogeneic stem cells (eg including lymphocytes) or with allogeneic T cells or with allogeneic lymphocytes from a foreign stem cell transplant donor (eg bone marrow). The named cells (not AP cells), which originate from the recipient of the transplant, cannot contain cancer cells, even in cancer patients with leukemia, since they do not come from the haematopoetic line. In one embodiment of the invention, they can be treated before incubation with the transplant in such a way that these cells will not proliferate. This is preferably achieved by irradiating the cells. The non-alloreactive but allogeneic T cells positively selected by one of the aforementioned embodiments or any sequence of the same are infused to the patient together with the allogeneic stem cells (eg bone marrow). In this way, one obtains a graft-versus-leukemia effect (GvL), but none, or a significantly reduced graft-versus-host disease effect (GvHD).

The methods that can be used make particular use of genetic engineering interventions on the patient's own cells. The interventions are performed using suitable probes and produce a protein that induces MHC II expression, preferably CIITA.

Transfection of the non-antigen presenting cells may also result in the production of B7-1 and / or B7-2 CIITA.

This is achieved in particular by nucleic acids which code for CIITA, B7-1 and B7-2.

The nucleic acids can be DNA, RNA, oligonucleotides, polynucleotides.

The DNA preferably contains regulatory elements such as enhancers, promoters, polyA-coding 3 'ends for the transcription of the DNA into RNA. The RNA in turn should contain regulatory elements for translating the RNA into protein.

The cells mentioned can be ex vivo in a manner known per se by treatment with viruses, viral vectors, bacterial vectors, plasmids which are suitable for introducing molecules into eukaryotic cells by viral gene transfer, electroporation techniques, iontophoresis, ballistic methods and / or other techniques, be transfected.

Said cell can be treated with viruses, viral vectors, bacterial vectors, plasmids by viral gene transfer, electroporation techniques, iontophoresis, ballistic methods and / or other techniques for introducing molecules into a cell with increased production of CIITA and / or B7-1 and / or B7-2 are transfected, whereby T cells which bind to the cell according to the invention via allogeneic antigens, which are presented, for example, on MHC molecules, are switched off.

CIITA, B7-1, B7-2 and / or constituents (proteins, peptides, peptidomimetics) and / or combinations of these molecules can be used as molecules. These molecules hinder stimulation and / or co-stimulation of T cells which take place in the presence of an alloantigen presentation and are brought into contact with the cell according to the invention.

The molecules (CIITA, B7-1, B7-2) can be introduced by vehicles such as liposomes, hydrogels, cyclodextrins, nanocapsules, nanoparticles, bio-adhesive microspheres and / or by electroporation techniques, iontophoresis, ballistic methods and / or other techniques for introducing Molecules are transferred into the cell according to the invention.

Nucleic acids can be transferred in particular by viruses, viral vectors, bacterial vectors, plasmids, which are transferred into the non-antigen-presenting cell by electroporation techniques, iontophoresis, ballistic methods and / or other techniques for introducing molecules.

According to the invention, cells can be infused into the patient. A medicament containing the preparation according to the invention is therefore also claimed according to the invention. The medicament according to the invention is preferably formulated as an infusion solution for intravenous or intraperitoneal administration. The formulation is chosen so that when the drug is administered there is no significant impairment of the effectiveness of the cell presenting the antigen according to the invention.

Physiological saline is preferred as the infusion solution. In principle, other solutions with a pH of 5.5 to 8.5 are also suitable. Serum, for example human serum, autologous or allologous serum or serum of other species, solutions with plasma substitutes, such as polyvinylpyrrolidone, are also suitable. Typically 0.5 ml to 500 ml should be applied. These quantities and pH values are of course not absolute, but can vary from Specialist, depending on conditions and requirements, is varied and adapted to the specific needs of a patient.

The cell presenting the antigen to be used according to the invention can be used according to the invention in particular for the production of a medicament for the treatment of immune reactions against allologic tissue features or for the prevention of GvHD.

In particular, the immune reactions to be treated are related to allologic tissue features, their gene sequences and / or partial sequences, in particular major histocompatibility complexes, MHC I, MHC II, rhesus factor, minor histocompatibility antigens (minor histocompatibility antigens).

The ex vivo selected cells can be infused into the patient and then subjected to an in vivo treatment with IL-2. The cells selected ex vivo can also be incubated ex vivo with IL-2 or other stimulants such as phytohemaglutinin (PHA) and then infused into the patient. It has been shown that the GvL effect can be enhanced in this way. This effect is described in the literature (Klingemann HG 1995; Sykes M 1994; Uharek L 1998; Vourka-Karussis U 1995; Weiss L 1995; Weiss L 1999; Xun CQ 1995).

tries

The humane model system

T cell proliferation on artificial APC

FIG. 7: HEK293 cells (human embryo kidney) were transfected with plasmids which code for either CIITA or B7-2 or for both genes. After the selection, stable clones expressing MHCII and / or B7-2 were selected and used for a mixed leukocyte reaction (MLR). CFSE-labeled human T cells from different donors were cultured for six days together with irradiated 293 cells. This results in an allogeneic system, since the HEK293 cells carry different histocompatibility antigens (mainly MHC) than the T cells used. However, T cells need a primary stimulus above MHC II and B7 to proliferate. The Proliferation of the T cells was determined by FACS analysis of the decreasing CFSE staining during cell division; the total number of cells was calculated using Truecount Beads. Figure 8 shows a representative experiment.

Figure 8 shows a T cell proliferation assay (human) with artificial APC (293 / MHCII-B7.2) and control cells (293, 293-B7-2 and 293-MHCII). 10 ⁵ T cells were incubated with different amounts of artificial APC, control cells or alone for 6 days. The absolute number of proliferated CD4 and CD8 T cells was then determined.

It can be seen that 293 cells expressing both MHCII and B7.2 can stimulate the proliferation of naive CD4 T cells in a dose dependent manner. CD4 T cells cultured on 293 cells or 293 cells expressing either MHCII or B7-2 show no increased proliferation. The proliferation of CD8 T cells is dose-dependent for all 293 clones used. T cell proliferation was highest on the double positive 293 clones (MHCII and B7-2).

This raised the question of whether T cells which are brought into contact with such artificial APC subsequently increase or decrease allogeneic immune reactions. For this purpose, the T cells were separated from the 293 after the first incubation described above and brought together a second time with different 293 clones.

Fig. 9: Scheme for the experiment

Execution :

Day 0 (isolation)

• Isolation of T cells from 50 ml whole blood (donor BH) via MACS → 5 x 10 ⁷ T cells

• plated overnight in RF medium (RPMI-10% FCS) day 1 (pre-stimulation, cell number ratio 1: 1) 1 x 10 ⁶ 293 MHCII / B7-2 or 293 MHCII per well in 6-well plate plated in 3 ml DF (DMEM-10% FCS)

• 2 h at 37 ° C → to settle the cells

• 150 s radiation to kill the 293 clones so that they do not overgrow the T cells (21.8 Gy)

• 1 x 10 ⁵ 293 MHCII / B7-2 or 293 MHCII + 1 x 10 ⁶ MACS-sorted T cells in 5 ml RF

• 3 days incubation in incubator day 4 (separation 293 and T cells)

1. MACS-CD3 positive sorting

2. CFSE staining for 4 minutes, then wash twice with RF, then incubate overnight in RF, then plated in RF in defined amounts

Day 6 (proliferation assay with differently pre-stimulated T cells)

• 293MHCII / B7-2: 2.4 x 10 ⁶ T cells → 12 approaches, see plate layout

• 293MHCII: 2 x 10 ⁶ T cells → 10 approaches, see plate layout

• 6 days incubation

Day 12 (analysis of proliferation assay ^)

CD4 / CD8 staining cell characterization

CFSE proliferation marker

Pl (propidium iodide) dead cells

Measurement at FACS with Cellquest from Becton Dickinson

Evaluation: with Attractors from Becton Dickinson

Plattenlavout:

Fig 10: Plate layout 1 T cells only

2 T cells only

3 Positive control: 293MHCII / B7-2 + ConA + T cells

4 Positive control: 293MHCII / B7-2 + ConA + T cells

5 2 x 10 ⁵ 293MHCII / B7-2 + 2 x 10 ⁵ T cells

6 2 x 10 ⁵ 293MHCII / B7-2 + 2 x 10 ⁵ T cells

7 2 x 10 ⁵ 293MHCII + 2 x 10 ⁵ T cells

8 2 x 10 ⁵ 293MHCII + 2 x 10 ⁵ T cells

9 2 x 10 ⁵ 293B7-2 + 2 x 10 ⁵ T cells

10 2 x 10 ⁵ 293B7-2 + 2 x 10 ⁵ T cells

11 2 x 10 ⁵ 293-2 + 2 x 10 ⁵ T cells

12 2 x 10 ⁵ 293-2 + 2 x 10 ⁵ T cells

2. Plate 293MHCII pre-stimulated T cells as above, without the approach 2 x 10 ⁵ 293B7-2 + 2 x 10 ⁵ T cells

Figure 11 shows CD4 T cell proliferation of T cells matured on double transgenic (MHC II / B7-2) or single positive (MHC II) 293. These T cells were incubated with various 293 clones. The ConA positive control was not shown for the 293-MHC II / B7-2, since this value (142962.9 +/- 11974.3) is far above the others.

Figure 12 shows CD8 T cell proliferation of T cells that have been matured to double transgenic (MHC II / B7-2) or single positive (MHC II) 293. These T cells were incubated with various 293 clones. The ConA positive control was not shown for the 293-MHC II / B7-2, since this value (64307.9 +/- 367.7) is far above the others. This experiment shows that neither the CD4 and CDS T cells preincubated on 293 MHC II nor on 293-MHC II / B7-2 are able to carry out immune reactions beyond background proliferation. It can also be seen that T cells which were incubated on first contact with 293MHCII could no longer be stimulated even with ConA. Therefore, the contact of the T cells with MHCII-positive, B7-negative cells seems to ensure a general shutdown of all T cells. Contact with MHCII and B7 on the 293, on the other hand, reduces the alloreactions, while the T cells remain stimulable, since they react normally to ConA. The selection affects both CD4 + and CD8 + T cells.

The mouse model system

In the same way as in the human ex Vo system, proliferation assays with non-APC and allogeneic T cells should be carried out. It was found that 10% of the MACS-sorted T cells were contaminated with other cells, 3% of which were not B220 positive. Due to the processing and appearance in the FACS, it was probably macrophages and DC. In order to investigate the question of how such contamination with donor DC affects the proliferation of the allogeneic T cells, these "impure" T cells were used in the assay. While we can assume that the T cells only proliferate after contact with MHCII and B7, i.e. "ignore" cells without both features, we assume in this assay that the T cells with all variants of the non-APC ( L929) can come to proliferation. Because the presence of donor DC is likely to cause L929 or its components to be phagocytosed by this DC and thus presented. This stimulates alloreactions. We have visualized the experiment in two schemes. The first scheme shows the planned trial and the expected outcome. The second shows the actual attempt.

The experiment also showed that the T cells proliferated in all batches. Looking at the CD8 T cells, it was shown that these cells are in all batches proliferated most strongly but not in the presence of MHCII-B7-L929. In the CD4 T cells, on the other hand, proliferation is strongest in the presence of the MHCII-positive L929. Examination of another marker protein (CD25) showed that CD25 + cells were among the proliferated CD4 T cells when the L929 expressed MHCII. The percentage of CD4 + CD25 + T cells proliferated was highest in the MHCII-B7-L929. In the batches with L929 without MHCII, no "new" CD25 + T cells were produced.

Interesting conclusions about the ratio of CD4 to CD8 T cells can also be drawn from this data. This is balanced in the MHCII-positive L929, while in the MHCII-negative approaches, the CD8 T cells preferentially proliferated. In addition, the non-APC were able to generate regulatory T cells despite the presence of the DC donor. This effect is surprising since we expected that the non-APC are inferior to the donor DC and therefore do not affect the differentiation of the T cells. It should also be borne in mind that we only looked at the "new", proliferated T cells, but not the previously existing CD25 + T cells, which make up up to 8% of the population. The function of the CD25 + T cells generated has yet to be determined. For this reason, we carried out the proliferation assays described below when this population was again contacted and the corresponding controls were contacted with the allogeneic L929 cells.

objective:

T cells primed by non-APC should be tested for their proliferation / activation in a second MLR. A reduced activation of L929-CIITA-B7.2, L929-CIITA primed T cells was expected upon contact with L929-CIITA and L929-CIITA-B7.2. L929 and L929-B7.2 primed T cells were included as controls.

Fig. 17: Scheme of the experiments Execution:

Preparation of the T cells:

T cells were prepared from Balb / c spleens 3 days before the initial incubation (primary MLR). After 3 days of cultivation, the T cells were control stained for CD3-APC, CD4-PE, CD8-APC and PL

Starting the first incubation:

Irradiated non-APC (L929-CIITA, L929-CIITA-B7.2, as controls L929 and L929-B7.2) were treated with Balb / cT cells for three days in the ratio shown in Table 1 in 12-well plates incubated.

Tab. 1: Incubation ratios and cell counts / well of a 12-well plate.

After three days of initial incubation, the T cells were carefully removed from the wells and transferred to 6-well plates. There they were cultivated for 2 days (rest phase).

Starting the second incubation:

The non-APC primed T cells were stained with CFSE and again irradiated with non-APC (L929-CIITA, L929-CIITA-B7.2, as controls L929 and L929-B7.2) in the ratio shown in Table 2 in 48-well plates incubated.

Tab. 2: Incubation ratios and cell counts / well of a 48-well plate.

After 6 days of incubation, the cells (T cells and non-APC) were removed from the wells by pipetting and transferred to Eppis. The cells were then washed with PBA and then taken up in 50 μl PBA / sample. 0.5 μl CD4-PE, CD8-APC were added to this approach. Samples were stained on ice for about 30 minutes and then washed with PBA.

TruCOUNT beads tubes (bead count: 50267) were filled with 2 ml PBA, vortexed and combined in a 50 ml falcon. The bead suspension was distributed to the samples to be measured with 500 μl per sample. Shortly before the FACS measurement, 2.5 μl PI (exclusion of dead cells) were added to the samples, vortexed and measured in the FACS.

The total number of cells could be determined on the basis of the known number of beads used and measured per batch.

The evaluation was carried out with Attractors (Becton Dickinson).

Results:

Summary, discussion and conclusion:

MACS-sorted Balb / c spleen T cells were incubated with artificial, allogeneic APC (L929-CIITA-B7.2, L929-CIITA, L929-B7.2 and L929) in a ratio of 1: 1 for 3 days. After separation of the T cells from the non-APC and a 2-day rest phase, the now primed T cells were stained with CFSE and again combined with the various non-APC in a ratio of 1: 1 for 6 days. The T cell proliferation could be measured by diluting the CFSE with each cell division.

The cell expansion of the differently primed T cells on contact with non-APC showed large differences in the absolute numbers. So L929 proliferated and L929-B7.2 primed CD4 + and CD8 + cells in contact with L929-CIITA-B7.2 twice as strong as L929-CIITA or L929-CIITA-B7.2 pre-stimulated T cells (see FIG. 14). This result suggests that T-cells with L929-CIITA (corresponds to APC without Costimulus) and L929-CIITA-B7.2 (corresponds to immature DC) have generated regulatory or anerergic T cells. Anergic T cells would no longer proliferate and regulatory ones could shut down other alloreactive T cells.

Benefits of fibroblasts

1. Fibroblasts are easily removed from the patient (e.g. from the skin)

2. They are not leukaemic cells

3. they do not present tumor antigens

4. They can be transfected with great efficiency

5. They have a long shelf life in culture

Claims

claims 1. Use of non-antigen-presenting cells which are stimulated by CIITA (CΙI transactivator) transfection or INFγ for the synthesis of MHC II for the ex-vivo selection of T-lymphocytes. 2. Use of non-antigen presenting cells according to claim 1, which are stimulated in addition to the synthesis of CD80 and / or CD86. 3. Use of non-antigen presenting cells according to claim 1 and / or 2, by the additional synthesis of CIITA, CD80 and / or CD86 by transfection of the non-AP cells with genetic information for CD80 and / or CD86. 4. Use according to claim 1 and / or 2, wherein the non-AP cell is selected from the group consisting of fibroblasts, epithelial cells, muscle cells, keratinocytes, hepatocytes, parenchymal cells, chondrocytes, melanocytes or combinations thereof. 5. A method for producing a composition containing T cells for a recipient, comprising the following steps: a) removal of T cells from a donor, b) contacting these T cells with non-AP cells , which are modified according to claims 1 to 3, wherein the non-AP cells originate from the recipient. 6. The method according to claim 5, wherein the T cells originate from a bone marrow donation or a bone marrow donor. 7. The method of claim 6, wherein the bone marrow donation is allogeneic. 8. The method according to at least one of claims 5 to 7, wherein the non-AP cell is selected from the group consisting of fibroblasts, epithelial cells, muscle cells, keratinocytes, hepatocytes, parenchymal cells, chondrocytes, melanocytes or combinations thereof. 9. The method according to at least one of claims 5 to 8, wherein the non-AP cells are separated from T cells after being brought into contact with T cells. 10. The method of claim 5, wherein the stem cells are separated from T cells. 11. Composition with T cells obtainable by a method according to any one of claims 5-10. 12. Medicament containing a composition according to claim 11. 13. Use of a composition according to claim 11, for the manufacture of a medicament for suppressing immune reactions in transplants, in particular bone marrow transplants. 14. Use according to one of claims 1 to 3 for the treatment or prevention of immune reactions against allogeneic tissue features. 15. CIITA according to at least one of claims 1 to 12, characterized in that it is used as a protein, polypeptide, oligopeptide, peptide, peptidomimetic or nucleic acid, which are DNA, RNA, oligonucleotides, polynucleotides, ribozymes, peptide nucleic acids (PNA). 16. Cell according to at least one of claims 1 to 13, characterized in that the DNA regulatory elements such as enhancers, promoters, polyA-coding 3 'ends for transcription of the DNA into RNA contains the RNA regulatory elements for translating the RNA into protein. 17. A method for the transfection of non-antigen presenting cells for use according to one of claims 1-14 by ex vivo treatment with viruses, viral vectors, bacterial vectors, plasmids, plasmids by viral gene transfer, electroporation techniques, iontophoresis, ballistic methods and / or other techniques have been introduced into eukaryotic cells. 18. The method of claim 17, wherein said cell is increased by treatment with viruses, viral vectors, bacterial vectors, plasmids by viral gene transfer, electroporation techniques, iontophoresis, ballistic methods and / or other techniques for introducing molecules into a cell Production of CIITA and possibly CD80 and / or CD86 is transfected, whereby T cells, which have allogeneic antigens z. B. are presented on MHC molecules to which the antigen-presenting cell bind, switched off. 19. The method according to claim 18, wherein the molecules according to claim 15 by vehicles such as liposomes, hydrogels, cyclodextrins, nanocapsules, nanoparticles, bio-adhesive microspheres and / or by electroporation techniques, iontophoresis, ballistic methods and / or other techniques for introducing molecules are transferred into the cell according to the invention. 20. The method according to at least one of claims 18 and or 19, wherein the molecules by viruses, viral vectors, bacterial vectors, plasmids by viral gene transfer, electroporation techniques, iontophoresis, ballistic methods and / or other techniques for introducing molecules into said Cells are transferred. 21. Medicament containing at least one cell according to the invention or according to at least one of claims 1-16. 22. Medicament according to claim 17, wherein at least one cell according to the invention is formulated for ex vivo application. 23. Use according to claim 1 to 4 for the manufacture of a medicament for the treatment of immune reactions in connection with allogeneic tissue characteristics, their gene sequences and / or partial sequences, in particular major histocompatibility complexes, MHC I, MHC II, rhesus factor, minor histocompatibility antigens (minor histocompatibility antigens). 24. Use according to claim 1 to 23, wherein the ex vivo selected cells are infused into the patient. 25. Infusion of IL-2 ex or in vivo and infusion of the selected T cells.