IMMUNOSUPPRESSIVE BLOOD CELLS AND METHODS OF PRODUCING THE SAME

Abstract

The present invention refers to a method of producing immunosuppressive bleed cells that can be used for the treatment of autoimmune diseases, in particular multiple sclerosis, organ graft rejection and graft-versus-host disease.

Claims

1-15. (canceled)

16. A method for treating an autoimmune disease, organ graft rejection, or graft-versus-host disease in a patient, said method comprising a) obtaining a blood cell sample and, optionally, preparing Peripheral Blood Mononuclear Cells (PBMCs) from said blood cell sample; b) treating said blood cell sample or PBMCs derived therefrom with a chemotherapeutic agent; and c) administering the treated blood cells or PBMCs of b) to said patient; and, thereby, treating an autoimmune disease, organ graft rejection, or graft-versus-host disease.

17. The method of claim 16, wherein said treating is treating autoimmune disease, wherein said blood cell sample is obtained from said patient, and wherein step b) comprises treating said blood cell sample or PBMCs derived therefrom with an autoantigen and with a chemotherapeutic agent.

18. The method of claim 17, wherein said treated blood cells or PBMCs ameliorate said autoimmune disease in said patient.

19. The method of claim 17, wherein the whole blood cell or PBMC sample is first treated with the autoantigen and subsequently with the chemotherapeutic agent.

20. The method of claim 17, wherein the whole blood cell or PBMC sample is first treated with the autoantigen and subsequently with the chemotherapeutic agent.

21. The method of claim 16, wherein the chemotherapeutic agent is at least one of mitomycin C, C2 ceramide, tunicamycin, mycophenolate-mofetil, tryptophan metabolites and semisynthetic derivates thereof, and proteasome inhibitors.

22. The method of claim 16, wherein the chemotherapeutic agent is mitomycin C.

23. The method of claim 17, wherein the autoantigen is selected from the group consisting of natural antigens, synthetic peptides and natural peptides.

24. The method of claim 17, wherein the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosis, hyperimmunoglobulin E, Hashimoto's thyroiditis, multiple sclerosis, progressive systemic sclerosis, myasthenia gravis, type I diabetes, uveitis, allergic encephalomyelitis, or glomerulonephritis.

25. The method of claim 17, wherein the autoimmune disease is type I diabetes and wherein the autoantigen is a diabetogenic autoantigen.

26. The method of claim 17, wherein the autoimmune disease is multiple sclerosis and wherein the autoantigen is a synthetic peptide of the myelin basic protein, preferably copaxone.

27. The method of claim 16, wherein said patient is a graft recipient and wherein said blood cell sample is obtained from a blood cell donor.

28. The method of claim 27, wherein said treating is prophylactically or symptomaticaily treating graft-versus-host disease and wherein the donor is the graft recipient; and/or wherein said treating is prophylactically or symptomatically treating organ graft rejection and wherein the donor is the from the graft donor.

29. The method of claim 27, wherein said treated donor blood cells or PBMCs ameliorate the risk of developing graft rejection and/or graft-versus-host disease in said patient.

30. A method for treating an autoimmune disease, organ graft rejection, or graft-versus-host disease in a patient, said method comprising administering to the patient a composition comprising a) isolated blood cells treated with a therapeutically effective amount of a chemotherapeutic agent and optionally an autoantigen or a derivative thereof, and b) optionally, a pharmaceutically acceptable carrier.

Description

DESCRIPTION OF THE DRAWINGS

[0069] FIGS. 1A-1D. Effect of MMC-treated BCs loaded with MBP on resting or activated T cells from MS patients. Primary stimulation (A, B): MBP-MMC-BCs are co-incubated with resting (A) (n=8) or activated peripheral lymphocytes (B) (n=7). Negative controls: MBP-BCs or lymphocytes only; positive controls: MMC-untreated MBP-BCs plus lymphocytes. Restimulation (C, D): CD4+ cells are isolated and re-stimulated with MBP-BCs of the same donor. Abscissa shows CD4 cells pretreatment. Ordinate shows T-cell proliferation. The first column represents BCs only (negative control). Data represent mean±SD and are expressed as percentage of positive control values (MBP-loaded BCs+lymphocytes=100%).

[0070] FIG. 2. Influence of MMC-BCs culture medium on stimulated T cells. MMC-treated or untreated BCs are cultivated for 72 h. Supematants (conditioned medium) are collected and used for cultures of anti-CD3 antibody-stimulated T cells with or without autologous BCs. Controls consisted of BCs and lymphocytes only. Data represent mean±SD and are expressed as percentage of positive control values (BC+anti-CD3+lymphocytes=100%) (n=4).

[0071] FIGS. 3A-3B. Cell cycle analysis of T celis exposed to MMC-BCs. Anti-CD3 antibody-stimulated T cells are co-cultured with (A) untreated and (B) MMC-treated (25 μg MMC/ml) autologous BCs. Twenty four hrs after supplementation with 10 μM BrdU the cells are analyzed by flow cytometry using a BrdU-Flow-Kit. The displayed quadrants show the percentage of cells in the corresponding cell cycle (G0/G1, G2/M, S) as well as that of dead cells.

[0072] FIG. 4. Flowcytometric analysis of apoptosis following treatment of BCs with MMC. BCs are treated with 50 μg/ml MMC and labelled with annexin-V-FITC and 7-AAD after 2, 6 and 24 hrs of incubation. Controls consisted of untreated BCs (-MMC). The lower and upper right quadrant show apoptosis. Percentages of apoptotic cells are displayed.

[0073] FIGS. 5A-5B. Effect of MMC-treated, MBP-presenting BC's in vivo. (A) Tg4 mice are injected i.v. with either MBP-BCs (.diamond-solid.) or MMC-treated MBP-BCs (.square-solid.). (B) Mice immunized in experiment A with MMC-treated MBP-BCs (.square-solid.) are challenged on day 28 with MBP-loaded BCs. Mice immunized in experiment A with MBP-BCs (.diamond-solid.) served as controls. Evaluation of EAE was performed according to the Coligan score. Data are shown as mean values±SEM (n=8 per group).

[0074] FIGS. 6A-6B. Prophylactic vaccination against EAE with MBP-loaded MMC-treated BC's (A) Tg4 mice are repetitively immunized with MBP-MMC-BCs. On day 0 these (.square-solid.), as well as non-vaccinated mice (.diamond-solid.), are challenged with MBP-pulsed BCs. (B) The EAE severity (Coligan score) is shown, starting from day 10 after MBP-BC challenge. Data are displayed as mean±SEM (n=8 non-vaccinated, n=10 vaccinated group).

[0075] FIG. 7. Phenotype of MMC-treated BCs. MMC-treated (50 μg/ml) and -untreated human BCs are labelled with FITC- or PE-conjugated specific monoclonal antibodies to MHC class II, CD80, CD86, and expression of molecules was analyzed by flow cytometry with a FACScalibur. The binding of antibodies is displayed in the histograms: filled grey histogram represents the isotype control, the red line binding to MMC-treated BCs, and the blue line to MMC-untreated BCs.

[0076] FIG. 8. Quantitative Real Time PCR of MMC-treated dendritic cells. Total RNA of MMC-treated and untreated BCs was extracted 18 hours after treatment, reverse transcribed and analyzed by quantitative real time PCR based on the SYBR green method. Data are normalized to RNA polymerase II. The ordinate shows the fold change of RNA expression levels (±SEM) of MMC-treated BCs as compared to the untreated counterparts (for each gene n≧5)

[0077] FIG. 9. Effect of MMC-treated blood cells on CD4.sup.+-T cells in vivo. MBP-specific TG4 T cells are labelled with CFSE-dye and transferred i.v. to syngeneic B10.PL mice. After 24 h PBS (negative control)(top), MBP-loaded blood cells (positive control )(middle), or MBP-loaded/MMC-treated blood cells (bottom) are injected i.v. After 4 days total lymph node cells are isolated and analyzed for CFSE-staining of F23.1.sup.+ (Vβ8.2) CD4.sup.+ T cells. Data represent mean percentages of CFSE.sup.low proliferating T cells (MBP-MMC-BCs vs. PBS-group: p=0.031; MBP-MMC-BCs vs. MBP-BCs group: p=not significant).

[0078] The invention is further explained by the following examples without being bound to it by any theory.

EXAMPLES

Examples 1

Experimental Procedures

[0079] (a) Mice

[0080] B10.PL mice were obtained from Jackson Laboratories (Bar Harbor, Me.). TCR-transgenic Tg4 mice (I-Au.sup.u background) express a TCR derived from an encephalitogenic CD4+ T cell clone specific for a MBP peptide (aa 1-9)(9).

[0081] (b) Generation of Dendritic Cells

[0082] Murine DCs are generated from bone marrow cells of B10.PL mice according to the protocol of Lutz et al. (10). For activation, 0.5 μM CpG-ODN 1668 is added. 90 min later non-adherent BMDC's are obtained. WMC (50 μg/ml) is added for 30 min of culture and the cells (10.sup.6/ml) are extensively washed. N-terminal acetylated MBP.sub.1-10 peptide Ac-ASQKRPSQRS (Ac1-10) is added at a concentration of 5 μM in combination with CpG-ODN. Human DCs are generated according to a standard protocol as previously described (11). For MBP-specific T cell studies, 30 μg/ml MSP (Sigma-Aldrich) is added to immature DCs until maturation. MMC (10-100 μg/ml) is added to the DC culture medium; after 30 min of incubation the cells are washed.

[0083] (c) T-Cell Studies In Vitro

[0084] Murine lymphocytes are cultured with BM-derived BCs. Human peripheral blood lymphocytes of MS patients are co-incubated with autologous MBP-loaded BCs. In a parallel experiment, DRB1*0301-BCs from healthy donors are loaded with MSP and co-incubated with MBP-specific CD4.sup.+ T cells (clone ES-BP8T) as previously described (11). In a control experiment, the BCs are loaded with an irrelevant peptide with comparable interaction with DRB1*0301. The HLA restriction can be calculated by the SYFPEITHI software (www.uni-tuebingen.de/uni/kxi). Co-cultures are performed at a BC:T cell ratio of 1:10. T cell proliferation is measured by [.sup.3H]thymidine incorporation.

[0085] (d) Supernatants of MMC-BCs

[0086] Mature BCs are treated with 25, 50 or 75 μg/ml MMC and washed. After 72 h of additional incubation the supernatants are collected and used as cell culture medium in a T-cell proliferation assay. 2×10.sup.5 cells/well are stimulated for 4 days with anti-CD3 monoclonal antibody (dilution 1:6400).

[0087] (e) Cell Cycle Analysis

[0088] Lymphocytes are stimulated for 2 days with anti-CD3 monoclonal antibody and cell cycle analysis is performed using the BrdU Flow Kit (BD Pharmingen, Heidelberg, Germany). Cells (10.sup.6) are incubated overnight with 10 μM BrdU. After fixation, permeabilization and treatment with 300 μg/ml DNase, the cells are stained with FITC-labeled anti-BrdU antibody and 7-AAD and analyzed by flow cytometry

[0089] (f) EAE Model

[0090] EAE is induced by i.v. Injection of 5×10.sup.6 activated BCs (in 0.2 ml) pulsed with 5 μM of autoantigenic MBP peptide (+/−MMC). On day 1 and 2 after immunization, each mouse is injected i.p. with 200 ng pertussis toxin (Calbiochem, Darmstadt, Germany) in 500 μl DPBS. Symptoms are evaluated daily according to the Coligan score.

[0091] (g) Affymetrix Microarray

[0092] RNA is converted into ds-cDNA using T7-(dT).sub.24 primers and the Superscript Choice system (invitrogen, Karlsruhe, Germany). Biotin-labeled cRNA is generated from the cDNA sample, hybridized to U133 Plus 2.0 gene chips (Affymetrix, High Wycombe, UK), stained with streptavidin-phycoerythrin (MoBiTec, Göttingen Germany) and scanned using the GeneArray scanner (Affymetrix). Microarray data of samples derived from 3 unrelated BC donors are analyzed using the Affymetrix Data Mining tool (DMT 4.0), the Affymetrix publishing tool (MDB 3.0), and the statistical data analysis software (Affymetrix Microarray Suite 5.0). Comparisons between MMC-treated and untreated BCs are done for genes with a positive detection call in at least one experimental group and with a fold change of at least 3 (corresponding to a signal log ratio between the two experimental groups of less than −1.5 or more than 1.5). Functional classifications from Gene Ontology (GO) Consortium (http://www.geneontology.org) are assigned to each identified gene.

[0093] (h) FACS Analysis

[0094] Human BCs are stained with fluorescein isothiocyanate (FITC)-labefed annexin V and 7-amino-actinomycin (7-AAD) to confirm apoptotic cell death.

[0095] BC staining is performed with fluorescence (FITC, PE)-labeled monoclonal antibodies (to MHC II, CD80, CD86,) to concentrations indicated by the manufacturers (BD Biosciences).

[0096] (i) Approval for Animal and Human Studies

[0097] Animal experiments are approved by the Animal Welfare Board of the Governmental Office Karlsruhe. Studies of human sera and cells are approved by the University of Heidelberg Ethics Committee

[0098] (k) Statistics

[0099] Results are shown as mean±SD or SEM as indicated. Single values of T cell proliferation represent the mean [.sup.3H]thymidine incorporation (cpm) of triplicate cultures and are given in percentage of the positive control (=100% proliferation) or cpm. P values are calculated by the unpaired Student's t-test using SigmaStat software (SPSS). Statistical significance is set at p<0.05.

Example 2

Results

[0100] Myelin-specific T cells have been proposed to play a role in the pathogenesis of MS (1,4). Therefore, MBP represents a candidate antigen for specific immunotherapy in MS. In the following experiments we studied the capacity of MMC-BCs loaded with MBP to control the activity of specific T cells derived from MS patients in cell cultures, and we tested their action in a mouse EAE model.

[0101] (a) Myelin-Basic-Protein-Loaded MMC-BCs Inhibit Specific T Cells of Multiple Sclerosis Patients In Vitro

[0102] BCs derived from MS patients are loaded with MBP and co-incubated with autologous T cells. In a parallel experiment, the BCs are loaded with MBP and treated with MMC. Whereas untreated BCs induce strong T cell stimulation, MMC-BCs do not (FIG. 1A).

[0103] Therapy of patients with active MS should address already activated autoreactive T cells. In order to find out whether these lymphocytes can be controlled by inhibitory BCs, pre-activated MBP-specific T cells derived from a MS patient are incubated with HLA-DR-matched, MBP-loaded MMC-BCs. The response of these T cells was significantly reduced (FIG. 1B).

[0104] In the next experiment we investigated whether suppressed T cells can be restimulated with naïve MBP-loaded BCs of the same donor. The results show that, once suppressed by MMC-BCs, T cells cannot or can only weakly be re-activated (FIG. 1C, FIG. 1D).

[0105] (b) Supernatants of Mitomycin-Treated BCs do not Inhibit the T Cells

[0106] Upon treatment of BCs with MMC a certain amount of substance might have diffused from the intracellular compartment into the culture medium and blocked T cell proliferation, in order to exclude that, supernatants of MMC-treated BCs are collected and used as medium in T cell proliferation assays. The results showed that T cell proliferation is not affected (FIG. 2), indicating that leakage of MMC from treated cells is not the reason for suppression.

[0107] (c) T Cells are Blocked in the G0/G1 Phase

[0108] When analyzing the mechanism of suppression, two aspects must be considered: the reaction of T cells to inhibitory BCs and the molecular changes of BCs induced by MMC-treatment.

[0109] As shown in the previous experiment, re-stimulation of suppressed T cells is not or only partially possible, indicating that the cells either became areactive or died. Cell cycle analysis revealed a significant accumulation in the G0/G1 phase of T cells co-incubated with MMC-BCs (FIG. 3). This finding argues for induction of T cell areactivity.

[0110] (d) Mitomycin C Does Not Inhibit the Expression of MHC-II and CD80/86 on BC's

[0111] MMC might have changed the expression of MHC II or CD80/86. FACS analysis showed that MHC II and CD80/86 are not down-regulated upon incubation with MMC (mean channel of MMC-treated/untreated BCs: MHC II=757/745; CD80=759/728; CD86=751/744); therefore, reduced antigen presentation by lower MHC II density, or less co-stimulation by lower C80/CD86 expression, cannot serve as an explanation for the inhibited T cell proliferation.

[0112] (e) Mitomycin C Modulates the Expression of Apoptotic and Immunoregulatory Genes of BC's

[0113] A comprehensive gene scan of 47,000 transcripts and additional variants was carried out by affymetrix microarray analysis. Genes whose expression was changed more than 3-fold upon treatment of BCs with MMC in 3 independent experiments are further analyzed. Based on this criterion, 116 genes are identified. Among the affected genes, too main clusters are found: one involved in apoptosis and the other mediating immunosuppression. Among apoptosis-related genes, 6 pro-apoptotic genes are upregulated (LRDD, TNFRSF 10b, PERP, FDXR, TRAF4, DDIT3) and 5 anti-apoptotic genes downregulated (NRG2, CFLAR, I-FLICE, Usurpin, FLAME-1), pointing to induction of cell death by apoptosis. It has been speculated that apoptotic cells are tolerogenic (12, 13). Therefore, it was important to verify by FACS whether the changed expression of these genes had repercussions on cell viability. As shown in FIG. 4, MMC-treated BCs enter earlier into apoptosis than untreated BCs. Interestingly, in parallel to apoptotic genes, well known immunosuppressive genes (ADM, TSC22D3, LILRB4) (14-16) are upregulated along with a series of potentially inhibitory genes (MAFB, CSF2RA, MAP4K4, GAB2) (17-21). Taken together, these findings indicate induction of apoptosis and increased expression of immunosuppressive genes in BCs treated with MMC.

[0114] (f) Myelin-Basic-Protein-Loaded MMC-BCs Inhibit Specific Mouse T Cells In Vitro

[0115] We addressed the question whether the suppressive effect mediated by MMC-BCs in vitro also works in vivo. For clarifying this point, a mouse EAE model is chosen—a setting in which MBP-specific T cells cause an inflammatory disease, similar to the inflammation of MS in humans. A prerequisite for their effectiveness in vivo is that, similarly to human BCs, MMC-treated mouse BCs are T-cell suppressive in vitro. Cell culture studies showed that mouse BCs loaded with MBP and treated with MMC significantly suppress specific syngeneic T cells of Tg4 mice (MBP-MMC-BCs+T cells=12,653±923 vs. MBP-BCs+T cells=24,727±3197; naïve BCs+T cells=7007±1591) (mean of cpm±SEM) (p=0.022).

[0116] Previous observations of Liu et al. (13) showed that, when injected into mice, antigen-loaded tolerogenic cells first drive antigen-specific T cells into cell-cycle, and subsequently the T cells are inactivated. This finding prompted us to trace the fate of the deleterious autoreactive T cells in animals treated with inhibitory BCs, MBP-specific T cells are labelled ex vivo with CFSE and injected into syngeneic mice. Thereafter, the animals are injected i.v. with MBP-loaded BCs treated with MMC, and T cells are isolated and analyzed by FACS. The MBP-specific T cells showed a significant degree of proliferation (MBP-MMC-BC=25% vs. MBP-BC=22%). Evidently, in spite of initial stimulation, the T cells must have been subsequently inactivated because, as shown in the following in vivo experiment, they are not able to cause EAE. This finding is in line with the observation described by Liu et al. (13).

[0117] (g) Vaccination with Myelin-Basic-Protein-Loaded MMC-BCs Protects Mice from Experimental Autoimmune Encephalitis

[0118] MBP-ioaded untreated BCs are injected into animals and, as expected, severe EAE occurs within 2-3 weeks (FIG. 5A). If MBP-loaded BCs are preireated with MMC and then injected, however, the animals remain completely free of symptoms, showing that MBP-specific T cells are not activated. An interesting question is whether the symptom-free animals became resistant to EAE. To this end, treated animals are re-challenged with MBP-BCs. One must keep in mind that the transgenic Tg4 mice used in our study carry>90% MBP-specific T cells, in contrast to only <0.0001% in normal rodents (9, 22). We suspect that it might be difficult to inactivate such a large number of T cells with one injection only. In a subsequent experiment, therefore, the animals are treated 5× with MBP-MMC-BCs and then re-challenged (FIG. 6A). As shown in FIG. 6B, this time the result is positive: whereas controls, which had not been vaccinated with MBP-MMC-BCs, developed severe EAE with lethal outcome, pre-vaccinated mice recovered after a mild episode of disease.

Example 3

Discussion

[0119] Attempts to generate regulatory blood cells, in particular dendritic cells (DCs) for control of autoimmune reactions have recently been described. Enk's group generated suppressive DCs by incubating the cells in vitro with IL-10 and inhibited ovalbumin-specific CD4 T-cell responses in naive and sensitized mice (23). Huang et al. (24) observed that a subpopulation of immature bone marrow-derived DCs, if pulsed with MBP and injected into syngeneic rats, protected from clinical EAE. Others showed that, to the contrary, mature but not immature DCs injected into mice with EAE reduced the severity of clinical signs and inflammation in the CNS (25). These conflicting findings stress the functional plasticity, from immunostimulation to suppression, of DCs under various conditions. Clinical signs of disease could aiso be reduced if rats or mice with incipient EAE are injected with interferon-γ-treated DCs (26). In neither of the iatter two studies are antigen-specific DCs used. By contrast, Menges et al. (27) used tumor-necrosis-factor-α matured DCs pulsed with auto-antigenic peptide and observed protection from EAE if the mice are injected before inductive immunization. Another experimental study suggested that inhibition of NF-κB by pharmacological agents enhanced the capacity of immature DCs to induce antigen-specific suppression to seif antigens in mice (28). When murine DCs are transduced with the gene for suppressor of cytokine signaling (SOCS)-3, they exhibited a DC2 phenotype that promoted Th2 cell differentiation and weakly influenced autoimmune reactions in vivo (29). The severity of EAE in mice couid aiso be reduced with autoantigen-loaded DCs expressing TRAIL or PDL1 transgenes (30).

[0120] Different functional behaviors of DCs belonging to the same maturational stage (5, 6), the difficulty to standardize the generation of suppressive DCs by biological agents, and reversible modifications induced by cytokines or other biological agents, all are hurdles for the use of suppressive DCs in clinical trials, entailing the risk of stimulating instead of inhibiting the immune response. Ideally, inhibitory DCs for clinical application should be easily and reproducibly generated, stable in their suppressive action, and capable of irreversibly inactivating autoreactive T cells.

[0121] Based on our previous experience in rats (8), in which allograft rejection was successfully controlled by MMC-BCs, stably inhibitory DCs for control of autoimmunity are generated in the present study by treating the cells with MMC and loading with autoantigen. These cells protected animals from lethal EAE, showing that, in principle, effective prophylactic vaccination against T cell mediated autoaggression is possible. MMC is an alkylating agent used in cancer therapy which strongly binds to distinct DNA sites, cross-links the double helical strands, inhibits DNA synthesis, and consequently suppresses cell proliferation. In addition, MMC inhibits RNA and protein synthesis. Interestingly, alkylating agents not only inhibit but also activate pathways usually triggered by stimulatory agents (31). Therefore, it is not surprising that in our model the expression of certain DC-genes was up- and not down-regulated. Because of the irreversible interaction of MMC with intracellular compounds, cells do not release MMC upon incubation with this substance. This was confirmed by our finding that supernatants of MMC-treated DCs do not suppress T cell reactions. Most importantly, in contrast to manipulations of DCs with biological agents (e.g. cytokines), MMC-treatment induces irreversibly suppressive DCs by induction of apoptosis, a feature that offers a potential for developing a stable therapeutic tool. Another advantage of this model is the use of non-toxic doses of a clinically approved drug. The therapeutic dose of MMC is 10-20 mg/m.sup.2; the concentration of MMC used in this study for incubation of cells was 0.05-0.100 mg/ml. Our analyses showed that after extensive washing the cell suspension contained, if at all, non-active traces of MMC. No clinical side effects are expected at these minimal amounts of free MMC in the injected solution.

[0122] Our findings demonstrate that MMC-BCs are effective in controlling both mouse and human autoreactive T cells. Previous studies in our laboratory showed that MMC-BCs are strongly inhibitory in rats (8). In difference to other models, the therapeutic tool described herein works across species. Moreover, in the present study the in vivo effect was tested under aggravating conditions. Normal rodents carry less than 10.sup.−6 MBP-reactive T cells in their repertoire (22). We used TG4 transgenic mice with >90% MBP-reactive T cells and consequently with an extreme proneness to EAE (9). If this huge number of “dangerous” T cells can be kept in check, we can expect a reliable effect when lower numbers of autoreactive T cells are involved.

[0123] Concerning the mechanism of suppression of autoreactive T cells derived from MS patients, we were concerned that MMC-BCs simply might “lose” their stimulatory capacity due to cell death. Our findings, however, show that suppressed T cells cannot be reactivated. Moreover, animals vaccinated with autoantigen-loaded inhibitory DCs became areactive to re-stimulation with the same antigen. These are signs of active suppression instead of a lacking immune response.

[0124] A previous study showed that exposure to necrotic tumor cells, in contrast to exposure to apoptotic cells, induces immunostimulation (12). This, as well as other observations (13), led to the hypothesis that necrotic cell death is immunogenic, whereas apoptotic cell death is poorly immunogenic or even tolerogenic. From a physiological standpoint of view this makes sense, because apoptosis is the normal process of cell death in our tissues. Would apoptosis induce immune responses, it would lead to inflammation and autoimmunity. Liu et al. (13) used this phenomenon to actively induce tolerance. Dying apoptotic splenocytes are loaded with ovalbumin and injected into syngeneic mice. After an initial phase of T cell stimulation the recipients became tolerant to ovalbumin (13). It is interesting to note the reports showing that DC lifespan has important consequences for DC-T-cell interaction, and thus determines the immunological outcome. Hugues et al. concluded that stable interactions favor T cell priming, whereas brief contacts between DCs and T cells may contribute to the induction of T cell tolerance (32). In the present series of experiments, MMC accelerated the natural process of apoptosis, shortening the lifespan of injected DCs and thus their contact with T cells. This provides a possible explanation for the observed tolerogenic effect. Obeid et al. (33) has recently analyzed the immunogenic potential of tumor cells rendered apoptotic by various chemotherapeutic drugs and observed that anthracyclins generate stimulatory cells whereas other drugs, such as mitomycin C, do not. If anthracyclins are used, the chaperon protein calreticulin was upregulated and responsible for the stimulatory action. This finding is important for tumor therapy, which aims at killing malignant cells and concomitantly stimulating the immune response against the tumor. Our findings are interesting in this context by demonstrating that treatment with MMC renders the cells apoptotic but—as shown by affymetrix microarray—does not upregulate calreticulin; instead, it upregulates immunosuppressive molecules. Whereas the observation of Obeid et al. (33) may be used for improving chemotherapy in cancer, our observation has a therapeutic potential for controlling autoimmune disease or graft rejection.

[0125] In the present study, induction of apoptosis was suggested by upregulation of pro-apoptotic genes, including LRDD (coding for PIDD), TNFRSF10b (coding for TRAIL-R2), PERP, FDXR, TRAF4, and DDIT3. Additionally, we noted downregulation of genes which protect from apoptosis, such as NRG2 and CFLAR (coding for cFLIP and its variants I-FLICE, usurpin, FLAME-1). Most importantly, apoptosis of MMC-treated DCs was demonstrated by FACS.

[0126] Gene expression analysis showed that, concomitantly with induction of apoptosis, a series of strongly immunosuppressive genes are upregulated. ADM (adrenomedullin), whose expression was increased 10 times, is a peptide that prevents sepsis-induced mortality, abrogates colitis, and provides highly effective therapy of arthritis by decreasing the presence of autoreactive Th1 cells, inducing regulatory T cells and inhibiting autoimmune and inflammatory responses (14). Another gene whose expression was upregulated by MMC was TSC22D3 (coding for GILZ). Interestingly, the same gene is upregulated upon exposure of DCs to glucocorticoids, IL-10, or TGF-β, all well-known immunological inhibitors (15). GILZ confers a suppressive phenotype to DCs and prevents them from activating T cells (15). A molecule induced by GILZ is LILRB4 (coding for ILT3), a protein which renders monocytes and DCs tolerogenic and has clinical relevance (16). Human heart transplant recipients with stable grafts have circulating T suppressor cells which upregulate ILT3 in donor antigen-presenting cells (16). These findings demonstrate an important immunoregulatory function of ILT3. We found a significant increase of ILT3expression in MMC-BCs. Other functionally relevant genes whose expression was modulated by MMC are MAFB—which directs differentiation away from DCs towards monocytes (17), CSF2RA—which transduces GM-CSF signals (18), MAP4K4—which mediates TNF-α signalling and cell migration (19, 20), and GAB2—which transmits signals delivered by cytokine-, growth factor-, and antigen-receptors (21). All of them might play a role in immunosuppression induced by MMC-treated DCs.

[0127] Taken together, the observations of Obeid et al. (33) and ours suggest that induction of apoptosis with concomitant upregulation of activatory molecules renders cells immunogenic, whereas apoptosis and upregulation of inhibitory molecules renders cells immunosuppressive.

[0128] In the 1970s, a random copolymer of amino acids, termed glatiramer acetate, was developed to mimic the composition of MBP. In clinical trials, glatiramer slowed progression of disability and significantly reduced the relapse rate of MS (34). Studies showed that the copolymer tolerized against a variety of different myelin antigens. More recently, altered peptide ligands of MBP and other auto-antigens constructed by substituting amino acids at the contact sites of these epitopes with the T cell receptor showed similar effects in animal models (4). These and other observations (35) indicate that the use of a single epitope can inhibit a disease caused by reactivity to multiple self-epitopes by directing unspecific regulatory mechanisms towards a certain organ. Apparently, in contrast to epitope “spreading”, epitope “containment” is also possible. Based on these observations, it is conceivable that MMC-BCs loaded with MBP, although addressing the immune response to one antigen, also can control reactions to neighboring molecules. An elegant variant of our model would be to load the inhibitory DCs with glatiramer acetate or other altered peptides derived from autoantigens. The suppressive action of peptides would be expected to be amplified.

[0129] Our in vivo data are derived from studies in the murine EAE model. It has been questioned to which extent this model reflects the pathogenesis of MS in humans (1). Of course, no mouse data, including those derived from EAE studies, can be automatically extrapolated to humans. It is worth mentioning, however, that in spite of all criticism 3 therapeutic compounds approved for use in MS—glatiramer acetate, mitoxantrone and natlizumab—emerged directly from findings in the EAE model (36). Our observations in mice gain additional relevance by the finding that T cells of MS patients are also suppressed by MBP-loaded MMC-BCs.

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