Granulocyte-macrophage colony-stimulating factor prevents diabetes development in NOD mice by inducing tolerogenic dendritic cells that sustain the suppressive function of CD4+CD25+ regulatory T cells

Autoimmune diabetes results from a breakdown of self-tolerance that leads to T cell-mediated beta-cell destruction. Abnormal maturation and other defects of dendritic cells (DCs) have been associated with the development of diabetes. Evidence is accumulating that self-tolerance can be restored and maintained by semimature DCs induced by GM-CSF. We have investigated whether GM-CSF is a valuable strategy to induce semimature DCs, thereby restoring and sustaining tolerance in NOD mice. We found that treatment of prediabetic NOD mice with GM-CSF provided protection against diabetes. The protection was associated with a marked increase in the number of tolerogenic immature splenic DCs and in the number of Foxp3+CD4+CD25+ regulatory T cells (Tregs). Activated DCs from GM-CSF-protected mice expressed lower levels of MHC class II and CD80/CD86 molecules, produced more IL-10 and were less effective in stimulating diabetogenic CD8+ T cells than DCs of PBS-treated NOD mice. Adoptive transfer experiments showed that splenocytes of GM-CSF-protected mice did not transfer diabetes into NOD.SCID recipients. Depletion of CD11c+ DCs before transfer released diabetogenic T cells from the suppressive effect of CD4+CD25+ Tregs, thereby promoting the development of diabetes. These results indicated that semimature DCs were required for the sustained suppressive function of CD4+CD25+ Tregs that were responsible for maintaining tolerance of diabetogenic T cells in NOD mice.     

GM-CSF induces bone marrow precursors of NOD mice to skew into tolerogenic dendritic cells that protect against diabetes

We have reported that GM-CSF treatment of NOD mice suppressed diabetes by increasing the number of tolerogenic dendritic cells (tDCs) and Tregs in the periphery. Here, we have investigated whether GM-CSF acted on NOD bone marrow DCs precursors to skew their differentiation to tDCs. DCs were generated from the bone marrow of GM-CSF-treated (GM.BMDCs) and PBS-treated (PBS.BMDCs) NOD mice and were assessed for their ability to acquire tolerogenic properties. Upon LPS stimulation, GM.BMDCs became fully mature, expressed high levels of PD-L1 and produced more IL-10 and less IL-12p70 and IFN-γ than PBS.BMDCs. In addition, LPS-stimulated GM.BMDCs possessed a reduced capacity to activate diabetogenic CD8⁺ T cells in a PD-1/PD-L1-dependent manner. A single injection of LPS-stimulated GM.BMDCs in NOD mice resulted in long-term protection from diabetes, in contrast to LPS-stimulated PBS.BMDCs. Our results showed that GM-CSF-treatment acted on bone marrow precursors to skew their differentiation into tDCs that protected NOD mice against diabetes.     

HLA-A*0201-restricted T cells from humanized NOD mice recognize autoantigens of potential clinical relevance to type 1 diabetes

In both humans and NOD mice, particular MHC genes are primary contributors to development of the autoreactive CD4+ and CD8+ T cell responses against pancreatic beta cells that cause type 1 diabetes (T1D). Association studies have suggested, but not proved, that the HLA-A*0201 MHC class I variant is an important contributor to T1D in humans. In this study, we show that transgenic expression in NOD mice of HLA-A*0201, in the absence of murine class I MHC molecules, is sufficient to mediate autoreactive CD8+ T cell responses contributing to T1D development. CD8+ T cells from the transgenic mice are cytotoxic to murine and human HLA-A*0201-positive islet cells. Hence, the murine and human islets must present one or more peptides in common. Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is one of several important T1D autoantigens in standard NOD mice. Three IGRP-derived peptides were identified as targets of diabetogenic HLA-A*0201-restricted T cells in our NOD transgenic stock. Collectively, these results indicate the utility of humanized HLA-A*0201-expressing NOD mice in the identification of T cells and autoantigens of potential relevance to human T1D. In particular, the identified antigenic peptides represent promising tools to explore the potential importance of IGRP in the development of human T1D.     

TGF-beta-dependent mechanisms mediate restoration of self-tolerance induced by antibodies to CD3 in overt autoimmune diabetes

CD3-specific antibodies have the unique capacity to restore self-tolerance in established autoimmunity. They induce long-term remission of overt diabetes in nonobese diabetic (NOD) mice and in human type I diabetes. The underlying mechanisms had been unclear until now. Here we report that treatment with CD3epsilon-specific antibodies induces transferable T-cell-mediated tolerance involving CD4+CD25+ cells. However, these CD4+CD25+ T cells are distinct from naturally occurring regulatory T cells that control physiological autoreactivity. CD3-specific antibody treatment induced remission in NOD Cd28-/- mice that were devoid of such regulatory cells. Remission of diabetes was abrogated by coadministration of a neutralizing transforming growth factor (TGF)-beta-specific antibody. The central role of TGF-beta was further suggested by its increased, long-lasting production by CD4+ T cells from tolerant mice. These data explain the intriguing tolerogenic effect of CD3-specific antibodies and position them as the first clinically applicable pharmacological stimulant of TGF-beta-producing regulatory CD4+ T cells.     

NOD bone marrow-derived dendritic cells are modulated by analogs of 1,25-dihydroxyvitamin D3

The immune effects of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) are mainly mediated through dendritic cells (DCs). In vitro, 1,25(OH)(2)D(3) treatment renders murine bone marrow (BM)-derived DCs more tolerogenic, indirectly altering behavior and fate of T lymphocytes. In vivo, treatment with 1,25(OH)(2)D(3) or its analogs prevents diabetes in NOD mice. The aim of this study was to investigate the effects of the 1,25(OH)(2)D(3)-analog TX527 on the expression of antigen-presenting and costimulatory/migratory molecules on BM-derived DCs from NOD mice. After culture with 20 ng/ml GM-CSF + 20 ng/ml IL-4 (8 days) followed by 1000 ng/ml LPS + 100 U/ml IFN-gamma (2 days), with or without 10(-8)M TX527, cells were counted and analyzed by FACS for MHC II, CD86, CD40 and CD54 expression within the CD11c(+) DC population. Upon TX527 treatment, cell recovery was significantly reduced whereas the CD11c(+) DC fraction remained constant. On CD11c(+) DCs, MHC II, CD86 and CD54 were significantly down-regulated and CD40 was twofold upregulated. Globally, BM-derived DCs from NOD mice become more tolerogenic upon TX527 treatment, confirming the effects of 1,25(OH)(2)D(3) on murine DCs and possibly explaining the protective effects of 1,25(OH)(2)D(3) and its analogs from diabetes in NOD mice.     

Dynamics of pathogenic and suppressor T cells in autoimmune diabetes development

In the nonobese diabetic (NOD) mouse, pathogenic and suppressor CD4(+) T cells can be distinguished by the constitutive expression of CD25. In this study, we demonstrate that the progression of autoimmune diabetes in NOD mice reflects modifications in both T cell subsets. CD4(+)CD25(+) suppressor T cells from 8-, but not 16-wk-old NOD mice delayed the onset of diabetes transferred by 16-wk-old CD25-depleted spleen cells. These results were paralleled by the inhibition of alloantigen-induced proliferation of CD4(+)CD25(-) cells, indicating an age-dependent decrease in suppressive activity. In addition, CD4(+)CD25(-) pathogenic T cells became progressively less sensitive to immunoregulation by CD4(+)CD25(+) T cells during diabetes development. CD4(+)CD25(-) T cells showed a higher proliferation and produced more IFN-gamma, but less IL-4 and IL-10, whereas CD4(+)CD25(+) T suppressor cells produced significantly lower levels of IL-10 in 16- compared with 8-wk-old NOD mice. Consistent with these findings, a higher frequency of Th1 cells was observed in the pancreas of 16-wk-old compared with 8-wk-old NOD mice. An increased percentage of CD4(+)CD25(-) T cells expressing CD54 was present in 16-wk-old and in diabetic NOD, but not in BALB/c mice. Costimulation via CD54 increased the proliferation of CD4(+)CD25(-) T cells from 16-, but not 8-wk-old NOD mice, and blocking CD54 prevented their proliferation, consistent with the role of CD54 in diabetes development. Thus, the pathogenesis of autoimmune diabetes in NOD mice is correlated with both an enhanced pathogenicity of CD4(+)CD25(-) T cells and a decreased suppressive activity of CD4(+)CD25(+) T cells.     

Phenotype and topography of human thymic B cells

Using single and double labeling immunohistochemical techniques and a large panel of monoclonal antibodies against B-cell differentiation antigens, including those newly defined at the Fourth International Leucocyte Typing Workshop, we have examined the immunophenotype and tissue distribution of human thymic B-cells. The existence of a distinct B-cell population as a constant constituent of the thymic microenvironment has been noted only recently. We found a singificant population of B-lymphocytes in the thymic medulla expressing the B-cell restricted antigens CD19, CD20, CD22, CD37, CD72, CD76 and IgM and IgD. As with other extrafollicular B-lymphocytes, they differ significantly from both follicle mantle and germinal center cells in morphology and immunophenotype, which points to alternative modes of B-cell differentiation. Thymic B-cells themselves show considerable heterogeneity and a subpopulation with dendritic features and the expression of CD23 has been referred to as “asteroid” cells. Their close association with T-cells and medullary epithelial cells points to a functional role for B-cells in the thymus. A second population of B-lymphocytes together with frequent lymph follicles is found within the extrathymic perviascular space. Though separated from the medulla by a layer of epithelial cells, a clear distinction between the B-cells of these two compartments is not always possible. The intramedullary B-cell compartment shows a parallel numeric increase with the occurrence of germinal centers in the perivascular space, mostly due to an accumulation of B-cells in the medulla adjacent to these lymph follicles. Thus a close relationship between the intra-and extramedullary B-cell population of the thymus seems likely. Presented in part in Leucocyte Typing IV (1989) Knapp W et al. (eds) Oxford University Press, Oxford, pp 221–222     

Phenotype and topography of human thymic B cells. An immunohistologic study.

Using single and double labeling immunohistochemical techniques and a large panel of monoclonal antibodies against B-cell differentiation antigens, including those newly defined at the Fourth International Leucocyte Typing Workshop, we have examined the immunophenotype and tissue distribution of human thymic B-cells. The existence of a distinct B-cell population as a constant constituent of the thymic microenvironment has been noted only recently. We found a significant population of B-lymphocytes in the thymic medulla expressing the B-cell restricted antigens CD19, CD20, CD22, CD37, CD72, CD76 and IgM and IgD. As with other extrafollicular B-lymphocytes, they differ significantly from both follicle mantle and germinal center cells in morphology and immunophenotype, which points to alternative modes of B-cell differentiation. Thymic B-cells themselves show considerable heterogeneity and a subpopulation with dendritic features and the expression of CD23 has been referred to as "asteroid" cells. Their close association with T-cells and medullary epithelial cells points to a functional role for B-cells in the thymus. A second population of B-lymphocytes together with frequent lymph follicles is found within the extrathymic perviascular space. Though separated from the medulla by a layer of epithelial cells, a clear distinction between the B-cells of these two compartments is not always possible. The intramedullary B-cell compartment shows a parallel numeric increase with the occurrence of germinal centers in the perivascular space, mostly due to an accumulation of B-cells in the medulla adjacent to these lymph follicles. Thus a close relationship between the intra- and extramedullary B-cell population of the thymus seems likely.     

Modulation of Dendritic Cell Immunobiology via Inhibition of 3-Hydroxy-3-Methylglutaryl-CoA (HMG-CoA) Reductase

The maturation status of dendritic cells determines whether interacting T cells are activated or if they become tolerant. Previously we could induce T cell tolerance by applying a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor (HMGCRI) atorvastatin, which also modulates MHC class II expression and has therapeutic potential in autoimmune disease. Here, we aimed at elucidating the impact of this therapeutic strategy on T cell differentiation as a consequence of alterations in dendritic cell function. We investigated the effect of HMGCRI during differentiation of peripheral human monocytes and murine bone marrow precursors to immature DC in vitro and assessed their phenotype. To examine the stimulatory and tolerogenic capacity of these modulated immature dendritic cells, we measured proliferation and suppressive function of CD4+ T cells after stimulation with the modulated immature dendritic cells. We found that an HMGCRI, atorvastatin, prevents dendrite formation during the generation of immature dendritic cells. The modulated immature dendritic cells had a diminished capacity to take up and present antigen as well as to induce an immune response. Of note, the consequence was an increased capacity to differentiate naïve T cells towards a suppressor phenotype that is less sensitive to proinflammatory stimuli and can effectively inhibit the proliferation of T effector cells in vitro. Thus, manipulation of antigen-presenting cells by HMGCRI contributes to an attenuated immune response as shown by promotion of T cells with suppressive capacities.     

Tolerogenic Vaccination Reduced Effector Memory CD4 T Cells and Induced Effector Memory Treg Cells for Type I Diabetes Treatment

Background

Vaccination could induce immune tolerance and protected NOD mice from the development of type I diabetes (T1D). We previously demonstrated that insulin peptide (B9-23) combined with dexamethasone (DEX) stimulated the expansion of antigen specific regulatory T (Treg) cells which in turn effectively prevented T1D in NOD mice. Here, we aimed to investigate the therapeutic effect of tolerogenic vaccination for T1D treatment.

Methodology/Principal Findings

The diabetic NOD mice (Blood glucose level ≧250 mg/dl) were treated with B9-23 and DEX twice. The tolerance was restored by blocking maturation of dendritic cells (DCs) and inducing Treg cells in treated NOD mice. Remarkably, the reduction of autoreactive effector memory CD4 T (Tm) cells and the induction of functional effector memory Treg (mTreg) cells contributed to the improvement of T1D in treated NOD mice.

Conclusions/Significance

Tolerogenic vaccination restored tolerance and ameliorated T1D by suppressing effector CD4 Tm cells and inducing effector mTreg cells. Our findings implicate the potential of tolerogenic vaccination for T1D treatment.     

Immunologic competence of T- and B-lymphocytes in tolerance induced by cyclophosphamide]

A study was made of immunological competence of T- and B-lymphocytes of mice subjected to tolerogenic treatment (administration of a massive dose of sheep erythrocytes and cyclophosphamide 7 days before the experiment). The capacity of lymphocytes of tolerant mice to influence the interaction of normal T- and B-lymphocytes was also investigated. This form of tolerance was caused not by T-suppressors, but by a true deficiency of T-cells-helpers (both in the thymus and in the spleen), and partially of B-cells (in the spleen). Some lack of B-cells in the bone marrow was connected with a nonspecific action of cyclophosphamide. Cyclophosphamide is supposed to selectively eliminate cells proliferating in response to the antigen.     

Liver Accumulation of Plasmodium chabaudi-Infected Red Blood Cells and Modulation of Regulatory T Cell and Dendritic Cell Responses

It is postulated that accumulation of malaria-infected Red Blood Cells (iRBCs) in the liver could be a parasitic escape mechanism against full destruction by the host immune system. Therefore, we evaluated the in vivo mechanism of this accumulation and its potential immunological consequences. A massive liver accumulation of P. c. chabaudi AS-iRBCs (Pc-iRBCs) was observed by intravital microscopy along with an over expression of ICAM-1 on day 7 of the infection, as measured by qRT-PCR. Phenotypic changes were also observed in regulatory T cells (Tregs) and dendritic cells (DCs) that were isolated from infected livers, which indicate a functional role for Tregs in the regulation of the liver inflammatory immune response. In fact, the suppressive function of liver-Tregs was in vitro tested, which demonstrated the capacity of these cells to suppress naive T cell activation to the same extent as that observed for spleen-Tregs. On the other hand, it is already known that CD4+ T cells isolated from spleens of protozoan parasite-infected mice are refractory to proliferate in vivo. In our experiments, we observed a similar lack of in vitro proliferative capacity in liver CD4+ T cells that were isolated on day 7 of infection. It is also known that nitric oxide and IL-10 are partially involved in acute phase immunosuppression; we found high expression levels of IL-10 and iNOS mRNA in day 7-infected livers, which indicates a possible role for these molecules in the observed immune suppression. Taken together, these results indicate that malaria parasite accumulation within the liver could be an escape mechanism to avoid sterile immunity sponsored by a tolerogenic environment.     

Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance

This study shows that the normal thymus produces immunoregulatory CD25+4+8- thymocytes capable of controlling self-reactive T cells. Transfer of thymocyte suspensions depleted of CD25+4+8- thymocytes, which constitute approximately 5% of steroid-resistant mature CD4+8- thymocytes in normal naive mice, produces various autoimmune diseases in syngeneic athymic nude mice. These CD25+4+8- thymocytes are nonproliferative (anergic) to TCR stimulation in vitro, but potently suppress the proliferation of other CD4+8- or CD4-8+ thymocytes; breakage of their anergic state in vitro by high doses of IL-2 or anti-CD28 Ab simultaneously abrogates their suppressive activity; and transfer of such suppression-abrogated thymocyte suspensions produces autoimmune disease in nude mice. These immunoregulatory CD25+4+8- thymocytes/T cells are functionally distinct from activated CD25+4+ T cells derived from CD25-4+ thymocytes/T cells in that the latter scarcely exhibits suppressive activity in vitro, although both CD25+4+ populations express a similar profile of cell surface markers. Furthermore, the CD25+4+8- thymocytes appear to acquire their anergic and suppressive property through the thymic selection process, since TCR transgenic mice develop similar anergic/suppressive CD25+4+8- thymocytes and CD25+4+ T cells that predominantly express TCRs utilizing endogenous alpha-chains, but RAG-2-deficient TCR transgenic mice do not. These results taken together indicate that anergic/suppressive CD25+4+8- thymocytes and peripheral T cells in normal naive mice may constitute a common T cell lineage functionally and developmentally distinct from other T cells, and that production of this unique immunoregulatory T cell population can be another key function of the thymus in maintaining immunologic self-tolerance.     

B- and T-cell-specific inactivation of thioredoxin reductase 2 does not impair lymphocyte development and maintenance

Thioredoxin reductases (Txnrds) are a group of selenoenzymes participating in cellular redox regulation. Three Txnrd isoforms are known, each of which exhibits distinct cellular localisation and tissue-specific expression pattern. Txnrd1 is found in the cytoplasm, expression of Txnrd2 is restricted to mitochondria and Txnrd3 shows testis-specific expression. Recently, it was shown that Txnrd2 strongly affects the development of blood cells, since mouse embryos deficient for Txnrd2 are severely anaemic, show increased apoptosis in foetal liver and possess haematopoietic liver stem cells of reduced capacity to proliferate in vitro. However, because Txnrd2-deficient mice die at embryonic day 13.5, it was not known how this enzyme affects blood cell function in the adult animal. In the present study we show that conditional Txnrd2 knockouts generated using CD4- and CD19Cre transgenic mice lack Txnrd2 expression in CD4-- and CD19-positive T- and B-lymphocytes, respectively. However, the development and differentiation of both cell types in thymus and bone marrow was not significantly impaired. In addition, B-cell proliferation and activation in response to CD40 and IL-4 was unaltered in Txnrd2-deficient B-cells.     

CD79a detected by ZL7.4 separates chronic lymphocytic leukemia from mantle cell lymphoma in the leukemic phase.

Both B-cell chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are characterized by a lymphoproliferation of neoplastic CD5+ B-cells, but an accurate differential diagnosis between these two malignancies is vitally important for guiding treatment options. Because CD79a has been identified as a pan-B marker, we intended to use it in place of CD19 to identify B-cells and to use CD23 to distinguish between CLL and MCL in the leukemic phase. Anti-CD79a (clone ZL7.4) was used to detect the Igalpha/mb1 protein in fresh CD5+ B-lymphocytes by dual-channel flow cytometry. Expression of CD19 and CD23 were similarly assessed. As expected, CD19 was expressed in all specimens, whereas CD23 expression was zero in 3/4 MCLs, weak in 1/4 MCLs, and 2/8 CLLs (10-19%) and stronger in 6/8 CLLs (> or =45%). However, although all the CD19+/CD5+ cells of MCL expressed high CD79a levels, CD79a expression was negligible or absent in 8/8 CLL specimens (mean positivity for CD79a = 2.41 +/- 2.71%). CD79a (ZL7.4) levels may provide a more reliable distinction than CD23 levels between CLL and MCL. If these results hold up in a larger series, we recommend that the ZL7.4 antibody should be considered in routine marker panels for CLL and low-grade lymphoma.     

Forced expression of HoxB4 enhances hematopoietic differentiation by human embryonic stem cells

HoxB4 has been shown to enhance hematopoietic engraftment by hematopoietic stem cells (HSC) from differentiating mouse embryonic stem cell (mESC) cultures. Here we examined the effect of ectopic expression of HoxB4 in differentiated human embryonic stem cells (hESCs). Stable HoxB4-expressing hESCs were established by lentiviral transduction, and the forced expression of HoxB4 did not affect stem cell features. HoxB4-expressing hESC-derived CD34+ cells generated higher numbers of erythroid and blast-like colonies than controls. The number of CD34+ cells increased but CD45+ and KDR+ cell numbers were not significantly affected. When the hESC derived CD34+ cells were transplanted into NOD/SCID beta 2m-/- mice, the ectopic expression of HoxB4 did not alter their repopulating capacity. Our findings show that overexpression of HoxB4 in differentiating hESCs increases hematopoietic colony formation and hematopoietic cell formation in vitro, but does not affect in vivo repopulation in adult mice hosts.     

A Lactobacillus rhamnosus Strain Induces a Heme Oxygenase Dependent Increase in Foxp3+ Regulatory T Cells

We investigated the consequences of feeding with a Lactobacillus species on the immune environment in GALT, and the role of dendritic cells and heme oxygenase-1 in mediating these responses. Feeding with a specific strain of Lactobacillus rhamnosus induced a significant increase in CD4+CD25+Foxp3+ functional regulatory T cells in GALT. This increase was greatest in the mesenteric lymph nodes and associated with a marked decrease in TNF and IFNγ production. Dendritic cell regulatory function and HO-1 expression was also increased. The increase in Foxp3+ T cells could be prevented by treatment with a heme oxygenase inhibitor. However, neither inhibition of heme oxygenase nor blockade of IL-10 and TGFβ prevented the inhibition of inflammatory cytokine production. In conclusion Lactobacillus feeding induced a tolerogenic environment in GALT. HO-1 was critical to the enhancement of Foxp3+ regulatory T cells while additional, as yet unknown, pathways were involved in the down-regulation of inflammatory cytokine production by T cells.     

Local Effects of Regulatory T Cells in MUC1 Transgenic Mice Potentiate Growth of MUC1 Expressing Tumor Cells In Vivo

MUC1 transgenic (MUC1.Tg) mice have widely been used as model recipients of cancer immunotherapy with MUC1. Although MUC1.Tg mice have previously been shown to be immunologically tolerant to MUC1, the involvement of regulatory T (Treg) cells in this phenotype remains unclear. Here, we showed that numbers of Treg cells in MUC1-expressing tumors were greater in MUC1.Tg mice than in control C57BL/6 (B6) mice, and that the growth of tumor cells expressing MUC1, but not that of control cells, in MUC1. Tg mice was faster than in B6 mice. The MUC1.Tg mice appeared to develop MUC1-specific peripheral tolerance, as transferred MUC1-specific T cells were unable to function in MUC1.Tg mice but were functional in control B6 mice. The suppressive function of CD4⁺CD25^high cells from MUC1.Tg mice was more potent than that of cells from control B6 mice when Treg cell activity against MUC1-specific T cells was compared in vitro. Therefore, the enhanced growth of MUC1-expressing tumor cells in MUC1.Tg mice is likely due to the presence of MUC1-specific Treg cells.     

Intrinsic tolerance in autologous collagen-induced arthritis is generated by CD152-dependent CD4+ suppressor cells

Collagen-induced arthritis is a mouse model of rheumatoid arthritis (RA) and is commonly induced after immunization with type II collagen (CII) of a non-mouse origin. T cell recognition of heterologous CII epitopes has been shown to be critical in development of arthritis, as mice with cartilage-restricted transgenic expression of the heterologous T cell epitope (MMC mice) are partially tolerized to CII. However, the mechanism responsible for tolerance and arthritis resistance in these mice is unclear. The present study investigated the regulatory mechanisms in naturally occurring self-tolerance in MMC mice. We found that expression of heterologous rat CII sequence in the cartilage of mice positively selects autoreactive CD4(+) T cells with suppressive capacity. Although CD4(+)CD25(+) cells did not play a prominent role in this suppression, CD152-expressing T cells played a crucial role in this tolerance. MMC CD4(+) T cells were able to suppress proliferation of wild-type cells in vitro where this suppression required cell-to-cell contact. The suppressive capability of MMC cells was also demonstrated in vivo, as transfer of such cells into wild-type arthritis susceptible mice delayed arthritis onset. This study also determined that both tolerance and disease resistance were CD152-dependent as demonstrated by Ab treatment experiments. These findings could have relevance for RA because the transgenic mice used express the same CII epitope in cartilage as humans and because autoreactive T cells, specific for this epitope, are present in transgenic mice as well as in patients with RA.