CD8alpha+ and CD11b+ dendritic cell-restricted MHC class II controls Th1 CD4+ T cell immunity

The activation, proliferation, differentiation, and trafficking of CD4 T cells is central to the development of type I immune responses. MHC class II (MHCII)-bearing dendritic cells (DCs) initiate CD4(+) T cell priming, but the relative contributions of other MHCII(+) APCs to the complete Th1 immune response is less clear. To address this question, we examined Th1 immunity in a mouse model in which I-A(beta)(b) expression was targeted specifically to the DCs of I-A(beta)b-/- mice. MHCII expression is reconstituted in CD11b(+) and CD8alpha(+) DCs, but other DC subtypes, macrophages, B cells, and parenchymal cells lack of expression of the I-A(beta)(b) chain. Presentation of both peptide and protein Ags by these DC subsets is sufficient for Th1 differentiation of Ag-specific CD4(+) T cells in vivo. Thus, Ag-specific CD4(+) T cells are primed to produce Th1 cytokines IL-2 and IFN-gamma. Additionally, proliferation, migration out of lymphoid organs, and the number of effector CD4(+) T cells are appropriately regulated. However, class II-negative B cells cannot receive help and Ag-specific IgG is not produced, confirming the critical MHCII requirement at this stage. These findings indicate that DCs are not only key initiators of the primary response, but provide all of the necessary cognate interactions to control CD4(+) T cell fate during the primary immune response.     

Intercellular exchange of class II major histocompatibility complex/peptide complexes is a conserved process that requires activation of T cells but is constitutive in other types of antigen presenting cell

Activated T cells acquire antigen presenting cell- (APC) derived class II major histocompatibility complex glycoproteins (MHCII) but the role of TCR in this process is controversial. This study provides additional evidence that ligation of TCR initiates activation-dependent processes that independently mediate acquisition of APC-derived molecules. First, intercellular exchange of MHCII resulted in the constitutive accumulation of xenogeneic rat I-A on murine B cells, whereas na?ve murine T cells required activation to adsorb xenogeneic I-A. Likewise, continuous lines of B cells, basophils, and M? from various species such as rat, mouse, and human constitutively acquired xenogeneic I-A. Second, inhibitors of T-cell activation such as wortmannin, EGTA, or mAb against I-A, TCR, LFA-1, or CD4 inhibited I-A acquisition by rested T cells but not by preactivated T cells. In conclusion, exchange of MHCII is a conserved process that requires activation of T cells but is constitutive in other types of APC.     

Major histocompatibility complex-linked control of the murine immune response to myelin basic protein

Recent experiments have shown that different regions of myelin basic protein (MBP) are encephalitogenic for different inbred strains of mice. It was therefore of interest to determine whether the immune response to MBP was MHC associated, and if so, what subregion controlled this response. Because PL/J and A/J mice were good responders to mouse MBP and C57Bl/10SN were not, B10.PL(73NS) and B10.A mice were immunized with mouse MBP under conditions designed to induce EAE. These strains were found to be highly susceptible. Intra-H-2 recombinant mice were then assessed for susceptibility. B10.A(4R) and B10.MBR were susceptible, whereas B10.A(5R) were resistant. Thus, EAE induced by purified MBP is under the control of the MHC, and the response maps to the I-A subregion. Production of IL 2 in vitro by T cells from MBP-primed mice in the presence of antigen and adherent cells was blocked by monoclonal antibody to the I-A, but not the I-E, subregion. When the specificity of the encephalitogenic response was tested, peptide 1-37 was active in B10.PL(73NS) and B10.A mice, whereas peptide 89-169 was active in A.SW, SWR, and B10.T(6R) strains. Serum from mice immunized with MBP peptides was assayed for antibody content. PL, B10.PL, and B10.A mice made a good antibody response to peptides 1-37 and 43-88 but were nonresponsive to peptide 89-169. SJL, A.SW, SWR, and B10.T(6R) mice responded well to peptide 89-169 but were poorly responsive to peptides 1-37 and 43-88.     

The cellular basis for the Ia restriction in murine experimental autoimmune thyroiditis

Susceptibility to experimental autoimmune thyroiditis (EAT) in the mouse is linked to the I-A subregion of the major histocompatibility complex. EAT can be induced in susceptible strains of mice by immunization with mouse thyroglobulin (MTg) and adjuvant. We have described a cell transfer system wherein spleen cells from EAT-susceptible CBA/J mice primed in vivo with MTg and lipopolysaccharide (LPS) can be activated in vitro with MTg to transfer EAT to naive syngeneic recipients. This cell transfer system was used to elucidate the cellular basis for the I-A restriction in EAT. While the cell active in transferring EAT was Thy 1+ I-A-, depletion of I-A+ cells from the in vitro culture prevented the activation of EAT effector T cells. MTg-pulsed mitomycin C-treated naive syngeneic spleen cells as antigen-presenting cells (APCs) could replace the I-A+ cells in vitro. Allogeneic (Balb/c) APCs were ineffective. Using APCs from several recombinant inbred strains of mice, it was shown that C3H/HEN and B10.A(4R) APCs were effective in activating MTg/LPS-primed CBA/J spleen cells to transfer EAT while B10.A(5R) APCs were ineffective. This maps the H-2 restriction to the K or I-A subregions. Addition of polyclonal anti-Iak or monoclonal anti-I-Ak or anti-L3T4 during in vitro activation inhibited both the generation of EAT effector cells and the proliferative response to MTg. Irrelevant anti-Ia reagents, monoclonal anti-I-Ek, and monoclonal anti-I-Jk were ineffective. Thus the I-A restriction in murine EAT appears to result from an I-A restricted interaction between Ia+ APCs and Ia- EAT effector T cells.     

Intercellular exchange of class II MHC complexes: ultrastructural localization and functional presentation of adsorbed I-A/peptide complexes

Activated rat T cells, like human T cells, synthesize class II MHC glycoproteins (MHCII) and absorb MHCII from neighboring T cells. This study focused on interactions of myelin basic protein (MBP)-specific T cells that either synthesized MHCII or absorbed MHCII during activation to assess cellular structures associated with presentation of functional MHCII/peptide complexes. Synthesis of MHCII by CD4(+)TCR(+) T cells involved I-A(+) multivesicular MHC class II-like compartments (MIIC), release of MHCII(+) vesicles, and expression of MHCII on a dendritic arborization. T-cell-mediated adsorption of MHCII was a saturable process that required close cell proximity, actin polymerization, and a permissive temperature. Adsorbed MHCII existed on vesicles that were intimately associated with the responder cell membrane. T cells bearing adsorbed vesicular MHCII presented antigen and were specifically lysed by CD4(+) T cell responders, but when labeled with anti-MHCII antibody were not susceptible to complement-mediated lysis. In summary, this study reveals vesicular compartments associated with synthesis and intercellular exchange of functional MHCII/peptide complexes.     

A role for accessibility to self-peptide-self-MHC complexes in intrathymic negative selection

Whether intrathymic-positive and -negative selection of conventional alpha beta T cells occur in anatomically distinct sites is a matter of debate. By using a system composed of two distinct immune receptors, the Y-Ae mAb and the 1H3.1 (V alpha 1/V beta 6) TCR, both directed against the 52--68 fragment of the I-E alpha-chain (E alpha 52--68) bound to I-A(b), we examined the occurrence of negative selection imposed in vivo by a self-peptide-self-MHC class II complex with differential tissue expression. 1H3.1 TCR-transgenic (Tg) mice were bred to mice having an I-E alpha transgene with expression directed to all MHC class II-positive cells, restricted to thymic epithelial cells, or restricted to B cells, dendritic cells, and medullary thymic epithelial cells. All 1H3.1 TCR/I-E alpha double-Tg mice revealed a severely diminished thymic cellularity. Their lymph node cells were depleted of V beta 6(+)CD4(+) cells and were unresponsive to E alpha 52--68 in vitro. The absolute number of CD4(+)CD8(+) thymocytes was drastically reduced in all combinations, indicating that negative selection caused by an endogenously expressed self-determinant can effectively occur in the thymic cortex in vivo. Moreover, both cortical epithelial cells and, interestingly, the few cortical dendritic cells were able to support negative selection of CD4(+)CD8(+) thymocytes, albeit with a distinct efficiency. Collectively, these observations support a model where, in addition to the avidity of the thymocyte/stromal cell interaction, in vivo negative selection of autoreactive TCR-Tg T cells is determined by accessibility to self-peptide-self-MHC complexes regardless of the anatomical site.     

B cells are required for generation of protective effector and memory CD4 cells in response to Pneumocystis lung infection

B cell-deficient mice are susceptible to infection by Pneumocystis carinii f. sp. muris (PC). To determine whether this susceptibility is due to a requirement for B cells to prime T cells, we compared CD4 T cell responses to PC in bone marrow chimeric mice that express MHC class II (MHCII) on all APCs (wild-type (WT) chimeras) and in bone marrow chimeric mice that express MHCII on all APCs except B cells (MHCII(-/-) chimeras). Although PC was rapidly cleared by WT chimeric mice, PC levels remained high in chimeric mice that lacked MHCII on B cells. In addition, although T cells were primed in the draining lymph nodes of MHCII(-/-) chimeric mice, the number of activated CD4 T cells infiltrating the lungs of these mice was reduced relative to the number in the lungs of WT chimeras. We also adoptively transferred purified CD4 T cells from the draining lymph nodes of PC-infected normal or B cell-deficient mice into SCID mice. Mice that received CD4 cells from normal mice were able to mount a response to infection in the lungs and clear PC. However, mice that received CD4 cells from B cell-deficient mice had a delayed T cell response in the lungs and failed to control the infection. These data indicate that B cells play a vital role in generation of CD4(+) memory T cells in response to PC infection in the lungs.     

Peptide-MHC class II dimers as therapeutics to modulate antigen-specific T cell responses in autoimmune diabetes

Type 1 diabetes is an autoimmune disorder caused by autoreactive T cells that mediate destruction of insulin-producing beta cells of the pancreas. Studies have shown that T cell tolerance can be restored by inducing a partial or altered signal through the TCR. To investigate the potential of bivalent peptide-MHC class II/Ig fusion proteins as therapeutics to restore Ag-specific tolerance, we have developed soluble peptide I-A(g7) dimers for use in the nonobese diabetic mouse model of diabetes. I-A(g7) dimers with a linked peptide specific for islet-reactive BDC2.5 TCR transgenic CD4(+) T cells were shown to specifically bind BDC2.5 T cells as well as a small population of Ag-specific T cells in nonobese diabetic mice. In vivo treatment with BDC2.5 peptide I-A(g7) dimers protected mice from diabetes mediated by the adoptive transfer of diabetogenic BDC2.5 CD4(+) T cells. The dimer therapy resulted in the activation and increased cell death of transferred BDC2.5 CD4(+) T cells. Surviving cells were hypoproliferative to challenge by Ag and produced increased levels of IL-10 and decreased levels of IFN-gamma compared with cells from control I-A(g7) dimer-treated mice. Anti-IL-10R therapy reversed the tolerogenic effects of the dimer. Thus, peptide I-A(g7) dimers induce tolerance of BDC2.5 TCR T cells through a combination of the induction of clonal anergy and anti-inflammatory cytokines.     

Self-specific MHC class II-restricted CD4-CD8- T cells that escape deletion and lack regulatory activity

In the presence of the I-Ealpha protein, transgenic (Tg) mice expressing the 1H3.1 alphabeta TCR that is specific for the Ealpha52-68:I-A(b) complex display drastic intrathymic deletion. Although peripheral T cells from these mice remained unresponsive to the Ealpha52-68:I-A(b) complex, they contained a subpopulation able to specifically react to this complex in the presence of exogenous IL-2, indicating that some 1H3.1 alphabeta TCR Tg T cells have escaped clonal deletion and efficiently populated the periphery. IL-2-dependent, Ealpha52-68:I-A(b) complex-responsive T cells were CD4-CD8- and expressed the 1H3.1 alphabeta TCR. Such T cells could develop intrathymically, did not show sign of regulatory/suppressor activity, displayed a typical naive phenotype, and seemed to persist in vivo over time. CD4-CD8- TCR Tg T cells were also detected when the surface density of the deleting ligand was increased on MHC class II+ cells. In addition, the development of CD4-CD8- 1H3.1 alphabeta TCR Tg T cells could be supported by I-A(b) molecules. These observations indicate that CD4 surface expression neither specifies, nor is required for, the thymic export of mature thymocytes expressing a MHC class II-restricted alphabeta TCR. The data also show that, although the avidity of the interaction involved in intrathymic deletion is significantly lower than that involved in mature T cell activation, its range can be large enough to be influenced by the presence or absence of coreceptors. Finally, the margin created by the absence of CD4 coreceptor was substantial because it could accommodate various amounts of the deleting ligand on thymic stromal cells.     

Nonobese diabetic mice display elevated levels of class II-associated invariant chain peptide associated with I-Ag7 on the cell surface

Peptide presentation by MHC class II molecules plays a pivotal role in determining the peripheral T cell repertoire as a result of both positive and negative selection in the thymus. Homozygous I-A(g7) expression imparts susceptibility to autoimmune diabetes in the nonobese diabetic mouse, and recently, it has been proposed that this arises from ineffectual peptide binding. Following biosynthesis, class II molecules are complexed with class II-associated invariant chain peptides (CLIP), which remain associated until displaced by Ag-derived peptides. If I-A(g7) is a poor peptide binder, then this may result in continued occupation by CLIP to the point of translocation to the cell surface. To test this hypothesis we generated affinity-purified polyclonal antisera that recognized murine CLIP bound to class II molecules in an allele-independent fashion. We have found abnormally high natural levels of cell surface class II occupancy by CLIP on nonobese diabetic splenic B cells. Experiments using I-A-transfected M12.C3 cells showed that I-A(g7) alone was associated with elevated levels of CLIP, suggesting that this was determined solely by the amino acid sequence of the class II molecule. These results indicated that an intrinsic property of I-A(g7) would affect both the quantity and the repertoire of self-peptides presented during thymic selection.     

Effects of in vivo monoclonal anti-I-A antibody treatment in neonatal mice on intrathymic and peripheral class II antigen expression

Intrathymic, Ia-bearing antigen-presenting cells (APC) are believed to play an important role in the development of a mature, functional T-cell repertoire. We used chronic in vivo treatment of neonatal mice with anti-I-A monoclonal Ab (MAb) to examine the expression of I-A and I-E antigens on intrathymic and peripheral APC. Three weeks after continuous treatment with anti-I-A MAb, FACS analysis of unfractionated spleen cells revealed a 75-90% reduction in the number of I-A bearing cells. Splenic antigen-presenting capacity measured by the ability of unseparated or density gradient-enriched APC to stimulate I-A- or I-E-reactive T-cell hybridomas was also greatly reduced. In contrast to the expression of I-A and I-E molecules in the splenic APC, anti-I-A MAb treatment resulted in decreased thymic APC I-A expression without significant changes in I-E as measured by FACS analysis. This was confirmed in functional studies in which allo-I-A- or auto-I-A-reactive T-cell hybridomas could not be stimulated by treated thymic APC. Unlike splenic APC, anti-I-A-treated thymic APC did not differ significantly from normals in their ability to stimulate allo-I-E-reactive T hybridomas. This lack of linkage or comodulation of I-A and I-E expression on thymic but not splenic APC may allow us to study the role of I-A molecules and I-E molecules on the development and expansion of functional, mature T-cell repertoires.     

Identification of CD4+ T cell-specific epitopes of islet-specific glucose-6-phosphatase catalytic subunit-related protein: a novel beta cell autoantigen in type 1 diabetes

Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) has been identified as a novel CD8(+) T cell-specific autoantigen in NOD mice. This study was undertaken to identify MHC class II-specific CD4(+) T cell epitopes of IGRP. Peptides named P1, P2, P3, P4, P5, P6, and P7 were synthesized by aligning the IGRP protein amino acid sequence with peptide-binding motifs of the NOD MHC class II (I-A(g7)) molecule. Peptides P1, P2, P3, and P7 were immunogenic and induced both spontaneous and primed responses. IGRP peptides P1-, P2-, P3-, and P7-induced responses were inhibited by the addition of anti-MHC class II (I-A(g7)) Ab, confirming that the response is indeed I-A(g7) restricted. Experiments using purified CD4(+) and CD8(+) T cells from IGRP peptide-primed mice also showed a predominant CD4(+) T cell response with no significant activation of CD8(+) T cells. T cells from P1-, P3-, and P7-primed mice secreted both IFN-gamma and IL-10 cytokines, whereas P2-primed cells secreted only IFN-gamma. Peptides P3 and P7 prevented the development of spontaneous diabetes and delayed adoptive transfer of diabetes. Peptides P1 and P2 delayed the onset of diabetes in both these models. In summary, we have identified two I-A(g7)-restricted CD4(+) T cell epitopes of IGRP that can modulate and prevent the development of diabetes in NOD mice. These results provide the first evidence on the role of IGRP-specific, MHC class II-restricted CD4(+) T cells in disease protection and may help in the development of novel therapies for type 1 diabetes.     

Autoimmune T cells: immune recognition of normal and variant peptide epitopes and peptide-based therapy

Experimental autoimmune encephalomyelitis (EAE) results from T helper (TH) cell recognition of myelin basic protein (MBP). We have characterized TH cell reactivity in B10.PL and PL/J (H-2u) mice to 39 N-terminal MBP peptide derivatives of different lengths and with individual amino acid substitutions. The peptide determinant of murine MBP can be divided into a minimal stimulatory core region (residues 1-6) and a tail region (residues 7-20) that alters the structure of the core region to affect both T cell recognition and MHC binding. Core recognition by B10.PL and PL/J mice is highly similar but in one case strain dependent. Peptide analogs that do not stimulate MBP-specific TH cells but bind to the I-Au molecule competitively inhibit T cell reactivity to MBP in vitro and prevent the induction of EAE in vivo.     

Class II MHC/peptide complexes are released from APC and are acquired by T cell responders during specific antigen recognition.

T cell expression of class II MHC/peptide complexes may be important for maintenance of peripheral self-tolerance, but mechanisms underlying the genesis of class II MHC glycoproteins on T cells are not well resolved. T cell APC (T-APC) used herein were transformed IL-2-dependent clones that constitutively synthesized class II MHC glycoproteins. When pulsed with myelin basic protein (MBP) and injected into Lewis rats, these T-APC reduced the severity of experimental autoimmune encephalomyelitis, whereas unpulsed T-APC were without activity. Normal MBP-reactive clones cultured without APC did not express class II MHC even when activated with mitogens and exposed to IFN-gamma. However, during a 4-h culture with T-APC or macrophage APC, recognition of MBP or mitogenic activation of responder T cells elicited high levels of I-A and I-E expression on responders. Acquisition of class II MHC glycoproteins by responders was resistant to the protein synthesis inhibitor cycloheximide, coincided with transfer of a PKH26 lipophilic dye from APC to responders, and resulted in the expression of syngeneic and allogeneic MHC glycoproteins on responders. Unlike rested I-A- T cell clones, rat thymic and splenic T cells expressed readily detectable levels of class II MHC glycoproteins. When preactivated with mitogens, naive T cells acquired APC-derived MHC class II molecules and other membrane-associated proteins when cultured with xenogeneic APC in the absence of Ag. In conclusion, this study provides evidence that APC donate membrane-bound peptide/MHC complexes to Ag-specific T cell responders by a mechanism associated with the induction of tolerance.     

Thymic and postthymic regulation of diabetogenic CD8 T cell development in TCR transgenic nonobese diabetic (NOD) mice

Natural development of diabetes in nonobese diabetic (NOD) mice requires both CD4 and CD8 T cells. Transgenic NOD mice carrying alphabeta TCR genes from a class I MHC (Kd)-restricted, pancreatic beta cell Ag-specific T cell clone develop diabetes significantly faster than nontransgenic NOD mice. In these TCR transgenic mice, a large fraction of T cells express both transgene derived and endogenous TCR beta chains. Only T cells expressing two TCR showed reactivity to the islet Ag. Development of diabetogenic T cells is inhibited in mice with no endogenous TCR expression due to the SCID mutation. These results demonstrate that the expression of two TCRs is necessary for the autoreactive diabetogenic T cells to escape thymic negative selection in the NOD mouse. Further analysis with MHC congenic NOD mice revealed that diabetes development in the class I MHC-restricted islet Ag-specific TCR transgenic mice is still dependent on the presence of the homozygosity of the NOD MHC class II I-Ag7.     

Diabetes resistance/susceptibility in T cells of nonobese diabetic mice conferred by MHC and MHC-linked genes

Polymorphism of MHC and MHC-linked genes is tightly associated with susceptibility to type 1 diabetes (T1D) in human and animal models. Despite the extensive studies, however, the role of MHC and MHC-linked genes expressed by T cells on T1D susceptibility remains unclear. Because T cells develop from TCR(-) thymic precursor (pre-T) cells that undergo MHC restriction mediated by thymic stroma cells, we reconstituted the T cell compartment of NOD.scid-RIP-B7.1 mice using pre-T cells isolated from NOD, NOR, AKR, and C57BL/6 (B6) mice. T1D developed rapidly in the mice reconstituted with pre-T cells derived from NOD or NOR donors. In contrast, most of the NOD.scid-RIP-B7.1 mice reconstituted with pre-T cells from AKR or B6 donors were free of T1D. Further analysis revealed that genes within MHC locus of AKR or B6 origin reduced incidence of T1D in the reconstituted NOD.scid-RIP-B7.1 mice. The expression of MHC class I genes of k, but not b haplotype, in T cells conferred T1D resistance. Replacement of an interval near the distal end of the D region in T cells of B6 origin with an identical allele of 129.S6 origin resulted in T1D development in the reconstituted mice. These results provide evidence that the expression of MHC class I and MHC-linked genes in T cells of NOD mice indeed contributes to T1D susceptibility, while expression of specific resistance alleles of MHC or MHC-linked genes in T cells alone would effectively reduce or even prevent T1D.     

Ex vivo analysis of thymic CD4 T cells in nonobese diabetic mice with tetramers generated from I-A(g7)/class II-associated invariant chain peptide precursors

The MHC determines susceptibility and resistance to type 1 diabetes in humans and nonobese diabetic (NOD) mice. To investigate how a disease-associated MHC molecule shapes the T cell repertoire in NOD mice, we generated a series of tetramers from I-A(g7)/class II-associated invariant chain peptide precursors by peptide exchange. No CD4 T cell populations could be identified for two glutamic acid decarboxylase 65 peptides, but tetramers with a peptide mimetic recognized by the BDC-2.5 and other islet-specific T cell clones labeled a distinct population in the thymus of young NOD mice. Tetramer-positive cells were identified in the immature CD4(+)CD8(low) population that arises during positive selection, and in larger numbers in the more mature CD4(+)CD8(-) population. Tetramer labeling was specific based on the use of multiple control tetramers, including one with a single amino acid analog peptide in which a critical TCR contact residue was substituted. The T cell population was already present in the thymus of 2-wk-old NOD mice before the typical onset of insulitis and was detected in B10 mice congenic for the NOD MHC locus, but not B10 control mice. These results demonstrate that a T cell population can expand in the thymus of NOD mice to levels that are at least two to three orders of magnitude higher than estimated for a given specificity in the naive T cell pool. Based on these data, we propose a model in which I-A(g7) confers susceptibility to type 1 diabetes by biasing positive selection in the thymus and later presenting peptides from islet autoantigens to such T cells in the periphery.     

Expression of the B7.1 costimulatory molecule on pancreatic beta cells abrogates the requirement for CD4 T cells in the development of type 1 diabetes

Although HLA-DQ8 has been implicated as a key determinant of genetic susceptibility to human type 1 diabetes, spontaneous diabetes has been observed in HLA-DQ8 transgenic mice that lack expression of murine MHC class II molecules (mII(-/-)) only when the potent costimulatory molecule, B7.1, is transgenically expressed on pancreatic beta cells. To study the contribution of HLA-DQ8 to the development of diabetes in this model, we crossed RIP-B7.1mII(-/-) mice with a set of transgenic mouse lines that differed in their HLA-DQ8 expression patterns on APC subpopulations, in particular dendritic cells and cortical thymic epithelial cells. Surprisingly, we found that even in the absence of HLA-DQ8 and CD4 T cells, a substantial fraction of the RIP-B7.1mII(-/-) mice developed diabetes. This disease process was remarkable for not only showing insulitis, but also inflammatory destruction of the exocrine pancreas with diffusely up-regulated expression of MHC class I and ICAM-1 molecules. Expression of HLA-DQ8 markedly increased the kinetics and frequency of diabetes, with the most severe disease in the lines with the highest levels of HLA-DQ8 on cortical thymic epithelial cells and the largest numbers of CD4 T cells. However, the adoptive transfer of diabetes was not HLA-DQ8-dependent and disease could be rapidly induced with purified CD8 T cells alone. Expression of B7.1 in the target tissue can thus dramatically alter the cellular and molecular requirements for the development of autoimmunity.     

Inhibition of MHC class II expression and immune responses by c-MIR

We previously reported a novel E3 ubiquitin ligase (E3), designated as c-MIR, which targets B7-2 to lysosomal degradation and down-regulates the B7-2 surface expression through ubiquitination of its cytoplasmic tail. B7-2 is well known as a costimulatory molecule for Ag presentation, suggesting that the manipulation of c-MIR expression modulates immune responses in vivo. To examine this hypothesis, we generated genetically modified mice in which c-MIR was expressed under an invariant chain (Ii) promoter. Dendritic cells derived from genetically engineered mice showed low ability to present Ags. In addition, these mice showed resistance to the onset of experimental autoimmune encephalomyelitis and an impaired development of CD4 T cells in the thymus and the periphery. These findings led us to conclude that MHC class II (MHC II) is an additional target for c-MIR. Indeed, forced expression of c-MIR in several B cell lines down-regulated the surface expression of MHC II, and down-regulation was found to depend on the presence of a single lysine residue in the cytoplasmic tail of the I-A beta-chain. In a reconstitution system using 293T cells, we found that the lysine residue at position 225 in the I-A beta-chain was ubiquitinated by c-MIR. To our knowledge, c-MIR is the first example of an E3 that is capable of inhibiting MHC II expression. Our findings suggest that c-MIR might potently regulate immune responses in vivo.