Detection of vasostatin‐1‐specific CD8+ T cells in non‐obese diabetic mice that contribute to diabetes pathogenesis

Chromogranin A (ChgA) is an antigenic target of pathogenic CD4⁺ T cells in a non‐obese diabetic (NOD) mouse model of type 1 diabetes (T1D). Vasostatin‐1 is a naturally processed fragment of ChgA. We have now identified a novel H2‐K^d‐restricted epitope of vasostatin‐1, ChgA 36‐44, which elicits CD8⁺ T cell responses in NOD mice. By using ChgA 36‐44/K^d tetramers we have determined the frequency of vasostatin‐1‐specific CD8⁺ T cells in pancreatic islets and draining lymph nodes of NOD mice. We also demonstrate that vasostatin‐1‐specific CD4⁺ and CD8⁺ T cells constitute a significant fraction of islet‐infiltrating T cells in diabetic NOD mice. Adoptive transfer of T cells from ChgA 36‐44 peptide‐primed NOD mice into NOD/severe combined immunodeficiency (SCID) mice led to T1D development. These findings indicate that vasostatin‐1‐specific CD8⁺ T cells contribute to the pathogenesis of type 1 diabetes in NOD mice.     

Administration of a determinant of preproinsulin can induce regulatory T cells and suppress anti-islet autoimmunity in NOD mice

Antigen-specific immunotherapy is expected to be an ideal strategy for treating type 1 diabetes (T1D). We investigated the therapeutic efficacy of a peptide in the leader sequence of preproinsulin, which was selected because of its binding affinity to the MHC I-A^g7 molecule. Preproinsulin-1 L7–24 peptide (L7–24) emulsified in Freund's incomplete adjuvant was administered subcutaneously to NOD mice. Administration of L7–24 increased the proportion of regulatory T cells in the spleen. Splenocytes of NOD mice immunized with this peptide secreted IL-4 and IL-10 in response to L7–24. This peptide also significantly prevented the development of diabetes and cured some newly diabetic NOD mice without recurrence. L7–24 peptide, which has a high affinity for pockets of I-A^g7, induced regulatory T cells and showed therapeutic effects. This peptide may provide a new approach for developing antigen-specific immunotherapy for autoimmune diabetes.     

High‐level intrathymic thyrotrophin receptor expression in thyroiditis‐prone mice protects against the spontaneous generation of pathogenic thyrotrophin receptor autoantibodies

The thyrotrophin receptor (TSHR) A‐subunit is the autoantigen targeted by pathogenic autoantibodies that cause Graves' hyperthyroidism, a common autoimmune disease in humans. Previously, we reported that pathogenic TSHR antibodies develop spontaneously in thyroiditis‐susceptible non‐obese diabetic (NOD).H2^h4 mice bearing a human TSHR A‐subunit transgene, which is expressed at low levels in both the thyroid and thymus (Lo‐expressor transgene). The present study tested recent evidence that high intrathymic TSHR expression protects against the development of pathogenic TSHR antibodies in humans. By successive back‐crossing, we transferred to the NOD.H2^h4 background a human TSHR A‐subunit transgene expressed at high levels in the thyroid and thymus (Hi‐expressor transgene). In the sixth back‐cross generation (> 98% NOD.H2^h4 genome), only transgenic offspring produced spontaneously immunoglobulin (Ig)G class non‐pathogenic human TSHR A‐subunit antibodies. In contrast, both transgenic and non‐transgenic offspring developed antibodies to thyroglobulin and thyroid peroxidase. However, non‐pathogenic human TSHR antibody levels in Hi‐expressor offspring were lower than in Lo‐expressor transgenic mice. Moreover, pathogenic TSHR antibodies, detected by inhibition of TSH binding to the TSHR, only developed in back‐cross offspring bearing the Lo‐expressor, but not the Hi‐expressor, transgene. High versus low expression human TSHR A‐subunit in the NOD.H2^h4 thymus was not explained by the transgene locations, namely chromosome 2 (127–147 Mb; Hi‐expressor) and chromosome 1 (22.9–39.3 Mb; low expressor). Nevertheless, using thyroiditis‐prone NOD.H2^h4 mice and two transgenic lines, our data support the association from human studies that low intrathymic TSHR expression is associated with susceptibility to developing pathogenic TSHR antibodies, while high intrathymic TSHR expression is protective.     

Genetic deletion of granzyme B does not confer resistance to the development of spontaneous diabetes in non‐obese diabetic mice

Granzyme B (GzmB) and perforin are proteins, secreted mainly by natural killer cells and cytotoxic T lymphocytes that are largely responsible for the induction of apoptosis in target cells. Because type 1 diabetes results from the selective destruction of β cells and perforin deficiency effectively reduces diabetes in non‐obese diabetic (NOD) mice, it can be deduced that β cell apoptosis involves the GzmB/perforin pathway. However, the relevance of GzmB remains totally unknown in non‐obese diabetic (NOD) mice. In this study we have focused on GzmB and examined the consequence of GzmB deficiency in NOD mice. We found that NOD.GzmB^–/– mice developed diabetes spontaneously with kinetics similar to those of wild‐type NOD (wt‐NOD) mice. Adoptive transfer study with regulatory T cell (T_reg)‐depleted splenocytes (SPCs) into NOD‐SCID mice or in‐vivo T_reg depletion by anti‐CD25 antibody at 4 weeks of age comparably induced the rapid progression of diabetes in the NOD.GzmB^–/– mice and wt‐NOD mice. Expression of GzmA and Fas was enhanced in the islets from pre‐diabetic NOD.GzmB^–/– mice. In contrast to spontaneous diabetes, GzmB deficiency suppressed the development of cyclophosphamide‐promoted diabetes in male NOD mice. Cyclophosphamide treatment led to a significantly lower percentage of apoptotic CD4⁺, CD8⁺ and CD4⁺CD25⁺ T cells in SPCs from NOD.GzmB^–/– mice than those from wt‐NOD mice. In conclusion, GzmB, in contrast to perforin, is not essentially involved in the effector mechanisms for β cell destruction in NOD mice.     

B6.g7 mice reconstituted with BDC2·5 non‐obese diabetic (BDC2·5NOD) stem cells do not develop autoimmune diabetes

In BDC2·5 non‐obese diabetic (BDC2·5NOD) mice, a spontaneous model of type 1 diabetes, CD4⁺ T cells express a transgene‐encoded T cell receptor (TCR) with reactivity against a pancreatic antigen, chromogranin. This leads to massive infiltration and destruction of the pancreatic islets and subsequent diabetes. When we reconstituted lethally irradiated, lymphocyte‐deficient B6.g7 (I‐A^g7+) Rag^–/– mice with BDC2·5NOD haematopoietic stem and progenitor cells (HSPC; ckit⁺Lin^–Sca‐1^hi), the recipients exhibited hyperglycaemia and succumbed to diabetes. Surprisingly, lymphocyte‐sufficient B6.g7 mice reconstituted with BDC2·5NOD HSPCs were protected from diabetes. In this study, we investigated the factors responsible for attenuation of diabetes in the B6.g7 recipients. Analysis of chimerism in the B6.g7 recipients showed that, although B cells and myeloid cells were 98% donor‐derived, the CD4⁺ T cell compartment contained ～50% host‐derived cells. These host‐derived CD4⁺ T cells were enriched for conventional regulatory T cells (T_regs) (CD25⁺forkhead box protein 3 (FoxP3)⁺] and also for host‐ derived CD4⁺CD25^–FoxP3^– T cells that express markers of suppressive function, CD73, FR4 and CD39. Although negative selection did not eliminate donor‐derived CD4⁺ T cells in the B6.g7 recipients, these cells were functionally suppressed. Thus, host‐derived CD4⁺ T cells that emerge in mice following myeloablation exhibit a regulatory phenoytpe and probably attenuate autoimmune diabetes. These cells may provide new therapeutic strategies to suppress autoimmunity.     

A Conformationally-Constrained MHC Class II I-A-Derived Peptide Protects NOD Mice from the Development of Diabetes

Allele-specific peptide vaccination against disease-associated MHC class II molecules is a promising new strategy for modulating self-antigen presentation to autoreactive T cells in autoimmune diseases. To evaluate the potential of this approach for treatment of insulin-dependent diabetes mellitus (IDDM), we have designed a cyclic peptide vaccine, DiavaX, from the third hypervariable region of the β-chain of the NOD mouse MHC class II I-A^g7. NOD mice were treated at 5 and 9 weeks of age with 100 μg DiavaX emulsified in alum, a control peptide in alum, or alum alone. At the end of the study, 87% of alum treated mice had developed diabetes, compared with only 28% of DiavaX-treated mice. None of the control peptides, including a linear I-A^g7, a scrambled cyclic I-A^g7, or an analogous cyclic I-A^speptide, reduced the incidence of diabetes, demonstrating that the protective effect of DiavaX is conformationally dependent and both allele- and sequence-specific. DiavaX treatment did not cause any general immune suppression, but did induce peptide-specific antibodies and memory T cells. DiavaX-induced protection from diabetes was associated with the maintenance of a non-destructive islet-associated autoimmune response. These data indicate that a conformationally constrained peptide from the disease-associated MHC represents a potential vaccine candidate for the prevention of clinical IDDM.     

Generation of β cell‐specific human cytotoxic T cells by lentiviral transduction and their survival in immunodeficient human leucocyte antigen‐transgenic mice

Several β cell antigens recognized by T cells in the non‐obese diabetic (NOD) mouse model of type 1 diabetes (T1D) are also T cell targets in the human disease. While numerous antigen‐specific therapies prevent diabetes in NOD mice, successful translation of rodent findings to patients has been difficult. A human leucocyte antigen (HLA)‐transgenic mouse model incorporating human β cell‐specific T cells might provide a better platform for evaluating antigen‐specific therapies. The ability to study such T cells is limited by their low frequency in peripheral blood and the difficulty in obtaining islet‐infiltrating T cells from patients. We have worked to overcome this limitation by using lentiviral transduction to ‘reprogram’ primary human CD8 T cells to express three T cell receptors (TCRs) specific for a peptide derived from the β cell antigen islet‐specific glucose‐6‐phosphatase catalytic subunit‐related protein (IGRP_265–273) and recognized in the context of the human class I major histocompatibility complex (MHC) molecule HLA‐A2. The TCRs bound peptide/MHC multimers with a range of avidities, but all bound with at least 10‐fold lower avidity than the anti‐viral TCR used for comparison. One exhibited antigenic recognition promiscuity. The β cell‐specific human CD8 T cells generated by lentiviral transduction with one of the TCRs released interferon (IFN)‐γ in response to antigen and exhibited cytotoxic activity against peptide‐pulsed target cells. The cells engrafted in HLA‐A2‐transgenic NOD‐scid IL2rγ^null mice and could be detected in the blood, spleen and pancreas up to 5 weeks post‐transfer, suggesting the utility of this approach for the evaluation of T cell‐modulatory therapies for T1D and other T cell‐mediated autoimmune diseases.     

Antigenic stimulation with cytochrome P450 2J expressed in mouse hepatocellular carcinoma cells regulates host anti‐tumour immunity

Cytochrome P450 2J subfamily (CYP2J) enzymes expressed in mouse hepatocellular carcinoma (HCC) cells were identified as an antigen recognized by specific CD4⁺ T cells and the structure of its T cell epitope was determined by proteomics‐based exploration. The major histocompatibility complex (MHC) class II binding peptides were isolated from I‐A^k/peptide complex of dendritic cells (DCs) loaded or unloaded with MIH‐2 mouse HCC cells. MHC class II‐binding peptides found in MIH‐2‐loaded DCs but not in unloaded DCs were determined by tandem mass spectrometric analysis. The peptide, consisting of amino acid 276–290 (DFIDAFLKEMTKYPE) of mouse CYP2J enzymes, was identified as an antigenic peptide presented in the context of MHC class II. Preventive treatment of mice with CYP2J peptide stimulated interferon (IFN)‐γ production of splenocytes and suppressed the growth of implanted CYP2J‐positive MIH‐2 cells but not CYP2J‐negative murine bladder tumour cells. However, continuous treatment of MIH‐2‐bearing mice with CYP2J peptide significantly suppressed IFN‐γ production of splenocytes and accelerated the growth of implanted MIH‐2 tumours in vivo. Increased frequencies of CD4⁺forkhead box P3 regulatory T cells and CD11b⁺Gr‐1⁺ myeloid suppressor cells were observed in splenocytes from the continuously immunized mice. These results indicate that antigenecity of CYP2J isoforms expressed in HCC cells activate host anti‐tumour immunity at an initial stage of HCC, but suppress host anti‐tumour immunity with excessive antigenic stimulation at an advanced stage.     

Fine specificity of anti‐citrullinated peptide antibodies discloses a heterogeneous antibody population in rheumatoid arthritis

Anti‐citrullinated peptide antibodies (ACPA) are highly specific for rheumatoid arthritis (RA). However, the predominant B cell epitopes have not yet been defined. The aim of this study was to examine the reactivity of ACPA against different peptides derived from citrullinated proteins and to investigate whether or not these antibodies constitute a homogeneous population. For this purpose, sera from patients with RA (n = 141), systemic lupus erythematosus (SLE) (n = 60), Sjögren's syndrome (SS) (n = 54) and healthy controls (n = 100) were tested for their reactivity against six citrullinated peptides derived from peptidyl arginine deiminase (PAD), vimentin (vim), alpha‐enolase (enol), fibrin, type II collagen (col‐II) and filaggrin, respectively. A non‐citrullinated control peptide derived from PAD was used as control (ctrlPAD^621–40). Antibody reactivity against each individual peptide was evaluated by enzyme‐linked immunosorbent assay (ELISA). Specificity and cross‐reactivity of ACPA were tested by using two prototype sera with homologous and cross‐inhibition assays. Specificity of ACPA from two prototype sera was confirmed by purification of anti‐peptide antibodies and homologous‐inhibition experiments. We found that sera from patients with RA reacted diversely with the six citrullinated peptides. More specifically, PAD^211–30 displayed 29·08% sensitivity, vim^60–75 29·08%, enol^5–21 37·59%, fibrin^617–31 31·21%, col‐II^358–75 29·97% and filaggrin^306–24 28·37%, while control ctrlPAD^621–40 showed no reactivity. All reactive peptides were found to be highly specific for RA. A notable cross‐reaction (>70%) was found mainly between filaggrin and the majority of anti‐citrullinated peptide antibodies. We concluded that ACPA in RA constitute a heterogeneous population with limited cross‐reactivity and without a predominant epitope.     

Cholera toxin subunit B peptide fusion proteins reveal impaired oral tolerance induction in diabetes‐prone but not in diabetes‐resistant mice

The cholera toxin B subunit (CTB) has been used as adjuvant to improve oral vaccine delivery in type 1 diabetes. The effect of CTB/peptide formulations on Ag‐specific CD4⁺ T cells has remained largely unexplored. Here, using tetramer analysis, we investigated how oral delivery of CTB fused to two CD4⁺ T‐cell epitopes, the BDC‐2.5 T‐cell 2.5mi mimotope and glutamic acid decarboxylase (GAD) 286–300, affected diabetogenic CD4⁺ T cells in nonobese diabetic (NOD) mice. When administered i.p., CTB‐2.5mi activated 2.5mi⁺ T cells and following intragastric delivery generated Ag‐specific Foxp3⁺ Treg and Th2 cells. While 2.5mi⁺ and GAD‐specific T cells were tolerized in diabetes‐resistant NODxB6.Foxp3^EGFP F1 and nonobese resistant (NOR) mice, this did not occur in NOD mice. This indicated that NOD mice had a recessive genetic resistance to induce oral tolerance to both CTB‐fused epitopes. In contrast to NODxB6.Foxp3^EGFP F1 mice, oral treatment in NOD mice lead to strong 2.5mi⁺ T‐cell activation and the sequestration of these cells to the effector‐memory pool. Oral treatment of NOD mice with CTB‐2.5mi failed to prevent diabetes. These findings underline the importance of investigating the effect of oral vaccine formulations on diabetogenic T cells as in selected cases they may have counterproductive consequences in human patients.     

Antitumor cytotoxic T-lymphocyte response in human lung carcinoma: identification of a tumor-associated antigen

Summary: We have isolated several cytotoxic T lymphocyte (CTL) clones from lymphocytes infiltrating a lung carcinoma of a patient with long survival. These clones showed a CD3⁺, CD8⁺, CD4^–, CD28^– phenotype and expressed a T‐cell receptor (TCR) encoded either by Vβ8‐Jβ1.5 or Vβ22‐Jβ1.4 rearrangements. Functional studies indicated that these clones mediated a high human leukocyte antigen (HLA)‐A2.1‐restricted cytotoxic activity against the autologous tumor cell line. Interestingly, TCRβ chain gene usage indicated that CTL clones identified in vitro were selectively expandedin vivo at the tumor site as compared to autologous peripheral blood lymphocytes (PBL). These findings provide evidence that an immune response may take place in non‐small cell lung carcinoma and that effector T cells may contribute to tumor regression. Further study indicated that the CTL clones recognized the same decamer peptide encoded by a mutated α‐actinin‐4 gene. Using tetramers of soluble HLA‐A2 molecules loaded with the mutated antigenic peptide, we have derived several anti‐α‐actinin‐4 T‐cell clones from patient PBL. These CTL, recognizing a truly tumor‐specific antigen, may play a role in the clinical evolution of this lung cancer patient. Adoptive transfer of CTL clones in a SCID/NOD mice model transplanted with autologous tumor supported their antitumor effect in vivo.     

NOD1 and NOD2 regulation of pulmonary innate immunity to Legionella pneumophila

The role of nucleotide‐binding oligomerization domain‐1 (NOD1) and nucleotide‐binding oligomerization domain‐2 (NOD2), cytoplasmic receptors which detect bacterial cell wall molecules, in pulmonary innate immune responses is poorly understood. We determined that both NOD1 and NOD2 detect heat‐killed Legionella and stimulate NF‐κb and IFN‐β promoter activity using an in vitro luciferase reporter system. We next infected NOD1‐ and NOD2‐deficient animals with aerosolized Legionella pneumophila. At 3 days post infection, Nod1^?/? mice had impaired bacterial clearance compared to WT controls. In addition, at 4 h and 24 h, Nod1^?/? mice had impaired neutrophil recruitment to the alveolar space. In contrast, increased lung neutrophils were seen in the Nod2^?/? animals at 24 h. Analysis of cytokine production at 4 h post infection revealed a significant decrease in proinflammatory cytokines in the Nod1^?/? animals when compared to WT animals. In contrast, increased 4‐h proinflammatory cytokines were seen in the Nod2^?/? animals. Furthermore, the lungs of both Nod1^?/? and Nod2^?/? mice had significantly increased pro‐inflammatory cytokine levels at 24 h, suggesting possible suppressive roles for later stages of infection. Together, our data suggest that although both NOD1 and NOD2 can detect Legionella, these receptors modulate the in vivo pulmonary immune response differently.     

Reversal of type 1 diabetes by a new MHC II‐peptide chimera: “Single‐epitope‐mediated suppression” to stabilize a polyclonal autoimmune T‐cell process

Polyclonality of self‐reactive CD4⁺ T cells is the hallmark of several autoimmune diseases like type 1 diabetes. We have previously reported that a soluble dimeric MHC II‐peptide chimera prevents and reverses type 1 diabetes induced by a monoclonal diabetogenic T‐cell population in double Tg mice [Casares, S. et al., Nat. Immunol. 2002. 3 : 383–391]. Since most of the glutamic acid decarboxylase 65 (GAD65)‐specific CD4⁺ T cells in the NOD mouse are tolerogenic but unable to function in an autoimmune environment, we have activated a silent, monoclonal T‐regulatory cell population (GAD65_217–230‐specific CD4⁺ T cells) using a soluble I‐A/GAD65_217–230/Fcγ2a dimer, and measured the effect on the ongoing polyclonal diabetogenic T‐cell process. Activated GAD65_217–230‐specific T cells and a fraction of the diabetogenic (B_9–23‐specific) T cells were polarized toward the IL‐10‐secreting T‐regulatory type 1‐like function in the pancreas of diabetic NOD mice. More importantly, this led to the reversal of hyperglycemia for more than 2 months post‐therapy in 80% of mice in the context of stabilization of pancreatic insulitis and improved insulin secretion by the β cells. These findings argue for the stabilization of a polyclonal self‐reactive T‐cell process by a single epitope‐mediated bystander suppression. Dimeric MHC class II‐peptide chimeras‐like approach may provide rational grounds for the development of more efficient antigen‐specific therapies in type 1 diabetes.     

4–1BB signal stimulates the activation,expansion, and effector functions of γδ T cells in mice and humans

We show here that the expression of 4–1BB is rapidly induced in γδ T cells following antigenic stimulation in both mice and humans, and ligation of the newly acquired 4–1BB with an agonistic anti‐4–1BB augments cell division and cytokine production. We further demonstrate that γδ rather than αβ T cells protect mice from Listeria monocytogenes (LM) infection and 4–1BB stimulation enhances the γδ T‐cell activities in the acute phase of LM infection. IFN‐γ produced from γδ T cells was the major soluble factor regulating LM infection. Vγ1⁺ T cells were expanded in LM‐infected mice and 4–1BB signal triggered an exclusive expansion of Vγ1⁺ T cells and induced IFN‐γ in these Vγ1⁺ T cells. Similarly, 4–1BB was induced on human γδ T cells and shown to be fully functional. Combination treatment with human γδ T cells and anti‐hu4–1BB effectively protected against LM infection in human γδ T cell‐transferred NOD‐SCID mice. Taken together, these data provide evidence that the 4–1BB signal is an important regulator of γδ T cells and induces robust host defense against LM infection.     

Chromogranin A is a T cell antigen in human type 1 diabetes

Chromogranin A (ChgA) is a beta cell secretory granule protein and a peptide of ChgA, WE14, was recently identified as a ligand for diabetogenic CD4 T cell clones derived from the NOD mouse. In this study we compared responses of human CD4 T cells from recent onset type 1 diabetic (T1D) and control subjects to WE14 and to an enzymatically modified version of this peptide. T cell responders to antigens were detected in PBMCs from study subjects by an indirect CD4 ELISPOT assay for IFN-γ. T1D patients (n = 27) were recent onset patients within one year of diagnosis, typed for HLA-DQ8. Controls (n = 31) were either 1st degree relatives with no antibodies or from the HLA-matched general population cohort of DAISY/TEDDY. A second cohort of patients (n = 11) and control subjects (n = 11) was tested at lower peptide concentrations. We found that WE14 is recognized by T cells from diabetic subjects vs. controls in a dose dependent manner. Treatment of WE14 with transglutaminase increased reactivity to the peptide in some patients. This work suggests that ChgA is an important target antigen in human T1D subjects and that post-translational modification may play a role in its reactivity and relationship to disease.     

Anti‐PR3 immune responses induce segmental and necrotizing glomerulonephritis

Wegener's granulomatosis (WG) is a life‐threatening autoimmune vasculitis that affects lungs, kidneys and other organs. A hallmark of WG is the presence of classic anti‐neutrophil cytoplasmic antibodies (c‐ANCA) against self‐proteinase 3 (PR3). Little is known about the role of these antibodies and PR3‐specific immune responses in disease development. In this study, we demonstrate that PR3‐specific autoimmune responses are pathogenic in non‐obese diabetic (NOD) mice with an impaired regulatory arm of the immune response. Immunization of autoimmunity prone NOD mice with rmPR3 (recombinant mouse PR3) in complete Freund's adjuvant (CFA) resulted in high levels of c‐ANCA, without detectable disease development. However, when splenocytes from these immunized mice were transferred into immunodeficient NOD–severe combined immunodeficiency (SCID) mice, the recipient mice developed vasculitis and severe segmental and necrotizing glomerulonephritis. No disease developed in NOD–SCID mice that received splenocytes from the CFA‐alone‐immunized donors (controls), indicating that disease development depends upon PR3‐specific immune responses. In contrast to the pathology observed in NOD–SCID mice, no disease was observed when splenocytes from rmPR3‐immunized C57BL/6 mice were transferred into immunodeficient C57BL/6‐RAG‐1^–/– mice, suggesting that complex and probably multi‐genetic factors play a role in the regulation of disease development.     

Altered monocyte cyclo‐oxygenase response in non‐obese diabetic mice

Monocytes infiltrate islets in non‐obese diabetic (NOD) mice. Activated monocyte/macrophages express cyclo‐oxygenase‐2 (COX‐2) promoting prostaglandin‐E₂ (PGE₂) secretion, while COX‐1 expression is constitutive. We investigated in female NOD mice: (i) natural history of monocyte COX expression basally and following lipopolysaccharide (LPS) stimulation; (ii) impact of COX‐2 specific inhibitor (Vioxx) on PGE₂, insulitis and diabetes. CD11b⁺ monocytes were analysed for COX mRNA expression from NOD (n = 48) and C57BL/6 control (n = 18) mice. NOD mice were treated with either Vioxx (total dose 80mg/kg) (n = 29) or methylcellulose as control (n = 29) administered by gavage at 4 weeks until diabetes developed or age 30 weeks. In all groups, basal monocyte COX mRNA and PGE₂ secretion were normal, while following LPS, after 5 weeks of age monocyte/macrophage COX‐1 mRNA decreased (P < 0·01) and COX‐2 mRNA increased (P < 0·01). However, diabetic NOD mice had reduced COX mRNA response (P = 0·03). Vioxx administration influenced neither PGE₂, insulitis nor diabetes. We demonstrate an isoform switch in monocyte/macrophage COX mRNA expression following LPS, which is altered in diabetic NOD mice as in human diabetes. However, Vioxx failed to affect insulitis or diabetes. We conclude that monocyte responses are altered in diabetic NOD mice but COX‐2 expression is unlikely to be critical to disease risk.     

The type 1 diabetes resistance locus B10 Idd9.3 mediates impaired B‐cell lymphopoiesis and implicates microRNA‐34a in diabetes protection

NOD.B10 Idd9.3 mice are congenic for the insulin‐dependent diabetes (Idd) Idd9.3 locus, which confers significant type 1 diabetes (T1D) protection and encodes 19 genes, including microRNA (miR)‐34a, from T1D‐resistant C57BL/10 mice. B cells have been shown to play a critical role in the priming of autoantigen‐specific CD4⁺ T cells in T1D pathogenesis in non‐obese diabetic (NOD) mice. We show that early B‐cell development is impaired in NOD.B10 Idd9.3 mice, resulting in the profound reduction of transitional and mature splenic B cells as compared with NOD mice. Molecular analysis revealed that miR‐34a expression was significantly higher in B‐cell progenitors and marginal zone B cells from NOD.B10 Idd9.3 mice than in NOD mice. Furthermore, miR‐34a expression in these cell populations inversely correlated with levels of Foxp1, an essential regulator of B‐cell lymphopoiesis, which is directly repressed by miR‐34a. In addition, we show that islet‐specific CD4⁺ T cells proliferated inefficiently when primed by NOD.B10 Idd9.3 B cells in vitro or in response to endogenous autoantigen in NOD.B10 Idd9.3 mice. Thus, Idd9.3‐encoded miR‐34a is a likely candidate in negatively regulating B‐cell lymphopoiesis, which may contribute to inefficient expansion of islet‐specific CD4⁺ T cells and to T1D protection in NOD.B10 Idd9.3 mice.     

Identification of overlapping epitopes in mutant ras oncogene peptides that activate CD4+ and CD8+ T cell responses

Mutant ras p21 proteins contain sequences which distinguish them from normal endogenous ras and, thus, may represent unique epitopes for T cell recognition of antigen bearing tumor cells. Here, we examined the capacity of a mutant K-ras 9-mer peptide to induce in vivo CD8⁺ cytotoxic T lymphocytes (CTL). The peptide chosen reflected positions 4–12 of the point-mutated sequence of the K-ras oncogene encoding the Gly to Val substitution at codon 12. The overall rationale for selecting this particular 9-mer sequence was threefold: the mutant peptide contained a putative major histocompatibility complex (MHC) class I consensus anchor motif for murine H-2K^d; specific binding to MHC class I may then create an immunogenic complex for the induction of anti-ras CD8⁺ CTL; and finally, the mutant sequence overlapped with a newly characterized anti-ras CD4⁺ T helper type 1 epitope, which may have implications for the coordination and activation of both anti-ras immune mechanisms against the same target cell antigenic determinant. A functional interaction with H-2K^d was demonstrated with the mutant ras4–12(V12) peptide, but not the normal ras4–12(G12) peptide, which specifically inhibited an H-2K^d-restricted, anti-nucleoprotein NP147–155 CTL response in a dose-dependent fashion. An anti-ras CD8⁺ T cell line was then established from immune splenocytes of BALB/c (H-2^d) mice injected with ras4–12(V12) in adjuvant, which mediated peptide-specific lysis of syngeneic P815 tumor targets. Cytotoxicity was restricted by H-2K^d and strongly specific for the mutant ras peptide. Importantly, these anti-ras CTL specifically lysed a syngeneic tumor line (i.e. A20 lymphoma) transduced with the corresponding point-mutated ras oncogene, suggesting T cell receptor recognition of endogenously derived antigen. Overall, these data demonstrated that mutant ras p21 at codon 12 (Gly → Val) contained a peptide sequence which exhibited specific functional binding to a murine MHC class I molecule; the ability of the mutant, but not the normal sequence to bind selectively to murine MHC class I likely reflected the generation of a C-terminal anchor residue; and the ras 4–12(V12) peptide was immunogenic for the production of antigen-specific CD8⁺ CTL, which lysed in vitro a syngeneic tumor cell line harboring the mutant K-ras oncogene.     

Abrogation of ICOS/ICOS ligand costimulation in NOD mice results in autoimmune deviation toward the neuromuscular system

NOD mice spontaneously develop insulin‐dependent diabetes around 10–40 wk of age. Numerous immune gene variants contribute to the autoimmune process. However, genes that direct the autoimmune response toward β cells remain ill defined. In this study, we provide evidence that the Icos and Icosl genes contribute to the diabetes process. Protection from diabetes in ICOS^?/? and ICOSL^?/? NOD mice was unexpectedly associated with the development of an autoimmune disorder of the neuro‐muscular system, characterized by myositis, sensory ganglionitis and, to a reduced extent, inflammatory infiltrates in the CNS. This syndrome was reproduced upon adoptive transfer of CD4⁺ and CD8⁺ T cells from diseased donors to naïve NOD.scid recipients. Our data further show that protection from diabetes results from defective activation of autoimmune diabetogenic effector T cells in ICOS^?/? NOD mice, whereas acceleration of diabetes in BDC2.5 ICOS^?/? NOD mice is induced by a dominant defect in Treg. Taken together, our findings indicate that costimulation signals play a key role in regulating immune tolerance in peripheral tissues and that the ICOS/ICOSL costimulatory pathway influences the balance between Treg and diabetogenic effector T cells.