Pancreatic IL-4 expression results in islet-reactive Th2 cells that inhibit diabetogenic lymphocytes in the nonobese diabetic mouse.

When immunological tolerance breaks down, autoimmune destruction of insulin-producing beta cells in the pancreas can cause insulin-dependent diabetes mellitus. We previously showed that transgenic nonobese diabetic (NOD) mice expressing IL-4 in the pancreas (NOD-IL-4 mice) were protected from insulitis and diabetes. Here we have characterized the avoidance of pathological autoimmunity in these mice. The absence of disease did not result from a lack of T cell priming, because T cells responding to dominant islet Ags were present. These islet Ag-specific T cells displayed a Th2 phenotype, indicating that Th2 responses could account for the observed tolerance. Interestingly, islet Ag-specific Th1 T cells were present and found to be functional, because neutralization of the Th2 effector cytokines IL-4 and IL-10 resulted in diabetes. Histological examination revealed that NOD-IL-4 splenocytes inhibited diabetogenic T cells in cotransfer experiments by limiting insulitis and delaying diabetes. Neutralization of IL-4 in this system abrogated the ability of NOD-IL-4 splenocytes to delay the onset of diabetes. These results indicate that IL-4 expressed in the islets does not prevent the generation of pathogenic islet responses but induces islet Ag-specific Th2 T cells that block the action of diabetogenic T cells in the pancreas.     

TLR2 agonist ameliorates murine experimental allergic conjunctivitis by inducing CD4 positive T-cell apoptosis rather than by affecting the Th1/Th2 balance

Innate immune responses that operate through Toll-like receptors (TLRs) are actively involved in the development of diseases predominantly mediated by adaptive immune responses. This is true also for allergic disease, as TLRs have been found to be involved in the development of allergic airway inflammation. We investigated whether stimulating TLR2 also abrogates murine allergic conjunctivitis by upregulating Th1 responses. We found that treating mice during the efferent phase with the TLR2 agonist Pam3CSK4 significantly suppressed eosinophil infiltration into the conjunctiva. However, Pam3CSK4 treatment inhibited both the Th1 and Th2 responses in the mice, and also suppressed eosinophil infiltration in IFN-gamma knockout mice. Flow cytometric analysis demonstrated that Pam3CSK4 treatment significantly elevated the numbers of annexin V-positive splenocytes, especially CD4 positive T cells. Thus, the stimulation of TLR2 during the efferent phase of murine allergic conjunctivitis suppresses eosinophil infiltration by inducing CD4 positive T-cell apoptosis rather than upregulating Th1 responses.     

A20 Is Critical for the Induction of Pam3CSK4-Tolerance in Monocytic THP-1 Cells

A20 functions to terminate Toll-like receptor (TLR)-induced immune response, and play important roles in the induction of lipopolysacchride (LPS)-tolerance. However, the molecular mechanism for Pam3CSK4-tolerance is uncertain. Here we report that TLR1/2 ligand Pam3CSK4 induced tolerance in monocytic THP-1 cells. The pre-treatment of THP-1 cells with Pam3CSK4 down-regulated the induction of pro-inflammatory cytokines induced by Pam3CSK4 re-stimulation. Pam3CSK4 pre-treatment also down-regulated the signaling transduction of JNK, p38 and NF-κB induced by Pam3CSK4 re-stimulation. The activation of TLR1/2 induced a rapid and robust up-regulation of A20, suggesting that A20 may contribute to the induction of Pam3CSK4-tolerance. This hypothesis was proved by the observation that the over-expression of A20 by gene transfer down-regulated Pam3CSK4-induced inflammatory responses, and the down-regulation of A20 by RNA interference inhibited the induction of tolerance. Moreover, LPS induced a significant up-regulation of A20, which contributed to the induction of cross-tolerance between LPS and Pam3CSK4. A20 was also induced by the treatment of THP-1 cells with TNF-α and IL-1β. The pre-treatment with TNF-α and IL-1β partly down-regulated Pam3CSK4-induced activation of MAPKs. Furthermore, pharmacologic inhibition of GSK3 signaling down-regulated Pam3CSK4-induced A20 expression, up-regulated Pam3CSK4-induced inflammatory responses, and partly reversed Pam3CSK4 pre-treatment-induced tolerance, suggesting that GSK3 is involved in TLR1/2-induced tolerance by up-regulation of A20 expression. Taken together, these results indicated that A20 is a critical regulator for TLR1/2-induced pro-inflammatory responses.     

Implication of NOD1 and NOD2 for the differentiation of multipotent mesenchymal stem cells derived from human umbilical cord blood

Toll-like receptors (TLRs) and Nod-like receptors (NLRs) are known to trigger an innate immune response against microbial infection. Although studies suggest that activation of TLRs modulate the function of mesenchymal stem cells (MSCs), little is known about the role of NLRs on the MSC function. In this study, we investigated whether NOD1 and NOD2 regulate the functions of human umbilical cord blood-derived MSCs (hUCB-MSCs). The genes of TLR2, TLR4, NOD1, and NOD2 were expressed in hUCB-MSCs. Stimulation with each agonist (Pam(3)CSK(4) for TLR2, LPS for TLR4, Tri-DAP for NOD1, and MDP for NOD2) led to IL-8 production in hUCB-MSC, suggesting the expressed receptors are functional in hUCB-MSC. CCK-8 assay revealed that none of agonist influenced proliferation of hUCB-MSCs. We next examined whether TLR and NLR agonists affect osteogenic-, adipogenic-, and chondrogenic differentiation of hUCB-MSCs. Pam(3)CSK(4) and Tri-DAP strongly enhanced osteogenic differentiation and ERK phosphorylation in hUCB-MSCs, and LPS and MDP also slightly did. Treatment of U0126 (MEK1/2 inhibitor) restored osteogenic differentiation enhanced by Pam(3)CSK(4). Tri-DAP and MDP inhibited adipogenic differentiation of hUCB-MSCs, but Pam(3)CSK(4) and LPS did not. On chondrogenic differentiation, all TLR and NLR agonists could promote chondrogenesis of hUCB-MSCs with difference in the ability. Our findings suggest that NOD1 and NOD2 as well as TLRs are involved in regulating the differentiation of MSCs.     

A critical role for mast cells and mast cell-derived IL-6 in TLR2-mediated inhibition of tumor growth

Several TLR agonists are effective in tumor immunotherapy, but their early innate mechanisms of action, particularly those of TLR2 agonists, are unclear. Mast cells are abundant surrounding solid tumors where they are often protumorigenic and enhance tumor angiogenesis. However, antitumor roles for mast cells have also been documented. The impact of mast cells may be dependent on their activation status and mediator release in different tumors. Using an orthotopic melanoma model in wild-type C57BL/6 and mast cell-deficient Kit(W-sh/W-sh) mice and a complementary Matrigel-tumor model in C57BL/6 mice, mast cells were shown to be crucial for TLR2 agonist (Pam(3)CSK(4))-induced tumor inhibition. Activation of TLR2 on mast cells reversed their well-documented protumorigenic role. Tumor growth inhibition after peritumoral administration of Pam(3)CSK(4) was restored in Kit(W-sh/W-sh) mice by local reconstitution with wild-type, but not TLR2-deficient, mast cells. Mast cells secrete multiple mediators after Pam(3)CSK(4) activation, and in vivo mast cell reconstitution studies also revealed that tumor growth inhibition required mast cell-derived IL-6, but not TNF. Mast cell-mediated anticancer properties were multifaceted. Direct antitumor effects in vitro and decreased angiogenesis and recruitment of NK and T cells in vivo were observed. TLR2-activated mast cells also inhibited the growth of lung cancer cells in vivo. Unlike other immune cells, mast cells are relatively radioresistant making them attractive candidates for combined treatment modalities. This study has important implications for the design of immunotherapeutic strategies and reveals, to our knowledge, a novel mechanism of action for TLR2 agonists in vivo.     

IFN regulatory factor 8 restricts the size of the marginal zone and follicular B cell pools

This study examined the effect of TLR2 activation by its specific ligand, Pam3CSK4, on cerebral ischemia/reperfusion (I/R) injury. Mice (n = 8/group) were treated with Pam3CSK4 1 h before cerebral ischemia (60 min), followed by reperfusion (24 h). Pam3CSK4 was also given to the mice (n = 8) 30 min after ischemia. Infarct size was determined by triphenyltetrazolium chloride staining. The morphology of neurons in brain sections was examined by Nissl staining. Pam3CSK4 administration significantly reduced infarct size by 55.9% (p < 0.01) compared with untreated I/R mice. Therapeutic treatment with Pam3CSK4 also significantly reduced infarct size by 55.8%. Morphologic examination showed that there was less neuronal damage in the hippocampus of Pam3CSK4-treated mice compared with untreated cerebral I/R mice. Pam3CSK4 treatment increased the levels of Hsp27, Hsp70, and Bcl2, and decreased Bax levels and NF-κB-binding activity in the brain tissues. Administration of Pam3CSK4 significantly increased the levels of phospho-Akt/Akt and phospho-GSK-3β/GSK-3β compared with untreated I/R mice. More significantly, either TLR2 deficiency or PI3K inhibition with LY29004 abolished the protection by Pam3CSK4. These data demonstrate that activation of TLR2 by its ligand prevents focal cerebral ischemic damage through a TLR2/PI3K/Akt-dependent mechanism. Of greater significance, these data indicate that therapy with a TLR2-specific agonist during cerebral ischemia is effective in reducing injury.     

I-Ag7-mediated antigen presentation by B lymphocytes is critical in overcoming a checkpoint in T cell tolerance to islet beta cells of nonobese diabetic mice.

B cell-deficient nonobese diabetic (NOD) mice are protected from the development of spontaneous autoimmune diabetes, suggesting a requisite role for Ag presentation by B lymphocytes for the activation of a diabetogenic T cell repertoire. This study specifically examines the importance of B cell-mediated MHC class II Ag presentation as a regulator of peripheral T cell tolerance to islet beta cells. We describe the construction of NOD mice with an I-Ag7 deficiency confined to the B cell compartment. Analysis of these mice, termed NOD BCIID, revealed the presence of functionally competent non-B cell APCs (macrophages/dendritic cells) with normal I-Ag7 expression and capable of activating Ag-reactive T cells. In addition, the secondary lymphoid organs of these mice harbored phenotypically normal CD4+ and CD8+ T cell compartments. Interestingly, whereas control NOD mice harboring I-Ag7-sufficient B cells developed diabetes spontaneously, NOD BCIID mice were resistant to the development of autoimmune diabetes. Despite their diabetes resistance, histologic examination of pancreata from NOD BCIID mice revealed foci of noninvasive peri-insulitis that could be intentionally converted into a destructive process upon treatment with cyclophosphamide. We conclude that I-Ag7-mediated Ag presentation by B cells serves to overcome a checkpoint in T cell tolerance to islet beta cells after their initial targeting has occurred. Overall, this work indicates that the full expression of the autoimmune potential of anti-islet T cells in NOD mice is intimately regulated by B cell-mediated MHC class II Ag presentation.     

TLR2 agonist ameliorates established allergic airway inflammation by promoting Th1 response and not via regulatory T cells

TLRs are primary sensors of both innate and adaptive immune systems, where they play a pivotal role in the response directed against structurally conserved components of pathogens. Synthetic bacterial lipopeptide Pam3CSK4 is a TLR2 agonist capable of modulating Th1 and Th2 responses. This study examines the therapeutic effect of Pam3CSK4 in established airway inflammation in a murine model of asthma. In mice previously sensitized and challenged with OVA, Pam3CSK4 given i.p. markedly reduced the total inflammatory cell infiltrate and eosinophilia in bronchoalveolar lavage fluid. Pam3CSK4 therapy was associated with a reduction in OVA-induced IL-4 and IL-5 secretion from thoracic lymph node culture, airways inflammation, bronchial hyperresponsiveness, and serum levels of IgE. Pam3CSK4 therapy was also associated with an increase in OVA-induced IFN-gamma, IL-12, and IL-10 production. However, the anti-inflammatory effect of Pam3CSK4 was independent of IL-10 or TGF-beta, but was critically dependent on IL-12, the production of which by dendritic cells was enhanced by Pam3CSK4 in vitro. Our results provide direct evidence that Pam3CSK4 could represent a novel therapeutic agent in allergic airways disease.     

Cutting edge: vasostatin-1-derived peptide ChgA29-42 is an antigenic epitope of diabetogenic BDC2.5 T cells in nonobese diabetic mice

Mechanistic and therapeutic insights in autoimmune diabetes would benefit from a more complete identification of relevant autoantigens. BDC2.5 TCR transgenic NOD mice express transgenes for TCR Vα1 and Vβ4 chains from the highly diabetogenic BDC2.5 CD4(+) T cell clone, which recognizes pancreatic β cell membrane Ags presented by NOD I-A(g7) MHC class II molecules. The antigenic epitope of BDC2.5 TCR is absent in β cells that do not express chromogranin A (ChgA) protein. However, characterization of the BDC2.5 epitope in ChgA has given inconclusive results. We have now identified a ChgA29-42 peptide within vasostatin-1, an N-terminal natural derivative of ChgA as the BDC2.5 TCR epitope. Having the necessary motif for binding to I-A(g7), it activates BDC2.5 T cells and induces an IFN-γ response. More importantly, adoptive transfer of naive BDC2.5 splenocytes activated with ChgA29-42 peptide transferred diabetes into NOD/SCID mice.     

Expression of toll‐like receptors and their regulatory roles in murine cardiac telocytes

Telocytes, newly discovered in the last decade, are interstitial cells found in numerous organs, with multiple proposed potential biological functions. Toll‐like receptors (TLRs) play an important role in innate and adaptive immunity by recognizing pathogen‐associated molecular patterns (PAMPs). However, it is still unknown whether telocytes express these innate receptors. We sought to determine the expression and role of TLRs in telocytes. In our study, we primarily detected TLR1‐9 expression in telocytes. The proliferation, apoptosis and immunoregulatory activity of telocytes activated with or without TLR ligands were determined. Our results showed that purified telocytes expressed TLR2, TLR3 and TLR5. In particular, telocytes expressed high levels of TLR2 as observed using flow cytometry. When we stimulated telocytes with TLR2 or TLR3 agonists (Pam3CSK4, PolyI:C), iNOS expression was greatly increased after Pam3CSK4 treatment. Additionally, telocyte proliferation was reduced and cell apoptosis was increased after TLR agonist stimulation. A co‐culture experiment showed that supernatant from telocytes pretreated with Pam3CSK4 inhibited T cell activation much more than that from untreated telocytes and this effect was mediated by iNOS. Overall, our results demonstrated TLR expression on telocytes for the first time and provided evidence of an immunoregulatory role of telocytes, indicating their clinical potential.     

Uncoupling of anergy from developmental arrest in anti-insulin B cells supports the development of autoimmune diabetes

Loss of tolerance is considered to be an early event that is essential for the development of autoimmune disease. In contrast to this expectation, autoimmune (type 1) diabetes develops in NOD mice that harbor an anti-insulin Ig transgene (125Tg), even though anti-insulin B cells are tolerant. Tolerance is maintained in a similar manner in both normal C57BL/6 and autoimmune NOD mice, as evidenced by B cell anergy to stimulation through their Ag receptor (anti-IgM), TLR4 (LPS), and CD40 (anti-CD40). Unlike B cells in other models of tolerance, anergic 125Tg B cells are not arrested in development, and they enter mature subsets of follicular and marginal zone B cells. In addition, 125Tg B cells remain competent to increase CD86 expression in response to both T cell-dependent (anti-CD40) and T cell-independent (anti-IgM or LPS) signals. Thus, for anti-insulin B cells, tolerance is characterized by defective B cell proliferation uncoupled from signals that promote maturation and costimulator function. In diabetes-prone NOD mice, anti-insulin B cells in this novel state of tolerance provide the essential B cell contribution required for autoimmune beta cell destruction. These findings suggest that the degree of functional impairment, rather than an overt breach of tolerance, is a critical feature that governs B cell contribution to T cell-mediated autoimmune disease.     

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.     

IFN-gamma affects homing of diabetogenic T cells.

IFN-gamma is a cytokine with pleiotropic functions that participates in immune and autoimmune responses. The lack of IFN-gamma is known to delay the development of autoimmune diabetes in nonobese diabetic (NOD) mice. Splenocytes from diabetic NOD and IFN-gamma knockout (KO) NOD mice transfer diabetes into NOD recipients equally well. However, adoptive transfer of diabetogenic T cells from NOD mice into NOD.IFN-gamma-KO or NOD mice lacking beta-chain of IFN-gamma receptor (NOD.IFN-gammaRbeta-KO) appeared to be much less efficient. We found that IFN-gamma influences the ability of diabetogenic cells to penetrate pancreatic islets. Tracing in vivo of insulin-specific CD8+ T cells has shown that homing of these cells to the islets of Langerhans was affected by the lack of IFN-gamma. While adhesion of insulin-specific CD8+ cells to microvasculature was normal, the diapedesis was significantly impaired. This effect was reversible by treatment of the animals with rIFN-gamma. Thus, IFN-gamma may, among other effects, influence immune and autoimmune responses by supporting the homing of activated T cells.     

Differential Effects of the Toll-Like Receptor 2 Agonists,PGN and Pam3CSK4 on Anti-IgE Induced Human Mast Cell Activation

Mast cells are pivotal in the pathogenesis of allergy and inflammation. In addition to the classical IgE-dependent mechanism involving crosslinking of the high-affinity receptor for IgE (FcεRI), mast cells are also activated by Toll-like receptors (TLRs) which are at the center of innate immunity. In this study, we demonstrated that the response of LAD2 cells (a human mast cell line) to anti-IgE was altered in the presence of the TLR2 agonists peptidoglycan (PGN) and tripalmitoyl-S-glycero-Cys-(Lys)4 (Pam3CSK4). Pretreatment of PGN and Pam3CSK4 inhibited anti-IgE induced calcium mobilization and degranulation without down-regulation of FcεRI expression. Pam3CSK4 but not PGN acted in synergy with anti-IgE for IL-8 release when the TLR2 agonist was added simultaneously with anti-IgE. Studies with inhibitors of key enzymes implicated in mast cell signaling revealed that the synergistic release of IL-8 induced by Pam3CSK4 and anti-IgE involved ERK and calcineurin signaling cascades. The differential modulations of anti-IgE induced mast cell activation by PGN and Pam3CSK4 suggest that dimerization of TLR2 with TLR1 or TLR6 produced different modulating actions on FcεRI mediated human mast cell activation.     

IL-6 and maturation govern TLR2 and TLR4 induced TLR agonist tolerance and cross-tolerance in dendritic cells

Stimulation of naive mouse dendritic cells (DC) with LPS or Pam(3)CSK(4) (P3C) induces production of TNF-alpha via TLR4- or TLR2-signaling. Although tolerance in macrophages has been studied in detail, we investigated the role of TLR agonist concentration and IL-6 for tolerance in DC. P3C- or LPS-primed DC were nonresponsive to P3C or LPS restimulation in terms of TNF-alpha but not IL-6 production. The mechanisms involved in tolerance were dependent on the concentration of the TLR ligand used for DC priming. DC primed with LPS or P3C at high concentrations developed a maturation dependent, IL-6 independent tolerance associated with inhibition of TLR signaling upstream of IkappaB as indicated by decreased IkappaB degradation. In contrast, priming of DC with LPS or P3C at low concentrations resulted in IL-6-dependent tolerance, which was abolished in IL-6 deficient DC, and was not accompanied by maturation of DC or by down-regulation of TLR2 or TLR4. In homotolerogenic DC primed with LPS or P3C at high concentrations, degradation of IkappaB upon restimulation with LPS or P3C was inhibited suggesting tolerance mechanism(s) upstream of IkappaB; in contrast, cross-tolerance in DC primed with LPS or P3C at low concentrations was not associated with reduced IkappaB degradation suggesting tolerance mechanisms downstream of IkappaB. Our data indicate that in naive DC TLR4- and TLR2-stimulation results in homo- and cross-tolerance; the mechanisms involved in tolerance depend on the concentration of the TLR agonist used for DC priming and are governed by IL-6 and maturation.     

Raji, P3HR-1 and Namalwa cells as a model for the study of Epstein-Barr virus (EBV) reactivation

The aim of the study was to characterize Raji, P3HR-1 and Namalwa cell lines in the aspect of their usefulness for the research on virus Epstein-Barr (EBV) reactivation, with the participation of Toll-like receptors (TLR). During a 12-day experiment, optimal conditions of cultivation (RPMI with 10% FCS at 37 degrees C in 5% CO2) were determined. In these conditions cells showed logarithmic growth. The presence of the DNA EBV was confirmed by the PCR method, showing that 12-day long maintenance of cells does not cause the loss of the virus. The presence of genes encoding TLR2, TLR3 and TLR4 was also confirmed by PCR. The TLRs expression at the mRNA level in cells subjected to 24h stimulation with TLR2, TLR3 and TLR4 agonist (Pam3CSK4, Poly(I:C) and LPS, respectively) was determined by the RT PCR method. The presence ofTLR4 mRNA was confirmed in the case of Namalwa cells stimulated by Pam3CSK and LPS, and P3HR cells stimulated by Pam3CSK4. In the case of Raji cells the expression of none of the receptors was confirmed at the mRNA level in cells with and without stimulation.     

Lipopolysaccharide-activated B cells down-regulate Th1 immunity and prevent autoimmune diabetes in nonobese diabetic mice.

B cells can serve dual roles in modulating T cell immunity through their potent capacity to present Ag and induce regulatory tolerance. Although B cells are necessary components for the initiation of spontaneous T cell autoimmunity to beta cell Ags in nonobese diabetic (NOD) mice, the role of activated B cells in the autoimmune process is poorly understood. In this study, we show that LPS-activated B cells, but not control B cells, express Fas ligand and secrete TGF-beta. Coincubation of diabetogenic T cells with activated B cells in vitro leads to the apoptosis of both T and B lymphocytes. Transfusion of activated B cells, but not control B cells, into prediabetic NOD mice inhibited spontaneous Th1 autoimmunity, but did not promote Th2 responses to beta cell autoantigens. Furthermore, this treatment induced mononuclear cell apoptosis predominantly in the spleen and temporarily impaired the activity of APCs. Cotransfer of activated B cells with diabetogenic splenic T cells prevented the adoptive transfer of type I diabetes mellitus (T1DM) to NOD/scid mice. Importantly, whereas 90% of NOD mice treated with control B cells developed T1DM within 27 wk, <20% of the NOD mice treated with activated B cells became hyperglycemic up to 1 year of age. Our data suggest that activated B cells can down-regulate pathogenic Th1 immunity through triggering the apoptosis of Th1 cells and/or inhibition of APC activity by the secretion of TGF-beta. These findings provide new insights into T-B cell interactions and may aid in the design of new therapies for human T1DM.     

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.     

TNF receptor 1 deficiency increases regulatory T cell function in nonobese diabetic mice

TNF has been implicated in the pathogenesis of type 1 diabetes. When administered early in life, TNF accelerates and increases diabetes in NOD mice. However, when administered late, TNF decreases diabetes incidence and delays onset. TNFR1-deficient NOD mice were fully protected from diabetes and only showed mild peri-insulitis. To further dissect how TNFR1 deficiency affects type 1 diabetes, these mice were crossed to β cell-specific, highly diabetogenic TCR transgenic I-A(g7)-restricted NOD4.1 mice and Kd-restricted NOD8.3 mice. TNFR1-deficient NOD4.1 and NOD8.3 mice were protected from diabetes and had significantly less insulitis compared with wild type NOD4.1 and NOD8.3 controls. Diabetic NOD4.1 mice rejected TNFR1-deficient islet grafts as efficiently as control islets, confirming that TNFR1 signaling is not directly required for β cell destruction. Flow cytometric analysis showed a significant increase in the number of CD4(+)CD25(+)Foxp3(+) T regulatory cells in TNFR1-deficient mice. TNFR1-deficient T regulatory cells were functionally better at suppressing effector cells than were wild type T regulatory cells both in vitro and in vivo. This study suggests that blocking TNF signaling may be beneficial in increasing the function of T regulatory cells and suppression of type 1 diabetes.     

Regulation of diabetes development by regulatory T cells in pancreatic islet antigen-specific TCR transgenic nonobese diabetic mice

Nonobese diabetic (NOD) mice carrying a transgenic TCR from an islet Ag-specific CD4 T cell clone, BDC2.5, do not develop diabetes. In contrast, the same transgenic NOD mice on the SCID background develop diabetes within 4 wk after birth. Using a newly developed mAb specific for the BDC2.5 TCR, we examined the interaction between diabetogenic T cells and regulatory T cells in NOD.BDC transgenic mice. CD4 T cells from NOD.BDC mice, expressing high levels of the clonotype, transfer diabetes to NOD.SCID recipients. In contrast, CD4 T cells expressing low levels due to the expression of both transgenic and endogenous TCR alpha-chains inhibit diabetes transfer. The clonotype-low CD4 T cells appear late in the ontogeny in the thymus and peripheral lymphoid organs, coinciding with resistance to cyclophosphamide-induced diabetes. These results demonstrate that diabetic processes in NOD.BDC mice are regulated by a balance between diabetogenic T cells and regulatory T cells. In the absence of specific manipulation, regulatory T cell function seems to be dominant and mice remain diabetes free. Understanding of mechanisms by which regulatory T cells inhibit diabetogenic processes would provide means to prevent diabetes development in high-risk human populations.