Defect in activation-induced cell death in non-obese diabetic (NOD) T lymphocytes

Activation-induced cell death (AICD) represents a major means of peripheral tolerance induction, eliminating effector cells. NOD mice, a widely used model for autoimmune diabetes, are characterized by high levels of circulating T lymphocytes and by resistance to several apoptosis-inducing signals. The aim of this study was to analyse AICD in peripheral NOD T lymphocytes. First, we demonstrated in an in vitro AICD model that NOD T lymphocytes are more resistant to AICD (64+/-2%) compared to non-autoimmune C57BL/6 T lymphocytes (73+/-2%), but also diabetes-resistant NOR T lymphocytes (76+/-3%, P<0.05). Moreover, both CD4(+)and CD8(+)subsets were affected. Analysis of the cellular and molecular pathways revealed lower caspase 8 levels, a central caspase proximally involved in the AICD-pathway (fluorescence of 258+/-47 in NOD vs. 441+/-16 in NOR and 414+/-61 in C57BL/6 T lymphocytes, P<0.05). Gene expression analysis using real-time RT-PCR additionally revealed low expression of Fas and FasL, the death receptor system activating caspase 8 and contributing to AICD. Additionally, low IL-2 levels, together with high TGFbeta and Bclx-L levels, confirm the presence of a NOD-specific AICD-resistance profile. In conclusion, we present cellular and molecular evidence for disturbed AICD mechanisms in NOD T lymphocytes. This resistance in AICD may contribute to defective tolerance induction to autoantigens in NOD mice.     

Disruption of the homeostatic balance between autoaggressive (CD4+CD40+) and regulatory (CD4+CD25+FoxP3+) T cells promotes diabetes

Although regulatory T cells (Tregs) are well described, identifying autoaggressive effector T cells has proven more difficult. However, we identified CD4loCD40+ (Th40) cells as being necessary and sufficient for diabetes in the NOD mouse model. Importantly, these cells are present in pancreata of prediabetic and diabetic NOD mice, and Th40 cells but not CD4+CD40(-) T cells transfer progressive insulitis and diabetes to NOD.scid recipients. Nonobese-resistant (NOR) mice have the identical T cell developmental background as NOD mice, yet they are diabetes-resistant. The seminal issue is how NOR mice remain tolerant to diabetogenic self-antigens. We show here that autoaggressive T cells develop in NOR mice and are confined to the Th40 subset. However, NOR mice maintain Treg numbers equivalent to their Th40 numbers. NOD mice have statistically equal numbers of CD4+CD25+forkhead box P3+intrinsic Tregs compared with NOR or nonautoimmune BALB/c mice, and NOD Tregs are equally as suppressive as NOR Tregs. A critical difference is that NOD mice develop expanded numbers of Th40 cells. We suggest that a determinant factor for autoimmunity includes the Th40:Treg ratio. Mechanistically, NOD Th40 cells have low susceptibility to Fas-induced cell death and unlike cells from NOR and BALB/c mice, have predominantly low Fas expression. CD40 engagement of Th40 cells induces Fas expression but further confers resistance to Fas-mediated cell death in NOD mice. A second fundamental difference is that NOD Th40 cells undergo much more rapid homeostatic expansion than Th40 cells from NOR mice.     

1Alpha,25-dihydroxyvitamin D3 restores thymocyte apoptosis sensitivity in non-obese diabetic (NOD) mice through dendritic cells

AIMS/HYPOTHESIS: Resistance of NOD thymocytes to apoptosis-inducing signals is restored by 1alpha,25-dihydroxyvitamin D3 (1alpha,25OH2D3), a therapy preventing diabetes in NOD mice. We studied whether modulation of thymocyte apoptosis is due to direct effects on thymic T lymphocytes or indirect effects via thymic dendritic cells, since both cell types constitute known targets for 1alpha,25OH2D3. METHODS AND RESULTS: Female NOD mice were treated with 1alpha,25OH2D3 (5microg/kg/2d) from 21 to 70 days. Vehicle-treated NOD and NOR mice served as controls. Analysis of thymic T lymphocytes from 1alpha,25OH2D3)-treated mice revealed a decrease in number of apoptosis-resistant CD4+CD8+ and CD4+CD8-HSA(high) T lymphocyte subsets, higher pro-apoptotic IL-2 and FasL, and lower anti-apoptotic Bclx-L mRNA expression levels. Thymic dendritic cells from 1alpha,25OH2D3-treated NOD mice had increased CD8alpha+FasL+ and CD80+/86+ expression compared to control NOD mice. In a syngeneic co-culture system of thymocytes and thymic dendritic cells, apoptosis levels were 20% higher only in co-cultures where both T cell- and dendritic cell-compartments originated from 1alpha,25OH2D3-treated mice. Activation-induced cell death-sensitivity in peripheral T lymphocytes was comparable to levels present in NOR mice, confirming better thymic selection in 1alpha,25OH2D3-treated mice. CONCLUSION/INTERPRETATION: We conclude that 1alpha,25OH2D3 needs both thymic T cell- and dendritic cell-compartments to exert its apoptosis-restorative effects in NOD thymocytes.     

Peripheral CD4loCD40+ auto-aggressive T cell expansion during insulin-dependent diabetes mellitus

The generation of auto-aggressive T cells involves failure of central or peripheral tolerance. We previously demonstrated that peripheral CD4(lo)CD40(+) T cells give rise to pathogenic T cells in the non-obese diabetic (NOD) model. Here we show that peripheral CD4(+)CD40(+) T cells from diabetic or pre-diabetic NOD mice induce insulin-dependent diabetes mellitus. Consistent with breach of peripheral tolerance, CD4(lo)CD40(+) T cells expand with age in NOD mice but not in MHC-matched non-obese resistant (NOR) or BALB/c controls. Suggestive of a causal role for CD40 in autoimmunity, blocking CD40-CD154 interactions early during NOD development prevents autoaggressive T cell expansion while promoting increases in CD4(+)CD25(+) regulatory T cells. Importantly, CD40 signals promote expansion of V alpha 3.2(+) and V alpha 8.3(+) T cells. Furthermore, peripheral V alpha 3.2(+)CD40(+) T cells induce diabetes in NOD.scid recipients while V alpha 8.3(+) T cells or V alpha 3.2(+)-depleted T cell populations do not. This is the first demonstration that primary T cells transfer disease with the kinetics of auto-aggressive T cell clones and that specific TCR V alpha expansion promotes diabetes.     

Antigen-induced T cell death is regulated by CD4 expression

Activation induced cell death (AICD) is a major physiologic pathway that regulates T cell homeostasis. In CD4 T cells, AICD is mediated mainly through Fas/FasL interactions. Although TCR occupancy triggers AICD, the contribution of its tightly associated CD4 coreceptor to the process that leads to AICD is not known. Here we show that CD4 molecule plays an essential regulatory role of TCR dependent AICD. Loss of CD4 rendered activated 5kc T cell hybridoma resistant to AICD. The resistance of CD4 negative 5kc T cells to AICD was due to selective inhibition of FasL expression and it could be reversed by addition of recombinant FasL. Furthermore, a direct functional link between CD4 and FasL was demonstrated by induction of FasL upon CD4 crosslinking in a TCR independent fashion. The importance of CD4 interaction with MHC/peptide complex in mediating AICD was also evident in normal T cells that could survive chronic stimulation with anti-CD3 but died after short period of proliferation after stimulation with MHC/peptide. Thus it appears that AICD is controlled by the CD4 molecule via regulation of FasL expression. These findings have important implications for our understanding of mechanisms of peripheral tolerance as well as pathogenesis of autoimmune diseases.     

Antigen-Induced T Cell Death Is Regulated by CD4 Expression

Activation induced cell death (AICD) is a major physiologic pathway that regulates T cell homeostasis. In CD4 T cells, AICD is mediated mainly through Fas/FasL interactions. Although TCR occupancy triggers AICD, the contribution of its tightly associated CD4 coreceptor to the process that leads to AICD is not known. Here we show that CD4 molecule plays an essential regulatory role of TCR dependent AICD. Loss of CD4 rendered activated 5kc T cell hybridoma resistant to AICD. The resistance of CD4 negative 5kc T cells to AICD was due to selective inhibition of FasL expression and it could be reversed by addition of recombinant FasL. Furthermore, a direct functional link between CD4 and FasL was demonstrated by induction of FasL upon CD4 cross-linking in a TCR independent fashion. The importance of CD4 interaction with MHC/peptide complex in mediating AICD was also evident in normal T cells that could survive chronic stimulation with anti-CD3 but died after short period of proliferation after stimulation with MHC/peptide. Thus it appears that AICD is controlled by the CD4 molecule via regulation of FasL expression. These findings have important implications for our understanding of mechanisms of peripheral tolerance as well as pathogenesis of autoimmune diseases.     

CD4+CD25+ regulatory T cells generated in response to insulin B:9-23 peptide prevent adoptive transfer of diabetes by diabetogenic T cells

NOD mice have a relative deficiency of CD4+CD25+ regulatory T cells that could result in an inability to maintain peripheral tolerance. The aim of this study was to induce the generation of CD4+CD25+ regulatory T cells in response to autoantigens to prevent type 1 diabetes (T1D). We found that immunization of NOD mice with insulin B-chain peptide B:9-23 followed by 72 h in vitro culture with B:9-23 peptide induces generation of CD4+CD25+ regulatory T cells. Route of immunization has a critical role in the generation of these cells. Non-autoimmune mice BALB/c, C57BL/6 and NOR did not show up regulation of CD4+CD25+ regulatory T cells. These cells secreted large amounts of TGF-beta and TNF-alpha with little or no IFN-gamma and IL-10. Adoptive transfer of these CD4+CD25+ regulatory T cells into NOD-SCID mice completely prevented the adoptive transfer of disease by diabetogenic T cells. Although, non-self antigenic OVA (323-339) peptide immunization and in vitro culture with OVA (323-339) peptide does result in up regulation of CD4+CD25+ T cells, these cells did not prevent transfer of diabetes. Our study for the first time identified the generation of antigen-specific CD4+CD25+ regulatory T cells specifically in response to immunization with B:9-23 peptide in NOD mice that are capable of blocking adoptive transfer of diabetes. Our results suggest the possibility of using autoantigens to induce antigen-specific regulatory T cells to prevent and regulate autoimmune diabetes.     

Increased proliferation of CD8+ T cells in SAP-deficient mice is associated with impaired activation-induced cell death

Defective signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) is responsible for the human X-linked lymphoproliferative syndrome. Defects in T helper 2, natural killer, natural killer T and B cells have been demonstrated in SAP-deficient humans and mice, and increased proliferation of CD8+ T cells has been observed. In the current study, we investigated the properties of CD8+ T cell proliferation and activation-induced cell death (AICD), using OT-I T cell receptor (TCR)-transgenic mice on either wild-type (WT) or SAP-/- background. Interestingly, we found that ovalbumin peptide-activated SAP-/- CD8+ T cells have lower AICD compared to their WT counterparts. Furthermore, the induction of p73, a key mediator of TCR-induced apoptosis through the mitochondrial apoptotic pathway, was significantly reduced at both the mRNA and protein levels in the activated mutant cells. Meanwhile, a reduced level of activated caspase 9 was observed in the mutant cells. We conclude that reduced AICD in activated SAP-/- CD8+ T cells is associated with impaired p73 induction, indicating that the initiation of the mitochondrial apoptotic pathway might be impaired. Our data demonstrate an intrinsic defect in SAP-/- CD8+ T cells and shed light on the increased responsiveness of CD8+ T cells in SAP-/- mice.     

IFN-gamma regulates Fas ligand expression in human CD4+ T lymphocytes and controls their anti-mycobacterial cytotoxic functions

Fas and Fas Ligand (FasL) expression, activation-induced cell death (AICD) and mycobacterial antigen-specific cytotoxicity of peripheral T cells from patients with complete inherited IFN-gamma receptor 1 binding chain deficiency (IFN-gammaR1-/-) were investigated. Fas was equally expressed in both normal and deficient T lymphoblasts and they underwent apoptosis when stimulated with agonist anti-Fas mAb. By contrast, T lymphoblasts and CD4+ T cell clones (TCC) from deficient patients displayed a reduced surface FasL expression and resistance to AICD. CD8+ TCC from healthy and deficient patients displayed similar high level of FasL and susceptibility to AICD. In Jurkat CD4+ T cells competent to transduce IFN-gamma signaling, IFN-gamma induced surface FasL export and their Fas-dependent apoptosis. Effector T cells generated from a patient with a dominant negative mutation of IFN-gammaR1 (IFN-gammaR1DN) following stimulation with mycobacterial antigens were unable to kill MHC class II-matched, mycobacterial antigen-pulsed macrophages. Normal Fas expression in T cells and FasL in CD8+ cells may account for the absence of autoimmune disorders in these patients. Conversely, defective FasL expression on IFN-gammaR1DN CD4+ T cells impairs their cytotoxic functions and highlights a novel role for IFN-gamma signaling in the control of mycobacterial infection in humans.     

IL-4 influences the differentiation and the susceptibility to activation-induced cell death of human naive CD8+ T cells

It is now well established that the cytokine environment influences the activation, differentiation, proliferation and death of T lymphocytes during the primary response to antigen. Using an in vitro model, we investigated the influence of IL-4, added at the onset of TCR stimulation, on phenotypic and functional markers of naive CD8+ T cell activation including the up-regulation of activation markers, proliferation as well as the susceptibility to activation-induced cell death (AICD). We report that IL-4, unlike IL-2 added at the onset of repeated TCR stimulation of naive CD8+ T cells prevents AICD, in part due to its ability to maintain the level of the survival-related protein Bcl-2. Moreover, TCR-triggered activation of naive CD8+ T cells in the presence of IL-4 leads to the development of a CD8+ T cell subset that proliferates normally, but which fails to exhibit characteristic activation parameters such as the up-regulation of CD25 and Granzyme B. Taken together, these results demonstrate that exposure to IL-4 during primary activation influences CD8+ T cell differentiation by inducing the development of a sub-population of AICD-resistant, proliferation-competent cells that do not show some of the typical features of CD8+ T cell activation.     

The Expression of Cytokine Genes in the Peritoneal Macrophages and Splenic CD4- and CD8-Positive Lymphocytes of the Nonobese Diabetic Mice

Type 1 diabetes is an autoimmune disease that results from the destruction of the insulin-producing pancreatic beta islet cells, probably via the influence of cytokines. However, direct correlation between the expression of selected cytokines by various immune cells at different time points during the progression of the disease has not yet been clearly demonstrated. In this study, we showed that the mRNA expression of the pro-inflammatory cytokines, TNF-alpha, IL-1 beta, IL-6, and GM-CSF, were increased while the anti-inflammatory cytokine, TGF-beta, decreased in the peritoneal macrophages of nonobese diabetic (NOD) mice. IL-6 expression however decreased when the mice became diabetic. Surprisingly the expression of IFN-gamma and IL-2 by splenic CD4+ cells were lower in 5-week-old NOD mice as compared to the nonobese diabetic resistant (NOR) control mice, but their expression was higher in older NOD mice. The expression of IL-4 and IL-10 decreased in splenic CD4-positive lymphocytes. Splenic CD8-positive lymphocytes expressed increased levels of IFN-gamma and IL-10 but the latter decreased sharply when diabetes occurred. The relevance of these findings to the pathogenesis of type 1 diabetes is discussed.     

Resistance to activation-induced cell death and elevated FLIPL expression of CD4+ T cells in a polyI:C-induced primary biliary cirrhosis mouse model

Primary biliary cirrhosis (PBC) is a type of organ-specific autoimmune disease in which immune tolerance is impaired by an unknown mechanism. We established a PBC animal model by injecting C57BL/6 mice with polyI:C to study activation-induced cell death (AICD) in CD4+ T lymphocytes and changes of apoptosis-associated molecules as a first step to understand the immune tolerance of PBC mice. Obvious inflammatory cell infiltration was observed in the portal area of the liver tissues in model mice and antimitochondrial antibodies (AMA) positive rate was 80%. The AICD level in both splenic and hepatic CD4+ T cells in the model group were all lower than those in controls, and in the model group the level for hepatic CD4+ T cells were significantly lower than that for splenic CD4+ T cells. Quantitative PCR revealed that FasL mRNA and TRAIL expression in CD4+ T cells in the model group decreased significantly compared with that in the control group. Western blots revealed that the expression of the anti-apoptotic protein FLIP_L in the model group increased significantly with the FLIP_L expression in hepatic CD4+ T cells significantly higher than that in splenic CD4+ T cells. There was a positive linear correlation between the number of infiltrated portal areas and relative expression of FLIP_L in splenic CD4+ T cells in model group. There were no obvious changes for caspase-8 in either group. These results show that the anti-apoptotic ability of CD4+ T lymphocytes play an important role in immune tolerance in the PBC mouse model, and elevated FLIP_L expression may enhance this ability. The inhibition of FasL and TRAIL expression may also help enhance this anti-apoptotic ability in CD4+ T lymphocytes and contribute to the aggravation of portal area inflammation.     

Intracellular expression of MICA in activated CD4 T lymphocytes and protection from NK cell-mediated MICA-dependent cytotoxicity

MICA is a stress-regulated molecule recognized by the NK cell-activating receptor NKG2D. Previously, we demonstrated that MICA is induced on activated T cells but regulation by mitogenic cytokines and its biological consequences remain unexplored. Here, we show that IL-2, IL-4, and IL-15 but not TNF-alpha or IFN-alpha induced MICA expression in T lymphocytes present in peripheral blood mononuclear cells (PBMCs), as assessed by Western blot. IL-2 effect involved Jak3/STAT5, p38 MAPK, p70(56) kinase, Lck/fyn kinases, and NF-kappaB. MICA expression was also observed in Th1 and Th2 cells. However, surface expression was not detected. T lymphocytes present in PBMCs and isolated CD4+ T lymphocytes stimulated with phorbol-12-myristate-13-acetate and ionomycin also induced MICA expression as assessed by Western blot, but only low levels were expressed at the cell surface. Activated but not resting CD4+ T lymphocytes were lysed by IL-15- or IL-2-stimulated NK cells, and susceptibility was increased when HLA class I molecules were blocked. Also, cytokine-stimulated NK cells produced more IFN-gamma after culture with activated CD4+ T lymphocytes. However, the participation of MICA in these responses, if any, was marginal. Confocal microscopy revealed that MICA is retained mostly inside activated CD4+ T cells. Our results suggest that low surface expression of MICA on activated CD4+ T lymphocytes might be a safeguard mechanism to protect them from NK cells in an inflammatory, virus-infected, or tumor microenvironment, where NK and activated CD4+ T cells are recruited.     

TNFR1 delivers pro-survival signals that are required for limiting TNFR2-dependent activation-induced cell death (AICD) in CD8+ T cells

Members of the TNF and TNF receptor (TNFR) superfamily play important roles in the maintenance of homeostasis of the immune system. Furthermore, several members of the TNFR family participate in T-cell activation and sustaining T-cell responses. We have shown that TNFR2 regulates T-cell activation by lowering the activation threshold and providing costimulatory signaling. Furthermore, activated TNFR2(-/-) CD8(+) T cells are highly resistant to activation-induced cell death (AICD). Here, we showed that using anti-TNFR2 antibodies to block TNFR2 on activated WT CD8(+) T cells rendered them resistant to AICD. This resistance of activated TNFR2(-/-) CD8(+) T cells to AICD correlated with the accumulation of TNF receptor-associated factor 2 (TRAF2). Overexpression of TRAF2 by retroviral transfection and knockdown of TRAF2 by small interfering RNA also support this conclusion. Furthermore, neutralizing TNF-α reduced TRAF2 accumulation in activated TNFR2(-/-) CD8(+) T cells and increased their susceptibility to AICD. AICD-resistant TNFR2(-/-) CD8(+) T cells expressed elevated levels of phosphorylated IκBα and higher DNA-binding activity of the p65 NK-κB subunit and neutralization of TNF-α blocked this increase. Therefore, in activated TNFR2(-/-) CD8(+) T cells, TNFR1 functions as a survival receptor by utilizing high intracellular levels of TRAF2 to promote IκBα phosphorylation and NF-κB activation.     

Glutamine protects activated human T cells from apoptosis by up-regulating glutathione and Bcl-2 levels

Glutamine is the most abundant amino acid in the body. A decrease of plasma glutamine concentrations is found in catabolic stress and is related to susceptibility to infections. Glutamine is known to modulate lymphocyte activation; however, little is known about glutamine modulation of cell death of activated human T cells. Using Jurkat T cells, we investigated glutamine modulation of T-cell apoptosis activated by PMA plus ionomycin. We found that glutamine at various concentrations significantly enhanced IL-2 production, cell proliferation, and cell viability of Jurkat T cells. Glutamine also decreased the number of apoptotic cells stimulated with PMA plus ionomycin as demonstrated by flow cytometry. Meanwhile, glutamine down-regulated CD95 and CD95L expression, but up-regulated CD45RO and Bcl-2 expression in activated T cells. Further investigation of CD95-mediated caspase activities revealed that supplementation of glutamine significantly decreased caspase-3 and caspase-8 activities in activated T cells. Since oxidative stress is closely associated with induction of lymphocyte apoptosis, we found that glutamine significantly increased glutathione (GSH), but decreased reactive oxygen species levels in activated T cells. Blockade of intracellular GSH formation enhanced, but exogenous GSH supplementation decreased, activated T-cell apoptosis. Studying normal peripheral lymphoproliferation, we also found that the presence of glutamine increased lymphoproliferation as well as Bcl-2 and CD95 expression; but decreased CD95L and activation-induced T-cell death. Taken together, glutamine appeared to augment lymphoproliferation but suppressed activation-induced T-cell death in both Jurkat T cells and human peripheral T lymphocytes.     

Differential regulation of activation-induced cell death in individual human T cell clones

BACKGROUND: Restimulation of T lymphocytes via the TCR/CD3 complex can result in CD95/CD95L-dependent activation-induced cell death (AICD). Although the correlation of AICD sensitivity to the T helper 1 phenotype was confirmed in different studies, the underlying mechanism is still debated. Thus, it has been suggested that in Th2 cells, AICD resistance is controlled by a TCR-induced upregulation of the CD95-associated inhibitory phosphatase, FAP-1. We and others demonstrated that AICD resistance is associated with a reduced surface expression of CD95L upon restimulation. METHODS: Utilizing RT-PCR, Western blotting and flow cytometry, we analyzed time-dependent changes in levels of CD95L mRNA, cytosolic protein and surface expression in five long-term human T cell clones and polarized helper populations. RESULTS: We confirm that the inducible CD95L surface expression is lower or absent in all tested AICD-resistant clones as compared to sensitive cells. It is of interest that striking differences with respect to the activation-dependent inducibility of CD95L mRNA expression in individual resistant clones were observed. In addition, alterations in the expression of the inhibitory phosphatase FAP-1 or TCR-dependent changes in CD95 sensitivity in AICD-resistant clones could be ruled out as a mechanism for AICD resistance of human T cell clones. CONCLUSIONS: (1) The data presented strongly support the previous notion that AICD resistance of human T cell clones is mainly regulated by a differential expression of CD95L. (2) Differential expression of CD95L on individual resistant clones results from a lack of mRNA induction in one set and from a markedly decreased surface expression of translated protein in another set of clones.     

CD4+CD25-T细胞凋亡机制的研究

目的：探讨CD4^＋CD25^-T细胞AICD发生的机制.方法：磁性细胞分离器（MACS）分离CD4^＋CD25^- T细胞.以CD3/CD28单克隆抗体活化BALB/c小鼠CD4^＋CD25^-T细胞或以OVA323-339肽、抗原递呈细胞活化DO11.10小鼠CD4^＋CD25^-T细胞两种方式建立AICD模型.基因芯片检测CD4^＋CD25^-T细胞和CD4＋CD25＋T细胞凋亡相关基因的表达.流式细胞仪检测细胞的凋亡率.并观察FasL中和抗体、TRAIL中和抗体及zVAD-fmk对CD4^＋CD25^-T细胞凋亡的影响.结果：MACS成功分离CD4^＋CD25^-T细胞,纯度可达98%.建立了CD3/CD28抗体以及OVA特异性抗原活化的CD4^＋CD25^-T细胞AICD模型,CD4^＋CD25^-T细胞凋亡率达35%～40%.基因芯片分析发现CD4^＋CD25^-T细胞相对高表达TRAIL、FAS,而CD4^＋CD25^-T细胞相对高表达DR5、FasL.FasL、TRAIL中和抗体及zVAD-fmk可明显抑制CD4＋CD25＋T细胞的凋亡.结论：FasL、TRAIL及其它凋亡相关分子可能参与了CD4^＋CD25^-T细胞的凋亡.