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it is generally acknowledged that cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4/CD152) plays a pivotal role in the regulation of T-cell activation and the establishment of self-tolerance in the periphery. CTLA-4-deficient (CTLA-4KO) mice develop a lymphoproliferative disorder and die within 4 weeks of birth, suggesting a role for CTLA-4 in T-cell homeostasis or the development and activity of T-regulatory (Treg) cells. To study the role of CTLA-4 in the control of experimental autoimmune encephalomyelitis (EAE), we have generated a CTLA-4KO mouse in which >90% of all CD4+ cells bear a Vβ8.2 transgenic T-cell receptor that is specific for myelin basic protein peptide Ac1-9 (ASQKRPSQR). These mice do not develop spontaneous lymphoproliferative disease or EAE and are resistant to disease induction. This correlates with a higher frequency of functional FoxP3+ Treg cells in the spleen and thymus of CTLA-4KO mice. The absence of CTLA-4-mediated suppression of CD28 signaling resulted in the early expression of FoxP3 on double-positive cells in the thymic cortex. We conclude that CTLA-4 is not essential for the peripheral function of FoxP3+ Treg cells but plays a pivotal role in their thymic selection. [ABSTRACT FROM AUTHOR]

The immune system is a delicately balanced network of interacting cells. In recent years, the concept of immune regulation/suppression has been firmly established, and both natural and induced regulatory cells play vital roles in protection from autoimmune disease. Recent work has revealed the diverse nature of regulatory CD4+ T (Treg) cells and the molecules involved in their function. Innate and adaptive responses to infection are able to override the suppressive properties of such regulatory cells, whereas several reports point to deficiencies in regulatory cell function in autoimmune disease. Protocols have been developed that allow the expansion of Treg cells in vitro and their antigen-specific induction in vivo. A full understanding of Treg differentiation and function will facilitate the development of improved strategies for prevention and treatment of autoimmune diseases. [Copyright &y& Elsevier]

Although the association of a chronic low-grade inflammation with obesity has long been appreciated, its molecular basis is yet to be defined. The proven involvement of adaptive immunity, coupled with a phenotypic switch from autoimmune suppressive tolerogenic Treg to pro-inflammatory CD4+ Th1 and CD8+ T cells, during progression of obesity necessitates the presence of a triggering antigen as an activator of T and B cells. Not surprisingly in this context, it is found that visceral adipose tissue-specific T cells show severely biased T cell receptor Vα repertoires in diet induced obese mice (Winer et al. 2009), implying an antigen-specific clonal expansion of T cells during obesity. HSP60 is an evolutionary conserved mitochondrial chaperonin that assists the correct folding of other mitochondrial proteins. However, its occurrence is not restricted to mitochondria and it can be located in the cytosol or exposed on the cell membrane also. An increase in cell membrane HSP60, which may be accompanied by HSP60 release into circulation, is especially considered a signal of autoimmunity. HSP60 has been associated with a broad range of diseases so far, particularly those with an autoimmune component. More recently, HSP60 is also linked to obesity as a mediator of adipose tissue inflammation and insulin resistance. Moreover, circulating HSP60 levels are found to be higher in obese individuals than lean controls (Märker et al. 2012). We observed an adaptive immune response against HSP60 at both T cell and B cell (antibody) levels during continuous high fat feeding of C57bl6 mice. Hence HSP60 appears to be one of the mystery auto-antigens triggering the early T and B cell responses during obesity. Furthermore, we attempted a peptide therapy in a dose escalation protocol aiming to down-regulate the inflammatory related adverse effects of obesity by achieving tolerance in T cell populations and suppressing the pathogenic antibody response. Treatment with a mixture of three proven immunomodulatory HSP60 peptides did not reduce weight but completely reversed the increase in VLDL/LDL levels and partially reversed the glucose intolerance in obese mice, which encourages further research to improve peptide therapy. [ABSTRACT FROM AUTHOR]

Myocarditis is the principal cause of heart failure in young adults. Frequently triggered acutely by an episode of viral infection, its progression to dilated cardiomyopathy is associated with the development of auto-immunity, especially to human cardiac α-myosin (hCAM). Consistent with this, HLA genotype influences prevalence of the disease. Previous studies showed that humanised DQ8 transgenic non-obese diabetic mice spontaneously developed autoimmune cardiomyopathy, whereas the DR4 allele is over represented in patients and there is no association with diabetes. We therefore attempted to induce experimental autoimmune myocarditis in DR4 transgenic mice (DR4 mice) as a more relevant model of the human disease. DR4 mice were injected with purified hCAM or vehicle subcutaneously in complete Freund's adjuvant (CFA). After 3 weeks, anesthetized mice were subjected to cardiac ultrasonography, following which blood was obtained from the abdominal aorta under terminal anaesthesia. The hearts were then perfused fixed for histology and spleens were harvested for proliferation assay. Potential immunomodulatory peptides were predicted in silico. Peptides were then proven water soluble and effective in T-cell proliferation assays. For immunotherapy, mice were pre-dosed with escalating doses of mixtures of 3 each of 6 soluble hCAM-derived peptides (pools 1 and 2) according to an established protocol. DR4 mice did not develop spontaneous myocarditis. However, all mice immunized with hCAM developed high titres of both IgG1 and IgG2c antibodies. Consistent with this, splenic T-cell proliferation responses to hCAM significantly increased compared to un-immunized mice. DR4 mice immunized with hCAM failed to gain weight and by echocardiography showed a significant decline in cardiac output and fractional shortening and increase in diastolic dimension compared to those injected with PBS in CFA alone. 5/5 immunized vs 0/5 control mice showed cardiac inflammation based on histology. 3/5 immunized mice died if the experiment was prolonged for 6 weeks. Pre-treatment with hCAM derived peptide pools 1 or 2 blunted the T-cell proliferation response and pool 2 also decreased both IgG1 and IgG2c levels. Pools 1 and 2 significantly improved the left ventricular cardiac function by increasing the percentage of ejection fraction and fractional shortening. Pool 2 also significantly reduced cardiac inflammation. We have developed a novel, more relevant humanized mouse model of autoimmune cardiomyopathy and demonstrated its ability to validate the immunomodulatory activity of hCAM derived peptides. Further use of our approach should prove valuable in developing optimized, clinically applicable peptide cocktails to prevent the progression of myocarditis to dilated cardiomyopathy. [ABSTRACT FROM AUTHOR]

CD4+ CD25+ Foxp3+ regulatory T cells (Tregs) have a profound ability to control immune responses. We have previously shown that the liver is a major source of peripherally induced Tregs. Here, we investigate the liver cell types and molecular mechanisms responsible for hepatic Treg induction. Methods To assess the Treg-inducing potential of liver resident antigen-presenting cell types, we studied the conversion of Foxp3− non-Tregs into Foxp3+ Tregs induced by liver dendritic cells (DCs), liver sinusoidal endothelial cells (LSECs), or Kupffer cells (KCs). The dependency of Treg induction on TGF-β was tested in Treg conversion assays using T cells with reduced TGF-β sensitivity. The suppressive potential of liver cell-induced Tregs was assessed by an in vitro suppression assay and in vivo, in the model of experimental autoimmune encephalomyelitis (EAE). Results All tested liver cell types were capable of inducing Foxp3+ Tregs; however, LSECs were most efficient in inducing Tregs. Treg-induction was antigen-specific and depended on TGF-β. LSECs featured membrane-bound LAP/TGF-β and the anchor molecule GARP, which is required for tethering LAP/TGF-β to the cell membrane. LSEC-induced Tregs suppressed proliferation and cytokine secretion of effector T cells in vitro. LSEC-induced Tregs were also functional suppressors in vivo, as neuroantigen-specific Tregs induced by LSECs were able to suppress EAE. Conclusions We demonstrate that LSECs are the major liver cell type responsible for TGF-β dependent hepatic Treg induction. The extraordinary capacity of LSECs to induce Tregs was associated with their unique ability to tether TGF-β to their membrane. [ABSTRACT FROM AUTHOR]

Today it is generally accepted that B cells require cognate interactions with CD4+ T cells to develop high-affinity antibodies against proteins. CD4+ T ceils recognize peptides (epitopes) presented by MHC class II molecules that are expressed on antigen-presenting cells. Structural features of both the MHC class II molecule and the peptide determine the specificity of CD4+ T cells that can bind to the MHC class ll-peptide complex. We used a new humanized hemophilic mouse model to identify FVIII peptides presented by HLA-DRB1*1501. This model carries a knockout of all murine MHC class II molecules and expresses a chimeric murine-human MHC class II complex that contains the peptide-binding sites of the human HLA-DRB1*1501. When mice were treated with human FVIII, the proportion of mice that developed antibodies depended on the application route of FVIII and the activation state of the innate immune system. We identified 8 FVIII peptide regions that contained CD4+ T-ceil epitopes presented by HLA-DRB1*1501 to CD4+ T cells during immune responses against FVIII. CD4+ T-cell responses after intravenous and subcutaneous application of FVIII involved the same immunodominant FVIII epitopes. Interestingly, most of the 8 peptide regions contained promiscuous epitopes that bound to several different HLA-DR proteins in in vitro binding assays. [ABSTRACT FROM AUTHOR]

Tregs are important mediators of immune tolerance to self antigens, and it has been suggested that Treg inactivation may cause autoimmune disease. Therefore, immunotherapy approaches that aim to restore or expand autoantigen-specific Treg activity might be beneficial for the treatment of autoimmune disease. Here we report that Treg-mediated suppression of autoimmune disease can be achieved in vivo by taking advantage of the ability of the liver to promote immune tolerance. Expression of the neural autoantigen myelin basic protein (MBP) in the liver was accomplished stably in liver-specific MBP transgenic mice and transiently using gene transfer to liver cells in vivo. Such ectopic MBP expression induced protection from autoimmune neuroinflammation in a mouse model of multiple sclerosis. Protection from autoimmunity was mediated by MBP-specific CD4+CD25+Foxp3+ Tregs, as demonstrated by the ability of these cells to prevent disease when adoptively transferred into nontransgenic mice and to suppress conventional CD4+CD25- T cell proliferation after antigen-specific stimulation with MBP in vitro. The generation of MBP-specific CD4+CD25+Foxp3+ Tregs in vivo depended on expression of MBP in the liver, but not in skin, and occurred by TGF-beta-dependent peripheral conversion from conventional non-Tregs. Our findings indicate that autoantigen expression in the liver may generate autoantigen-specific Tregs. Thus, targeting of autoantigens to hepatocytes may be a novel approach to prevention or treatment of autoimmune diseases. [ABSTRACT FROM AUTHOR]

How T cell receptor (TCR) signal strength modulates T cell function and to what extent this is modified by immune checkpoint blockade (ICB) are key questions in immunology. Using Nr4a3 -Tocky mice, we characterized early quantitative and qualitative changes that occur in CD4+ T cells in relation to TCR signaling strength. We captured how dose- and time-dependent programming of distinct co-inhibitory receptors rapidly recalibrates T cell activation thresholds and visualized the immediate effects of ICB on T cell re-activation. Our findings reveal that anti-PD1 immunotherapy leads to an increased TCR signal strength. We defined a strong TCR signal metric of five genes upregulated by anti-PD1 in T cells (TCR.strong), which was superior to a canonical T cell activation gene signature in stratifying melanoma patient outcomes to anti-PD1 therapy. Our study therefore reveals how analysis of TCR signal strength—and its manipulation—can provide powerful metrics for monitoring outcomes to immunotherapy. [Display omitted] • TCR signal strength drives dynamic and time-dependent changes in CD4+ T cells • Inhibitory receptor expression recalibrates T cell activation thresholds • PD1 blockade leads to a strong TCR signal signature in T cells (TCR.strong) • TCR.strong can stratify melanoma patient responses to anti-PD1 therapy How antigen and immune checkpoint engagement regulate T cell function is not completely understood. Elliot et al. reveal how antigen abundance regulates immune checkpoint expression and recalibrates T cell activation thresholds. PD1 blockade lowers the T cell activation threshold, resulting in a transcriptional signature that stratifies responses to immunotherapy. [ABSTRACT FROM AUTHOR]

Peripheral immune regulation is critical for the maintenance of self-tolerance. Here we have investigated signaling processes that distinguish T cells with regulatory capability from effector T cells. The murine Tg4 T cell receptor recognizes a peptide derived from the self-antigen myelin basic protein. T cells from Tg4 T cell receptor transgenic mice can be used to generate effector T cells and three types of T cells with regulatory capability, inducible regulatory T cells, T cells tolerized by repeated in vivo antigenic peptide exposure or T cells treated with the tolerogenic drug UCB9608 (a phosphatidylinositol 4 kinase IIIβ inhibitor). We comparatively studied signaling in all of these T cells by activating them with the same antigen presenting cells presenting the same myelin basic protein peptide. Supramolecular signaling structures, as efficiently detected by large-scale live cell imaging, are critical mediators of T cell activation. The formation of a supramolecular signaling complex anchored by the adaptor protein linker for activation of T cells (LAT) was consistently terminated more rapidly in Tg4 T cells with regulatory capability. Such termination could be partially reversed by blocking the inhibitory receptors CTLA-4 and PD-1. Our work suggests that attenuation of proximal signaling may favor regulatory over effector function in T cells. [ABSTRACT FROM AUTHOR]

The article discusses a study on role of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) in the thymic development of conventional and regulatory T cells (Tconv and Treg). It examines the mechanism of the immunological process of central tolerance in the thymus, such as the selection of T-cell receptor (TCR) and negative selection. It states that the location of CTLA-4 in the corticomedullary region in mice suggests that it influences thymic development of Tconv and Treg cells.