Graft rejection by the immune system is a major cause of transplant failure. Lifelong immunosuppression decreases the incidence of graft rejection; however, nonspecific immunosuppression results in increased susceptibly to infection and cancer. Regulatory T cells (Tregs), which suppress the activation of the immune system and induce tolerance, are currently under evaluation for use in clinical transplantation. Ex vivo expanded polyclonal Tregs that are introduced into transplant recipients alter the balance of T effector cells to Tregs; however, experimental data suggest that alloantigen-specific Tregs would be more effective at preventing graft rejection. We have developed a method to enrich alloantigen-specific human Tregs based on the coexpression of activation markers, CD69 and CD71. These Tregs could be readily expanded in vitro and demonstrated potent antigen-specific suppression. In a humanized mouse model of alloimmune-mediated injury of human skin grafts, alloantigen-specific Tregs resulted in a significant reduction in clinically relevant indicators of dermal tissue injury when compared with polyclonal Tregs, restoring a histology comparable to healthy skin. This method of human allospecific Treg selection should be scalable to the clinic. The improved in vivo efficacy of alloantigen-specific Tregs over polyclonal Tregs shown here suggests that generating “customized” Tregs with defined anti-donor allospecificities may improve current practice in clinical immunotherapy.

Stable expression of Foxp3 in regulatory T cells (Tregs) depends on DNA demethylation at the Treg-specific demethylated region (TSDR), a conserved, CpG-rich region within the Foxp3 locus. The TSDR is selectively demethylated in ex vivo Tregs purified from secondary lymphoid organs, but it is unclear at which stage of Treg development demethylation takes place. In this study, we show that commitment to a stable lineage occurred during early stages of murine thymic Treg development by engraving of lineage-specific epigenetic marks in parallel with establishment of a Treg-specific gene expression profile. TSDR demethylation was achieved through an active mechanism and involved enzymes of the ten-eleven-translocation family and hydroxylation of methylated cytosines, a modification that is implicated as an initiating step of mitosis-independent DNA demethylation pathways and has not yet been observed at specific loci during immune cell differentiation. Together, our results demonstrate that initiating TSDR demethylation during early stages of thymic Treg development commences stabilization of Foxp3 expression and guarantees full functionality and long-term lineage stability of Tregs.

Numerous reports have demonstrated that CD4+CD25+regulatory T cells (Tregs) from individuals with a range of human autoimmune diseases, including Type 1 diabetes (T1D),are deficient in theirability to control autologous pro-inflammatory responses when compared to non-diseased, control individuals. Treg dysfunction could be a primary, causal event or may result from perturbations in the immune system during disease development.Polymorphisms in genes associated with Treg function, such as IL2RA, confer a higher risk of autoimmune disease. Although this suggests a primary role for defective Tregs in autoimmunity, a link between IL2RA gene polymorphisms and Treg function has not been examined. We addressed this by examining the impact of an IL2RA haplotype associated with T1D on Treg fitness and suppressive function. Studies were conducted using healthy human subjects to avoid any confounding effects of disease. We demonstrated that the presence of an autoimmune disease-associated IL2RA haplotype correlates with diminished interleukin (IL)-2-responsiveness in antigen-experienced CD4+ T cells, as measured by phosphorylation of STAT5a, and is associated with lower levels of FoxP3 expression by Tregs, and a reduction in their ability to suppress proliferation of autologous effector T cells. These data offer a rationale that contributes to the molecular and cellular mechanisms through which polymorphisms in the IL-2RA gene impact upon immune regulation, and consequently upon susceptibility to autoimmune and inflammatory diseases.

Foxp3 + regulatory T (Treg) cells restrict immune pathology in inflamed tissues; however, an inflammatory environment presents a threat to Treg cell identity and function. Here, we establish a transcriptional signature of central nervous system (CNS) Treg cells that accumulate during experimental autoimmune encephalitis (EAE) and identify a pathway that maintains Treg cell function and identity during severe inflammation. This pathway is dependent on the transcriptional regulator Blimp1, which prevents downregulation of Foxp3 expression and ''toxic'' gain-of-function of Treg cells in the inflamed CNS. Blimp1 negatively regulates IL-6-and STAT3-dependent Dnmt3a expression and function restraining methylation of Treg cell-specific conserved non-coding sequence 2 (CNS2) in the Foxp3 locus. Consequently, CNS2 is heavily methylated when Blimp1 is ablated, leading to a loss of Foxp3 expression and severe disease. These findings identify a Blimp1-dependent pathway that preserves Treg cell stability in inflamed non-lymphoid tissues.