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Maintenance of peripheral tolerance requires a balance between autoreactive conventional T cells (T conv ) and thymically derived forkhead box protein 3 (FoxP3) + regulatory T cells (tT regs ). Considerable controversy exists regarding the similarities/differences in T cell receptor (TCR) repertoires expressed by T conv and tT regs . We generated highly purified populations of human adult and cord blood T conv and tT regs based on the differential expression of CD25 and CD127. The purity of the sorted populations was validated by intracellular staining for FoxP3 and Helios. We also purified an overlap group of CD4 T cells from adult donors to ensure that considerable numbers of shared clonotypes could be detected when present. We used deep sequencing of entire TCR-β CDR3 sequences to analyse the TCR repertoire of T conv and tT regs . Our studies suggest that both neonatal and adult human T conv and tT reg cells are, in fact, entirely distinct CD4 T cell lineages., (Published 2016. This article is a U.S. Government work and is in the public domain in the USA.)

Short-term outcomes of kidney transplantation have improved dramatically, but chronic rejection and regimen-related toxicity continue to compromise overall patient outcomes. Development of regulatory T cells (Tregs) as a means to decrease alloresponsiveness and limit the need for pharmacologic immunosuppression is an active area of preclinical and clinical investigation. Nevertheless, the immunomodulatory effects of end-stage renal disease on the efficacy of various strategies to generate and expand recipient Tregs for kidney transplantation are incompletely characterized. In this study, we show that Tregs can be successfully generated from either freshly isolated or previously cryopreserved uremic recipient (responder) and healthy donor (stimulator) peripheral blood mononuclear cells using the strategy of ex vivo costimulatory blockade with belatacept during mixed lymphocyte culture. Moreover, these Tregs maintain a CD3(+) CD4(+) CD25(+) CD127(lo) surface phenotype, high levels of intracellular FOXP3 and significant demethylation of the FOXP3 Treg-specific demethylation region on allorestimulation with donor stimulator cells. These data support evaluation of this simple, brief Treg production strategy in clinical trials of mismatched kidney transplantation., (© Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.)