Deep sequencing of the TCR-ß repertoire of human forkhead box protein 3 (FoxP3)+ and FoxP3- T cells suggests that they are completely distinct and non-overlapping.

Maintenance of peripheral tolerance requires a balance between autoreactive conventional T cells (Tconv ) and thymically derived forkhead box protein 3 (FoxP3)+ regulatory T cells (tTregs ). Considerable controversy exists regarding the similarities/differences in T cell receptor (TCR) repertoires expressed by Tconv and tTregs . We generated highly purified populations of human adult and cord blood Tconv and tTregs 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-ß CDR3 sequences to analyse the TCR repertoire of Tconv and tTregs . Our studies suggest that both neonatal and adult human Tconv and tTreg cells are, in fact, entirely distinct CD4 T cell lineages.

Ex Vivo Costimulatory Blockade to Generate Regulatory T Cells From Patients Awaiting Kidney Transplantation.

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.