The suppressive function of regulatory T cells (Treg) is impaired in multiple sclerosis (MS) patients. The mechanism underlying the Treg functional defect is unknown. Treg mature in the thymus and the majority of cells circulating in the periphery rapidly adopt a memory phenotype. Because our own previous findings suggest that the thymic output of T cells is impaired in MS, we hypothesized that an altered Treg generation may contribute to the suppressive deficiency. We therefore determined the role of Treg that enter the circulation as recent thymic emigrants (RTE) and, unlike their CD45RO+ memory counterparts, express CD31 as typical surface marker. We show that the numbers of CD31+-coexpressing CD4+CD25+CD45RA+CD45RO−FOXP3+ Treg (RTE-Treg) within peripheral blood decline with age and are significantly reduced in MS patients. The reduced de novo generation of RTE-Treg is compensated by higher proportions of memory Treg, resulting in a stable cell count of the total Treg population. Depletion of CD31+ cells from Treg diminishes the suppressive capacity of donor but not patient Treg and neutralizes the difference in inhibitory potencies between the two groups. Overall, there was a clear correlation between Treg-mediated suppression and the prevalence of RTE-Treg, indicating that CD31-expressing naive Treg contribute to the functional properties of the entire Treg population. Furthermore, patient-derived Treg, but not healthy Treg, exhibit a contracted TCR Vβ repertoire. These observations suggest that a shift in the homeostatic composition of Treg subsets related to a reduced thymic-dependent de novo generation of RTE-Treg with a compensatory expansion of memory Treg may contribute to the Treg defect associated with MS.

† CD4 + CD25 hi Foxp3 + regulatory T cells (T regs ) are critical mediators of self-tolerance, which is crucial for the prevention of autoimmune disease, but T regs can also inhibit antitumor immunity. T regs inhibit the proliferation of CD4 + CD25 − conventional T cells (T cons ), as well as the ability of these cells to produce effector cytokines; however, the molecular mechanism of suppression remains unclear. Here, we showed that human T regs rapidly suppressed the release of calcium ions (Ca 2+ ) from intracellular stores in response to T cell receptor (TCR) activation in T cons . The inhibition of Ca 2+ signaling resulted in decreased dephosphorylation, and thus decreased activation, of the transcription factor nuclear factor of activated T cells 1 (NFAT1) and reduced the activation of nuclear factor kB (NF-kB). In contrast, Ca 2+ -independent events in T cons , such as TCR-proximal signaling and activation of the transcription factor activator protein 1 (AP-1), were not affected during coculture with T regs . Despite suppressing intracellular Ca 2+ mobilization, coculture with T regs did not block the generation of inositol 1,4,5-trisphosphate in TCR-stimulated T cons . The T reg -induced suppression of the activity of NFAT and NF-kB and of the expression of the gene encoding the cytokine interleukin-2 was reversed in T cons by increasing the concentration of intracellular Ca 2+ . Our results elucidate a previously unrecognized and rapid mechanism of T reg -mediated suppression. This increased understanding of T reg function may be exploited to generate possible therapies for the treatment of autoimmune diseases and cancer.

CD4+CD25++Foxp3+ regulatory T cells (Tregs) control self-reactive cells to maintain peripheral tolerance. Treg homeostasis has to be controlled tightly to ensure balanced Treg-mediated suppression. One mechanism that regulates the CD4+ T cell pool is activation-induced cell death (AICD). This is mimicked in vitro by TCR restimulation-induced expression of the death ligand CD95L (FasL/APO-1L/CD178) in expanded T cells. These cells express the death receptor CD95 (Fas/APO-1), and binding of CD95L to CD95 results in AICD. In contrast, Tregs do not undergo AICD upon TCR (re)stimulation in vitro despite a functional CD95 cell death pathway. In this study, we show that human and murine Tregs express low levels of CD95L upon stimulation. Knockdown of the transcriptional repressor Foxp3 partially rescues CD95L expression and AICD in human Tregs. Moreover, upon stimulation Foxp3-mutant Tregs from Scurfy mice express CD95L similar to conventional T cells. We further addressed whether exogenous CD95 stimulation provides a mechanism of Treg homeostatic control in vivo in mice. Triggering of CD95 reduced Treg numbers systemically as reflected by in vivo imaging and decreased GFP+ Treg numbers ex vivo. Our study reveals that Foxp3 negatively regulates CD95L expression in Tregs and demonstrates that Tregs are susceptible to homeostatic control by CD95 stimulation.