Thymectomy in mice on neonatal day 3 leads to the development of multiorgan autoimmune disease due to loss of a CD⁺CD25⁺ T cell regulatory population in their peripheral lymphoid tissues. Here, we report the identification of a CD4⁺ population of regulatory T cells in the circulation of humans expressing high levels of CD25 that exhibit in vitro characteristics identical with those of the CD4⁺CD25⁺ regulatory cells isolated in mice. With TCR cross-linking, CD4⁺CD25^high cells did not proliferate but instead totally inhibited proliferation and cytokine secretion by activated CD4⁺CD25⁻ responder T cells in a contact-dependent manner. The CD4⁺CD25^high regulatory T cells expressed high levels of CD45RO but not CD45RA, akin to the expression of CD45RB^low on murine CD4⁺CD25⁺ regulatory cells. Increasing the strength of signal by providing either costimulation with CD28 cross-linking or the addition of IL-2 to a maximal anti-CD3 stimulus resulted in a modest induction of proliferation and the loss of observable suppression in cocultures of CD4⁺CD25^high regulatory cells and CD4⁺CD25⁻ responder cells. Whereas higher ratios of CD4⁺CD25^high T cells are required to suppress proliferation if the PD-L1 receptor is blocked, regulatory cell function is shown to persist in the absence of the PD-1/PD-L1 or CTLA-4/B7 pathway. Thus, regulatory CD4 T cells expressing high levels of the IL-2 receptor are present in humans, providing the opportunity to determine whether alterations of these populations of T cells are involved in the induction of human autoimmune disorders.

Although implicated in antagonistic functions, both regulatory T cells (Tregs) and Th17 effector cells play an important role in controlling autoimmune pathogenesis. Paradoxically, recent studies indicate that Tregs have the capacity to produce interleukin-17 (IL-17), although the ability of these cells to retain their suppressive function remains unknown. Here we report that human Tregs within the CD4+CD45RA−CD25highCCR6+HLA-DR−FoxP3+ population produce IL-17 when activated in the presence of the proinflammatory cytokines IL-1β and IL-6, whereas IL-17 secretion was inhibited by TGFβ. To assess the ability of a single Treg to secrete IL-17 and to suppress in vitro immune function, we isolated clones from this population. We found that IL-17+/FoxP3+ Treg clones retain suppressive function and exhibit the plasticity to secrete IL-17 or suppress depending on the nature of the stimulus provided. IL-17 production by these Treg clones was accompanied by sustained FoxP3 expression and concomitant, but reversible, loss of suppressive activity. Our data demonstrate that at the single cell level a subset of in vitro suppressive FoxP3+ cells can be driven to secrete IL-17 under inflammatory conditions. These findings suggest a new mechanism by which inflammation can drive Tregs to secrete IL-17, thereby dampening suppression and promoting an inflammatory milieu.

It has been known for decades that circulating human CD4 cells can express functional MHC class II molecules that induce T cell nonresponsiveness with Ag presentation. Because there is significant expression of MHC class II (MHC-II) determinants (DR) on a subpopulation CD4⁺CD25^high regulatory T cells (Treg), we examined the function of CD4 cells expressing MHC-DR. We demonstrate that MHC-II expression on human CD4⁺CD25^high T cells identifies a functionally distinct population of Treg that induces early contact-dependent suppression that is associated with high Foxp3 expression. In striking contrast, MHC-II⁻ CD4⁺CD25^high Treg induce early IL-4 and IL-10 secretion and a late Foxp3-associated contact-dependent suppression. The DR expressing CD25^high Treg express higher levels of Foxp3 message and protein, compared with the DR⁻CD25^high Treg population. Direct single-cell cloning of CD4⁺CD25^high Treg revealed that, regardless of initial DR expression, ex vivo expression of CD25^high, and not DR, predicted which clones would exhibit contact-dependent suppression, high levels of Foxp3 message, and an increased propensity to become constitutive for DR expression. Thus, the direct ex vivo expression of MHC-II in the context of CD25^high identifies a mature, functionally distinct regulatory T cell population involved in contact-dependent in vitro suppression.