Regulatory CD4 T cells (Treg) control inflammatory reactions to commensal bacteria and opportunist pathogens. Activation of Treg functions during these processes might be mediated by host-derived proinflammatory molecules or directly by bacterial products. We tested the hypothesis that engagement of germline-encoded receptors expressed by Treg participate in the triggering of their function. We report that the subset of CD4 cells known to exert regulatory functions in vivo (CD45RB^low CD25⁺) selectively express Toll-like receptors (TLR)-4, -5, -7, and -8. Exposure of CD4⁺ CD25⁺ cells to the TLR-4 ligand lipopolysaccharide (LPS) induces up-regulation of several activation markers and enhances their survival/proliferation. This proliferative response does not require antigen-presenting cells and is augmented by T cell receptor triggering and interleukin 2 stimulation. Most importantly, LPS treatment increases CD4⁺ CD25⁺ cell suppressor efficiency by 10-fold and reveals suppressive activity in the CD4⁺ CD45RB^low CD25⁻ subset that when tested ex-vivo, scores negative. Moreover, LPS-activated Treg efficiently control naive CD4 T cell–dependent wasting disease. These findings provide the first evidence that Treg respond directly to proinflammatory bacterial products, a mechanism that likely contributes to the control of inflammatory responses.

Expression of the IL-2 receptor α chain (CD25) by peripheral CD4 T cells follows cellular activation. However, CD25 expression by CD4 cells is widely used as a marker to identify regulatory T cells (T_R), although cells with regulatory properties are also found in the CD4⁺CD25^–subset. By usingin vivofunctional assays and Foxp3 expression as a faithful marker of T_Rdifferentiation, we have evaluated the requirements for CD25 expression by peripheral T_R. We first show thatin vivodepletion of CD25⁺cells prevents the development of spontaneous encephalomyelitis in recombination-activating gene (RAG)-deficient anti-myelin basic protein T cell antigen receptor (TCR) transgenic mice, and allows disease induction in otherwise healthy RAG-competent transgenic mice. Similar treatment in normal thymectomized animals is followed by the fast recovery of a normal number of CD25⁺T_R. Consistently, Foxp3-expressing T_Rencompassed in the CD25^–cell population convert to CD25⁺after homeostatic expansion and are selectable by IL-2in vitro. Surface expression of CD25 on T_Ris controlled by the activity of conventional CD4 cells and is fully labile because it can be lost and regained without affecting the functional potential of the cells. These findings reveal that Foxp3-expressing CD25^–cells constitute a peripheral reservoir of differentiated T_R, recruited to the CD25⁺pool upon homeostatic expansion and/or activation. This analysis, together with the notion that physiological commitment of T_Rtakes place exclusively in the thymus should help for the interpretation of experiments assessing peripheral T_Rdifferentiation from naive CD4 T cells, defined as CD25^–.

The thymus generates a lineage-committed subset of Regulatory T-cells (Tregs), best identified by the expression of the transcription factor FOXP3. The development of thymus-derived Tregs is known to require high avidity interaction with MHC-self peptides leading to the generation of self-reactive Tregs fundamental for the maintenance of self-tolerance. Notwithstanding their crucial role in the control of immune responses, human thymic Treg differentiation remains poorly understood. In this mini-review we will focus on the developmental stages at which Treg lineage commitment occurs, and their spatial localization in the human thymus, reviewing the molecular requirements, including TCR and cytokine signalling, as well as the cellular interactions involved. An overview of the impact of described thymic defects on the Treg compartment will be provided, illustrating the importance of these in vivo models to investigate human Treg development.

Most Forkhead box P3 ⁺ (Foxp3 ⁺ ) CD4 regulatory T cell (Treg) precursors are newly formed thymocytes that acquire Foxp3 expression on antigen encounter in the thymus. Differentiation of Treg, however, can also occur in the periphery. What limits this second layer of self- and nonself-reactive Treg production in physiological conditions remains to be understood. In this work, we tested the hypothesis that, similarly to thymic Treg, the precursors of peripheral Treg are immature T cells. We show that CD4 ⁺ CD8 ⁻ Foxp3 ⁻ thymocytes and recent thymic emigrants (RTEs), contrarily to peripheral naïve mature cells, efficiently differentiate into Treg on transfer into lymphopenic mice. By varying donor and recipient mice and conducting ex vivo assays, we document that the preferential conversion of newly formed T cells does not require intrathymic preactivation, is cell-intrinsic, and correlates with low and high sensitivity to natural inhibitors and inducers of Foxp3 expression, such as IL-6, T-cell receptor triggering, and TGF-β. Finally, ex vivo analysis of human thymocytes and peripheral blood T cells revealed that human RTE and newly developed T cells share an increased potential to acquire a FOXP3 ^bright CD25 ^high Treg phenotype. Our findings indicating that RTEs are the precursors of Tregs differentiated in the periphery should guide the design of Treg-based therapies.

Treg cells, best identified by the expression of the transcription factor FOXP3, play a crucial role in maintaining self‐tolerance. Natural Treg cells constitute an independent thymus‐derived T‐cell lineage whose developmental program in humans is still ill‐defined. Here, we provide evidence of a Treg‐cell differentiation pathway at the double positive (DP) stage, prior to commitment to the CD4⁺ or CD8⁺ lineage, in pediatric thymuses. FOXP3⁺ DP cells displayed a functional IL‐7 receptor and increased Bcl‐2 levels that may protect them from cell death/negative selection, and an activated/suppressive phenotype that was lost as CD4 single positive (SP) cells matured and acquired egress markers. A subpopulation of FOXP3⁺ DP thymocytes expressing CD103 likely represents the precursor of FOXP3⁺ CD8SP cells, which homogeneously expressed this mucosal‐homing molecule. Finally, co‐cultures of DP thymocytes with primary thymic epithelial cells and multiple linear regression analyses support that FOXP3⁺ SP cells are largely derived from FOXP3⁺ DP thymocytes. Overall, our data suggest that human Treg‐cell lineage commitment significantly occurs at the DP stage with possible implications for the diversity and autoreactivity of the natural Treg‐cell repertoire.