While natural CD4+Foxp3+ regulatory T (nTREG) cells have long been viewed as a stable and distinct lineage that is committed to suppressive functions in vivo, recent evidence supporting this notion remains highly controversial. We sought to determine whether Foxp3 expression and the nTREG cell phenotype are stable in vivo and modulated by the inflammatory microenvironment. Here, we show that Foxp3+ nTREG cells from thymic or peripheral lymphoid organs reveal extensive functional plasticity in vivo. We show that nTREG cells readily lose Foxp3 expression, destabilizing their phenotype, in turn, enabling them to reprogram into Th1 and Th17 effector cells. nTREG cell reprogramming is a characteristic of the entire Foxp3+ nTREG population and the stable Foxp3NEG TREG cell phenotype is associated with a methylated foxp3 promoter. The extent of nTREG cell reprogramming is modulated by the presence of effector T cell-mediated signals, and occurs independently of variation in IL-2 production in vivo. Moreover, the gut microenvironment or parasitic infection favours the reprogramming of Foxp3+ TREG cells into effector T cells and promotes host immunity. IL-17 is predominantly produced by reprogrammed Foxp3+ nTREG cells, and precedes Foxp3 down-regulation, a process accentuated in mesenteric sites. Lastly, mTOR inhibition with the immunosuppressive drug, rapamycin, stabilizes Foxp3 expression in TREG cells and strongly inhibits IL-17 but not RORγt expression in reprogrammed Foxp3− TREG cells. Overall, inflammatory signals modulate mTOR signalling and influence the stability of the Foxp3+ nTREG cell phenotype.

The preTCR is associated with signal-transducing CD3␥, ␦, , and polypeptides. It is generally agreed that CD3 chains play redundant roles in the receptor-mediated signal transduction. In the present study, we show that the intracytoplasmic (IC) domain of CD3 is essential for early thymocyte maturation. We demonstrate that the IC domain-deleted CD3 fails to restore the double negative (DN) to double positive (DP) thymocyte development in CD3-deficient mice. Additional experiments show that the membrane proximal basic amino acid rich sequence in the IC domain of CD3 is sufficient for the DN to DP differentiation, whereas the proline rich sequence is required for efficient proliferation. This is probably due to impaired ligand independent recruitment of Nck to the proline rich sequence motif of CD3 within the context of the preTCR. The data presented in this study elucidates mechanistic basis for the preTCR-induced proliferation of the DN thymocytes and have identified distinct roles for individual motifs of CD3 in the preTCR-mediated differentiation and proliferation. These data provide the first genetic and phenotypic evidence for requirement of the IC domain of a CD3 chain in thymocyte development. ϩ (double positive or DP) and thymic selection of the DP thymocytes results in the generation of CD4ϩ (single positive or SP). The preTCR, which consists of a pT␣/TCR␤ heterodimer and signal transducing invariant CD3␥-, ␦-, -, and -chains, mediates the DN to DP transition (1-3). The preTCR is expressed at a low level on the DN thymocytes and is suggested to be due to the endoplasmic reticulum (ER) retention and constitutive internalization of the surface receptor (4 -7). It is generally agreed that the preTCR functions in a ligand-independent manner. The oligomerization of preTCR via the pT␣-chain has been proposed as the mechanism for the preTCR-mediated cell autonomous signal transduction (8). The surface expression of the preTCR initiates multiple intracellular signaling pathways, culminating in the DN to DP differentiation and clonal expansion, resulting in a several-fold increase in thymic cellularity (3). Several studies suggest that the preTCR-mediated differentiation and clonal expansion of the DN thymocytes are two distinct processes (reviewed in Ref. 9). Thus, transgenic expression of dominant-negative fas-associated death domain, or abrogation of either early growth response gene 3 or c-Myc transcription factor impairs proliferation, but not differentiation, of the DN thymocytes (10 -12). However, it is not clear whether the preTCR is capable of directly inducing multiple pathways each resulting in distinct biological processes.CD3 chains play multiple roles in the preTCR-mediated DN to DP thymocyte differentiation. These include ordered intracellular assembly and transport to the cell surface, receptor-induced differential signal transduction, and receptor internalization (ligand-induced and independent). CD3 chain specific transcripts are expressed as early as DN1 stage and almost all the DN3 thymocytes e...

No abstract