Summary Immune tolerance requires regulatory T (Treg) cells to prevent autoimmune disease, with the transcription factor Foxp3 functioning as the critical regulator of Treg cell development and function. We report here that Foxp3 was lethal to developing Treg cells in the thymus because it induced a unique pro-apoptotic protein signature (Puma++p-Bim++p-JNK++DUSP6-) and repressed expression of pro-survival Bcl-2 molecules. However, Foxp3 lethality was prevented by common gamma chain (γc)-dependent cytokine signals that were present in the thymus in limiting amounts sufficient to support only ~1 million Treg cells. Consequently, most newly arising Treg cells in the thymus were deprived of this signal and underwent Foxp3-induced death, with Foxp3+CD25- Treg precursor cells being the most susceptible. Thus, we identify Foxp3 as a pro-apoptotic protein that requires developing Treg cells to compete with one another for limiting amounts of γc-dependent survival signals in the thymus.

The thymus generates major histocompatibility complex (MHC)-restricted alphabetaT cells that only recognize antigenic ligands in association with MHC or MHC-like molecules. We hypothesized that MHC specificity might be imposed on a broader alphabetaTCR repertoire during thymic selection by CD4 and CD8 coreceptors that bind and effectively sequester the tyrosine kinase Lck, thereby preventing T cell receptor (TCR) signaling by non-MHC ligands that do not engage either coreceptor. This hypothesis predicts that, in coreceptor-deficient mice, alphabeta thymocytes would be signaled by non-MHC ligands to differentiate into alphabetaT cells lacking MHC specificity. We now report that MHC-independent alphabetaT cells were indeed generated in mice deficient in both coreceptors as well as MHC ("quad-deficient" mice) and that such mice contained a diverse alphabetaT cell repertoire whose MHC independence was confirmed at the clonal level. We conclude that CD4 and CD8 coreceptors impose MHC specificity on a broader alphabetaTCR repertoire during thymic selection by preventing thymocytes from being signaled by non-MHC ligands.

CTLA-4 proteins contribute to the suppressor function of regulatory T cells (Tregs), but the mechanism by which they do so remains incompletely understood. In the present study, we assessed CTLA-4 protein function in both Tregs and conventional (Tconv) CD4 ؉ T cells. We report that CTLA-4 proteins are responsible for all 3 characteristic Treg functions of suppression, TCR hyposignaling, and anergy. However, Treg suppression and anergy only required the external domain of CTLA-4, whereas TCR hyposignaling required its internal domain. Surprisingly, TCR hyposignaling was neither required for Treg suppression nor anergy because costimulatory blockade by the external domain of CTLA-4 was sufficient for both functions. We also report that CTLA-4 proteins were localized in Tregs in submembrane vesicles that rapidly recycled to/from the cell surface, whereas CTLA-4 proteins in naive Tconv cells were retained in Golgi vesicles away from the cell membrane and had no effect on Tconv cell function. However, TCR signaling of Tconv cells released CTLA-4 proteins from Golgi retention and caused activated Tconv cells to acquire suppressor function. Therefore, the results of this study demonstrate the importance of intracellular localization for CTLA-4 protein function and reveal that CTLA-4 protein externalization imparts suppressor function to both regulatory and conventional CD4 ؉ T cells. IntroductionT cells are selected in the thymus to express TCRs reactive against foreign pathogens but tolerant to self-ligands. However, thymic selection is imperfect, so small numbers of potentially autoreactive T cells invariably escape into the periphery, where their autoreactive potential must be muted by peripheral tolerance mechanisms. Most prominent of these peripheral tolerance mechanisms are T-regulatory cells (Tregs) that suppress the activation of autoreactive T cells in vivo. [1][2] Tregs are CD4 ϩ CD25 ϩ T cells that express the X-chromosome-linked transcription factor Foxp3. [3][4][5][6][7] Foxp3 ϩ CD4 ϩ CD25 ϩ Tregs possess several unique characteristics that distinguish them from nonregulatory CD4 ϩ T cells. In particular, in addition to possessing the ability to suppress the activation of naive T cells, Tregs themselves have impaired TCR signal transduction and fail to proliferate to antigenic stimulation in the absence of exogenously added IL-2. These 3 functions are characteristic of Tregs and are referred to as suppression, TCR hyposignaling, and anergy.A protein that is present in Tregs and the expression of which in Tregs is dependent on Foxp3 is CTLA-4. 5 Mice with Tregs that lack CTLA-4 protein expression were shown recently to develop lethal autoimmunity, revealing that Treg expression of CTLA-4 was necessary for immune suppression and prevention of in vivo autoimmunity. [8][9] A variety of molecular mechanisms for CTLA-4-mediated suppression have been proposed: (1) competition between CTLA-4 and the costimulatory molecule CD28 for binding to their shared APC ligands CD80 and CD86 10 ; (2) disruption of CD28 local...