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.

Summary Clonal deletion of autoreactive thymocytes is important for self-tolerance, but the intra-thymic signals that induce clonal deletion have not been clearly identified. We now report that clonal deletion during negative selection requires CD28 costimulation of autoreactive thymocytes at the CD4+CD8lo intermediate stage of differentiation. Autoreactive thymocytes were prevented from undergoing clonal deletion by either absent CD28 costimulation or transgenic over-expression of the anti-apoptotic factors Bcl-2 or Mcl-1, with surviving thymocytes differentiating into anergic T cell receptor αβ+ double negative thymocytes that preferentially migrated to the intestine where they re-expressed CD8α and were sequestered as CD8αα intraepithelial lymphocytes (IELs). This study identifies CD28 costimulation as the intrathymic signal required for clonal deletion and identifies CD8αα IELs as the developmental fate of autoreactive thymocytes that survive negative selection.

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...