Positive and negative selection of thymocytes in the thymus are critical for the development of a mature and self-tolerant T-cell repertoire. The proapoptotic Bcl-2 family member Bim is important for negative selection by inducing apoptosis in thymocytes receiving a strong signal through their antigen receptor. However, in the case of ubiquitous self-antigens (UbA), Bim is not required for the clonal deletion of self-reactive thymocytes, suggesting the existence of nonapoptotic clonal deletion mechanisms. Unlike UbA, clonal deletion to tissue-restricted antigens (TRAs) requires positive selection and CCR7-mediated migration to the medulla. This led us to hypothesize that Bim is required for the latter. To study the role of Bim in clonal deletion to TRA, we constructed bone marrow (BM) chimeras using OT-I Bim-deficient or -sufficient donor bone marrow and recipients that express membrane bound chicken ovalbumin under control of the rat insulin promoter (RipmOVA). We found that clonal deletion to TRA was completely abrogated in the absence of Bim and large numbers of mature OT-I CD8 T cells survived in the periphery. Despite the large numbers of autoreactive T cells, the chimeras did not develop diabetes and OT-I Bim-deficient T cells from these chimeras were functionally impaired. Collectively, these data provide unique evidence of a differential, thymocyte-intrinsic, molecular requirement downstream of the T-cell receptor (TCR) for clonal deletion to UbA versus TRA and highlight the profound ability of other tolerance mechanisms to control T-cell autoreactivity in the absence of thymic clonal deletion.autoimmunity | thymocyte development

Negative selection against highly self-reactive thymocytes is critical for preventing autoimmunity. Thymocyte deletion, anergy induction, and agonist selection are all forms of negative selection that can occur following a high-affinity TCR signal. Of Bim and Nur77, two TCR-induced proteins with proapoptotic function, Bim has been shown to be important for clonal deletion in several model systems, whereas Nur77 was often dispensable. However, Nur77 has been reported to influence other aspects of T cell development by mechanisms that may not be related to its proapoptotic function. In this study, we examined the role of Nur77 during thymocyte development in the presence and absence of Bim to separate apoptotic from nonapoptotic functions of Nur77. Polyclonal Bim and BimNur77 mice exhibited comparable accumulation of high-affinity signaled CD4CD8 double-positive thymocytes and CD8 and CD4 single-positive thymocytes. However, combined Bim and Nur77 deficiency increased the frequency of thymic Foxp3 T regulatory cells and Foxp3FR4CD73 anergic phenotype CD4 T cells compared with Bim mice, suggesting that Nur77 expression impairs the development of nonconventional tolerance-inducing cell fates. Using the OT-I RIP-mOVA model, we found that Nur77 deficiency did not substantially impact clonal deletion nor did it exacerbate the defect in clonal deletion in the absence of Bim. However, additional loss of Nur77 in the absence of Bim led to diabetes induction, suggesting that Nur77 promotes tolerance in this context. Together, these data reveal novel nondeletional roles for Nur77 that differ between T cell subsets and have implications for self-tolerance.

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) α and β loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders(1-4). T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the αβTCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the αβTCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC(5). Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events(6). For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes(7-9). Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCRα chain at the DN stage, resulting in premature negative selection. Our lab has developed the HY(cd4) model, in which the transgenic HY TCRα is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice(10). Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HY(cd4) mouse model. While negative selection in HY(cd4) mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

Highly self-reactive T cells are censored from the repertoire by both central and peripheral tolerance mechanisms upon receipt of high-affinity TCR signals. Clonal deletion is considered a major driver of central tolerance; however, other mechanisms such as induction of regulatory T cells and functional impairment have been described. An understanding of the interplay between these different central tolerance mechanisms is still lacking. We previously showed that impaired clonal deletion to a model tissue-restricted antigen (TRA) did not compromise tolerance. In this study, we determined that T cells that failed clonal deletion in this model were rendered functionally impaired in the thymus. PD-1 was induced in the thymus and established cell-intrinsic tolerance to TRA in CD8+ thymocytes independently of clonal deletion. PD-1 signaling in developing thymocytes was sufficient to induce tolerance but was dispensable for the initial maintenance of tolerance in the periphery. We showed that chronic exposure to high affinity antigen supported the long-term maintenance of tolerance in this model. Taken together, our study identifies a role for PD-1 in establishing central tolerance in autoreactive T cells that escape clonal deletion and sheds light on potential mechanisms of action of anti-PD-1 pathway immune checkpoint blockade and the development of immune-related adverse events.Significance StatementThe establishment of T cell tolerance is critical to prevent autoimmune diseases. Apoptosis of highly self-reactive thymocytes is an important mechanism that enforces central tolerance. However, not all self-reactive thymocytes undergo apoptosis during development, and the fate of cells that evade this process is under-examined. Using bone marrow chimera, adoptive transfer, and thymic transplant experiments, we found acute PD-1 signaling is required to establish tolerance to tissue-restricted antigen (TRA) independently of clonal deletion. This tolerance is maintained by chronic exposure to tolerizing antigen but persists in the absence of PD-1 until late time points. Overall, this study identifies a role for PD-1 in establishing central tolerance, and it provides insight into the mechanism of PD-1 pathwaytargeting cancer immunotherapies.

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