Deletion Of Thymocytes Research Articles

CD8 T cell tolerance results from eviction of immature autoreactive cells from the thymus.

CD8 T cell tolerance is thought to result from clonal deletion of autoreactive thymocytes before they differentiate into mature CD8 T cells in the thymus. However, we report that, in mice, CD8 T cell tolerance instead results from premature thymic eviction of immature autoreactive CD8 thymocytes into the periphery, where they differentiate into self-tolerant mature CD8 T cells. Premature thymic eviction is triggered by T cell receptor (TCR)-driven down-regulation of the transcriptional repressor Gfi1, which induces expression of sphingosine-1-phosphate receptor-1 (S1P1) on negatively selected immature CD8 thymocytes. Thus, premature thymic eviction is the basis for CD8 T cell tolerance and is the mechanism responsible for the appearance in the periphery of mature CD8 T cells bearing autoreactive TCRs that are absent from the thymus.

CCR4 and CCR7 differentially regulate thymocyte localization with distinct outcomes for central tolerance.

Central tolerance ensures autoreactive T cells are eliminated or diverted to the regulatory T cell lineage, thus preventing autoimmunity. To undergo central tolerance, thymocytes must enter the medulla to test their T-cell receptors (TCRs) for autoreactivity against the diverse self-antigens displayed by antigen-presenting cells (APCs). While CCR7 is known to promote thymocyte medullary entry and negative selection, our previous studies implicate CCR4 in these processes, raising the question of whether CCR4 and CCR7 play distinct or redundant roles in central tolerance. Here, synchronized positive selection assays, two-photon time-lapse microscopy, and quantification of TCR-signaled apoptotic thymocytes, demonstrate that CCR4 and CCR7 promote medullary accumulation and central tolerance of distinct post-positive selection thymocyte subsets in mice. CCR4 is upregulated within hours of positive selection signaling and promotes medullary entry and clonal deletion of immature post-positive selection thymocytes. In contrast, CCR7 is expressed several days later and is required for medullary localization and negative selection of mature thymocytes. In addition, CCR4 and CCR7 differentially enforce self-tolerance, with CCR4 enforcing tolerance to self-antigens presented by activated APCs, which express CCR4 ligands. Our findings show that CCR7 expression is not synonymous with medullary localization and support a revised model of central tolerance in which CCR4 and CCR7 promote early and late stages of negative selection, respectively, via interactions with distinct APC subsets.

CCR4 and CCR7 differentially regulate thymocyte localization with distinct outcomes for central tolerance

Abstract Central tolerance ensures autoreactive thymocytes are either eliminated via clonal deletion or are diverted to the regulatory T-cell lineage, thus minimizing T cell autoimmunity. To undergo central tolerance, thymocytes must enter the medulla to test their TCRs for autoreactivity against the diverse self-antigens displayed by medullary antigen presenting cells (APCs). While CCR7 is known to promote thymocyte medullary entry and negative selection against some self-antigens, our previous studies implicate CCR4 in these processes, raising the question of whether CCR4 and CCR7 play distinct or redundant roles in central tolerance. Here, we use synchronized positive selection assays and 2-photon timelapse microscopy to demonstrate that CCR4 and CCR7 are expressed sequentially after positive selection and promote medullary accumulation and central tolerance of distinct post-positive selection thymocyte subsets. CCR4 is upregulated within hours of positive selection signaling and promotes medullary entry and clonal deletion of immature post-positive selection thymocytes. In contrast, CCR7 is expressed several days later and promotes medullary entry and negative selection of mature thymocytes. Unlike CCR7 ligands, which are expressed by medullary thymic epithelial cells, CCR4 ligands are expressed by hematopoietic APCs, and we find that CCR4 enforces thymocyte tolerance to self-antigens presented by activated thymic APCs. Our findings demonstrate that CCR7 expression is neither necessary nor sufficient for thymocyte medullary entry and support a revised model of central tolerance in which CCR4 and CCR7 promote early and late stages of central tolerance, respectively, via interactions with distinct APC subsets. Supported by a grant from the NIH/NIAID (R01AI104870)

Characterisation of APS-1 Experimental Models Is Crucial for Development of Novel Therapies.

Autoimmune polyglandular syndrome type 1 (APS-1) is an inherited autosomal disorder. The most common clinical features of the disease include adrenocortical failure, hypoparathyroidism (HP), and chronic mucocutaneous candidiasis (CMC). APS-1 is caused by mutations in the autoimmune regulator (AIRE) gene. AIRE is a transcriptional factor involved in the regulation of thousands of genes in the thymus. It facilitates central tolerance by promoting the ectopic expression of tissue-specific antigens (TSAs) in medullary thymic epithelial cells (mTECs), leading to the deletion of self-reactive thymocytes. Several Aire-deficient mice were developed separately, on different backgrounds; seven published Aire knockout mice show a variety of phenotypes depending on the strain used to generate the experimental model. The first Aire-deficient mice were generated on a "black 6" background almost 20 years ago. The model showed mild phenotype with relatively modest penetrance compared to models generated on BALBc or NOD backgrounds. The generation of all these experimental models is crucial for development and testing new therapeutics as well as reading the response to treatments.

How Thymocyte Deletion in the Cortex May Curtail Antigen-Specific T-Regulatory Cell Development in the Medulla.

CD4+ T cell responses to self-antigens are pivotal for immunological self-tolerance. Activation of Foxp3– T-conventional (T-conv) cells can precipitate autoimmune disease, whereas activation of Foxp3+ T-regulatory (T-reg) cells is essential to prevent autoimmune disease. This distinction indicates the importance of the thymus in controlling the differentiation of self-reactive CD4+ T cells. Thymocytes and thymic antigen-presenting cells (APC) depend on each other for normal maturation and differentiation. In this Hypothesis and Theory article, we propose this mutual dependence dictates which self-antigens induce T-reg cell development in the thymic medulla. We postulate self-reactive CD4+ CD8– thymocytes deliver signals that stabilize and amplify the presentation of their cognate self-antigen by APC in the thymic medulla, thereby seeding a niche for the development of T-reg cells specific for the same self-antigen. By limiting the number of antigen-specific CD4+ thymocytes in the medulla, thymocyte deletion in the cortex may impede the formation of medullary T-reg niches containing certain self-antigens. Susceptibility to autoimmune disease may arise from cortical deletion creating a “hole” in the self-antigen repertoire recognized by T-reg cells.

Intrathymic SIRPa cDC subsets organization in normal and stress conditions reveal another level of cDCs heterogeneity

Three major subsets constitute the dendritic cells (DCs) pool in the thymus. They play key roles in self-antigen-specific thymocyte deletion and in the development of immunoregulatory T cells. Resident SIRPa- conventional DCs (cDCs, CD11c+ PDCA1lo ) are derived from intrathymic progenitors, whereas migratory SIRPa+ cDCs and plasmacytoid DCs (pDCs, CD11c+ PDCA1+ ) originate from extrathymic sites. Here, we describe the organization and the shaping of cDC populations at the steady state and under stress conditions in wild-type and mutant mice (CD3eKO, IL7RaKO, and Flt3LKO). In neonates, the thymus is mainly composed of SIRPa- -resident cDCs, whereas both cDC subsets are present in equal proportions in the adult. Upon thymus colonization, migratory SIRPa+ cDCs gain expression of phenotypic markers in a microenvironment dependent way. Here, we show that both processes are deeply impacted by mutations affecting T cell development. Under stress conditions such as sublethal irradiation, intrathymic resident SIRPa- cDCs are the first to regenerate the thymic cDC pool. Upon bone marrow transplantation, migratory SIRPa+ cDCs become the main source of thymic cDCs. These successive waves of regeneration eventually lead to a balance between resident and migratory DCs within the newly colonized thymus. These findings highlight an unrevealed division of labor between resident and migratory subsets for the organization/establishment of the thymic cDC compartment.

B7-CD28 co-stimulation enforces central tolerance by thymic clonal deletion and Treg cell generation through distinct mechanisms

Abstract To establish central T cell tolerance in the thymus, thymocytes expressing TCR strongly reactive to self-Ag-MHC complex are eliminated from the conventional T cell (Tconv) repertoire by two distinct processes, clonal deletion or clonal diversion to non-conventional T cell lineages such as Foxp3+ regulatory T (Treg) cells. We analyzed the requirements for B7-CD28 co-stimulation for thymic clonal deletion and Treg cell generation of endogenous tissue-restricted-antigen (TRA)-specific thymocytes. We found that: 1) both clonal deletion and Treg cell generation of TRA-specific thymocytes were B7-CD28-dependent, and that clonal deletion had a greater effect than Treg diversion in eliminating TRA-specific T cells from the Tconv repertoire; 2) B7-expressing APC requirements differed for clonal deletion and Treg generation; and 3) although B7-CD28 mediated both clonal deletion and Treg cell generation, CD28 cytoplasmic domain requirements for these two processes were different. These results indicate that qualitatively distinct CD28 signaling determines fates of strongly self-reactive thymocytes. To further characterize the CD28 signaling pathways involved in clonal deletion and Treg cell generation, single cell transcriptomic analyses of TRA-specific thymocyte are underway.

DOCK8 deficiency diminishes thymic T-regulatory cell development but not thymic deletion.

ObjectiveTo define the effect of DOCK8 deficiency on thymic tolerance in mice.MethodsThymocytes from wild‐type (Dock8+/+) and DOCK8‐deficient (Dock8pri/pri) mice were examined by flow cytometry. Some mice had transgenic expression of the BCL2 anti‐apoptotic protein in haemopoietic cells. Some mice expressed the transgenic 3A9 T‐cell receptor (TCR), which triggers thymocyte deletion in mice also expressing hen egg lysozyme under the insulin promoter.ResultsIn Dock8pr/pri mice, the proportion of thymocytes induced to acquire tolerance at the immature CCR7− stage was normal. Deletion of strongly self‐reactive CD4+ thymocytes occurred efficiently in Dock8pri/pri mice in a TCR‐transgenic model that requires self‐antigen transfer from epithelial cells to bone marrow (BM)‐derived antigen‐presenting cells. Thymic Foxp3+ T‐regulatory cells (TREG) and Helios+ Foxp3− TREG precursors were decreased in Dock8pri/pri mice, including when apoptosis was inhibited by BCL2 transgene expression. Dock8pri/pri thymic TREG expressed CD25 and CTLA‐4 at normal levels. The results suggest that DOCK8 deficiency does not affect the function of BM‐derived antigen‐presenting cells in the thymus, the TCR self‐reactivity threshold that activates tolerance mechanisms in thymocytes or the apoptotic deletion of these thymocytes. However, DOCK8 is required to prevent a subset of developing TREG cells from undergoing cell death via a mechanism that is distinct from apoptosis.ConclusionDOCK8 deficiency diminishes TREG development in the thymus without compromising thymocyte deletion.

Examination of Fas-Induced Apoptosis of Murine Thymocytes in Thymic Tissue Slices Reveals That Fas Is Dispensable for Negative Selection.

The death receptor Fas can induce cell death through the extrinsic pathway of apoptosis in a variety of cells, including developing thymocytes. Although Fas-induced cell death has been researched and modeled extensively, most of the studies have been done in vitro because of the lethality of Fas triggering in vivo. Thus, little is known about the time line of this type of cell death in vivo, specifically, how does the presence of macrophages and pro-survival cytokines affect apoptosis progression. In addition, although the sequence and timing of events during intrinsic pathway activation in thymocytes in situ have been described, no corresponding data for the extrinsic pathway are available. To address this gap in our knowledge, we established a novel system to study Fas-induced thymocyte cell death using tissue explants. We found that within 1 h of Fas ligation, caspase 3 was activated, within 2 h phosphatidylserine was externalized to serve as an “eat-me” signal, and at the same time, we observed signs of cell loss, likely due to efferocytosis. Both caspase 3 activation and phosphatidylserine exposure were critical for cell loss. Although Fas ligand (FasL) was delivered simultaneously to all cells, we observed significant variation in the entry into the cell death pathway. This model also allowed us to revisit the role of Fas in negative selection, and we ruled out an essential part for it in the deletion of autoreactive thymocytes. Our work provides a timeline for the apoptosis-associated events following Fas triggering in situ and confirms the lack of involvement of Fas in the negative selection of thymocytes.

Cutting Edge: Intestinal Mucus Limits the Clonal Deletion of Developing T Cells Specific for an Oral Antigen.

A layer of mucus functions to segregate contents of the intestinal lumen from the intestinal epithelium. The MUC2 mucin is the primary constituent of intestinal mucus and plays critical protective roles against luminal microbes and other noxious agents. In this study, we investigated whether MUC2 helps maintain CD8 T cell tolerance toward intestinal luminal Ags by gavaging wild-type and Muc2-/- mice with a model Ag and monitoring immune responses posttreatment. We report that orally delivered OVA rapidly disseminates through the blood of Muc2-/- (but not control) mice and causes immune activation of Ag-specific CD8 T cells at both local and distal sites. Further, the administration of oral OVA to Muc2-/- mice led to its presentation by thymic dendritic cells and the deletion of Ag-specific thymocytes. Collectively, our findings suggest that intestinal mucus helps limit the shaping of the TCR repertoire of developing thymocytes by intestinal luminal Ags.

GILT in Thymic Epithelial Cells Facilitates Central CD4 T Cell Tolerance to a Tissue-Restricted, Melanoma-Associated Self-Antigen.

Central tolerance prevents autoimmunity, but also limits T cell responses to potentially immunodominant tumor epitopes with limited expression in healthy tissues. In peripheral APCs, γ-IFN-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of disulfide bond-containing proteins, including the self-antigen and melanoma Ag tyrosinase-related protein 1 (TRP1). The role of GILT in thymic Ag processing and generation of central tolerance has not been investigated. We found that GILT enhanced the negative selection of TRP1-specific thymocytes in mice. GILT expression was enriched in thymic APCs capable of mediating deletion, namely medullary thymic epithelial cells (mTECs) and dendritic cells, whereas TRP1 expression was restricted solely to mTECs. GILT facilitated MHC class II-restricted presentation of endogenous TRP1 by pooled thymic APCs. Using bone marrow chimeras, GILT expression in thymic epithelial cells (TECs), but not hematopoietic cells, was sufficient for complete deletion of TRP1-specific thymocytes. An increased frequency of TRP1-specific regulatory T (Treg) cells was present in chimeras with increased deletion of TRP1-specific thymocytes. Only chimeras that lacked GILT in both TECs and hematopoietic cells had a high conventional T/Treg cell ratio and were protected from melanoma challenge. Thus, GILT expression in thymic APCs, and mTECs in particular, preferentially facilitates MHC class II-restricted presentation, negative selection, and increased Treg cells, resulting in a diminished antitumor response to a tissue-restricted, melanoma-associated self-antigen.

GILT in thymic epithelial cells facilitates central CD4 T cell tolerance to a tissue-restricted, melanoma-associated self antigen

Abstract Central tolerance prevents autoimmunity, but also limits T cell responses to potentially immunodominant tumor epitopes with limited expression in healthy tissues. In peripheral antigen presenting cells (APCs), gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of disulfide bond-containing proteins, including the self and melanoma antigen tyrosinase-related protein 1 (TRP1). The role of GILT in thymic antigen processing and generation of central tolerance has not been investigated. We found that GILT enhanced the negative selection of TRP1-specific thymocytes. GILT expression was enriched in thymic APCs capable of mediating deletion, namely medullary thymic epithelial cells (mTECs) and dendritic cells, while TRP1 expression was restricted solely to mTECs. GILT facilitated MHC class II-restricted presentation of endogenous TRP1 by pooled thymic APCs. Using bone marrow chimeras, GILT expression in thymic epithelial cells (TECs), but not hematopoietic cells, was sufficient for complete deletion of TRP1-specific thymocytes. An increased frequency of TRP1-specific regulatory T (Treg) cells was present in chimeras with increased deletion of TRP1-specific thymocytes. Only chimeras that lacked GILT in both TECs and hematopoietic cells had a high conventional T:Treg cell ratio and were protected from melanoma challenge. Thus, GILT expression in thymic APCs, and mTECs in particular, preferentially facilitates MHC class II-restricted presentation, negative selection and increased Treg cells, resulting in a diminished anti-tumor response to a tissue-restricted, melanoma-associated self antigen.

Ruxolitinib, a selective JAK1/2 inhibitor, for the treatment of serious diseases caused by Staphylococcus aureus superantigens

Abstract Staphylococcal superantigens (SSAgs), produced by Staphylococcus aureus, non-specifically activate 30–50% of all CD4+ and CD8+ T cells expressing certain T cell receptor (TCR) Vb gene families. SSAg-activated T cells rapidly produce large quantities of cytokines and chemokines resulting in a systemic inflammatory response syndrome (SIRS), which culminates in multi organ failure (MOF) and ultimately death. Through this process, SSAgs play a significant role in the immunopathogenesis of pneumonia, sepsis, and toxic shock syndrome. Because most cytokines produced during SIRS act through the Janus Kinase (JAK) signaling pathway, we hypothesized that therapeutics inhibiting JAK functions would attenuate SIRS, MOF, and lower mortality caused by SSAgs. We tested ruxolitinib (RUXO), an FDA-approved selective JAK 1/2 inhibitor. Several in vitro and in vivo studies were performed using HLA-DR3 transgenic mice because SSAgs bind to HLA class II molecules with higher affinity. In vitro, at 100, 10, and 1 mm concentrations, RUXO significantly inhibited SSAg-induced upregulation of CD25 and CD69 on both CD4+ and CD8+ T cell subsets as well as SSAg-induced production of IL-2, IL-17, and IFN-g in a dose-dependent manner at 24 and 48 hours. For in vivo studies, mice (5–6 per group) were gavaged with RUXO (100 mg/kg) or vehicle, twice daily on days −1, 0 and 1, challenged with SSAg (20 mg/mouse, i.p) on day 0 and euthanized on day 2. SSAg-induced SIRS, thymocyte deletion, upregulation of activation markers (CD69 and CD25), and immunopathology were significantly attenuated in RUXO-treated mice compared to vehicle-treated mice. Overall, JAK inhibitors such as ruxolitinib can be used as an adjunct to treat serious diseases caused by SSAg.

A37 DEFECTS IN INTESTINAL MUCUS DISRUPTS THE REPERTOIRE OF DEVELOPING T CELLS IN THE THYMUS

Severe, chronic inflammation of the intestinal tract is a defining hallmark of inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn’s disease. IBD is postulated to result from a complex combination of environmental and genetic factors that lead to excessive bacterial stimulation of the mucosal immune system. An overlying layer of mucus functions to segregate contents of the intestinal lumen from the intestinal epithelium and the underlying immune system. Importantly, decreased mucus production and impaired mucus function have been associated with UC pathogenesis. MUC2 is the primary intestinal mucin and acts as a structural scaffold for mucus formation - forming a physical barrier to shield the intestinal wall. However, recent work suggests that mucus may also function to maintain immunological tolerance towards luminal antigens by promoting the regulatory activity of intestinal antigen-presenting cells. Currently, it is unclear what role mucus plays in influencing the T cell repertoire and modulating T cell immunity both at local and distal sites. We hypothesize that mucus is essential for maintaining T cell tolerance towards intestinal luminal antigens and preventing disease caused by constitutive T cell activation. To investigate whether peripheral T cells become highly activated in young, mildly colitic Muc2-/- mice following oral antigen gavage. To test our hypothesis, young (6–10 week old) wild type and MUC2-deficient (Muc2-/-) mice were gavaged with the model antigen ovalbumin (OVA) and the elicited OVA-specific T cell responses monitored at various time points post-treatment. We found that oral administration of OVA resulted in the antigen rapidly escaping the gut and disseminating throughout the blood and lymphoid tissues of Muc2-/- but not wild type mice. Given the systemic spread of antigen in the Muc2-/- mice, we next sought to investigate whether the selection of developing T cells in the thymus of Muc2-/- mice was impacted by dietary antigens. Remarkably, we found that gavage of Muc2-/- mice led to presentation of OVA by thymic dendritic cells and the deletion of OVA-specific thymocytes whereas T cell development in wild type mice appeared unaffected. Based on our results, we concluded that oral antigens are rapidly disseminated throughout Muc2-/- mice. The heightened concentration of oral antigens induced rapid depletion of oral antigen-specific double positive (DP) thymocytes, and altered the T cell repertoire. Future studies will explore whether IBD patients suffering from impaired gut mucus structure/function also suffer defects in their T cell repertoires. CIHR

Central CD4+ T cell tolerance: deletion versus regulatory T cell differentiation.

The diversion of MHC class II-restricted thymocytes into the regulatory T (Treg) cell lineage is driven by intrathymic encounter of agonist self-antigens in a similar manner to the clonal deletion of thymocytes. Somewhat paradoxically, it thus seems that the expression of an autoreactive T cell receptor is a shared characteristic of T cells that are subject to clonal deletion and T cells that are diverted into the Treg cell lineage. Here, we discuss how thymocyte-intrinsic and thymocyte-extrinsic determinants may specify the choice between these two fundamentally different T cell fates.

GILT in thymic epithelial cells preferentially facilitates MHC class II-restricted presentation in generating central T cell tolerance to a self and melanoma antigen

Abstract Central tolerance prevents autoimmunity, but limits T cell responses to tumor antigens that are self antigens. In peripheral APCs, gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of disulfide bond-containing proteins, including the self and melanoma antigen tyrosinase-related protein 1 (TRP1). The role of GILT in thymic antigen processing and generation of central tolerance is unknown. We found that GILT enhanced the clonal deletion of TRP1-specific thymocytes. GILT expression was enriched in thymic APCs capable of mediating deletion, namely medullary thymic epithelial cells (mTECs) and dendritic cells. TRP1 expression was restricted to mTECs. GILT facilitated MHC class II-restricted presentation of endogenous TRP1 by pooled thymic APCs. Using bone marrow chimeras, GILT expression in thymic epithelial cells (TECs), but not hematopoietic cells, was sufficient for deletion of TRP1-specific thymocytes. Chimeras with GILT expression in TECs also had an increased frequency of TRP1-specific regulatory T (Treg) cells. These data demonstrate an advantage of TEC-derived GILT for antigen processing and subsequent T cell tolerance. Only chimeras that lacked GILT in both TECs and hematopoietic cells were protected from melanoma and had a high effector T cell:Treg cell ratio. Thus, GILT diminishes T cell-mediated protection from melanoma through promoting thymic deletion and Treg cells.

Indirect presentation in the thymus limits naive and regulatory T-cell differentiation by promoting deletion of self-reactive thymocytes.

Acquisition of T-cell central tolerance involves distinct pathways of self-antigen presentation to thymocytes. One pathway termed indirect presentation requires a self-antigen transfer step from thymic epithelial cells (TECs) to bone marrow-derived cells before the self-antigen is presented to thymocytes. The role of indirect presentation in central tolerance is context-dependent, potentially due to variation in self-antigen expression, processing and presentation in the thymus. Here, we report experiments in mice in which TECs expressed a membrane-bound transgenic self-antigen, hen egg lysozyme (HEL), from either the insulin (insHEL) or thyroglobulin (thyroHEL) promoter. Intrathymic HEL expression was less abundant and more confined to the medulla in insHEL mice compared with thyroHEL mice. When indirect presentation was impaired by generating mice lacking MHC class II expression in bone marrow-derived antigen-presenting cells, insHEL-mediated thymocyte deletion was abolished, whereas thyroHEL-mediated deletion occurred at a later stage of thymocyte development and Foxp3+ regulatory T-cell differentiation increased. Indirect presentation increased the strength of T-cell receptor signalling that both self-antigens induced in thymocytes, as assessed by Helios expression. Hence, indirect presentation limits the differentiation of naive and regulatory T cells by promoting deletion of self-reactive thymocytes.

Nur77 Regulates Nondeletional Mechanisms of Tolerance in T Cells.

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 Bim-/-Nur77-/- mice exhibited comparable accumulation of high-affinity signaled CD4+CD8+ 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 Foxp3-FR4hiCD73hi 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.

Essential role of CCL21 in establishment of central self-tolerance in T cells.

The chemokine receptor CCR7 directs T cell relocation into and within lymphoid organs, including the migration of developing thymocytes into the thymic medulla. However, how three functional CCR7 ligands in mouse, CCL19, CCL21Ser, and CCL21Leu, divide their roles in immune organs is unclear. By producing mice specifically deficient in CCL21Ser, we show that CCL21Ser is essential for the accumulation of positively selected thymocytes in the thymic medulla. CCL21Ser-deficient mice were impaired in the medullary deletion of self-reactive thymocytes and developed autoimmune dacryoadenitis. T cell accumulation in the lymph nodes was also defective. These results indicate a nonredundant role of CCL21Ser in the establishment of self-tolerance in T cells in the thymic medulla, and reveal a functional inequality among CCR7 ligands in vivo.

Dual TCRα expression alters agonist selection, redirecting Treg-biased insulin-specific thymocytes into the conventional T cell lineage

Abstract Despite accounting for ~10% of the T cell population in mice and humans, the role of dual TCR-expressing T cells in immunity remains poorly understood. It has been hypothesized that dual TCR T cells pose an “autoimmune hazard” by allowing self-reactive TCRs to escape negative selection. We revisited this hypothesis using the non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D). We bred NOD mice hemizygous at both TCRα and β (TCRα+/− β+/−) loci, rendering them incapable of producing dual TCR T cells. We found that NOD mice lacking dual TCRα expression were resistant to developing diabetes resulting from an increased insulin-specific Treg:Tconv ratio. Furthermore, early depletion of CD25-expressing cells was sufficient to induce diabetes in single TCR T cell NOD mice demonstrating that these mice are capable of developing diabetes and that Treg cells are responsible for their observed resistance to diabetes. Further investigation uncovered a previously-unrecognized impact of dual TCRα expression on agonist selection. Specifically, we found in single TCR T cell mice that single positive (SP) thymocytes had a higher Treg:CD4 ratio indicating increased Treg commitment, that insulin-specific thymocytes exhibited higher CD5 expression suggesting increased TCR signaling, and that deletion of SP thymocytes was reduced. Altogether these data demonstrate that dual TCRα expression perturbs agonist selection of the insulin-specific thymocyte subset, resulting in skewing of this population toward the diabetogenic conventional CD4+ T cell lineage and away from the regulatory T cell lineage. Thus, dual TCR expression can promote autoimmunity by reducing the ratio of protective Treg cells to pathogenic T cells.