Defective Negative Selection Research Articles

Cellular mechanisms and clinical applications for phenocopies of inborn errors of immunity: infectious susceptibility due to cytokine autoantibodies

ABSTRACT Introduction With a growing knowledge of Inborn errors immunity (IEI), immunological profiling and genetic predisposition to IEI phenocopies have been developed in recent years. Areas covered Here we summarized the correlation between various pathogen invasions, autoantibody profiles, and corresponding clinical features in the context of patients with IEI phenocopies. It has been extensively evident that patients with anti-cytokine autoantibodies underly impaired anti-pathogen immune responses and lead to broad unregulated inflammation and tissue damage. Several hypotheses of anti-cytokine autoantibodies production are summarized here, including a defective negative selection of autoreactive T cells, abnormal germinal center formation, molecular mimicry, HLA class II allele region, lack of auto-reactive lymphocyte apoptosis, and other possible hypotheses. Expert opinion Phenocopies of IEI associated with anti-cytokine autoantibodies are increasingly recognized as one of the causes of acquired immunodeficiency and susceptibility to certain pathogen infections, especially facing the current challenge of the COVID-19 pandemic. By investigating clinical, genetic, and pathogenesis autoantibodies profiles associated with various pathogens’ susceptibilities, we could better understand the IEI phenocopies with anti-cytokine autoantibodies, especially for those that underlie life-threatening SARS-CoV-2.

Editing T cell repertoire by thymic epithelial cell-directed gene transfer abrogates risk of type 1 diabetes development

Insulin is the primary autoantigen (Ag) targeted by T cells in type 1 diabetes (T1D). Although biomarkers precisely identifying subjects at high risk of T1D are available, successful prophylaxis is still an unmet need. Leaky central tolerance to insulin may be partially ascribed to the instability of the MHC-InsB9-23 complex, which lowers TCR avidity, thus resulting in defective negative selection of autoreactive clones and inadequate insulin-specific T regulatory cell (Treg) induction. We developed a lentiviral vector (LV)-based strategy to engineer thymic epithelial cells (TECs) to correct diabetogenic T cell repertoire. Intrathymic (it) LV injection established stable transgene expression in EpCAM+ TECs, by virtue of transduction of TEC precursors. it-LV-driven presentation of the immunodominant portion of ovalbumin allowed persistent and complete negative selection of responsive T cells in OT-II chimeric mice. We successfully applied this strategy to correct the diabetogenic repertoire of young non-obese diabetic mice, imposing the presentation by TECs of the stronger agonist InsulinB9-23R22E and partially depleting the existing T cell compartment. We further circumscribed LV-driven presentation of InsulinB9-23R22E by micro-RNA regulation to CD45− TECs without loss of efficacy in protection from diabetes, associated with expanded insulin-specific Tregs. Overall, our gene transfer-based prophylaxis fine-tuned the central tolerance processes of negative selection and Treg induction, correcting an autoimmune prone T cell repertoire.

Autoantigen specific T-cell receptor induces organ-specific autoimmunity by escaping T cell negative selection

Abstract Previously we reported a new model of spontaneous autoimmune uveitis in which mice with a hypomorphic mutation of Aire (AireGW/+) are combined with deletion of Lyn. The key autoantigen in this model is interphotoreceptor retinoid-binding protein (IRBP) as AireGW/+Lyn−/−IRBP−/− mice failed to develop uveitis. We performed single-cell RNA-seq with TCR sequencing for IRBP 271–290 (P2)-specific CD4+ T cells from eye-draining lymph nodes (LN) of these mice with and without uveitis. Both mice showed an expansion of these T cells in eye-draining LNs, but it was greater in mice with disease. A remarkable clonal diversity of TCRs was seen. Expansions of particular T cell clonotypes were more evident in LN of mice with disease, and even greater in the retinas of mice with disease. We reconstituted several P2–specific TCR clonotypes in a T cell hybridoma line and verified their specificity. One of the TCRs was expressed in thymocytes by retroviral transduction of bone marrow stem cells from Rag2−/− mice and transplanted into WT or AireGW/+ recipient mice. Remarkably, the small residual expression of IRBP in the thymus of AireGW/+ mice was sufficient to induce substantial negative selection of thymocytes with this TCR. In addition, we generated TCR transgenic mice with this TCR clonotype. The TCR transgenic mice spontaneously developed uveitis with expansion of P2-specific CD4+ T cells. Combined with AireGW/+ or IRBP−/−, there were more P2-specific CD4+ T cells in the thymus of TCR transgenic mice. Thus, suboptimal Aire function in AireGW/+Lyn−/− mice results in a partial defect in negative selection of T cells recognizing IRBP in the thymus, promoting development of uveitis. This disease is accelerated in mice expressing a TCR transgene recognizing IRBP. Supported by AI138479

Failures in thymus medulla regeneration during immune recovery cause tolerance loss and prime recipients for auto-GVHD.

Bone marrow transplantation (BMT) is a widely used therapy for blood cancers and primary immunodeficiency. Following transplant, the thymus plays a key role in immune reconstitution by generating a naive αβT cell pool from transplant-derived progenitors. While donor-derived thymopoiesis during the early post-transplant period is well studied, the ability of the thymus to synchronize T cell development with essential tolerance mechanisms is poorly understood. Using a syngeneic mouse transplant model, we analyzed T cell recovery alongside the regeneration and function of intrathymic microenvironments. We report a specific and prolonged failure in the post-transplant recovery of medullary thymic epithelial cells (mTECs). This manifests as loss of medulla-dependent tolerance mechanisms, including failures in Foxp3+ regulatory T cell development and formation of the intrathymic dendritic cell pool. In addition, defective negative selection enables escape of self-reactive conventional αβT cells that promote autoimmunity. Collectively, we show that post-transplant T cell recovery involves an uncoupling of thymopoiesis from thymic tolerance, which results in autoimmune reconstitution caused by failures in thymic medulla regeneration.

Transcriptome and IgH Repertoire Analyses Show That CD11chi B Cells Are a Distinct Population With Similarity to B Cells Arising in Autoimmunity and Infection.

A distinct B cell population marked by elevated CD11c expression is found in patients with systemic lupus erythematosus (SLE). Cells with a similar phenotype have been described during chronic infection, but variable gating strategies and nomenclature have led to uncertainty of their relationship to each other. We isolated CD11chi cells from peripheral blood and characterized them using transcriptome and IgH repertoire analyses. Gene expression data revealed the CD11chi IgD+ and IgD− subsets were highly similar to each other, but distinct from naive, memory, and plasma cell subsets. Although CD11chi B cells were enriched in some germinal center (GC) transcripts and expressed numerous negative regulators of B cell receptor (BCR) activation, they were distinct from GC B cells. Gene expression patterns from SLE CD11chi B cells were shared with other human diseases, but not with mouse age-associated B cells. IgH V-gene sequencing analysis showed IgD+ and IgD− CD11chi B cells had somatic hypermutation and were clonally related to each other and to conventional memory and plasma cells. However, the IgH repertoires expressed by the different subsets suggested that defects in negative selection during GC transit could contribute to autoimmunity. The results portray a pervasive B cell population that accumulates during autoimmunity and chronic infection and is refractory to BCR signaling.

Negative selection of human T cells recognizing a naturally-expressed tissue-restricted antigen in the human thymus

During T cell development in mice, thymic negative selection deletes cells with the potential to recognize and react to self-antigens. In human T cell-dependent autoimmune diseases such as Type 1 diabetes, multiple sclerosis, and rheumatoid arthritis, T cells reactive to autoantigens are thought to escape negative selection, traffic to the periphery and attack self-tissues. However, physiological thymic negative selection of autoreactive human T cells has not been previously studied. We now describe a human T-cell receptor-transgenic humanized mouse model that permits the study of autoreactive T-cell development in a human thymus. Our studies demonstrate that thymocytes expressing the autoreactive Clone 5 TCR, which recognizes insulin B:9–23 presented by HLA-DQ8, are efficiently negatively selected at the double and single positive stage in human immune systems derived from HLA-DQ8+ HSCs. In the absence of hematopoietic expression of the HLA restriction element, negative selection of Clone 5 is less efficient and restricted to the single positive stage. To our knowledge, these data provide the first demonstration of negative selection of human T cells recognizing a naturally-expressed tissue-restricted antigen. Intrathymic antigen presenting cells are required to delete less mature thymocytes, while presentation by medullary thymic epithelial cells may be sufficient to delete more mature single positive cells. These observations set the stage for investigation of putative defects in negative selection in human autoimmune diseases.

Identification of a genomic enhancer that enforces proper apoptosis induction in thymic negative selection

During thymic negative selection, autoreactive thymocytes carrying T cell receptor (TCR) with overtly strong affinity to self-MHC/self-peptide are removed by Bim-dependent apoptosis, but how Bim is specifically regulated to link TCR activation and apoptosis induction is unclear. Here we identify a murine T cell-specific genomic enhancer EBAB (Bub1-Acoxl-Bim), whose deletion leads to accumulation of thymocytes expressing high affinity TCRs. Consistently, EBAB knockout mice have defective negative selection and fail to delete autoreactive thymocytes in various settings, with this defect accompanied by reduced Bim expression and apoptosis induction. By contrast, EBAB is dispensable for maintaining peripheral T cell homeostasis via Bim-dependent pathways. Our data thus implicate EBAB as an important, developmental stage-specific regulator of Bim expression and apoptosis induction to enforce thymic negative selection and suppress autoimmunity. Our study unravels a part of genomic enhancer codes that underlie complex and context-dependent gene regulation in TCR signaling.

Th1 Biased Progressive Autoimmunity in Aged Aire-Deficient Mice Accelerated Thymic Epithelial Cell Senescence.

Although autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus, are frequently associated with premature aging of the thymus, a direct link is missing between autoimmunity and thymic atrophy. Here we monitored the progression of thymic involution in Aire-deficient mice, in which defective negative selection causes spontaneous and progressive development of autoimmunity. In young and middle-aged mice, Aire deficiency appeared to be protective as supported by the reduced β-gal+ epithelial cells and the enhanced thymic output. However, once the autoimmune phenotype was fully developed in aged Aire-deficient mice, their thymuses underwent accelerated involution. In comparison to the age-matched wildtype littermates, old Aire-deficient mice showed lower numbers of total thymocytes and recent thymic emigrants but more β-gal+ thymic epithelial cells. This phenomenon may partly be attributable to the increased number of activated Th1 cells homing to the thymus. This speculation was further supported by the enhanced thymic aging following repeated challenges with complete Freund’s adjuvant immunization. Taken together, the present study highlights a unique mechanism by which autoimmunity facilitates the senescence of thymic epithelial cells through returning Th1 cells.

Histone Deacetylase 7 mediates tissue-specific autoimmunity via control of innate effector function in invariant Natural Killer T Cells.

We report that Histone Deacetylase 7 (HDAC7) controls the thymic effector programming of Natural Killer T (NKT) cells, and that interference with this function contributes to tissue-specific autoimmunity. Gain of HDAC7 function in thymocytes blocks both negative selection and NKT development, and diverts Vα14/Jα18 TCR transgenic thymocytes into a Tconv-like lineage. Conversely, HDAC7 deletion promotes thymocyte apoptosis and causes expansion of innate-effector cells. Investigating the mechanisms involved, we found that HDAC7 binds PLZF and modulates PLZF-dependent transcription. Moreover, HDAC7 and many of its transcriptional targets are human risk loci for IBD and PSC, autoimmune diseases that strikingly resemble the disease we observe in HDAC7 gain-of-function in mice. Importantly, reconstitution of iNKT cells in these mice mitigated their disease, suggesting that the combined defects in negative selection and iNKT cells due to altered HDAC7 function can cause tissue-restricted autoimmunity, a finding that may explain the association between HDAC7 and hepatobiliary autoimmunity.

Capacity of tTreg generation is not impaired in the atrophied thymus.

Postnatal thymic epithelial cell (TEC) homeostatic defect- or natural aging-induced thymic atrophy results in a decline in central T-cell tolerance establishment, which is constituted by thymocyte negative selection and cluster of differentiation (CD) 4+ thymic regulatory T (tTreg) cell generation. Emerging evidence shows this decline mainly results from defects in negative selection, but there is insufficient evidence regarding whether tTreg cell generation is also impaired. We mechanistically studied tTreg cell generation in the atrophied thymus by utilizing both postnatal TEC-defective (resulting from FoxN1-floxed conditional knockout [cKO]) and naturally aged mouse models. We found that the capacity of tTreg cell generation was not impaired compared to CD4+ thymic conventional T cells, suggesting thymic atrophy positively influences tTreg cell generation. This is potentially attributed to decreased T cell receptor (TCR) signaling strength due to inefficiency in promiscuous expression of self-antigens or presenting a neo-self-antigen by medullary TECs, displaying decreased negative selection-related marker genes (Nur77 and CD5high) in CD4 single positive (SP) thymocytes. Our results provide evidence that the atrophied thymus attempts to balance the defective negative selection by enhancing tTreg cell generation to maintain central T-cell tolerance in the elderly. Once the balance is broken, age-related diseases could take place.

‘Interleukin-7 production by dendritics cells is critical for negative selection of CD4+ lymphocytes in the thymus’

Abstract Introduction IL-7 plays a crucial role in thymopoiesis, this cytokine is mainly expressed by thymic epithelial stroma cells and dendritics cells (DCs). During thymopoiesis, thymocytes undergo positive and negative selection which ensures the generation of a functional T-cell repertoire. In the thymus, Antigen presenting cells including medullary thymic epithelial cells (mTEC) and DCs, express a variety of self-peptides that contribute to purge the repertoire of self-reactive thymocytes. Studies have indicated that co-stimulation through cell surface molecules and cytokine mediated by DCs is important during negative selection. Given that DCs produce IL-7 and IL-7 can modulate TCR signaling, we hypothesized that IL-7 production by DCs might increases TCR sensitivity and modulates negative selection. Objective We limited IL-7 production to thymic epithelial cells by engineering chimeric Rag−/− mice transplanted with hematopoietic stem cells from IL-7−/− mice. Following bone marrow (BM) transplantation, control mice (C57BL/6 BM in Rag−/−) remains healthy whereas recipients of IL-7−/− BM developed signs of illness and dies within 5–6 weeks. Adoptive transfer of CD4+ but not CD8+ T cells recapitulated the disease host. We confirmed a defect in negative selection by observing the production of autoreactive anti-HY T cells TCR transgenic Marilyn male mice receiving IL-7−/− BM. Conclusion Our data identified an unsuspected role of DCs-derived IL-7 in negative selection of CD4+ T cells.

Central immune tolerance establishment under thymic involution (Supported by NIH/NIAID 1R01AI121147 to D-M Su)

Abstract The establishment of central immune tolerance in the thymus is occurs via thymocyte negative selection to deplete self-reactive T clones and thymic CD4+ regulatory T cell (tTreg) generation. Due to limited published data, it is largely unclear whether these two mechanisms are both defective in the involuted thymus, as a result of aging or other insults. We used mouse models with thymic epithelial cell (TEC) conditional defect (FoxN1 conditional knockout) and naturally aged defect to study mechanistic changes in central tolerance establishment. We identified a defect in thymocyte negative selection but not in tTreg generation. In comparison to tTreg with thymic conventional CD4+ T cells (tTcon) in the same thymus, the ratios were increased with severity of thymic involution, suggesting that activity of tTreg generation is increased in the involuted condition. We found that these changes were due to alterations in signaling strength received by T cell receptors, characterized by a decline of Nur77 and CD5 in CD4+ single positive thymocytes and an enhancement of Zap-70 in newly-generated tTregs. This is due to weakened signaling provided by MHC/self-peptide complex ligand from defective TECs, but not due to changes in tTreg development-required factors (TGFb, IL-2, IL-15, and NF-kB). Our results suggest that the involuted thymus attempts to balance the defective negative selection by enhancing tTreg generation to maintain central immune tolerance. If this balance is broken, age-related inflammatory diseases could take place.

CCR7 Controls Thymus Recirculation, but Not Production and Emigration, of Foxp3(+) T Cells.

SummaryCurrent models of Foxp3+ regulatory T cell (Treg) development involve CCR7-mediated migration of thymocytes into the thymus medulla to enable essential interactions with medullary epithelium. However, increased Foxp3+ thymic Treg numbers in Ccr7−/− mice challenge this view, and the role of CCR7 in Treg development, emigration, and/or recirculation is unknown. Here, we have examined CCR7 and Rag2pGFP levels during Treg development and generated Rag2pGFPCcr7−/− mice to study its impact on the intrathymic Treg pool. We reveal surprising developmental heterogeneity in thymocytes described as Treg precursors, showing that they contain recirculating CCR6+CCR7−Rag2pGFP− T cells. Although CCR7 defines bona fide Rag2GFP+ Treg precursors, it is not required for Treg production and emigration. Rather, we show that lack of CCR7 renders the thymus more receptive to Treg thymus homing. Our study reveals a role for CCR7 in limiting Treg recirculation back to the thymus and enables separation of the mechanisms controlling Treg production and thymic recirculation.

Autoimmune hepatitis in a murine autoimmune polyendocrine syndrome type 1 model is directed against multiple autoantigens

Autoimmune polyendocrine syndrome type 1 (APS-1) is caused by mutations of the autoimmune regulator (AIRE) gene. Mouse studies have shown that this results in defective negative selection of T cells and defective early seeding of peripheral organs with regulatory T cells (Tregs). Aire deficiency in humans and mice manifests as spontaneous autoimmunity against multiple organs, and 20% of patients develop an autoimmune hepatitis (AIH). To study AIH in APS-1, we generated a murine model of human AIH on a BALB/c mouse background, in which Aire is truncated at exon 2. A subgroup of 24% of mice is affected by AIH, characterized by lymphoplasmacytic and periportal hepatic infiltrates, autoantibodies, elevated aminotransferases, and a chronic and progressive course of disease. Disease manifestation was dependent on specific Aire mutations and the genetic background of the mice. Though intrahepatic Treg numbers were increased and hyperproliferative, the intrahepatic CD4/CD8 ratio was decreased. The targets of the adaptive autoimmune response were polyspecific and not focussed on essential autoantigens, as described for other APS-1-related autoimmune diseases. The AIH could be treated with prednisolone or adoptive transfer of polyspecific Tregs. Development of AIH in APS-1 is dependent on specific Aire mutations and genetic background genes. Autoimmune response is polyspecific and can be controlled by steroids or transfer with Tregs. This might enable new treatment options for patients with AIH.

T cell cross-reactivity to self peptide:MHCII shapes the foreign peptide: MHCII-specific CD4+ T cell repertoire (APP2P.112)

Abstract The sizes of naïve CD4+ T cell populations specific for different foreign peptide:major histocompatibility complex II (p:MHCII) ligands vary by several orders of magnitude. This variability is biologically significant because large naïve populations generate more effector cells during an immune response than small ones. We hypothesized that the size of a given foreign p:MHCII-specific population is influenced by negative selection due to cross-reactivity between the foreign peptide and self peptides. We found that the frequency of naïve T cells specific for a given foreign p:MHCII ligand was inversely related to the number of self peptides predicted to bind MHCII and sharing 4 TCR contact amino acids with the foreign peptide. In addition, foreign p:MHCII-specific populations that were very small in normal mice were preferentially increased in mice with defective negative selection. Furthermore, gene-targeted mice lacking individual self peptides predicted to be involved in negative selection showed increased responsiveness to cross-reactive foreign peptides. These results suggest that immunodominant foreign peptides have this property because the corresponding naïve CD4+ T cell population has been subjected to minimal negative selection on cross-reactive self peptides.

Enhancement of an anti-tumor immune response by transient blockade of central T cell tolerance.

Thymic central tolerance is a critical process that prevents autoimmunity but also presents a challenge to the generation of anti-tumor immune responses. Medullary thymic epithelial cells (mTECs) eliminate self-reactive T cells by displaying a diverse repertoire of tissue-specific antigens (TSAs) that are also shared by tumors. Therefore, while protecting against autoimmunity, mTECs simultaneously limit the generation of tumor-specific effector T cells by expressing tumor self-antigens. This ectopic expression of TSAs largely depends on autoimmune regulator (Aire), which is expressed in mature mTECs. Thus, therapies to deplete Aire-expressing mTECs represent an attractive strategy to increase the pool of tumor-specific effector T cells. Recent work has implicated the TNF family members RANK and RANK-Ligand (RANKL) in the development of Aire-expressing mTECs. We show that in vivo RANKL blockade selectively and transiently depletes Aire and TSA expression in the thymus to create a window of defective negative selection. Furthermore, we demonstrate that RANKL blockade can rescue melanoma-specific T cells from thymic deletion and that persistence of these tumor-specific effector T cells promoted increased host survival in response to tumor challenge. These results indicate that modulating central tolerance through RANKL can alter thymic output and potentially provide therapeutic benefit by enhancing anti-tumor immunity.

Lymphopenia-Induced Proliferation in Aire-Deficient Mice Helps to Explain Their Autoimmunity and Differences from Human Patients

Studies on autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) and its mouse model – both caused by mutant AIRE – have greatly advanced the understanding of thymic processes that generate a self-tolerant T-cell repertoire. Much is now known about the molecular mechanisms by which AIRE induces tissue-specific antigen expression in thymic epithelium, and how this leads to negative selection of auto-reactive thymocytes. However, we still do not understand the processes that lead to the activation of any infrequent naïve auto-reactive T-cells exported by AIRE-deficient thymi. Also, the striking phenotypic differences between APECED and its mouse models have puzzled researchers for years. The aim of this review is to suggest explanations for some of these unanswered questions, based on a fresh view of published experiments. We review evidence that auto-reactive T-cells can be activated by the prolonged neonatal lymphopenia that naturally develops in young Aire-deficient mice due to delayed export of mature thymocytes. Lymphopenia-induced proliferation (LIP) helps to fill the empty space; by favoring auto-reactive T-cells, it also leads to lymphocyte infiltration in the same tissues as in day 3 thymectomized animals. The LIP becomes uncontrolled when loss of Aire is combined with defects in genes responsible for anergy induction and Treg responsiveness, or in signaling from the T-cell receptor and homeostatic cytokines. In APECED patients, LIP is much less likely to be involved in activation of naïve auto-reactive T-cells, as humans are born with a more mature immune system than in neonatal mice. We suggest that human AIRE-deficiency presents with different phenotypes because of additional precipitating factors that compound the defective negative selection of potentially autoaggressive tissue-specific thymocytes.

Late-onset myasthenia gravis – CTLA4low genotype association and low-for-age thymic output of naïve T cells

Late-onset myasthenia gravis (LOMG) has become the largest MG subgroup, but the underlying pathogenetic mechanisms remain mysterious. Among the few etiological clues are the almost unique serologic parallels between LOMG and thymoma-associated MG (TAMG), notably autoantibodies against acetylcholine receptors, titin, ryanodine receptor, type I interferons or IL-12. This is why we checked LOMG patients for two further peculiar features of TAMG – its associations with the CTLA4high/gain-of-function +49A/A genotype and with increased thymic export of naïve T cells into the blood, possibly after defective negative selection in AIRE-deficient thymomas. We analyzed genomic DNA from 116 Caucasian LOMG patients for CTLA4 alleles by PCR/restriction fragment length polymorphism, and blood mononuclear cells for recent thymic emigrants by quantitative PCR for T cell receptor excision circles. In sharp contrast with TAMG, we now find that: i) CTLA4low +49G(+) genotypes were more frequent (p = 0.0029) among the 69 LOMG patients with age at onset ≥60 years compared with 172 healthy controls; ii) thymic export of naïve T cells from the non-neoplastic thymuses of 36 LOMG patients was lower (p = 0.0058) at diagnosis than in 77 age-matched controls. These new findings are important because they suggest distinct initiating mechanisms in TAMG and LOMG and hint at aberrant immuno-regulation in the periphery in LOMG. We therefore propose alternate defects in central thymic or peripheral tolerance induction in TAMG and LOMG converging on similar final outcomes. In addition, our data support a 60-year-threshold for onset of ‘true LOMG’ and an LOMG/early-onset MG overlapping group of patients between 40 and 60.

Redox balance of mouse medullary CD4 single‐positive thymocytes

After positive selection, the newly differentiated single-positive (SP) thymocytes undergo negative selection to eliminate autoreactive T cells, functional maturation to acquire immunocompetence and egress capability. To investigate whether the intracellular reduction/oxidation (redox) balance has an important role on SP maturation, the levels of intracellular reactive oxygen species (ROS) and the expression of proteins that regulate ROS were compared among the four subsets of mouse TCRαβ(+)CD4(+)CD8(-) thymocytes (SP1-SP4) that represent sequential stages of SP differentiation program. A gradual increase of ROS and a gradual decrease of thioredoxin were revealed along the SP maturation process. The high ROS level at the mature SP stage did not result from a specific enrichment at this stage of natural regulatory T cells and SP thymocytes undergoing negative selection (Helios positive). An increase of ROS in the most mature SP4 cells resulted in enhanced cytokine production upon stimulation, whereas an early increase of ROS in the immature SP1 thymocytes resulted in enhanced apoptosis. Aire(-/-) mice that have defects in negative selection and a developmental blockage at the SP3-SP4 transition showed significantly less ROS in SP3 thymocytes. Thymic epithelial cells that have been shown to promote SP maturation in vitro also increased the ROS level of SP thymocytes. These results suggest that ROS may be involved in promoting the functional maturation of CD4(+) SPs and thymic medullary microenvironment contributes to the pro-oxidant shift of SP thymocytes.

Aire Deficiency Promotes TRP-1–Specific Immune Rejection of Melanoma

The thymic transcription factor autoimmune regulator (Aire) prevents autoimmunity in part by promoting expression of tissue-specific self-antigens, which include many cancer antigens. For example, AIRE-deficient patients are predisposed to vitiligo, an autoimmune disease of melanocytes that is often triggered by efficacious immunotherapies against melanoma. Therefore, we hypothesized that Aire deficiency in mice may elevate immune responses to cancer and provide insights into how such responses might be triggered. In this study, we show that Aire deficiency decreases thymic expression of TRP-1 (TYRP1), which is a self-antigen in melanocytes and a cancer antigen in melanomas. Aire deficiency resulted in defective negative selection of TRP-1-specific T cells without affecting thymic numbers of regulatory T cells. Aire-deficient mice displayed elevated T-cell immune responses that were associated with suppression of melanoma outgrowth. Furthermore, transplantation of Aire-deficient thymic stroma was sufficient to confer more effective immune rejection of melanoma in an otherwise Aire wild-type host. Together, our work showed how Aire deficiency can enhance immune responses against melanoma and how manipulating TRP-1-specific T-cell negative selection may offer a logical strategy to enhance immune rejection of melanoma.