Early developing B cells undergo negative selection by central nervous system-specific antigens in the meninges.

Self-reactive B cell progenitors are eliminated through central tolerance checkpoints, a process thought to be restricted to the bone marrow in mammals. Here, we identified a consecutive trajectory of B cell development in the meninges of mice and non-human primates. The meningeal B cells were located predominantly at the dural sinuses, where endothelial cells expressed essential niche factors to support B cell development. Parabiosis experiments together with lineage tracing showed that meningeal developing B cells were replenished continuously from hematopoietic stem cell (HSC)-derived progenitors via a circulation-independent route. Autoreactive immature B cells that recognized myelin oligodendrocyte glycoprotein (MOG), a central nervous system-specific antigen, were eliminated specifically from the meninges. Furthermore, genetic deletion of the Mog gene restored the self-reactive B cell population in the meninges. These findings identify the meninges as a distinct reservoir for B cell development, allowing in situ negative selection to ensure a locally non-self-reactive immune repertoire.

Type III interferon drives thymic B cell activation and regulatory T cell generation.

The activation of thymic B cells is critical for their licensing as antigen presenting cells and resulting ability to mediate T cell central tolerance. The processes leading to licensing are still not fully understood. By comparing thymic B cells to activated Peyer's patch B cells at steady state, we found that thymic B cell activation starts during the neonatal period and is characterized by TCR/CD40-dependent activation, followed by immunoglobulin class switch recombination (CSR) without forming germinal centers. Transcriptional analysis also demonstrated a strong interferon signature, which was not apparent in the periphery. Thymic B cell activation and CSR were primarily dependent on type III IFN signaling, and loss of type III IFN receptor in thymic B cells resulted in reduced thymocyte regulatory T cell (Treg) development. Finally, from TCR deep sequencing, we estimate that licensed B cells induce development of a substantial fraction of the Treg cell repertoire. Together, these findings reveal the importance of steady-state type III IFN in generating licensed thymic B cells that induce T cell tolerance to activated B cells.

Memory IgM protects endogenous insulin from autoimmune destruction.

The enormous diversity of antibody specificities is generated by random rearrangement of immunoglobulin gene segments and is important for general protection against pathogens. Since random rearrangement harbors the risk of producing self-destructive antibodies, it is assumed that autoreactive antibody specificities are removed during early B-cell development leading to a peripheral compartment devoid of autoreactivity. Here, we immunized wild-type mice with insulin as a common self-antigen and monitored diabetes symptoms as a measure for autoimmune disease. Our results show that autoreactive anti-insulin IgM and IgG antibodies associated with autoimmune diabetes can readily be generated in wild-type animals. Surprisingly, recall immunizations induced increased titers of high-affinity insulin-specific IgM, which prevented autoimmune diabetes. We refer to this phenomenon as adaptive tolerance, in which high-affinity memory IgM prevents autoimmune destruction by competing with self-destructive antibodies. Together, this study suggests that B-cell tolerance is not defined by the absolute elimination of autoreactive specificities, as harmful autoantibody responses can be generated in wild-type animals. In contrast, inducible generation of autoantigen-specific affinity-matured IgM acts as a protective mechanism preventing self-destruction.

The insulin secretory granule is a hotspot for autoantigen formation in type 1 diabetes.

In type 1 diabetes, the insulin-producing beta cells of the pancreas are destroyed through the activity of autoreactive T cells. In addition to strong and well-documented HLA class II risk haplotypes, type 1 diabetes is associated with noncoding polymorphisms within the insulin gene locus. Furthermore, autoantibody prevalence data and murine studies implicate insulin as a crucial autoantigen for the disease. Studies identify secretory granules, where proinsulin is processed into mature insulin, stored and released in response to glucose stimulation, as a source of antigenic epitopes and neoepitopes. In this review, we integrate established concepts, including the role that susceptible HLA and thymic selection of the T cell repertoire play in setting the stage for autoimmunity, with emerging insights about beta cell and insulin secretory granule biology. In particular, the acidic, peptide-rich environment of secretory granules combined with its array of enzymes generates a distinct proteome that is unique to functional beta cells. These factors converge to generate non-templated peptide sequences that are recognised by autoreactive T cells. Although unanswered questions remain, formation and presentation of these epitopes and the resulting immune responses appear to be key aspects of disease initiation. In addition, these pathways may represent important opportunities for therapeutic intervention.

Aire-dependent transcripts escape Raver2-induced splice-event inclusion in the thymic epithelium.

Aire allows medullary thymic epithelial cells (mTECs) to express and present a large number of self-antigens for central tolerance. Although mTECs express a high diversity of self-antigen splice isoforms, the extent and regulation of alternative splicing events (ASEs) in their transcripts, notably in those induced by Aire, is unknown. In contrast to Aire-neutral genes, we find that transcripts of Aire-sensitive genes show only a low number of ASEs in mTECs, with about a quarter present in peripheral tissues excluded from the thymus. We identify Raver2, as a splicing-related factor overexpressed in mTECs and dependent on H3K36me3 marks, that promotes ASEs in transcripts of Aire-neutral genes, leaving Aire-sensitive ones unaffected. H3K36me3 profiling reveals its depletion at Aire-sensitive genes and supports a mechanism that is preceding Aire expression leading to transcripts of Aire-sensitive genes with low ASEs that escape Raver2-induced alternative splicing. The lack of ASEs in Aire-induced transcripts would result in an incomplete Aire-dependent negative selection of autoreactive T cells, thus highlighting the need of complementary tolerance mechanisms to prevent activation of these cells in the periphery.

Deep generative selection models of T and B cell receptor repertoires with soNNia.

Subclasses of lymphocytes carry different functional roles to work together and produce an immune response and lasting immunity. Additionally to these functional roles, T and B cell lymphocytes rely on the diversity of their receptor chains to recognize different pathogens. The lymphocyte subclasses emerge from common ancestors generated with the same diversity of receptors during selection processes. Here, we leverage biophysical models of receptor generation with machine learning models of selection to identify specific sequence features characteristic of functional lymphocyte repertoires and subrepertoires. Specifically, using only repertoire-level sequence information, we classify CD4+ and CD8+ T cells, find correlations between receptor chains arising during selection, and identify T cell subsets that are targets of pathogenic epitopes. We also show examples of when simple linear classifiers do as well as more complex machine learning methods.

Normal neonatal TREC and KREC levels in early onset juvenile idiopathic arthritis.

OBJECTIVE: Dysregulated central tolerance predisposes to autoimmune diseases. Reduced thymic output as well as compromised central B cell tolerance checkpoints have been proposed in the pathogenesis of juvenile idiopathic arthritis (JIA). The aim of this study was to investigate neonatal levels of T-cell receptor excision circles (TRECs) and kappa-deleting element excision circles (KRECs), as markers of T- and B-cell output at birth, in patients with early onset JIA. METHODS: TRECs and KRECs were quantitated by multiplex qPCR from dried blood spots (DBS), collected 2-5 days after birth, in 156 children with early onset JIA and in 312 matched controls. RESULTS: When analysed from neonatal dried blood spots, the median TREC level was 78 (IQR 55-113) in JIA cases and 88 (IQR 57-117) copies/well in controls. The median KREC level was 51 (IQR 35-69) and 53 (IQR 35-74) copies/well, in JIA cases and controls, respectively. Stratification by sex and age at disease onset did not reveal any difference in the levels of TRECs and KRECs. CONCLUSION: T- and B-cell output at birth, as measured by TREC and KREC levels in neonatal dried blood spots, does not differ in children with early onset JIA compared to controls.

Butyrophilin 2a2 (Btn2a2) expression on thymic epithelial cells promotes central T cell tolerance and prevents autoimmune disease.

Butyrophilins are surface receptors belonging to the immunoglobulin superfamily. While several members of the butyrophilin family have been implicated in the development of unconventional T cells, butyrophilin 2a2 (Btn2a2) has been shown to inhibit conventional T cell activation. Here, we demonstrate that in steady state, the primary source of Btn2a2 are thymic epithelial cells (TEC). Absence of Btn2a2 alters thymic T cell maturation and bypasses central tolerance mechanisms. Furthermore, Btn2a2-/- mice develop spontaneous autoimmunity resembling human primary Sjögren's Syndrome (pSS), including formation of tertiary lymphoid structures (TLS) in target organs. Ligation of Btn2a2 on developing thymocytes is associated with reduced TCR signaling and CD5 levels, while absence of Btn2a2 results in increased TCR signaling and CD5 levels. These results define a novel role for Btn2a2 in promoting central tolerance by modulating TCR signaling strength and indicate a potential mechanism of pSS development.

APECED and the place of AIRE in the puzzle of the immune network associated with autoimmunity.

In the last 20 years, discoveries about the autoimmune regulator (AIRE) protein and its critical role in immune tolerance have provided fundamental insights into understanding the molecular basis of autoimmunity. This review provides a comprehensive overview of the effect of AIRE on immunological tolerance and the characteristics of autoimmune diseases in Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy (APECED), which is caused by biallelic AIRE mutations. A better understanding of the immunological mechanisms of AIRE deficiency may enlighten immune tolerance mechanisms and new diagnostic and treatment strategies for autoimmune diseases. Considering that not all clinical features of APECED are present in a given follow-up period, the diagnosis is not easy in a patient at the first visit. Longer follow-up and a multidisciplinary approach are essential for diagnosis. It is challenging to prevent endocrine and other organ damage compared with other diseases associated with multiple autoimmunities, such as FOXP3, LRBA, and CTLA4 deficiencies. Unfortunately, no curative therapy like haematopoietic stem cell transplantation or specific immunomodulation is present that is successful in the treatment.

Rectangular tolerance regions and multivariate normal reference regions in laboratory medicine.

Reference intervals are widely used in the interpretation of results of biochemical and physiological tests of patients. When there are multiple biochemical analytes measured from each subject, a multivariate reference region is needed. Because of their greater specificity against false positives, such reference regions are more desirable than separate univariate reference intervals that disregard the cross-correlations between variables. Traditionally, under multivariate normality, reference regions have been constructed as ellipsoidal regions. This approach suffers from a major drawback: it cannot detect component-wise extreme observations. In the present work, procedures are developed to construct rectangular reference regions in the multivariate normal setup. The construction is based on the criteria for tolerance intervals. The problems addressed include the computation of a rectangular tolerance region and simultaneous tolerance intervals. Also addressed is the computation of mixed reference intervals that include both two-sided and one-sided limits, simultaneously. A parametric bootstrap approach is used in the computations, and the accuracy of the proposed methodology is assessed using estimated coverage probabilities. The problem of sample size determination is also addressed, and the results are illustrated using examples that call for the computation of reference regions.

Lessons from primary immunodeficiencies: Autoimmune regulator and autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy.

The discovery of the autoimmune regulator (AIRE) protein and the delineation of its critical contributions in the establishment of central immune tolerance has significantly expanded our understanding of the immunological mechanisms that protect from the development of autoimmune disease. The parallel identification and characterization of patient cohorts with the monogenic disorder autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), which is typically caused by biallelic AIRE mutations, has underscored the critical contribution of AIRE in fungal immune surveillance at mucosal surfaces and in prevention of multiorgan autoimmunity in humans. In this review, we synthesize the current clinical, genetic, molecular and immunological knowledge derived from basic studies in Aire-deficient animals and from APECED patient cohorts. We also outline major advances and research endeavors that show promise for informing improved diagnostic and therapeutic approaches for patients with APECED.

Impaired central tolerance induces changes in the gut microbiota that exacerbate autoimmune hepatitis.

Medullary thymic epithelial cells (mTECs) induce T cell tolerance in the thymus through the elimination of self-reactive thymocytes. Commensal bacteria are also critical for shaping T cell responses in the gut and distal organs. We previously showed that mice depleted of mTECs (Traf6ΔTEC) generated autoreactive T cells and developed autoimmune hepatitis (AIH). In this report, we found that Toll-like receptor (TLR)-mediated microbial sensing on liver hematopoietic cells and the gut microbiota contributed to AIH development in Traf6ΔTEC mice. While adoptive transfer of thymic Traf6ΔTEC T cells in immune-deficient mice was sufficient for AIH development, colonization of germ-free mice with Traf6ΔTEC microbiota failed to induce AIH, suggesting that the gut microbiota contributes to but is not sufficient for AIH development. Microbiota-mediated exacerbation of AIH associated with increased numbers of hepatic Foxp3+ T cells and their increase was proportional to the degree of inflammation. The contribution of the gut microbiota to AIH development associated with an altered microbial signature whose composition was influenced by the qualitative nature of the thymic T cell compartment. These results suggest that aberrant selection of T cells in the thymus can induce changes in the gut microbiota that lead to exacerbation of organ-specific autoimmunity and AIH. Our results add to our understanding of the mechanisms of AIH development and create a platform towards developing novel therapeutic approaches for treating this disease.

Enteric α-defensins on the verge of intestinal immune tolerance and inflammation.

The gut is the biggest immune organ in the body that encloses commensal microbiota which aids in food digestion. Paneth cells, positioned at the frontline of host-microbiota interphase, can modulate the composition of microbiota. Paneth cells achieve this via the delivery of microbicidal substances, among which enteric α-defensins play the primary role. If microbiota is dysregulated, it can impact the function of the local mucosal immune system. Importantly, this system is also exposed to an enormous number of antigens which are derived from the gut-resident microbiota and processed food, and may potentially trigger undesirable local inflammatory responses. To understand the intricate regulations and liaisons between Paneth cells, microbiota and the immune system in this intestinal-specific setting, one must consider their mode of interaction in a wider context of regulatory processes which impose immune tolerance not only to self, but also to microbiota and food-derived antigens. These include, but are not limited to, tolerogenic mechanisms of central tolerance in the thymus and peripheral tolerance in the secondary lymphoid organs, and the intestine itself. Defects in these processes can compromise homeostasis in the intestinal mucosal immunity. In this review, which is focused on tolerance to intestinal antigens and its relevance for the pathogenesis of gut immune diseases, we provide an outline of such multilayered immune control mechanisms and highlight functional links that underpin their cooperative nature.

Thymus machinery for T-cell selection.

An immunocompetent and self-tolerant pool of naive T cells is formed in the thymus through the process of repertoire selection. T cells that are potentially capable of responding to foreign antigens are positively selected in the thymic cortex and are further selected in the thymic medulla to help prevent self-reactivity. The affinity between T-cell antigen receptors expressed by newly generated T cells and self-peptide-major histocompatibility complexes displayed in the thymic microenvironments plays a key role in determining the fate of developing T cells during thymic selection. Recent advances in our knowledge of the biology of thymic epithelial cells have revealed unique machinery that contributes to positive and negative selection in the thymus. In this article, we summarize recent findings on thymic T-cell selection, focusing on the machinery unique to thymic epithelial cells.

Integrity of IKK/NF-κB Shields Thymic Stroma That Suppresses Susceptibility to Autoimmunity, Fungal Infection, and Carcinogenesis.

A pathogenic connection between autoreactive T cells, fungal infection, and carcinogenesis has been demonstrated in studies of human autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) as well as in a mouse model in which kinase-dead Ikkα knock-in mice develop impaired central tolerance, autoreactive T cell-mediated autoimmunity, chronic fungal infection, and esophageal squamous cell carcinoma, which recapitulates APECED. IκB kinase α (IKKα) is one subunit of the IKK complex required for NF-κB activation. IKK/NF-κB is essential for central tolerance establishment by regulating the development of medullary thymic epithelial cells (mTECs) that facilitate the deletion of autoreactive T cells in the thymus. In this review, we extensively discuss the pathogenic roles of inborn errors in the IKK/NF-κB loci in the phenotypically related diseases APECED, immune deficiency syndrome, and severe combined immunodeficiency; differentiate how IKK/NF-κB components, through mTEC (stroma), T cells/leukocytes, or epithelial cells, contribute to the pathogenesis of infectious diseases, autoimmunity, and cancer; and highlight the medical significance of IKK/NF-κB in these diseases.

Contributions of Age-Related Thymic Involution to Immunosenescence and Inflammaging.

Immune system aging is characterized by the paradox of immunosenescence (insufficiency) and inflammaging (over-reaction), which incorporate two sides of the same coin, resulting in immune disorder. Immunosenescence refers to disruption in the structural architecture of immune organs and dysfunction in immune responses, resulting from both aged innate and adaptive immunity. Inflammaging, described as a chronic, sterile, systemic inflammatory condition associated with advanced age, is mainly attributed to somatic cellular senescence-associated secretory phenotype (SASP) and age-related autoimmune predisposition. However, the inability to reduce senescent somatic cells (SSCs), because of immunosenescence, exacerbates inflammaging. Age-related adaptive immune system deviations, particularly altered T cell function, are derived from age-related thymic atrophy or involution, a hallmark of thymic aging. Recently, there have been major developments in understanding how age-related thymic involution contributes to inflammaging and immunosenescence at the cellular and molecular levels, including genetic and epigenetic regulation, as well as developments of many potential rejuvenation strategies. Herein, we discuss the research progress uncovering how age-related thymic involution contributes to immunosenescence and inflammaging, as well as their intersection. We also describe how T cell adaptive immunity mediates inflammaging and plays a crucial role in the progression of age-related neurological and cardiovascular diseases, as well as cancer. We then briefly outline the underlying cellular and molecular mechanisms of age-related thymic involution, and finally summarize potential rejuvenation strategies to restore aged thymic function.

Medullary thymic epithelial stem cells: role in thymic epithelial cell maintenance and thymic involution.

The thymus consists of two distinct anatomical regions, the cortex and the medulla; medullary thymic epithelial cells (mTECs) play a crucial role in establishing central T-cell tolerance for self-antigens. Although the understanding of mTEC development in thymic organogenesis as well as the regulation of their differentiation and maturation has improved, the mechanisms of postnatal maintenance remain poorly understood. This issue has a central importance in immune homeostasis and physiological thymic involution as well as autoimmune disorders in various clinicopathological settings. Recently, several reports have demonstrated the existence of TEC stem or progenitor cells in the postnatal thymus, which are either bipotent or unipotent. We identified stem cells specified for mTEC-lineage that are generated in the thymic ontogeny and may sustain mTEC regeneration and lifelong central T-cell self-tolerance. This finding suggested that the thymic medulla is maintained autonomously by its own stem cells. Although several issues, including the relationship with other putative TEC stem/progenitors, remain unclear, further examination of mTEC stem cells (mTECSCs) and their regulatory mechanisms may contribute to the understanding of postnatal immune homeostasis. Possible relationships between decline of mTECSC activity and early thymic involution as well as various autoimmune disorders are discussed.

The Autoimmune Regulator (Aire) transactivates HLA-G gene expression in thymic epithelial cells.

The Autoimmune Regulator (Aire) protein coordinates the negative selection of developing thymocytes by inducing the expression of hundreds of tissue-specific antigens within the thymic medulla, which is also a primary site of the expression of the immune checkpoint HLA-G molecule. Considering the immunomodulatory properties of Aire and HLA-G, and considering that the role of the constitutive thymus expression of HLA-G has not been elucidated, we studied the effect of AIRE cDNA transfection on HLA-G expression in 4D6 thymic cells and in the HLA-G-positive JEG-3 choriocarcinoma cells. Aire promoted the transactivation of HLA-G gene by increasing the overall transcription, inducing the transcription of at least G1 and G2/G4 isoforms, and incrementing the occurrence and distribution of intracellular HLA-G protein solely in 4D6 thymic cells. Luciferase-based assays and chromatin immunoprecipitation experiments performed in 4D6 cells revealed that Aire targeted at least two regions within the 5'-untranslated regulatory region (5'-URR) extending 1·4 kb from the first ATG initiation codon. The interaction occurs independently of three putative Aire-binding sites. These results indicate that the Aire-induced upregulation of HLA-G in thymic cells is likely to act through the interaction of Aire with specific HLA-G 5'-URR DNA-binding factors. Such a multimeric transcriptional complex might operate in the thymus during the process of promiscuous gene expression.

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.

EBI2 contributes to the induction of thymic central tolerance in mice by promoting rapid motility of medullary thymocytes.

Maturing thymocytes enter the thymic medulla, where they encounter numerous self-antigens presented by antigen presenting cells (APCs). Those thymocytes that are strongly self-reactive undergo either negative selection or diversion into the regulatory T-cell lineage. Although the majority of the proteome is expressed in the medulla, many self-antigens are expressed by only a minor fraction of medullary APCs; thus, thymocytes must efficiently enter the medulla and scan APCs to ensure central tolerance. Chemokine receptors promote lymphocyte migration, organization within tissues, and interactions with APCs in lymphoid organs. The chemokine receptor EBI2 governs localization of T cells, B cells, and dendritic cells (DCs) during immune responses in secondary lymphoid organs. However, the role of EBI2 in thymocyte development has not been elucidated. Here, we demonstrate that EBI2 is expressed by murine CD4+ single positive (CD4SP) thymocytes and thymic DCs. EBI2 deficiency alters the TCR repertoire, but does not grossly impact thymocyte cellularity or subset distribution. EBI2 deficiency also impairs negative selection of OT-II TCR transgenic thymocytes responding to an endogenous self-antigen. Two-photon imaging revealed that EBI2 deficiency results in reduced migration and impaired medullary accumulation of CD4SP thymocytes. These data identify a role for EBI2 in promoting efficient thymic central tolerance.