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Although CD8 + T-cell-mediated autoimmune β cell destruction occurs in type 1 diabetes (T1D), the target epitopes processed and presented by β cells are unknown. To identify them, we combined peptidomics and transcriptomics strategies. Inflammatory cytokines increased peptide presentation in vitro, paralleling upregulation of human leukocyte antigen (HLA) class I expression. Peptide sources featured several insulin granule proteins and all known β cell antigens, barring islet-specific glucose-6-phosphatase catalytic subunit-related protein. Preproinsulin yielded HLA-A2-restricted epitopes previously described. Secretogranin V and its mRNA splice isoform SCG5-009, proconvertase-2, urocortin-3, the insulin gene enhancer protein ISL-1, and an islet amyloid polypeptide transpeptidation product emerged as antigens processed into HLA-A2-restricted epitopes, which, as those already described, were recognized by circulating naive CD8 + T cells in T1D and healthy donors and by pancreas-infiltrating cells in T1D donors. This peptidome opens new avenues to understand antigen processing by β cells and for the development of T cell biomarkers and tolerogenic vaccination strategies., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The thymus is responsible for generating a diverse yet self-tolerant pool of T cells 1 . Although the thymic medulla consists mostly of developing and mature AIRE + epithelial cells, recent evidence has suggested that there is far greater heterogeneity among medullary thymic epithelial cells than was previously thought 2 . Here we describe in detail an epithelial subset that is remarkably similar to peripheral tuft cells that are found at mucosal barriers 3 . Similar to the periphery, thymic tuft cells express the canonical taste transduction pathway and IL-25. However, they are unique in their spatial association with cornified aggregates, ability to present antigens and expression of a broad diversity of taste receptors. Some thymic tuft cells pass through an Aire-expressing stage and depend on a known AIRE-binding partner, HIPK2, for their development. Notably, the taste chemosensory protein TRPM5 is required for their thymic function through which they support the development and polarization of thymic invariant natural killer T cells and act to establish a medullary microenvironment that is enriched in the type 2 cytokine, IL-4. These findings indicate that there is a compartmentalized medullary environment in which differentiation of a minor and highly specialized epithelial subset has a non-redundant role in shaping thymic function.

The study of organogenesis, tissue-homeostasis and regeneration requires the precise assessment of in vivo cell proliferation. To this end a host of methods have been developed to detect and quantify DNA synthesis in proliferating cells. These include cell labeling with various nucleotide analogues and fluorescence reporter-based animal models with each method presenting its idiosyncratic shortcomings. Quantitative assessment of epithelial cell turnover has been partly hampered due to their variable and limited in vivo accessibility and the requirement for harsher isolation procedures to procure single cells for FACS analysis. Here, we report a reliable protocol to study in vivo cell proliferation of epithelial cells in mice by repeatedly injecting EdU intravenously for an extended 12-day period. EdU incorporation was quantitated ex vivo by FACS after tissue dissociation in order to obtain single epithelial cell suspensions. As a lead population, we analyzed thymic epithelial cells (TECs), where we were able to label compartmentalized TEC subsets to saturation without apparent toxic effects on the thymus architecture or stress-sensitive TEC lineage differentiation. The data is in concordance with the prevailing model of medullary TEC terminal differentiation that includes the post-Aire stage. The same protocol was successfully applied to epithelial cells of various other organs - skin, lymph node, kidney and small intestine - tissues with widely varying frequencies and rates of proliferating epithelial cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The human leukocyte antigen-A2 (HLA-A2)-restricted zinc transporter 8 186-194 (ZnT8 186-194 ) and other islet epitopes elicit interferon-γ secretion by CD8 + T cells preferentially in type 1 diabetes (T1D) patients compared with controls. We show that clonal ZnT8 186-194 -reactive CD8 + T cells express private T cell receptors and display equivalent functional properties in T1D and healthy individuals. Ex vivo analyses further revealed that CD8 + T cells reactive to ZnT8 186-194 and other islet epitopes circulate at similar frequencies and exhibit a predominantly naïve phenotype in age-matched T1D and healthy donors. Higher frequencies of ZnT8 186-194 -reactive CD8 + T cells with a more antigen-experienced phenotype were detected in children versus adults, irrespective of disease status. Moreover, some ZnT8 186-194 -reactive CD8 + T cell clonotypes were found to cross-recognize a Bacteroides stercoris mimotope. Whereas ZnT8 was poorly expressed in thymic medullary epithelial cells, variable thymic expression levels of islet antigens did not modulate the peripheral frequency of their cognate CD8 + T cells. In contrast, ZnT8 186-194 -reactive cells were enriched in the pancreata of T1D patients versus nondiabetic and type 2 diabetic individuals. Thus, islet-reactive CD8 + T cells circulate in most individuals but home to the pancreas preferentially in T1D patients. We conclude that the activation of this common islet-reactive T cell repertoire and progression to T1D likely require defective peripheral immunoregulation and/or a proinflammatory islet microenvironment., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Establishing effective central tolerance requires the promiscuous expression of tissue-restricted antigens by medullary thymic epithelial cells. However, whether central tolerance also extends to post-translationally modified proteins is not clear. Here we show a mouse model of autoimmunity in which disease development is dependent on post-translational modification (PTM) of the tissue-restricted self-antigen collagen type II. T cells specific for the non-modified antigen undergo efficient central tolerance. By contrast, PTM-reactive T cells escape thymic selection, though the PTM variant constitutes the dominant form in the periphery. This finding implies that the PTM protein is absent in the thymus, or present at concentrations insufficient to induce negative selection of developing thymocytes and explains the lower level of tolerance induction against the PTM antigen. As the majority of self-antigens are post-translationally modified, these data raise the possibility that T cells specific for other self-antigens naturally subjected to PTM may escape central tolerance induction by a similar mechanism.

The basic two-step terminal differentiation model of the medullary thymic epithelial cell (mTEC) lineage from immature MHC class II (MHCII) lo to mature MHCII hi mTECs has recently been extended to include a third stage, namely the post-Aire MHCII lo subset as identified by lineage-tracing models. However, a suitable surface marker distinguishing the phenotypically overlapping pre- from the post-Aire MHCII lo stage has been lacking. In this study, we introduce the lectin Tetragonolobus purpureas agglutinin (TPA) as a novel cell surface marker that allows for such delineation. Based on our data, we derived the following sequence of mTEC differentiation: TPA lo MHCII lo → TPA lo MHCII hi → TPA hi MHCII hi → TPA hi MHCII lo Surprisingly, in the steady-state postnatal thymus TPA lo MHCII lo pre-Aire rather than terminally differentiated post-Aire TPA hi MHCII lo mTECs were marked for apoptosis at an exceptionally high rate of ∼70%. Hence, only the minor cycling fraction of the MHCII lo subset (<20%) potentially qualified as mTEC precursors. FoxN1 expression inversely correlated with the fraction of slow cycling and apoptotic cells within the four TPA subsets. TPA also further subdivided human mTECs, although with different subset distribution. Our revised road map emphazises close parallels of terminal mTEC development with that of skin, undergoing an alternative route of cell death, namely cornification rather than apoptosis. The high rate of apoptosis in pre-Aire MHCII lo mTECs points to a "quality control" step during early mTEC differentiation., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Immunization with myelin components can elicit experimental autoimmune encephalomyelitis (EAE). EAE susceptibility varies between mouse strains, depending on the antigen employed. BL/6 mice are largely resistant to EAE induction with proteolipid protein (PLP), probably a reflection of antigen-specific tolerance. However, the extent and mechanism(s) of tolerance to PLP remain unclear. Here, we identified three PLP epitopes in PLP-deficient BL/6 mice. PLP-sufficient mice did not respond against two of these, whereas tolerance was "leaky" for an epitope with weak predicted MHCII binding, and only this epitope was encephalitogenic. In TCR transgenic mice, the "EAE-susceptibility-associated" epitope was "ignored" by specific CD4 T cells, whereas the "resistance-associated" epitope induced clonal deletion and T reg induction in the thymus. Central tolerance was autoimmune regulator dependent and required expression and presentation of PLP by thymic epithelial cells (TECs). TEC-specific ablation of PLP revealed that peripheral tolerance, mediated by dendritic cells through recessive tolerance mechanisms (deletion and anergy), could largely compensate for a lack of central tolerance. However, adoptive EAE was exacerbated in mice lacking PLP in TECs, pointing toward a non-redundant role of the thymus in dominant tolerance to PLP. Our findings reveal multiple layers of tolerance to a central nervous system autoantigen that vary among epitopes and thereby specify disease susceptibility. Understanding how different modalities of tolerance apply to distinct T cell epitopes of a target in autoimmunity has implications for antigen-specific strategies to therapeutically interfere with unwanted immune reactions against self.

The origin of the thymic epithelium, i.e. the cortical (cTEC) and medullary (mTEC) epithelial cells, from bipotent stem cells through TEC progenitors and lineage-specific progeny still remains poorly understood. We sought to obtain an unbiased view of the incipient emergence of TEC subsets by following embryonic TEC development based on co-expression of EpCAM, CD80 and MHC class II (MHCII) on non-hematopoietic (CD45 - ) thymic stromal cells in wild-type BL6 mice. Using a combination of ex vivo analysis, Re-aggregate Thymic Organ Culture (RTOC) reconstitution assays and mathematical modeling, we traced emergent lineage commitment in murine embryonic TECs. Both experimental and mathematical datasets supported the following developmental sequence: MHCII - CD80 - → MHCII lo CD80 - → MHCII hi CD80 - → MHCII hi CD80 hi TECs, whereby MHCII hi CD80 - and MHCII hi CD80 hi TECs bear features of cTECs and mTECs respectively. These emergent MHCII hi CD80 - cTECs directly generate mature MHCII hi CD80 hi mTECs in vivo and in vitro, thus supporting the asynchronous model of TEC lineage commitment., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Adult tissue-specific stem cells (SCs) mediate tissue homeostasis and regeneration and can give rise to all lineages in the corresponding tissue, similar to the early progenitors that generate organs in the first place. However, the developmental origins of adult SCs are largely unknown. We recently identified thymosphere-forming stem cells (TSFCs) in the adult mouse thymus, which display genuine stemness features and can generate the two major thymic epithelial cell lineages. Here, we show that embryonic TSFCs possess stemness features but differ from adult TSFCs in surface marker profile. Our findings support the model of a continuous thymic SC lineage that is maintained throughout ontogeny. TGF-β signaling differentially affects embryonic versus adult thymosphere formation, suggesting that thymic epithelial SC potency depends on both developmental stage and environmental signals. Collectively, our findings suggest that embryonic TSFCs contribute to an adult SC pool and that TSFC plasticity is controlled by TGF-β signaling., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

T-cell polyspecificity, predicting that individual T cells recognize a continuum of related ligands, implies that multiple antigens can tolerize T cells specific for a given self-antigen. We previously showed in C57BL/6 mice that part of the CD4(+) T-cell repertoire specific for myelin oligodendrocyte glycoprotein (MOG) 35-55 also recognizes the neuronal antigen neurofilament medium (NF-M) 15-35. Such bi-specific CD4(+) T cells are frequent and produce inflammatory cytokines after stimulation. Since T cells recognizing two self-antigens would be expected to be tolerized more efficiently, this finding prompted us to study how polyspecificity impacts tolerance. We found that similar to MOG, NF-M is expressed in the thymus by medullary thymic epithelial cells, a tolerogenic population. Nevertheless, the frequency, phenotype, and capacity to transfer experimental autoimmune encephalomyelitis (EAE) of MOG35-55 -reactive CD4(+) T cells were increased in MOG-deficient but not in NF-M-deficient mice. We found that presentation of NF-M15-35 by I-A(b) on dendritic cells is of short duration, suggesting unstable MHC class II binding. Consistently, introducing an MHC-anchoring residue into NF-M15-35 (NF-M15-35 T20Y) increased its immunogenicity, activating a repertoire able to induce EAE. Our results show that in C57BL/6 mice bi-specific encephalitogenic T cells manage to escape tolerization due to inefficient exposure to two self-antigens., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Recent studies have leveraged MHC tetramer and TCR sequencing approaches towards a more precise characterization of the peripheral T cell repertoire, providing important insight into both the contribution of self-reactive T cells to the overall repertoire and their function. The peripheral T cell repertoire of healthy individuals contains a high frequency of diverse, self-reactive T cells. Furthermore, self-reactive T cells can perform essential beneficial physiological functions. We review these recent findings here, and discuss their implications to the current understanding of peripheral tolerance and the role of self-reactive T cells in autoimmune disease. We outline gaps in understanding, and argue that an important step forward is to revise the definition of self-reactive T cells to incorporate new concepts regarding the nature and physiological functions of different populations of T cells capable of recognizing self-antigens., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Promiscuous expression of a plethora of tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential for central tolerance. This promiscuous gene expression (pGE) is characterized by inclusion of a broad range of TRAs and by its mosaic expression patterns, i.e. each antigen is only expressed in 1-3% of mTECs. It is currently unclear to which extent random and/or deterministic mechanisms are involved in the regulation of pGE. In order to address this issue, we deconstructed the transcriptional heterogeneity in mTEC to minor subsets expressing a particular TRA. We identified six delineable co-expression groups in mouse mTECs. These co-expression groups displayed a variable degree of mutual overlap and mapped to different stages of mTEC development. Co-expressed genes showed chromosomal preference and clustered within delimited genomic regions. Moreover, co-expression groups in mice and humans selected by a pair of orthologous genes preferentially co-expressed sets of orthologous genes attesting to the species conservation of pGE between mouse and human. Furthermore, co-expressed genes were enriched for specific transcription factor binding motifs concomitant with up-regulation of the corresponding transcription factors, implicating additional factors in the regulation of pGE besides the Autoimmune Regulator (Aire). Thus promiscuous transcription of self-antigens in mTECs entails a highly coordinated process, which is evolutionary strictly conserved between species., (Copyright © 2015 Elsevier Ltd. All rights reserved.)