Aire mediates tolerance to insulin through thymic trimming of high-affinity T cell clones.

Insulin is a central autoantigen in the pathogenesis of T1D, and thymic epithelial cell expression of insulin under the control of the Autoimmune Regulator (Aire) is thought to be a key component of maintaining tolerance to insulin. In spite of this general working model, direct detection of this thymic selection on insulin-specific T cells has been somewhat elusive. Here, we used a combination of highly sensitive T cell receptor transgenic models for detecting thymic selection and sorting and sequencing of Insulin-specific CD4+ T cells from Aire-deficient mice as a strategy to further define their selection. This analysis revealed a number of unique t cell receptor (TCR) clones in Aire-deficient hosts with high affinity for insulin/major histocompatibility complex (MHC) ligands. We then modeled the thymic selection of one of these clones in Aire-deficient versus wild-type hosts and found that this model clone could escape thymic negative selection in the absence of thymic Aire. Together, these results suggest that thymic expression of insulin plays a key role in trimming and removing high-affinity insulin-specific T cells from the repertoire to help promote tolerance.

The self-reactive FVIII T cell repertoire in healthy individuals relies on a short set of epitopes and public clonotypes.

Non-mutated FVIII-specific CD4 T cell epitopes have been recently found to contribute to the development of inhibitors in patients with hemophilia A (HA), while auto-reactive CD4 T cells specific to FVIII circulate in the blood of healthy individuals at a frequency close to the foreign protein ovalbumin. Thus, although FVIII is a self-protein, the central tolerance raised against FVIII appears to be low. In this study, we conducted a comprehensive analysis of the FVIII CD4 T cell repertoire in 29 healthy donors. Sequencing of the CDR3ß TCR region from isolated FVIII-specific CD4 T cells revealed a limited usage and pairing of TRBV and TRBJ genes as well as a mostly hydrophobic composition of the CDR3ß region according to their auto-reactivity. The FVIII repertoire is dominated by a few clonotypes, with only 13 clonotypes accounting for half of the FVIII response. Through a large-scale epitope mapping of the full-length FVIII sequence, we identified 18 immunodominant epitopes located in the A1, A3, C1, and C2 domains and covering half of the T cell response. These epitopes exhibited a broad specificity for HLA-DR or DP molecules or both. T cell priming with this reduced set of peptides revealed that highly expanded clonotypes specific to these epitopes were responsible individually for up to 32% of the total FVIII repertoire. These FVIII T cell epitopes and clonotypes were shared among HLA-unrelated donors tested and previously reported HA patients. Our study highlights the role of the auto-reactive T cell response against FVIII in HA and its similarity to the response observed in healthy individuals. Thus, it provides valuable insights for the development of new tolerance induction and deimmunization strategies.

A robust deep learning workflow to predict CD8 + T-cell epitopes.

BACKGROUND: T-cells play a crucial role in the adaptive immune system by triggering responses against cancer cells and pathogens, while maintaining tolerance against self-antigens, which has sparked interest in the development of various T-cell-focused immunotherapies. However, the identification of antigens recognised by T-cells is low-throughput and laborious. To overcome some of these limitations, computational methods for predicting CD8 + T-cell epitopes have emerged. Despite recent developments, most immunogenicity algorithms struggle to learn features of peptide immunogenicity from small datasets, suffer from HLA bias and are unable to reliably predict pathology-specific CD8 + T-cell epitopes. METHODS: We developed TRAP (T-cell recognition potential of HLA-I presented peptides), a robust deep learning workflow for predicting CD8 + T-cell epitopes from MHC-I presented pathogenic and self-peptides. TRAP uses transfer learning, deep learning architecture and MHC binding information to make context-specific predictions of CD8 + T-cell epitopes. TRAP also detects low-confidence predictions for peptides that differ significantly from those in the training datasets to abstain from making incorrect predictions. To estimate the immunogenicity of pathogenic peptides with low-confidence predictions, we further developed a novel metric, RSAT (relative similarity to autoantigens and tumour-associated antigens), as a complementary to 'dissimilarity to self' from cancer studies. RESULTS: TRAP was used to identify epitopes from glioblastoma patients as well as SARS-CoV-2 peptides, and it outperformed other algorithms in both cancer and pathogenic settings. TRAP was especially effective at extracting immunogenicity-associated properties from restricted data of emerging pathogens and translating them onto related species, as well as minimising the loss of likely epitopes in imbalanced datasets. We also demonstrated that the novel metric termed RSAT was able to estimate immunogenic of pathogenic peptides of various lengths and species. TRAP implementation is available at: https://github.com/ChloeHJ/TRAP . CONCLUSIONS: This study presents a novel computational workflow for accurately predicting CD8 + T-cell epitopes to foster a better understanding of antigen-specific T-cell response and the development of effective clinical therapeutics.

Quantifying changes in the T cell receptor repertoire during thymic development.

One of the feats of adaptive immunity is its ability to recognize foreign pathogens while sparing the self. During maturation in the thymus, T cells are selected through the binding properties of their antigen-specific T-cell receptor (TCR), through the elimination of both weakly (positive selection) and strongly (negative selection) self-reactive receptors. However, the impact of thymic selection on the TCR repertoire is poorly understood. Here, we use transgenic Nur77-mice expressing a T-cell activation reporter to study the repertoires of thymic T cells at various stages of their development, including cells that do not pass selection. We combine high-throughput repertoire sequencing with statistical inference techniques to characterize the selection of the TCR in these distinct subsets. We find small but significant differences in the TCR repertoire parameters between the maturation stages, which recapitulate known differentiation pathways leading to the CD4+ and CD8+ subtypes. These differences can be simulated by simple models of selection acting linearly on the sequence features. We find no evidence of specific sequences or sequence motifs or features that are suppressed by negative selection. These results favour a collective or statistical model for T-cell self non-self discrimination, where negative selection biases the repertoire away from self recognition, rather than ensuring lack of self-reactivity at the single-cell level.

What's self got to do with it: Sources of heterogeneity among naive T cells.

There is a long-standing assumption that naive CD4+ and CD8+ T cells are largely homogeneous populations despite the extraordinary diversity of their T cell receptors (TCR). The self-immunopeptidome plays a key role in the selection of the naive T cell repertoire in the thymus, and self-peptides are also an important driver of differences between individual naive T cells with regard to their subsequent functional contributions to an immune response. Accumulating evidence suggests that as early as the ß-selection stage of T cell development, when only one of the recombined chains of the mature TCR is expressed, signaling thresholds may be established for positive selection of immature thymocytes. Stochastic encounters subsequently made with self-ligands during positive selection in the thymus imprint functional biases that a T cell will carry with it throughout its lifetime, although ongoing interactions with self in the periphery ensure a level of plasticity in the gene expression wiring of naive T cells. Identifying the sources of heterogeneity in the naive T cell population and which functional attributes of T cells can be modulated through post-thymic interventions versus those that are fixed during T cell development, could enable us to better select or generate T cells with particular traits to improve the efficacy of T cell therapies.

LAMP2 regulates autophagy in the thymic epithelium and thymic stroma-dependent CD4 T cell development.

Within the thymus, thymic epithelial cells (TECs) provide dedicated thymic stroma microenvironments for T cell development. Because TEC functionality is sensitive to aging and cytoablative therapies, unraveling the molecular elements that coordinate their thymopoietic role has fundamental and clinical implications. Particularly, the selection of CD4 T cells depends on interactions between TCRs expressed on T cell precursors and self-peptides:MHC II complexes presented by cortical TECs (cTECs). Although the macroautophagy/autophagy-lysosomal protein degradation pathway is implicated in CD4 T cell selection, the molecular mechanism that controls the generation of selecting MHC II ligands remains elusive. LAMP2 (lysosomal-associated membrane protein 2) is a well-recognized mediator of autolysosome (AL) maturation. We showed that LAMP2 is highly expressed in cTECs. Notably, genetic inactivation of Lamp2 in thymic stromal cells specifically impaired the development of CD4 T cells that completed positive selection, without misdirecting MHC II-restricted cells into the CD8 lineage. Mechanistically, defects in autophagy in lamp2-deficient cTECs were linked to alterations in MHC II processing, which was associated with a marked reduction in CD4 TCR repertoire diversity selected within the lamp2-deficient thymic stroma. Together, our findings suggest that LAMP2 interconnects the autophagy-lysosomal axis and the processing of selecting self-peptides:MHC II complexes in cTECs, underling its implications for the generation of a broad CD4 TCR repertoire.Abbreviations: AIRE: autoimmune regulator (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy); AL: autolysosome; AP: autophagosome; Baf-A1: bafilomycin A1; B2M: beta-2 microglobulin; CTSL: cathepsin L; CD74/Ii: CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated); CFSE: carboxyfluorescein succinimidyl ester; CFU: colony-forming unit; CLIP: class II-associated invariant chain peptides; cTECs: cortical TECs dKO: double knockout; DN: double negative; DP: double positive; ENPEP/LY51: glutamyl aminopeptidase; FOXP3: forkhead box; P3 IFNG/IFNγ: interferon gamma; IKZF2/HELIOS: IKAROS family zinc finger 2; IL2RA/CD25: interleukin 2 receptor, alpha chain; KO: knockout; LAMP2: lysosomal-associated membrane protein 2; LIP: lymphopenia-induced proliferation; Lm: Listeria monocytogenes; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MHC: major histocompatibility complex; mTECs: medullary TECs; PRSS16/TSSP: protease, serine 16 (thymus); SELL/CD62L: selectin, lymphocyte; SP: single positive; TCR: T cell receptor; TCRB: T cell receptor beta chain; TECs: thymic epithelial cells; UEA-1: Ulex europaeus agglutinin-1; WT: wild-type.

H2-O deficiency promotes regulatory T cell differentiation and CD4 T cell hyperactivity.

Regulatory T cells (Treg) are crucial immune modulators, yet the exact mechanism of thymic Treg development remains controversial. Here, we present the first direct evidence for H2-O, an MHC class II peptide editing molecular chaperon, on selection of thymic Tregs. We identified that lack of H2-O in the thymic medulla promotes thymic Treg development and leads to an increased peripheral Treg frequency. Single-cell RNA-sequencing (scRNA-seq) analysis of splenic CD4 T cells revealed not only an enrichment of effector-like Tregs, but also activated CD4 T cells in the absence of H2-O. Our data support two concepts; a) lack of H2-O expression in the thymic medulla creates an environment permissive to Treg development and, b) that loss of H2-O drives increased basal auto-stimulation of CD4 T cells. These findings can help in better understanding of predispositions to autoimmunity and design of therapeutics for treatment of autoimmune diseases.

MHC-independent αßT cells: Lessons learned about thymic selection and MHC-restriction.

Understanding the generation of an MHC-restricted T cell repertoire is the cornerstone of modern T cell immunology. The unique ability of αßT cells to only recognize peptide antigens presented by MHC molecules but not conformational antigens is referred to as MHC restriction. How MHC restriction is imposed on a very large T cell receptor (TCR) repertoire is still heavily debated. We recently proposed the selection model, which posits that newly re-arranged TCRs can structurally recognize a wide variety of antigens, ranging from peptides presented by MHC molecules to native proteins like cell surface markers. However, on a molecular level, the sequestration of the essential tyrosine kinase Lck by the coreceptors CD4 and CD8 allows only MHC-restricted TCRs to signal. In the absence of Lck sequestration, MHC-independent TCRs can signal and instruct the generation of mature αßT cells that can recognize native protein ligands. The selection model thus explains how only MHC-restricted TCRs can signal and survive thymic selection. In this review, we will discuss the genetic evidence that led to our selection model. We will summarize the selection mechanism and structural properties of MHC-independent TCRs and further discuss the various non-MHC ligands we have identified.

Type of PaperY192 within the SH2 Domain of Lck Regulates TCR Signaling Downstream of PLC-γ1 and Thymic Selection.

Signaling via the TCR, which is initiated by the Src-family tyrosine kinase Lck, is crucial for the determination of cell fates in the thymus. Because of its pivotal role, ablation of Lck results in a profound block of T-cell development. Here, we show that, in addition to its well-known function in the initiation of TCR signaling, Lck also acts at a more downstream level. This novel function of Lck is determined by the tyrosine residue (Y192) located in its SH2 domain. Thymocytes from knock-in mice expressing a phosphomimetic Y192E mutant of Lck initiate TCR signaling upon CD3 cross-linking up to the level of PLC-γ1 phosphorylation. However, the activation of downstream pathways including Ca2+ influx and phosphorylation of Erk1/2 are impaired. Accordingly, positive and negative selections are blocked in LckY192E knock-in mice. Collectively, our data indicate that Lck has a novel function downstream of PLCγ-1 in the regulation of thymocyte differentiation and selection.

Genetic Modifiers of Thymic Selection and Central Tolerance in Type 1 Diabetes.

Type 1 diabetes (T1D) is caused by the T cell-driven autoimmune destruction of insulin-producing cells in the pancreas. T1D served as the prototypical autoimmune disease for genome wide association studies (GWAS) after having already been the subject of many linkage and association studies prior to the development of GWAS technology. Of the many T1D-associated gene variants, a minority appear disease-specific, while most are shared with one or more other autoimmune condition. Shared disease variants suggest defects in fundamental aspects of immune tolerance. The first layer of protective tolerance induction is known as central tolerance and takes place during the thymic selection of T cells. In this article, we will review candidate genes for type 1 diabetes whose function implicates them in central tolerance. We will describe examples of gene variants that modify the function of T cells intrinsically and others that indirectly affect thymic selection. Overall, these insights will show that a significant component of the genetic risk for T1D - and autoimmunity in general - pertains to the earliest stages of tolerance induction, at a time when protective intervention may not be feasible.

High Throughput Human T Cell Receptor Sequencing: A New Window Into Repertoire Establishment and Alloreactivity.

Recent advances in high throughput sequencing (HTS) of T cell receptors (TCRs) and in transcriptomic analysis, particularly at the single cell level, have opened the door to a new level of understanding of human immunology and immune-related diseases. In this article, we discuss the use of HTS of TCRs to discern the factors controlling human T cell repertoire development and how this approach can be used in combination with human immune system (HIS) mouse models to understand human repertoire selection in an unprecedented manner. An exceptionally high proportion of human T cells has alloreactive potential, which can best be understood as a consequence of the processes governing thymic selection. High throughput TCR sequencing has allowed assessment of the development, magnitude and nature of the human alloresponse at a new level and has provided a tool for tracking the fate of pre-transplant-defined donor- and host-reactive TCRs following transplantation. New insights into human allograft rejection and tolerance obtained with this method in combination with single cell transcriptional analyses are reviewed here.

Peptides for T cell selection in the thymus.

Major histocompatibility complex (MHC)-associated peptides generated and displayed by antigen-presenting cells in the thymus are essential for the generation of functional and self-tolerant T cells that protect our body from various pathogens. The peptides displayed by cortical thymic epithelial cells (cTECs) are generated by unique enzymatic machineries including the thymoproteasomes, and are involved in the positive selection of self-protective T cells. On the other hand, the peptides displayed by medullary thymic epithelial cells (mTECs) and thymic dendritic cells (DCs) are involved in further selection to establish self-tolerance in T cells. Although the biochemical nature of the peptide repertoire displayed in the thymus remains unclear, many studies have suggested a thymus-specific mechanism for the generation of MHC-associated peptides in the thymus. In this review, we summarize basic knowledge and recent advances in MHC-associated thymic peptides, focusing on the generation and function of thymoproteasome-dependent peptides specifically displayed by cTECs.

Drug and Chemical Allergy: A Role for a Specific Naive T-Cell Repertoire?

Allergic reactions to drugs and chemicals are mediated by an adaptive immune response involving specific T cells. During thymic selection, T cells that have not yet encountered their cognate antigen are considered naive T cells. Due to the artificial nature of drug/chemical-T-cell epitopes, it is not clear whether thymic selection of drug/chemical-specific T cells is a common phenomenon or remains limited to few donors or simply does not exist, suggesting T-cell receptor (TCR) cross-reactivity with other antigens. Selection of drug/chemical-specific T cells could be a relatively rare event accounting for the low occurrence of drug allergy. On the other hand, a large T-cell repertoire found in multiple donors would underline the potential of a drug/chemical to be recognized by many donors. Recent observations raise the hypothesis that not only the drug/chemical, but also parts of the haptenated protein or peptides may constitute the important structural determinants for antigen recognition by the TCR. These observations may also suggest that in the case of drug/chemical allergy, the T-cell repertoire results from particular properties of certain TCR to recognize hapten-modified peptides without need for previous thymic selection. The aim of this review is to address the existence and the role of a naive T-cell repertoire in drug and chemical allergy. Understanding this role has the potential to reveal efficient strategies not only for allergy diagnosis but also for prediction of the immunogenic potential of new chemicals.

Distinguishing Signal From Noise in Immunopeptidome Studies of Limiting-Abundance Biological Samples: Peptides Presented by I-Ab in C57BL/6 Mouse Thymus.

Antigen presentation by MHC-II proteins in the thymus is central to selection of CD4 T cells, but analysis of the full repertoire of presented peptides responsible for positive and negative selection is complicated by the low abundance of antigen presenting cells. A key challenge in analysis of limiting abundance immunopeptidomes by mass spectrometry is distinguishing true MHC-binding peptides from co-eluting non-specifically bound peptides present in the mixture eluted from immunoaffinity-purified MHC molecules. Herein we tested several approaches to minimize the impact of non-specific background peptides, including analyzing eluates from isotype-control antibody-conjugated beads, considering only peptides present in nested sets, and using predicted binding motif analysis to identify core epitopes. We evaluated these methods using well-understood human cell line samples, and then applied them to analysis of the I-Ab presented immunopeptidome of the thymus of C57BL/6 mice, comparing this to the more easily characterized splenic B cell and dendritic cell populations. We identified a total of 3473 unique peptides eluted from the various tissues, using a data dependent acquisition strategy with a false-discovery rate of <1%. The immunopeptidomes presented in thymus as compared to splenic B cells and DCs identified shared and tissue-specific epitopes. A broader length distribution was observed for peptides presented in the thymus as compared to splenic B cells or DCs. Detailed analysis of 61 differentially presented peptides indicated a wider distribution of I-Ab binding affinities in thymus as compared to splenic B cells. These results suggest different constraints on antigen processing and presentation pathways in central versus peripheral tissues.

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.

The Role of Proteasomes in the Thymus.

The thymus provides a microenvironment that supports the generation and selection of T cells. Cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells (mTECs) are essential components of the thymic microenvironment and present MHC-associated self-antigens to developing thymocytes for the generation of immunocompetent and self-tolerant T cells. Proteasomes are multicomponent protease complexes that degrade ubiquitinated proteins and produce peptides that are destined to be associated with MHC class I molecules. cTECs specifically express thymoproteasomes that are essential for optimal positive selection of CD8+ T cells, whereas mTECs, which contribute to the establishment of self-tolerance in T cells, express immunoproteasomes. Immunoproteasomes are also detectable in dendritic cells and developing thymocytes, additionally contributing to T cell development in the thymus. In this review, we summarize the functions of proteasomes expressed in the thymus, focusing on recent findings pertaining to the functions of the thymoproteasomes and the immunoproteasomes.

Modeling the Dynamics of T-Cell Development in the Thymus.

The thymus hosts the development of a specific type of adaptive immune cells called T cells. T cells orchestrate the adaptive immune response through recognition of antigen by the highly variable T-cell receptor (TCR). T-cell development is a tightly coordinated process comprising lineage commitment, somatic recombination of Tcr gene loci and selection for functional, but non-self-reactive TCRs, all interspersed with massive proliferation and cell death. Thus, the thymus produces a pool of T cells throughout life capable of responding to virtually any exogenous attack while preserving the body through self-tolerance. The thymus has been of considerable interest to both immunologists and theoretical biologists due to its multi-scale quantitative properties, bridging molecular binding, population dynamics and polyclonal repertoire specificity. Here, we review experimental strategies aimed at revealing quantitative and dynamic properties of T-cell development and how they have been implemented in mathematical modeling strategies that were reported to help understand the flexible dynamics of the highly dividing and dying thymic cell populations. Furthermore, we summarize the current challenges to estimating in vivo cellular dynamics and to reaching a next-generation multi-scale picture of T-cell development.

Development of double-positive thymocytes at single-cell resolution.

BACKGROUND: T cells generated from thymopoiesis are essential for the immune system, and recent single-cell studies have contributed to our understanding of the development of thymocytes at the genetic and epigenetic levels. However, the development of double-positive (DP) T cells, which comprise the majority of thymocytes, has not been well investigated. METHODS: We applied single-cell sequencing to mouse thymocytes and analyzed the transcriptome data using Seurat. By applying unsupervised clustering, we defined thymocyte subtypes and validated DP cell subtypes by flow cytometry. We classified the cell cycle phases of each cell according to expression of cell cycle phase-specific genes. For immune synapse detection, we used immunofluorescent staining and ImageStream-based flow cytometry. We studied and integrated human thymocyte data to verify the conservation of our findings and also performed cross-species comparisons to examine species-specific gene regulation. RESULTS: We classified blast, rearrangement, and selection subtypes of DP thymocytes and used the surface markers CD2 and Ly6d to identify these subtypes by flow cytometry. Based on this new classification, we found that the proliferation of blast DP cells is quite different from that of double-positive cells and other cell types, which tend to exit the cell cycle after a single round. At the DP cell selection stage, we observed that CD8-associated immune synapses formed between thymocytes, indicating that CD8sp selection occurred among thymocytes themselves. Moreover, cross-species comparison revealed species-specific transcription factors (TFs) that contribute to the transcriptional differences of thymocytes from humans and mice. CONCLUSIONS: Our study classified DP thymocyte subtypes of different developmental stages and provided new insight into the development of DP thymocytes at single-cell resolution, furthering our knowledge of the fundamental immunological process of thymopoiesis.

Role of the thymus in spontaneous development of a multi-organ autoimmune disease in human immune system mice.

We evaluated the role of the thymus in development of multi-organ autoimmunity in human immune system (HIS) mice. T cells were essential for disease development and the same T cell clones with varying phenotypes infiltrated multiple tissues. De novo-generated hematopoietic stem cell (HSC)-derived T cells were the major disease drivers, though thymocytes pre-existing in grafted human thymi contributed if not first depleted. HIS mice with a native mouse thymus developed disease earlier than thymectomized mice with a thymocyte-depleted human thymus graft. Defective structure in the native mouse thymus was associated with impaired negative selection of thymocytes expressing a transgenic TCR recognizing a self-antigen. Disease developed without direct recognition of antigens on recipient mouse MHC. While human thymus grafts had normal structure and negative selection, failure to tolerize human T cells recognizing mouse antigens presented on HLA molecules may explain eventual disease development. These new insights have implications for human autoimmunity and suggest methods of avoiding autoimmunity in next-generation HIS mice.