Peripheral T cell activation, not thymic selection, expands the T follicular helper repertoire in a lupus-prone murine model.

Many autoimmune diseases are characterized by the activation of autoreactive T cells. The T cell repertoire is established in the thymus; it remains uncertain whether the presence of disease-associated autoreactive T cells reflects abnormal T cell selection in the thymus or aberrant T cell activation in the periphery. Here, we describe T cell selection, activation, and T cell repertoire diversity in female mice deficient for B lymphocyte-induced maturation protein (BLIMP)-1 in dendritic cells (DCs) (Prdm1 CKO). These mice exhibit a lupus-like phenotype with an expanded population of T follicular helper (Tfh) cells having a more diverse T cell receptor (TCR) repertoire than wild-type mice and, in turn, develop a lupus-like pathology. To understand the origin of the aberrant Tfh population, we analyzed the TCR repertoire of thymocytes and naive CD4 T cells from Prdm1 CKO mice. We show that early development and selection of T cells in the thymus are not affected. Importantly, however, we observed increased TCR signal strength and increased proliferation of naive T cells cultured in vitro with antigen and BLIMP1-deficient DCs compared to control DCs. Moreover, there was increased diversity in the TCR repertoire in naive CD4+ T cells stimulated in vitro with BLIMP1-deficient DCs. Collectively, our data indicate that lowering the threshold for peripheral T cell activation without altering thymic selection and naive T cell TCR repertoire leads to an expanded repertoire of antigen-activated T cells and impairs peripheral T cell tolerance.

T cell deletional tolerance restricts AQP4 but not MOG CNS autoimmunity.

Aquaporin-4 (AQP4)-specific Th17 cells are thought to have a central role in neuromyelitis optica (NMO) pathogenesis. When modeling NMO, only AQP4-reactive Th17 cells from AQP4-deficient (AQP4-/-), but not wild-type (WT) mice, caused CNS autoimmunity in recipient WT mice, indicating that a tightly regulated mechanism normally ensures tolerance to AQP4. Here, we found that pathogenic AQP4 T cell epitopes bind MHC II with exceptionally high affinity. Examination of T cell receptor (TCR) α/ß usage revealed that AQP4-specific T cells from AQP4-/- mice employed a distinct TCR repertoire and exhibited clonal expansion. Selective thymic AQP4 deficiency did not fully restore AQP4-reactive T cells, demonstrating that thymic negative selection alone did not account for AQP4-specific tolerance in WT mice. Indeed, AQP4-specific Th17 cells caused paralysis in recipient WT or B cell-deficient mice, which was followed by complete recovery that was associated with apoptosis of donor T cells. However, donor AQP4-reactive T cells survived and caused persistent paralysis in recipient mice deficient in both T and B cells or mice lacking T cells only. Thus, AQP4 CNS autoimmunity was limited by T cell-dependent deletion of AQP4-reactive T cells. In contrast, myelin oligodendrocyte glycoprotein (MOG)-specific T cells survived and caused sustained disease in WT mice. These findings underscore the importance of peripheral T cell deletional tolerance to AQP4, which may be relevant to understanding the balance of AQP4-reactive T cells in health and in NMO. T cell tolerance to AQP4, expressed in multiple tissues, is distinct from tolerance to MOG, an autoantigen restricted in its expression.

Autoimmunity in motion: Mechanisms of immune regulation and destruction revealed by in vivo imaging.

Autoimmunity arises when mechanisms of immune tolerance fail. Here we discuss mechanisms of T cell activation and tolerance and the dynamics of the autoimmune response at the site of disease. Live imaging of autoimmunity provides the ability to analyze immune cell dynamics at the single-cell level within the complex intact environment where disease occurs. These analyses have revealed mechanisms of T cell activation and tolerance in the lymph nodes, mechanisms of T cell entry into sites of autoimmune disease, and mechanisms leading to pathogenesis or protection in the autoimmune lesions. The overarching conclusions point to stable versus transient T cell antigen presenting cell interactions dictating the balance between T cell activation and tolerance, and T cell restimulation as a driver of pathogenesis at the site of autoimmunity. Findings from models of multiple sclerosis and type 1 diabetes are highlighted, however, the results have implications for basic mechanisms of T cell regulation during immune responses, tumor immunity, and autoimmunity.

NR4A nuclear receptors in T and B lymphocytes: Gatekeepers of immune tolerance.

Random VDJ recombination early in T and B cell development enables the adaptive immune system to recognize a vast array of evolving pathogens via antigen receptors. However, the potential of such randomly generated TCRs and BCRs to recognize and respond to self-antigens requires layers of tolerance mechanisms to mitigate the risk of life-threatening autoimmunity. Since they were originally cloned more than three decades ago, the NR4A family of nuclear hormone receptors have been implicated in many critical aspects of immune tolerance, including negative selection of thymocytes, peripheral T cell tolerance, regulatory T cells (Treg), and most recently in peripheral B cell tolerance. In this review, we discuss important insights from many laboratories as well as our own group into the function and mechanisms by which this small class of primary response genes promotes self-tolerance and immune homeostasis to balance the need for host defense against the inherent risks posed by the adaptive immune system.

Thymic mimetic cells: tolerogenic masqueraders.

Medullary thymic epithelial cells (mTECs) clonally delete or divert autoreactive T cells by ectopically expressing a diverse array of peripheral-tissue antigens (PTAs) within the thymus. Although thymic stromal cells with histological features of extra-thymic cell types, like myocytes or neurons, have been observed by light microscopy since the mid-1800s, most modern work on PTA expression has focused on the transcription factor Aire. Here, we highlight recent work that has refocused attention on such 'misplaced' thymic cells, referred to collectively as thymic mimetic cells. We review the molecular underpinnings of mimetic cells and their roles in establishing T cell tolerance, and we propose that mimetic cells play important roles in autoimmunity. Finally, we suggest future directions for this emerging area.

Biologically indeterminate yet ordered promiscuous gene expression in single medullary thymic epithelial cells.

To induce central T-cell tolerance, medullary thymic epithelial cells (mTEC) collectively express most protein-coding genes, thereby presenting an extensive library of tissue-restricted antigens (TRAs). To resolve mTEC diversity and whether promiscuous gene expression (PGE) is stochastic or coordinated, we sequenced transcriptomes of 6,894 single mTEC, enriching for 1,795 rare cells expressing either of two TRAs, TSPAN8 or GP2. Transcriptional heterogeneity allowed partitioning of mTEC into 15 reproducible subpopulations representing distinct maturational trajectories, stages and subtypes, including novel mTEC subsets, such as chemokine-expressing and ciliated TEC, which warrant further characterisation. Unexpectedly, 50 modules of genes were robustly defined each showing patterns of co-expression within individual cells, which were mainly not explicable by chromosomal location, biological pathway or tissue specificity. Further, TSPAN8+ and GP2+ mTEC were randomly dispersed within thymic medullary islands. Consequently, these data support observations that PGE exhibits ordered co-expression, although mechanisms underlying this instruction remain biologically indeterminate. Ordered co-expression and random spatial distribution of a diverse range of TRAs likely enhance their presentation and encounter with passing thymocytes, while maintaining mTEC identity.

Single-cell fate mapping reveals widespread clonal ignorance of low-affinity T cells exposed to systemic infection.

T cell ignorance is a specific form of immunological tolerance. It describes the maintenance of naivety in antigen-specific T cells in vivo despite the presence of their target antigen. It is thought to mainly play a role during the steady state, when self-antigens are presented in absence of costimulatory signals and at low density or to T cells of low affinity. In how far antigen-specific T cells can also remain clonally ignorant to foreign antigens, presented in the inflammatory context of systemic infection, remains unclear. Using single-cell in vivo fate mapping and high throughput flow cytometric enrichment, we find that high-affinity antigen-specific CD8+ T cells are efficiently recruited upon systemic infection. In contrast, most low-affinity antigen-specific T cells ignore the priming antigen and persist in the naïve state while remaining fully responsive to subsequent immunization with a high-affinity ligand. These data establish the widespread clonal ignorance of low-affinity T cells as a major factor shaping the composition of antigen-specific CD8+ T cell responses to systemic infection.

Highlight of 2023: Thymic B cells-important players in the establishment of T-cell tolerance.

In this article for the Highlights of 2023 Series, we discuss four recent articles that investigated thymic B cells, in both mice and humans. These studies provide important novel insights into the biology of this unique B-cell population, from their activation and differentiation to their role in promoting the negative selection of thymocytes and the generation of regulatory T cells.

Activation of T cell checkpoint pathways during ß-cell antigen presentation by engineered dendritic cells promotes protection from type 1 diabetes.

Defective immune regulation has been recognized in type 1 diabetes (T1D). Immune regulatory T cell check-point receptors, which are generally upregulated on activated T cells, have been the molecules of attention as therapeutic targets for enhancing immune response in tumour therapy. Here, we show that pancreatic ß-cell antigen (BcAg) presentation by engineered tolerogenic dendritic cells (tDCs) that express CTLA4 selective ligand (B7.1wa) or a combination of CTLA4, PD1 and BTLA selective ligands (B7.1wa, PD-L1 and HVEM-CRD1 respectively; multiligand-DCs) causes an increase in regulatory cytokine and T cell (Treg) responses and suppression of the effector T cell function as compared with engineered control-DCs. Non-obese diabetic mice treated with BcAg-pulsed CTLA4-ligand-DCs and multiligand-DCs at pre-diabetic and early-hyperglycaemic stages showed significantly lower degree of insulitis, higher frequencies of insulin-positive islets, profound delay in and reversal of hyperglycaemia for a significant duration. Immune cells from the tDC-treated mice not only produced lower amounts of IFNγ and higher amounts of IL10 and TGFß1 upon BcAg challenge, but also failed to induce hyperglycaemia upon adoptive transfer. While both CTLA4-ligand-DCs and multiligand-DCs were effective in inducing tolerance, multiligand-DC treatment produced an overall higher suppressive effect on effector T cell function and disease outcome. These studies show that enhanced engagement of T cell checkpoint receptors during BcAg presentation can modulate T cell function and suppress autoimmunity and progression of the disease in T1D.

Tolerance induction by liposomes targeting a single CD8 epitope IGRP206-214 in a model of type 1 diabetes is impeded by co-targeting a CD4+ islet epitope.

The autoimmune disease type 1 diabetes is predominantly mediated by CD8+ cytotoxic T-cell destruction of islet beta cells, of which islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206-214 is a dominant target antigen specificity. Previously, we found that a liposome-based antigen-specific immunotherapy encapsulating the CD4+ T-cell islet epitope 2.5mim together with the nuclear factor-κB inhibitor calcitriol induced regulatory T cells and protected from diabetes in NOD mice. Here we investigated whether the same system delivering IGRP206-214 could induce antigen-specific CD8+ T-cell-targeted immune regulation and delay diabetes. Subcutaneous administration of IGRP206-214 /calcitriol liposomes transiently activated and expanded IGRP-specific T-cell receptor transgenic 8.3 CD8+ T cells. Liposomal co-delivery of calcitriol was required to optimally suppress endogenous IGRP-specific CD8+ T-cell interferon-γ production and cytotoxicity. Concordantly, a short course of IGRP206-214 /calcitriol liposomes delayed diabetes progression and reduced insulitis. However, when IGRP206-214 /calcitriol liposomes were delivered together with 2.5mim /calcitriol liposomes, disease protection was not observed and the regulatory effect of 2.5mim /calcitriol liposomes was abrogated. Thus, tolerogenic liposomes that target either a dominant CD8+ or a CD4+ T-cell islet epitope can delay diabetes progression but combining multiple epitopes does not enhance protection.

Restoring tolerance to ß-cells in Type 1 diabetes: Current and emerging strategies.

Type 1 diabetes (T1D) results from insulin insufficiency due to islet death and dysfunction following T cell-mediated autoimmune attack. The technical feasibility of durable, functional autologous islet restoration is progressing such that it presents the most likely long-term cure for T1D but cannot succeed without the necessary counterpart of clinically effective therapeutic strategies that prevent grafted islets' destruction by pre-existing anti-islet T cells. While advances have been made in broad immunosuppression to lower off-target effects, the risk of opportunistic infections and cancers remains a concern, especially for well-managed T1D patients. Current immunomodulatory strategies in development focus on autologous Treg expansion, treatments to decrease antigen presentation and T effector (Teff) activation, and broad depletion of T cells with or without hematopoietic stem cell transplants. Emerging strategies harnessing the intensified DNA damage response present in expanding T cells, exacerbating their already high sensitivity to apoptosis to abate autoreactive Teff cells.

Inventories of naive and tolerant mouse CD4 T cell repertoires reveal a hierarchy of deleted and diverted T cell receptors.

Deletion or Treg cell differentiation are alternative fates of autoreactive MHCII-restricted thymocytes. How these different modes of tolerance determine the size and composition of polyclonal cohorts of autoreactive T cells with shared specificity is poorly understood. We addressed how tolerance to a naturally expressed autoantigen of the central nervous system shapes the CD4 T cell repertoire. Specific cells in the tolerant peripheral repertoire either were Foxp3+ or displayed anergy hallmarks and, surprisingly, were at least as frequent as in the nontolerant repertoire. Despite this apparent lack of deletional tolerance, repertoire inventories uncovered that some T cell receptors (TCRs) were lost from the CD4 T cell pool, whereas others mediated Treg cell differentiation. The antigen responsiveness of these TCRs supported an affinity model of central tolerance. Importantly, the contribution of different diverter TCRs to the nascent thymic Treg cell population reflected their antigen reactivity rather than their frequency among precursors. This reveals a multilayered TCR hierarchy in CD4 T cell tolerance that separates deleted and diverted TCRs and assures that the Treg cell compartment is filled with cells of maximal permissive antigen reactivity.

Blocking Entry of Hepatitis B and D Viruses to Hepatocytes as a Novel Immunotherapy for Treating Chronic Infections.

BACKGROUND: Chronic hepatitis B and D virus (HBV/HDV) infections can cause cancer. Current HBV therapy using nucleoside analogues (NAs) is life-long and reduces but does not eliminate the risk of cancer. A hallmark of chronic hepatitis B is a dysfunctional HBV-specific T-cell response. We therefore designed an immunotherapy driven by naive healthy T cells specific for the HDV antigen (HDAg) to bypass the need for HBV-specific T cells in order to prime PreS1-specific T cells and PreS1 antibodies blocking HBV entry. METHODS: Ten combinations of PreS1 and/or HDAg sequences were evaluated for induction of PreS1 antibodies and HBV- and HDV-specific T cells in vitro and in vivo. Neutralization of HBV by PreS1-specific murine and rabbit antibodies was evaluated in cell culture, and rabbit anti-PreS1 were tested for neutralization of HBV in mice repopulated with human hepatocytes. RESULTS: The best vaccine candidate induced T cells to PreS1 and HDAg, and PreS1 antibodies blocking HBV entry in vitro. Importantly, adoptive transfer of PreS1 antibodies prevented, or modulated, HBV infection after a subsequent challenge in humanized mice. CONCLUSIONS: We here describe a novel immunotherapy for chronic HBV/HDV that targets viral entry to complement NAs and coming therapies inhibiting viral maturation.

'Nur'turing tumor T cell tolerance and exhaustion: novel function for Nuclear Receptor Nur77 in immunity.

The nuclear receptor Nur77 is expressed in a multitude of tissues, regulating cell differentiation and homeostasis. Dysregulation of Nur77 signaling is associated with cancer, cardiovascular disease, and disorders of the CNS. The role of Nur77 in T cells has been studied for almost 30 years now. There is a clear appreciation that Nur77 is crucial for apoptosis of self-reactive T cells. However, the regulation and function of Nur77 in mature T cells remains largely unclear. In an exciting development, Nur77 has been recently demonstrated to impinge on cancer immunotherapy involving chimeric antigen receptor (CAR) T cells and tumor infiltrating lymphocytes (TILs). These studies indicated that Nur77 deficiency reduced T cell tolerance and exhaustion, thus raising the effectiveness of immune therapy in mice. Based on these novel insights, it may be proposed that regulation of Nur77 activity holds promise for innovative drug development in the field of cellular immunotherapy in cancer. In this review, we therefore summarize the role of Nur77 in T cell selection and maturation; and further develop the idea of targeting its activity in these cells as a potential strategy to augment current cancer immunotherapy treatments.

A Question of Tolerance-Antigen-Specific Immunotherapy for Type 1 Diabetes.

PURPOSE OF REVIEW: Antigen-specific immunotherapy (ASI) is a long sought-after goal for type 1 diabetes (T1D), with the potential of greater long-term safety than non-specific immunotherapy. We review the most recent advances in identification of target islet epitopes, delivery platforms and the ongoing challenges. RECENT FINDINGS: It is now recognised that human proinsulin contains a hotspot of epitopes targeted in people with T1D. Beta-cell neoantigens are also under investigation as ASI target epitopes. Consideration of the predicted HLA-specificity of the target antigen for subject selection is now being incorporated into trial design. Cell-free ASI approaches delivering antigen with or without additional immunomodulatory agents can induce antigen-specific regulatory T cell responses, including in patients and many novel nanoparticle-based platforms are under development. ASI for T1D is rapidly advancing with a number of modalities currently being trialled in patients and many more under development in preclinical models.

Reduced positive selection of a human TCR in a swine thymus using a humanized mouse model for xenotolerance induction.

BACKGROUND: Tolerance-inducing approaches to xenotransplantation would be optimal and may be necessary for long-term survival of transplanted pig organs in human patients. The ideal approach would generate donor-specific unresponsiveness to the pig organ without suppressing the patient's normal immune function. Porcine thymus transplantation has shown efficacy in promoting xenotolerance in humanized mice and large animal models. However, murine studies demonstrate that T cells selected in a swine thymus are positively selected only by swine thymic epithelial cells, and therefore, cells expressing human HLA-restricted TCRs may not be selected efficiently in a transplanted pig thymus. This may lead to suboptimal patient immune function. METHODS: To assess human thymocyte selection in a pig thymus, we used a TCR transgenic humanized mouse model to study positive selection of cells expressing the MART1 TCR, a well-characterized human HLA-A2-restricted TCR, in a grafted pig thymus. RESULTS: Positive selection of T cells expressing the MART1 TCR was inefficient in both a non-selecting human HLA-A2- or swine thymus compared with an HLA-A2+ thymus. Additionally, CD8 MART1 TCRbright T cells were detected in the spleens of mice transplanted with HLA-A2+ thymi but were significantly reduced in the spleens of mice transplanted with swine or HLA-A2- thymi. [Correction added on October 15, 2019, after first online publication: The missing superscript values +, -, and bright have been included in the Results section.] CONCLUSIONS: Positive selection of cells expressing a human-restricted TCR in a transplanted pig thymus is inefficient, suggesting that modifications to improve positive selection of cells expressing human-restricted TCRs in a pig thymus may be necessary to support development of a protective human T-cell pool in future patients.

Thymus-specific serine protease, a protease that shapes the CD4 T cell repertoire.

The lifespan of T cells is determined by continuous interactions of their T cell receptors (TCR) with self-peptide-MHC (self-pMHC) complexes presented by different subsets of antigen-presenting cells (APC). In the thymus, developing thymocytes are positively selected through recognition of self-pMHC presented by cortical thymic epithelial cells (cTEC). They are subsequently negatively selected by medullary thymic epithelial cells (mTEC) or thymic dendritic cells (DC) presenting self-pMHC complexes. In the periphery, the homeostasis of mature T cells is likewise controlled by the interaction of their TCR with self-pMHC complexes presented by lymph node stromal cells while they may be tolerized by DC presenting tissue-derived self-antigens. To perform these tasks, the different subsets of APC are equipped with distinct combination of antigen processing enzymes and consequently present specific repertoire of self-peptides. Here, we discuss one such antigen processing enzyme, the thymus-specific serine protease (TSSP), which is predominantly expressed by thymic stromal cells. In thymic DC and TEC, TSSP edits the repertoire of peptide presented by class II molecules and thus shapes the CD4 T cell repertoire.

Regulation of immune responses by E3 ubiquitin ligase Cbl-b.

Casitas B lymphoma-b (Cbl-b), a RING finger E3 ubiquitin ligase, has been identified as a critical regulator of adaptive immune responses. Cbl-b is essential for establishing the threshold for T cell activation and regulating peripheral T cell tolerance through various mechanisms. Intriguingly, recent studies indicate that Cbl-b also modulates innate immune responses, and plays a key role in host defense to pathogens and anti-tumor immunity. These studies suggest that targeting Cbl-b may represent a potential therapeutic strategy for the management of human immune-related disorders such as autoimmune diseases, infections, tumors, and allergic airway inflammation. In this review, we summarize the latest developments regarding the roles of Cbl-b in innate and adaptive immunity, and immune-mediated diseases.

T-cell tolerance as a potential effect of congenital leishmaniasis on offspring immunity.

Congenital transmission of leishmaniasis is recognized in cases detected by passive surveillance. Most cases are from low-resource countries, limiting the study of several important aspects of this route of infection, including the offspring's immune response. Studies on natural and experimentally infected animals suggest that parasites might be transmitted to the embryo or foetus at any time during pregnancy. As immune system undergoes sequential stages of development, an infection before the time of self-recognition could lead to central tolerance, making an individual specifically tolerant and susceptible to infection. In the alternative scenario, infection after self-recognition would allow the proper development of T-lymphocyte clones in response to Leishmania antigens, providing resistance to the disease. Newborns undergo a transient period of low expression of several immune surface molecules and a naïve adaptive immune response with no memory, which together might contribute to slow elimination of the parasite over several months. This insight is a proposed independent mechanism of the previously proven T-cell exhaustion and must be investigated. Analyses of infected placenta, cord blood and infant immunity are required for a better understanding of immunity in congenital leishmaniasis infection.

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.