MHC heterozygosity limits T cell receptor variability in CD4 T cells.

αß T cell receptor (TCR) V(D)J genes code for billions of TCR combinations. However, only some appear on peripheral T cells in any individual because, to mature, thymocytes must react with low affinity but not high affinity with thymus expressed major histocompatibility (MHC)/peptides. MHC proteins are very polymorphic. Different alleles bind different peptides. Therefore, any individual might express many different MHC alleles to ensure that some peptides from an invader are bound to MHC and activate T cells. However, most individuals express limited numbers of MHC alleles. To explore this, we compared the TCR repertoires of naïve CD4 T cells in mice expressing one or two MHC alleles. Unexpectedly, the TCRs in heterozygotes were less diverse that those in the sum of their MHC homozygous relatives. Our results suggest that thymus negative selection cancels out the advantages of increased thymic positive selection in the MHC heterozygotes.

Dysfunctional states of unconventional T-cell subsets in cancer.

Unconventional T cells represent a promising therapeutic agent to overcome the current limitations of immunotherapies due to their universal T-cell receptors, ability to respond directly to cytokine stimulation, and capacity to recruit and modulate conventional immune cells in the tumor microenvironment. Like conventional T cells, unconventional T cells can enter a dysfunctional state, and the functional differences associated with this state may provide insight into the discrepancies observed in their role in antitumor immunity in various cancers. The exhaustive signature of unconventional T cells differs from conventional αß T cells, and understanding the differences in the mechanisms underlying exhaustive differentiation in these cell types may aid in the discovery of new treatments to improve sustained antitumor responses. Ongoing clinical trials investigating therapies that leverage unconventional T-cell populations have shown success in treating hematologic malignancies and reducing the immunosuppressive tumor environment. However, several hurdles remain to extend these promising results into solid tumors. Here we discuss the current knowledge on unconventional T-cell function/dysfunction and consider how the incorporation of therapies that modulate unconventional T-cell exhaustion may aid in overcoming the current limitations of immunotherapy. Additionally, we discuss how components of the tumor microenvironment alter the functions of unconventional T cells and how these changes can affect tumor infiltration by lymphocytes and alter conventional T-cell responses.

MAIT cells drive chronic inflammation in a genetically diverse murine model of spontaneous colitis.

Background & aims: Lymphocytes that produce IL-17 can confer protective immunity during infections by pathogens, yet their involvement in inflammatory diseases is a subject of debate. Although these cells may perpetuate inflammation, resulting in tissue damage, they are also capable of contributing directly or indirectly to tissue repair, thus necessitating more detailed investigation. Mucosal-Associated-Invariant-T (MAIT) cells are innate-like T cells, acquiring a type III phenotype in the thymus. Here, we dissected the role of MAIT cells in vivo using a spontaneous colitis model in a genetically diverse mouse strain. Methods: Multiparameter spectral flow cytometry and scRNAseq were used to characterize MAIT and immune cell dynamics and transcriptomic signatures respectively, in the collaborative-cross strain, CC011/Unc and CC011/Unc- Traj33 -/- . Results: In contrast to many conventional mouse laboratory strains, the CC011 strain harbors a high baseline population of MAIT cells. We observed an age-related increase in colonic MAIT cells, Th17 cells, regulatory T cells, and neutrophils, which paralleled the development of spontaneous colitis. This progression manifested histological traits reminiscent of human IBD. The transcriptomic analysis of colonic MAIT cells from CC011 revealed an activation profile consistent with an inflammatory milieu, marked by an enhanced type-III response. Notably, IL-17A was abundantly secreted by MAIT cells in the colons of afflicted mice. Conversely, in the MAIT cell-deficient CC011-Traj33-/- mice, there was a notable absence of significant colonic histopathology. Furthermore, myeloperoxidase staining indicated a substantial decrease in colonic neutrophils. Conclusions: Our findings suggest that MAIT cells play a pivotal role in modulating the severity of intestinal pathology, potentially orchestrating the inflammatory process by driving the accumulation of neutrophils within the colonic environment.

Unraveling the Phenotypic States of Human innate-like T Cells: Comparative Insights with Conventional T Cells and Mouse Models.

The "innate-like" T cell compartment, known as Tinn, represents a diverse group of T cells that straddle the boundary between innate and adaptive immunity, having the ability to mount rapid responses following activation. In mice, this ability is acquired during thymic development. We explored the transcriptional landscape of Tinn compared to conventional T cells (Tconv) in the human thymus and blood using single cell RNA sequencing and flow cytometry. We reveal that in human blood, the majority of Tinn cells, including iNKT, MAIT, and Vδ2+Vγ9+ T cells, share an effector program characterized by the expression of unique chemokine and cytokine receptors, and cytotoxic molecules. This program is driven by specific transcription factors, distinct from those governing Tconv cells. Conversely, only a fraction of thymic Tinn cells displays an effector phenotype, while others share transcriptional features with developing Tconv cells, indicating potential divergent developmental pathways. Unlike the mouse, human Tinn cells do not differentiate into multiple effector subsets but develop a mixed type I/type III effector potential. To conduct a comprehensive cross-species analysis, we constructed a murine Tinn developmental atlas and uncovered additional species-specific distinctions, including the absence of type II Tinn cells in humans, which implies distinct immune regulatory mechanisms across species. The study provides insights into the development and functionality of Tinn cells, emphasizing their role in immune responses and their potential as targets for therapeutic interventions.

Current insights in mouse iNKT and MAIT cell development using single cell transcriptomics data.

Innate T (Tinn) cells are a collection of T cells with important regulatory functions that have a crucial role in immunity towards tumors, bacteria, viruses, and in cell-mediated autoimmunity. In mice, the two main αß Tinn cell subsets include the invariant NKT (iNKT) cells that recognize glycolipid antigens presented by non-polymorphic CD1d molecules and the mucosal associated invariant T (MAIT) cells that recognize vitamin B metabolites presented by the non-polymorphic MR1 molecules. Due to their ability to promptly secrete large quantities of cytokines either after T cell antigen receptor (TCR) activation or upon exposure to tissue- and antigen-presenting cell-derived cytokines, Tinn cells are thought to act as a bridge between the innate and adaptive immune systems and have the ability to shape the overall immune response. Their swift response reflects the early acquisition of helper effector programs during their development in the thymus, independently of pathogen exposure and prior to taking up residence in peripheral tissues. Several studies recently profiled, in an unbiased manner, the transcriptomes of mouse thymic iNKT and MAIT cells at the single cell level. Based on these data, we re-examine in this review how Tinn cells develop in the mouse thymus and undergo effector differentiation.

Circulating CD8+ mucosal-associated invariant T cells correlate with improved treatment responses and overall survival in anti-PD-1-treated melanoma patients.

OBJECTIVES: While much of the research concerning factors associated with responses to immune checkpoint inhibitors (ICIs) has focussed on the contributions of conventional peptide-specific T cells, the role of unconventional T cells, such as mucosal-associated invariant T (MAIT) cells, in human melanoma remains largely unknown. MAIT cells are an abundant population of innate-like T cells expressing a semi-invariant T-cell receptor restricted to the MHC class I-like molecule, MR1, presenting vitamin B metabolites derived from bacteria. We sought to characterise MAIT cells in melanoma patients and determined their association with treatment responses and clinical outcomes. METHODS: In this prospective clinical study, we analysed the frequency and functional profile of circulating and tumor-infiltrating MAIT cells in human melanoma patients. Using flow cytometry, we compared these across metastatic sites and between ICI responders vs. non-responders as well as healthy donors. RESULTS: We identified tumor-infiltrating MAIT cells in melanomas across metastatic sites and found that the number of circulating MAIT cells is reduced in melanoma patients compared to healthy donors. However, circulating MAIT cell frequencies are restored by ICI treatment in responding patients, correlating with treatment responses, in which patients with high frequencies of MAIT cells exhibited significantly improved overall survival. CONCLUSION: Our results suggest that MAIT cells may be a potential predictive marker of responses to immunotherapies and provide rationale for testing MAIT cell-directed therapies in combination with current and next-generation ICIs.

Single cell analysis of host response to helminth infection reveals the clonal breadth, heterogeneity, and tissue-specific programming of the responding CD4+ T cell repertoire.

The CD4+ T cell response is critical to host protection against helminth infection. How this response varies across different hosts and tissues remains an important gap in our understanding. Using IL-4-reporter mice to identify responding CD4+ T cells to Nippostrongylus brasiliensis infection, T cell receptor sequencing paired with novel clustering algorithms revealed a broadly reactive and clonally diverse CD4+ T cell response. While the most prevalent clones and clonotypes exhibited some tissue selectivity, most were observed to reside in both the lung and lung-draining lymph nodes. Antigen-reactivity of the broader repertoires was predicted to be shared across both tissues and individual mice. Transcriptome, trajectory, and chromatin accessibility analysis of lung and lymph-node repertoires revealed three unique but related populations of responding IL-4+ CD4+ T cells consistent with T follicular helper, T helper 2, and a transitional population sharing similarity with both populations. The shared antigen reactivity of lymph node and lung repertoires combined with the adoption of tissue-specific gene programs allows for the pairing of cellular and humoral responses critical to the orchestration of anti-helminth immunity.

CD1a autoreactivity: When size does matter.

CD1a-autoreactive T cells represent a significant proportion of circulating αß T cells in humans and appear to be enriched in the skin. How their autoreactivity is regulated remains unclear. In this issue of JEM, Cotton et al. (2021. J. Exp. Med.https://doi.org/10.1084/jem.20202699) show that CD1a molecules do not randomly survey cellular lipids but instead capture certain lipid classes that broadly interfere with the binding of autoreactive T cell antigen receptors to the target CD1a. These findings provide new potential therapeutic avenues for manipulating CD1a autoreactive T cell responses.

MAIT Cells: Partners or Enemies in Cancer Immunotherapy?

A recent boom in mucosal-associated invariant T (MAIT) cell research has identified relationships between MAIT cell abundance, function, and clinical outcomes in various malignancies. As they express a variety of immune checkpoint receptors and ligands, and possess strong cytotoxic functions, MAIT cells are an attractive new subject in the field of tumor immunology. MAIT cells are a class of innate-like T cells that express a semi-invariant T cell antigen receptor (TCR) that recognizes microbially derived non-peptide antigens presented by the non-polymorphic MHC class-1 like molecule, MR1. In this review, we outline the current (and often contradictory) evidence exploring MAIT cell biology and how MAIT cells impact clinical outcomes in different human cancers, as well as what role they may have in cancer immunotherapy.

Tolerance induction in memory CD4 T cells is partial and reversible.

Memory T cells respond rapidly in part because they are less reliant on a heightened levels of costimulatory molecules. This enables rapid control of secondary infecting pathogens but presents challenges to efforts to control or silence memory CD4 T cells, for example in antigen-specific tolerance strategies for autoimmunity. We have examined the transcriptional and functional consequences of reactivating memory CD4 T cells in the absence of an adjuvant. We find that memory CD4 T cells generated by infection or immunisation survive secondary activation with antigen delivered without adjuvant, regardless of their location in secondary lymphoid organs or peripheral tissues. These cells were, however, functionally altered following a tertiary immunisation with antigen and adjuvant, proliferating poorly but maintaining their ability to produce inflammatory cytokines. Transcriptional and cell cycle analysis of these memory CD4 T cells suggests they are unable to commit fully to cell division potentially because of low expression of DNA repair enzymes. In contrast, these memory CD4 T cells could proliferate following tertiary reactivation by viral re-infection. These data indicate that antigen-specific tolerogenic strategies must examine multiple parameters of Tcell function, and provide insight into the molecular mechanisms that may lead to deletional tolerance of memory CD4 T cells.

Type II Natural Killer T Cells Contribute to Protection Against Systemic Methicillin-Resistant Staphylococcus aureus Infection.

Methicillin-resistant Staphylococcus aureus (SA) bacteremia is responsible for over 10,000 deaths in the hospital setting each year. Both conventional CD4+ T cells and γδ T cells play protective roles in SA infection through secretion of IFN-γ and IL-17. However, the role of other unconventional T cells in SA infection is largely unknown. Natural killer T (NKT) cells, a subset of innate-like T cells, are activated rapidly in response to a wide range of self and microbial lipid antigens presented by MHC I-like molecule CD1d. NKT cells are divided into two groups, invariant NKT (iNKT) and type II NKT cells, based on TCR usage. Using mice lacking either iNKT cells or both types of NKT cells, we show that both NKT cell subsets are activated after systemic SA infection and produce IFN-γ in response to SA antigen, however type II NKT cells are sufficient to control bacterial burden and inflammatory infiltrate in infected organs. This protective capacity was specific for NKT cells, as mice lacking mucosal associated invariant T (MAIT) cells, another innate-like T cell subset, had no increased susceptibility to SA systemic infection. We identify polar lipid species from SA that induce IFN-γ production from type II NKT cells, which requires both CD1d-TCR engagement and IL-12 production by antigen presenting cells. We also demonstrate that a population of T cells enriched for type II NKT cells are increased in PBMC of SA bacteremic patients compared to healthy controls. Therefore, type II NKT cells perform effector functions that enhance control of SA infection prior to conventional T cell activation and recognize SA-derived lipid antigens. As CD1d is highly conserved in humans, these CD1d-restricted SA lipid antigens could be used in the design of next generation SA vaccines targeting cell-mediated immunity.

Thymic iNKT single cell analyses unmask the common developmental program of mouse innate T cells.

Most T lymphocytes leave the thymus as naïve cells with limited functionality. However, unique populations of innate-like T cells differentiate into functionally distinct effector subsets during their development in the thymus. Here, we profiled >10,000 differentiating thymic invariant natural killer T (iNKT) cells using single-cell RNA sequencing to produce a comprehensive transcriptional landscape that highlights their maturation, function, and fate decisions at homeostasis. Our results reveal transcriptional profiles that are broadly shared between iNKT and mucosal-associated invariant T (MAIT) cells, illustrating a common core developmental program. We further unmask a mutual requirement for Hivep3, a zinc finger transcription factor and adapter protein. Hivep3 is expressed in early precursors and regulates the post-selection proliferative burst, differentiation and functions of iNKT cells. Altogether, our results highlight the common requirements for the development of innate-like T cells with a focus on how Hivep3 impacts the maturation of these lymphocytes.

Contribution of the SYK Tyrosine kinase expression to human iNKT self-reactivity.

Invariant Natural Killer T (iNKT) cells are particular T lymphocytes at the frontier between innate and adaptative immunities. They participate in the elimination of pathogens or tumor cells, but also in the development of allergic reactions and autoimmune diseases. From their first descriptions, the phenomenon of self-reactivity has been described. Indeed, they are able to recognize exogenous and endogenous lipids. However, the mechanisms underlying the self-reactivity are still largely unknown, particularly in humans. Using a CD1d tetramer-based sensitive immunomagnetic approach, we generated self-reactive iNKT cell lines from blood circulating iNKT cells of healthy donors. Analysis of their functional characteristics in vitro showed that these cells recognized endogenous lipids presented by CD1d molecules through their TCR that do not correspond to α-glycosylceramides. TCR sequencing and transcriptomic analysis of T cell clones revealed that a particular TCR signature and an expression of the SYK protein kinase were two mechanisms supporting human iNKT self-reactivity. The SYK expression, strong in the most self-reactive iNKT clones and variable in ex vivo isolated iNKT cells, seems to decrease the activation threshold of iNKT cells and increase their overall antigenic sensitivity. This study indicates that a modulation of the TCR intracellular signal contributes to iNKT self-reactivity.

Inherent reactivity of unselected TCR repertoires to peptide-MHC molecules.

The repertoire of αß T cell antigen receptors (TCRs) on mature T cells is selected in the thymus where it is rendered both self-tolerant and restricted to the recognition of major histocompatibility complex molecules presenting peptide antigens (pMHC). It remains unclear whether germline TCR sequences exhibit an inherent bias to interact with pMHC prior to selection. Here, we isolated TCR libraries from unselected thymocytes and upon reexpression of these random TCR repertoires in recipient T cell hybridomas, interrogated their reactivities to antigen-presenting cell lines. While these random TCR combinations could potentially have reacted with any surface molecule on the cell lines, the hybridomas were stimulated most frequently by pMHC ligands. The nature and CDR3 loop composition of the TCRß chain played a dominant role in determining pMHC-reactivity. Replacing the germline regions of mouse TCRß chains with those of other jawed vertebrates preserved reactivity to mouse pMHC. Finally, introducing the CD4 coreceptor into the hybridomas increased the proportion of cells that could respond to pMHC ligands. Thus, αß TCRs display an intrinsic and evolutionary conserved bias for pMHC molecules in the absence of any selective pressure, which is further strengthened in the presence of coreceptors.

How C-terminal additions to insulin B-chain fragments create superagonists for T cells in mouse and human type 1 diabetes.

In type 1 diabetes (T1D), proinsulin is a major autoantigen and the insulin B:9-23 peptide contains epitopes for CD4+ T cells in both mice and humans. This peptide requires carboxyl-terminal mutations for uniform binding in the proper position within the mouse IAg7 or human DQ8 major histocompatibility complex (MHC) class II (MHCII) peptide grooves and for strong CD4+ T cell stimulation. Here, we present crystal structures showing how these mutations control CD4+ T cell receptor (TCR) binding to these MHCII-peptide complexes. Our data reveal stricking similarities between mouse and human CD4+ TCRs in their interactions with these ligands. We also show how fusions between fragments of B:9-23 and of proinsulin C-peptide create chimeric peptides with activities as strong or stronger than the mutated insulin peptides. We propose transpeptidation in the lysosome as a mechanism that could accomplish these fusions in vivo, similar to the creation of fused peptide epitopes for MHCI presentation shown to occur by transpeptidation in the proteasome. Were this mechanism limited to the pancreas and absent in the thymus, it could provide an explanation for how diabetogenic T cells escape negative selection during development but find their modified target antigens in the pancreas to cause T1D.

Development of T cell lines sensitive to antigen stimulation.

Immortalized T cells such as T cell hybridomas, transfectomas, and transductants are useful tools to study tri-molecular complexes consisting of peptide, MHC, and T cell receptor (TCR) molecules. These cells have been utilized for antigen discovery studies for decades due to simplicity and rapidness of growing cells. However, responsiveness to antigen stimulation is typically less sensitive compared to primary T cells, resulting in occasional false negative outcomes especially for TCRs having low affinity to a peptide-MHC complex (pMHC). To overcome this obstacle, we genetically engineered T cell hybridomas to express additional CD3 molecules as well as CD4 with two amino acid substitutions that increase affinity to MHC class II molecules. The manipulated T cell hybridomas that were further transduced with retroviral vectors encoding TCRs of interest responded to cognate antigens more robustly than non-manipulated cells without evoking non-antigen specific reactivity. Of importance, the manipulation with CD3 and mutated human CD4 expression was effective in increasing responsiveness of T cell hybridomas to a wide variety of TCR, peptide, and MHC combinations across class II genetic loci (i.e. HLA-DR, HLA-DQ, HLA-DP, and murine H2-IA) and species (i.e. both humans and mice), and thus will be useful to identify antigen specificity of T cells.

Invariant Natural Killer T Cell Subsets-More Than Just Developmental Intermediates.

Invariant natural killer T (iNKT) cells are a CD1d-restricted T cell population that can respond to lipid antigenic stimulation within minutes by secreting a wide variety of cytokines. This broad functional scope has placed iNKT cells at the frontlines of many kinds of immune responses. Although the diverse functional capacities of iNKT cells have long been acknowledged, only recently have distinct iNKT cell subsets, each with a marked functional predisposition, been appreciated. Furthermore, the subsets can frequently occupy distinct niches in different tissues and sometimes establish long-term tissue residency where they can impact homeostasis and respond quickly when they sense perturbations. In this review, we discuss the developmental origins of the iNKT cell subsets, their localization patterns, and detail what is known about how different subsets specifically influence their surroundings in conditions of steady and diseased states.

TCR signal strength controls thymic differentiation of iNKT cell subsets.

During development in the thymus, invariant natural killer T (iNKT) cells commit to one of three major functionally different subsets, iNKT1, iNKT2, and iNKT17. Here, we show that T cell antigen receptor (TCR) signal strength governs the development of iNKT cell subsets, with strong signaling promoting iNKT2 and iNKT17 development. Altering TCR diversity or signaling diminishes iNKT2 and iNKT17 cell subset development in a cell-intrinsic manner. Decreased TCR signaling affects the persistence of Egr2 expression and the upregulation of PLZF. By genome-wide comparison of chromatin accessibility, we identify a subset of iNKT2-specific regulatory elements containing NFAT and Egr binding motifs that is less accessible in iNKT2 cells that develop from reduced TCR signaling. These data suggest that variable TCR signaling modulates regulatory element activity at NFAT and Egr binding sites exerting a determinative influence on the dynamics of gene enhancer accessibility and the developmental fate of iNKT cells.

Characterization of Thymic Development of Natural Killer T Cell Subsets by Multiparameter Flow Cytometry.

Natural killer T (NKT) cells are a subset of αß T cells that recognize lipid antigens presented by the nonclassical MHC molecule CD1d. Although numerically small, these cells have been shown to play an important role in the regulation of multiple immune responses, including microbial infection, autoimmunity, and cancer. Even in the steady state, cytokine production by NKT cells influences the basal status and function of other immune cells, including dendritic cells and CD8 T cells. To fully understand their biology and harness them in the clinic, it is imperative to dissect the molecular mechanisms involved in the acquisition of their functionality. Unlike conventional αß T cells, NKT cells acquire their effector function during development in the thymus. At this time, precursors commit to one of three functionally different effector lineages: NKT1, NKT2, and NKT17. These subsets are characterized by the secretion of different cytokines upon antigenic stimulation and by the expression of the master transcription factors Tbet, promyelocytic leukemia zinc finger (PLZF), and retinoic orphan receptor γ t (RORγt). Here we describe a multicolor flow cytometry protocol to identify NKT cell subsets and interrogate the progression of NKT precursors through their development in the thymus.

Differing roles of CD1d2 and CD1d1 proteins in type I natural killer T cell development and function.

MHC class I-like CD1 molecules have evolved to present lipid-based antigens to T cells. Differences in the antigen-binding clefts of the CD1 family members determine the conformation and size of the lipids that are presented, although the factors that shape CD1 diversity remain unclear. In mice, two homologous genes, CD1D1 and CD1D2, encode the CD1d protein, which is essential to the development and function of natural killer T (NKT) cells. However, it remains unclear whether both CD1d isoforms are equivalent in their antigen presentation capacity and functions. Here, we report that CD1d2 molecules are expressed in the thymus of some mouse strains, where they select functional type I NKT cells. Intriguingly, the T cell antigen receptor repertoire and phenotype of CD1d2-selected type I NKT cells in CD1D1-/- mice differed from CD1d1-selected type I NKT cells. The structures of CD1d2 in complex with endogenous lipids and a truncated acyl-chain analog of α-galactosylceramide revealed that its A'-pocket was restricted in size compared with CD1d1. Accordingly, CD1d2 molecules could not present glycolipid antigens with long acyl chains efficiently, favoring the presentation of short acyl chain antigens. These results indicate that the two CD1d molecules present different sets of self-antigen(s) in the mouse thymus, thereby impacting the development of invariant NKT cells.