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The Collaborative Cross (CC) is an emerging panel of recombinant inbred (RI) mouse strains. Each strain is genetically distinct but all descended from the same eight inbred founders. In 66 strains from incipient lines of the CC (pre-CC), as well as the 8 CC founders and some of their F1 offspring, we examined subsets of lymphocytes and antigen-presenting cells. We found significant variation among the founders, with even greater diversity in the pre-CC. Genome-wide association using inferred haplotypes detected highly significant loci controlling B-to-T cell ratio, CD8 T-cell numbers, CD11c and CD23 expression. Comparison of overall strain effects in the CC founders with strain effects at QTL in the pre-CC revealed sharp contrasts in the genetic architecture of two traits with significant loci: variation in CD23 can be explained largely by additive genetics at one locus, whereas variation in B-to-T ratio has a more complex etiology. For CD23, we found a strong QTL whose confidence interval contained the CD23 structural gene Fcer2a. Our data on the pre-CC demonstrate the utility of the CC for studying immunophenotypes and the value of integrating founder, CC and F1 data. The extreme immunophenotypes observed could have pleiotropic effects in other CC experiments.

T helper (Th) 17 cells encompass a spectrum of cell states, including cells that maintain homeostatic tissue functions and pro-inflammatory cells that can drive autoimmune tissue damage. Identifying regulators that determine Th17 cell states can identify ways to control tissue inflammation and restore homeostasis. Here, we found that interleukin (IL)-23, a cytokine critical for inducing pro-inflammatory Th17 cells, decreased transcription factor T cell factor 1 (TCF1) expression. Conditional deletion of TCF1 in mature T cells increased the pro-inflammatory potential of Th17 cells, even in the absence of IL-23 receptor signaling, and conferred pro-inflammatory potential to homeostatic Th17 cells. Conversely, sustained TCF1 expression decreased pro-inflammatory Th17 potential. Mechanistically, TCF1 bound to RORγt, thereby interfering with its pro-inflammatory functions, and orchestrated a regulatory network that determined Th17 cell state. Our findings identify TCF1 as a major determinant of Th17 cell state and provide important insight for the development of therapies for Th17-driven inflammatory diseases., Competing Interests: Declaration of interests A.C.A. is a member of the SAB for Tizona Therapeutics, Trishula Therapeutics, Compass Therapeutics, and Zumutor Biologics. A.C.A. is also a paid consultant for Excepgen. V.K.K. is a co-founder, has an ownership interest in, and is a member of SAB of Celsius Therapeutics, Tizona Therapeutics, Larkspur Biosciences, and Trishula Therapeutics. V.K.K. is also the chair of the board and has equity interests in Bicara Therapeutics. H.L.W. is a member of the SAB for Tiziana Life Sciences and vTv Therapeutics and has stock in vTv Therapeutics. A.C.A.’s, V.K.K.’s, and H.L.W.’s interests were reviewed and managed by the Brigham and Women’s Hospital and Mass General Brigham in accordance with their conflict-of-interest policies. The authors have filed a patent related to this work., (Copyright © 2024 Elsevier Inc. All rights reserved.)

CD8 + T cells must persist and function in diverse tumor microenvironments to exert their effects. Thus, understanding common underlying expression programs could better inform the next generation of immunotherapies. We apply a generalizable matrix factorization algorithm that recovers both shared and context-specific expression programs from diverse datasets to a single-cell RNA sequencing (scRNA-seq) compendium of 33,161 CD8 + T cells from 132 patients with seven human cancers. Our meta-single-cell analyses uncover a pan-cancer T cell dysfunction program that predicts clinical non-response to checkpoint blockade in melanoma and highlights CXCR6 as a pan-cancer marker of chronically activated T cells. Cxcr6 is trans-activated by AP-1 and repressed by TCF1. Using mouse models, we show that Cxcr6 deletion in CD8 + T cells increases apoptosis of PD1 + TIM3 + cells, dampens CD28 signaling, and compromises tumor growth control. Our study uncovers a TCF1:CXCR6 axis that counterbalances PD1-mediated suppression of CD8 + cell responses and is essential for effective anti-tumor immunity., Competing Interests: Declaration of interests A.C.A. is a member of the SAB for Tizona Therapeutics, Trishula Therapeutics, Compass Therapeutics, ExcepGen, and Zumutor Biologics, which have interests in cancer immunotherapy. A.C.A. is also a paid consultant for iTeos Therapeutics and Larkspur Biosciences. A.C.A.’s interests were reviewed and managed by the Brigham and Women’s Hospital and Partners Healthcare in accordance with their conflict-of-interest policies. O.R.-R. is an employee of Genentech. A.R. is a co-founder and equity holder of Celsius Therapeutics, an equity holder in Immunitas, and was an SAB member of Thermo Fisher Scientific, Syros Pharmaceuticals, Neogene Therapeutics, and Asimov. From August 1, 2020, A.R. is an employee of Genentech and has equity in Roche. When at the Broad, A.R.’s interests were reviewed and managed by the Broad Institute, MIT and HHMI in accordance with their conflict-of-interest policies. A provisional patent application was filed including work in this manuscript., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Autoimmune diseases are characterized by dysfunctional immune systems that misrecognize self as non-self and cause tissue destruction. Several cell types have been implicated in triggering and sustaining disease. Due to a strong association of major histocompatibility complex II (MHC-II) proteins with various autoimmune diseases, CD4 + T lymphocytes have been thoroughly investigated for their roles in dictating disease course. CD4 + T cell activation is a coordinated process that requires three distinct signals: Signal 1, which is mediated by antigen recognition on MHC-II molecules; Signal 2, which boosts signal 1 in a costimulatory manner; and Signal 3, which helps to differentiate the activated cells into functionally relevant subsets. These signals are disrupted during autoimmunity and prompt CD4 + T cells to break tolerance. Herein, we review our current understanding of how each of the three signals plays a role in three different autoimmune diseases and highlight the genetic polymorphisms that predispose individuals to autoimmunity. We also discuss the drawbacks of existing therapies and how they can be addressed to achieve lasting tolerance in patients., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Innate T (T inn ) 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 αβ T inn 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, T inn 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 T inn cells develop in the mouse thymus and undergo effector differentiation., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The identification of the peptide epitopes presented by major histocompatibility complex class II (MHCII) molecules that drive the CD4 T cell component of autoimmune diseases has presented a formidable challenge over several decades. In type 1 diabetes (T1D), recent insight into this problem has come from the realization that several of the important epitopes are not directly processed from a protein source, but rather pieced together by fusion of different peptide fragments of secretory granule proteins to create new chimeric epitopes. We have proposed that this fusion is performed by a reverse proteolysis reaction called transpeptidation, occurring during the catabolic turnover of pancreatic proteins when secretory granules fuse with lysosomes (crinophagy). Here, we demonstrate several highly antigenic chimeric epitopes for diabetogenic CD4 T cells that are produced by digestion of the appropriate inactive fragments of the granule proteins with the lysosomal protease cathepsin L (Cat-L). This pathway has implications for how self-tolerance can be broken peripherally in T1D and other autoimmune diseases., Competing Interests: Disclosures: K. Hansen reported other from Omix Technologies outside the submitted work. No other disclosures were reported., (© 2020 Reed et al.)

The repertoire of αβ 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 TCRβ chain played a dominant role in determining pMHC-reactivity. Replacing the germline regions of mouse TCRβ 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, αβ 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., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Leukemias typically arise in the bone marrow and then spread to the blood and into other tissues. To disseminate into tissues, leukemia cells migrate into the blood stream and then exit the circulation by migrating across vascular endothelial barriers. Formin proteins regulate cytoskeletal remodeling and cell migration of normal and malignant cells. The Formin mDia1 is highly expressed in transformed lymphocytes and regulates lymphocyte migration. However, the role of mDia1 in regulating leukemia progression in vivo is unknown. Here, we investigated how mDia1 mediates the ability of leukemia cells to migrate and disseminate in vivo . For these studies, we used a mouse model of Bcr-Abl pre-B cell acute lymphoblastic leukemia. Our data showed that mDia1-deficient leukemia cells have reduced chemotaxis and ability to complete transendothelial migration in vitro . In vivo , mDia1 deficiency reduced the ability of leukemia cells to engraft in recipient mice. Furthermore, leukemia dissemination to various tissues and leukemia progression were inhibited by mDia1 depletion. Finally, mDia1 depletion in leukemia cells resulted in prolonged survival of recipient mice in a leukemia transfer model. Overall, our data show that the Formin mDia1 mediates leukemia cell migration, and drives leukemia engraftment and progression in vivo , suggesting that targeting mDia1 could provide a new method for treatment of leukemia.

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.

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.

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., Competing Interests: The authors declare no conflict of interest.

Mature T cells bearing αβ T cell receptors react with foreign antigens bound to alleles of major histocompatibility complex proteins (MHC) that they were exposed to during their development in the thymus, a phenomenon known as positive selection. The structural basis for positive selection has long been debated. Here, using mice expressing one of two different T cell receptor β chains and various MHC alleles, we show that positive selection-induced MHC bias of T cell receptors is affected both by the germline encoded elements of the T cell receptor α and β chain and, surprisingly, dramatically affected by the non germ line encoded portions of CDR3 of the T cell receptor α chain. Thus, in addition to determining specificity for antigen, the non germline encoded elements of T cell receptors may help the proteins cope with the extremely polymorphic nature of major histocompatibility complex products within the species.

The interaction of αβ T-cell antigen receptors (TCRs) with peptides bound to MHC molecules lies at the center of adaptive immunity. Whether TCRs have evolved to react with MHC or, instead, processes in the thymus involving coreceptors and other molecules select MHC-specific TCRs de novo from a random repertoire is a longstanding immunological question. Here, using nuclease-targeted mutagenesis, we address this question in vivo by generating three independent lines of knockin mice with single-amino acid mutations of conserved class II MHC amino acids that often are involved in interactions with the germ-line-encoded portions of TCRs. Although the TCR repertoire generated in these mutants is similar in size and diversity to that in WT mice, the evolutionary bias of TCRs for MHC is suggested by a shift and preferential use of some TCR subfamilies over others in mice expressing the mutant class II MHCs. Furthermore, T cells educated on these mutant MHC molecules are alloreactive to each other and to WT cells, and vice versa, suggesting strong functional differences among these repertoires. Taken together, these results highlight both the flexibility of thymic selection and the evolutionary bias of TCRs for MHC., Competing Interests: The authors declare no conflict of interest.

Polymorphic major histocompatibility complex (MHC) molecules play a central role in the vertebrate adaptive immune system. By presenting short peptides derived from pathogen-derived proteins, these "classical" MHC molecules can alert the T cell branch of the immune system of infected cells and clear the pathogen. There exist other "non-classical" MHC molecules, which while similar in structure to classical MHC proteins, are contrasted by their limited polymorphism. While the functions of many class Ib MHC molecules have still to be elucidated, the nature and diversity of antigens (if any) that some of them might present to the immune system is expected to be more restricted and might function as another approach to distinguish self from non-self. The MHC-related 1 (MR1) molecule is a member of this family of non-classical MHC proteins. It was recently shown to present unique antigens in the form of vitamin metabolites found in certain microbes. MR1 is strongly conserved genetically, structurally, and functionally through mammalian evolution, indicating its necessity in ensuring an effective immune system for members of this class. Although MR1 will be celebrating 21 years this year since its discovery, most of our understanding of how this molecule functions has only been uncovered in the past decade. Herein, we discuss where MR1 is expressed, how it selectively is able to bind to its appropriate antigens and how it, then, is able to specifically activate a distinct population of T cells.

Invariant Natural Killer T (iNKT) cells are a unique subset of T lymphocytes that have been implicated in both promoting and suppressing a multitude of immune responses. In mice, iNKT cells express T cell antigen receptors (TCRs) comprising a unique TCRα rearrangement between the Trav11 and Traj18 gene segments. When paired with certain Trbv TCRβ chains, these TCRs recognize lipid antigens presented by the major histocompatibility complex (MHC) class I-like molecule, CD1d. Until recently, the sole model of iNKT deficiency targeted the Jα18, which is absolutely required to form the TCR with the appropriate antigenic specificity. However, these mice were demonstrated to have a large reduction in TCR repertoire diversity, which could confound results arising from studies using these mice. Here, we have created a new NKT-deficient mouse strain using transcription activator-like effector nuclease (TALEN) technology to only disrupt the expression of Jα18, leaving the remaining Jα repertoire unperturbed. We confirm that these mice lack iNKT cells and do not respond to lipid antigen stimulation while the development of conventional T cells, regulatory T cells, and type Ib NKT cells is normal. This new mouse strain will serve as a new model of iNKT cell deficiency to facilitate our understanding of iNKT biology.

Conventional T cells have historically been linked to exacerbating allergy. By efficiently generating primarily TH 2 cells, allergens skew the immune response to produce IL-4, IL-13, and IgE. Previously, CD1a-responsive T cells were shown to functionally respond to bee and wasp venom allergens. In this issue of the European Journal of Immunology, Subramaniam et al. [Eur. J. Immunol. 2016. 46: 242-252] show that more functionally active CD1a-restricted cells are present in bee venom-allergic patients than in healthy patients. Additionally, the authors show that these cells are not as frequently found in individuals receiving venom immunotherapy. Consequently, this study implicates CD1a-reactive cells as the primary responders to venom allergy, which considerably regulate the downstream immune response., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)