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Cell death mechanisms in T lymphocytes vary according to their developmental stage, cell subset and activation status. The cell death control mechanisms of mucosal-associated invariant T (MAIT) cells, a specialized T cell population, are largely unknown. Here we report that MAIT cells express key necroptotic machinery; receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) protein, in abundance. Despite this, we discovered that the loss of RIPK3, but not necroptotic effector MLKL or apoptotic caspase-8, specifically increased MAIT cell abundance at steady-state in the thymus, spleen, liver and lungs, in a cell-intrinsic manner. In contrast, over the course of infection with Francisella tularensis, RIPK3 deficiency did not impact the magnitude of the expansion nor contraction of MAIT cell pools. These findings suggest that, distinct from conventional T cells, the accumulation of MAIT cells is restrained by RIPK3 signalling, likely prior to thymic egress, in a manner independent of canonical apoptotic and necroptotic cell death pathways.

Tissue-resident memory T (TRM) cells are non-recirculating cells that exist throughout the body. Although TRM cells in various organs rely on common transcriptional networks to establish tissue residency, location-specific factors adapt these cells to their tissue of lodgment. Here we analyze TRM cell heterogeneity between organs and find that the different environments in which these cells differentiate dictate TRM cell function, durability and malleability. We find that unequal responsiveness to TGFβ is a major driver of this diversity. Notably, dampened TGFβ signaling results in CD103- TRM cells with increased proliferative potential, enhanced function and reduced longevity compared with their TGFβ-responsive CD103+ TRM counterparts. Furthermore, whereas CD103- TRM cells readily modified their phenotype upon relocation, CD103+ TRM cells were comparatively resistant to transdifferentiation. Thus, despite common requirements for TRM cell development, tissue adaptation of these cells confers discrete functional properties such that TRM cells exist along a spectrum of differentiation potential that is governed by their local tissue microenvironment.

S1PR5 impairs TRM cell differentiation by limiting T cell entry and promoting T cell egress from peripheral tissues. Local TGF-β signaling coordinates suppression of the T-bet–ZEB2–S1PR5 emigration axis, thus enforcing tissue residency., Tissue-resident memory T (TRM) cells provide long-lasting immune protection. One of the key events controlling TRM cell development is the local retention of TRM cell precursors coupled to downregulation of molecules necessary for tissue exit. Sphingosine-1-phosphate receptor 5 (S1PR5) is a migratory receptor with an uncharted function in T cells. Here, we show that S1PR5 plays a critical role in T cell infiltration and emigration from peripheral organs, as well as being specifically downregulated in TRM cells. Consequentially, TRM cell development was selectively impaired upon ectopic expression of S1pr5, whereas loss of S1pr5 enhanced skin TRM cell formation by promoting peripheral T cell sequestration. Importantly, we found that T-bet and ZEB2 were required for S1pr5 induction and that local TGF-β signaling was necessary to promote coordinated Tbx21, Zeb2, and S1pr5 downregulation. Moreover, S1PR5-mediated control of tissue residency was conserved across innate and adaptive immune compartments. Together, these results identify the T-bet–ZEB2–S1PR5 axis as a previously unappreciated mechanism modulating the generation of tissue-resident lymphocytes., Graphical Abstract

Type I and type II natural killer T (NKT) cells are restricted to the lipid antigen-presenting molecule CD1d. While we have an understanding of the antigen reactivity and function of type I NKT cells, our knowledge of type II NKT cells in health and disease remains unclear. Here we describe a population of type II NKT cells that recognise and respond to the microbial antigen, α-glucuronosyl-diacylglycerol (α-GlcADAG) presented by CD1d, but not the prototypical type I NKT cell agonist, α-galactosylceramide. Surprisingly, the crystal structure of a type II NKT TCR-CD1d-α-GlcADAG complex reveals a CD1d F’-pocket-docking mode that contrasts sharply with the previously determined A’-roof positioning of a sulfatide-reactive type II NKT TCR. Our data also suggest that diverse type II NKT TCRs directed against distinct microbial or mammalian lipid antigens adopt multiple recognition strategies on CD1d, thereby maximising the potential for type II NKT cells to detect different lipid antigens., Natural killer T (NKT) cells include type I that express semi-invariant T cell receptor (TCR), and type II that cover a broader repertoire. Here the authors describe the crystal structure of a type II NKT TCR complexed with CD1d/antigen to propose that type II NKT TCRs may adapt multiple CD1d docking modes to maximise antigen recognition efficacy.

Mucosal‐associated invariant T (MAIT) cells are unconventional T cells that recognize antigens derived from riboflavin biosynthesis. In addition to anti‐microbial functions, human MAIT cells are associated with cancers, autoimmunity, allergies and inflammatory disorders, although their role is poorly understood. Activated MAIT cells are well known for their rapid release of Th1 and Th17 cytokines, but we have discovered that chronic stimulation can also lead to potent interleukin (IL)‐13 expression. We used RNA‐seq and qRT‐PCR to demonstrate high expression of the IL‐13 gene in chronically stimulated MAIT cells, and directly identify IL‐13 using intracellular flow cytometry and multiplex bead analysis of MAIT cell cultures. This unexpected finding has important implications for IL‐13‐dependent diseases, such as colorectal cancer (CRC), that occur in mucosal areas where MAIT cells are abundant. We identify MAIT cells near CRC tumors and show that these areas and precancerous polyps express high levels of the IL‐13 receptor, which promotes tumor progression and metastasis. Our data suggest that MAIT cells have a more complicated role in CRC than currently realized and that they represent a promising new target for immunotherapies where IL‐13 can be a critical factor., Mucosal‐associated invariant T (MAIT) cells are regarded as proinflammatory lymphocytes with a Th1/Th17 cytokine response. This study shows human MAIT cells can also be prominent IL‐13‐producing cells. The findings shed new light on the potential role of MAIT cells in tumor immunity and their potential as new targets for immunotherapies in colorectal cancer and other IL‐13‐dependent diseases.

Natural killer T (NKT) cells detect lipids presented by CD1d. Most studies focus on type I NKT cells that express semi-invariant αβ T cell receptors (TCR) and recognize α-galactosylceramides. However, CD1d also presents structurally distinct lipids to NKT cells expressing diverse TCRs (type II NKT cells), but our knowledge of the antigens for type II NKT cells is limited. An early study identified a nonlipidic NKT cell agonist, phenyl pentamethyldihydrobenzofuransulfonate (PPBF), which is notable for its similarity to common sulfa drugs, but its mechanism of NKT cell activation remained unknown. Here, we demonstrate that a range of pentamethylbenzofuransulfonates (PBFs), including PPBF, activate polyclonal type II NKT cells from human donors. Whereas these sulfa drug–like molecules might have acted pharmacologically on cells, here we demonstrate direct contact between TCRs and PBF-treated CD1d complexes. Further, PBF-treated CD1d tetramers identified type II NKT cell populations expressing αβTCRs and γδTCRs, including those with variable and joining region gene usage (TRAV12-1–TRAJ6) that was conserved across donors. By trapping a CD1d–type II NKT TCR complex for direct mass-spectrometric analysis, we detected molecules that allow the binding of CD1d to TCRs, finding that both selected PBF family members and short-chain sphingomyelin lipids are present in these complexes. Furthermore, the combination of PPBF and short-chain sphingomyelin enhances CD1d tetramer staining of PPBF-reactive T cell lines over either molecule alone. This study demonstrates that nonlipidic small molecules, which resemble sulfa drugs implicated in systemic hypersensitivity and drug allergy reactions, are targeted by a polyclonal population of type II NKT cells in a CD1d-restricted manner.

CD1c presents lipid-based antigens to CD1c-restricted T cells which are thought to be a major component of the human T cell pool. The study of CD1c-restricted T cells, however, is hampered by the presence of an abundantly expressed CD1c-binding partner on blood cells distinct to the T cell receptor (TCR), confounding analysis of TCR-mediated CD1c tetramer staining. Here, we identify the CD36 family (CD36, CD36-L1 and CD36-L2) as novel ligands for CD1c, CD1b and CD1d proteins, and show that CD36 is the receptor responsible for non-TCR-mediated CD1c tetramer staining of blood cells. Moreover, CD36-blockade enables tetramer-based identification of CD1c-restricted T cells and clarifies identification of CD1b- and CD1d-restricted T cells. We use this technique to characterise CD1c-restricted T cells ex vivo and show diverse phenotypic features, TCR repertoire and antigen-specific subsets. Accordingly, this work will enable further studies into the biology of CD1 and human CD1-restricted T cells.One Sentence SummaryCD1 molecules bind CD36 family members and blockade of this interaction facilitates the study of CD1-restricted T cells.

Natural Killer T (NKT) cells detect lipids presented by CD1d. Most studies focus on type I NKT cells that express semi-invariant αβ T cell receptors (TCR) and recognise α-galactosylceramides. However, CD1d also presents structurally distinct lipids to NKT cells expressing diverse TCRs (type II NKT cells) but our knowledge of the antigens for type II NKT cells is limited. An early study identified an NKT cell agonist, phenyl pentamethyldihydrobenzofuransulfonate (PPBF), which is notable for its similarity to common sulfa-drugs, but its mechanism of NKT-cell activation remained unknown. Here we demonstrate that a range of pentamethylbenzofuransulfonate (PBFs), including PPBF, activate polyclonal type II NKT cells from human donors. Whereas these sulfa drug-like molecules might have acted pharmacologically on cells, here we demonstrate direct contact between TCRs and PBF-treated CD1d complexes. Further, PBF-treated CD1d-tetramers identified type II NKT cell populations cells expressing αβ and γδTCRs, including those with variable and joining region gene usage (TRAV12-1–TRAJ6) that was conserved across donors. By trapping a CD1d-type II NKT TCR complex for direct mass spectrometric analysis, we detected molecules that allow binding of CD1d to TCRs, finding that both PBF and short-chain sphingomyelin lipids are present in these complexes. Furthermore, the combination of PPBF and short-chain sphingomyelin enhances CD1d tetramer staining of PPBF-reactive T cell lines over either molecule alone. This study demonstrates that non-lipidic small molecules, that resemble sulfa-drugs implicated in systemic hypersensitivity and drug allergy reactions, activate a polyclonal population of type II NKT cells in a CD1d-restricted manner.Significance StatementWhereas T cells are known to recognize peptide, vitamin B metabolite or lipid antigens, we identify several non-lipidic small molecules, pentamethylbenzofuransulfonates (PBFs), that activate a population of CD1d-restricted NKT cells. This represents a breakthrough in the field of NKT cell biology. This study also reveals a previously unknown population of PBF-reactive NKT cells in healthy individuals with stereotyped receptors that paves the way for future studies of the role of these cells in immunity, including sulfa-drug hypersensitivity.

CD1c presents lipid-based antigens to CD1c-restricted T cells, which are thought to be a major component of the human T cell pool. However, the study of CD1c-restricted T cells is hampered by the presence of an abundantly expressed, non-T cell receptor (TCR) ligand for CD1c on blood cells, confounding analysis of TCR-mediated CD1c tetramer staining. Here, we identified the CD36 family (CD36, SR-B1, and LIMP-2) as ligands for CD1c, CD1b, and CD1d proteins and showed that CD36 is the receptor responsible for non-TCR-mediated CD1c tetramer staining of blood cells. Moreover, CD36 blockade clarified tetramer-based identification of CD1c-restricted T cells and improved identification of CD1b- and CD1d-restricted T cells. We used this technique to characterize CD1c-restricted T cells ex vivo and showed diverse phenotypic features, TCR repertoire, and antigen-specific subsets. Accordingly, this work will enable further studies into the biology of CD1 and human CD1-restricted T cells.

Antibody-mediated modulation of major histocompatibility complex (MHC) molecules, or MHC class I-like molecules, could constitute an effective immunotherapeutic approach. We describe how single-domain antibodies (VHH), specific for the human MHC class I-like molecule CD1d, can modulate the function of CD1d-restricted T cells and how one VHH (1D12) specifically induced strong type I natural killer T (NKT) cell activation. The crystal structure of the VHH1D12-CD1d(α-GalCer)-NKT T-cell receptor (TCR) complex revealed that VHH1D12 simultaneously contacted CD1d and the type I NKT TCR, thereby stabilizing this interaction through intrinsic bispecificity. This led to greatly enhanced type I NKT cell-mediated antitumor activity in in vitro, including multiple myeloma and acute myeloid leukemia patient-derived bone marrow samples, and in vivo models. Our findings underscore the versatility of VHH molecules in targeting composite epitopes, in this case consisting of a complexed monomorphic antigen-presenting molecule and an invariant TCR, and represent a generalizable antitumor approach.

Summary: Vδ2+ T cells play a critical role in immunity to micro-organisms and cancer but exhibit substantial heterogeneity in humans. Here, we demonstrate that CD26 and CD94 define transcriptionally, phenotypically, and functionally distinct Vδ2+ T cell subsets. Despite distinct antigen specificities, CD26hiCD94lo Vδ2+ cells exhibit substantial similarities to CD26hi mucosal-associated invariant T (MAIT) cells, although CD26− Vδ2+ cells exhibit cytotoxic, effector-like profiles. At birth, the Vδ2+Vγ9+ population is dominated by CD26hiCD94lo cells; during adolescence and adulthood, Vδ2+ cells acquire CD94/NKG2A expression and the relative frequency of the CD26hiCD94lo subset declines. Critically, exposure of the CD26hiCD94lo subset to phosphoantigen in the context of interleukin-23 (IL-23) and CD26 engagement drives the acquisition of a cytotoxic program and concurrent loss of the MAIT cell-like phenotype. The ability to modulate the cytotoxic potential of CD26hiCD94lo Vδ2+ cells, combined with their adenosine-binding capacity, may make them ideal targets for immunotherapeutic expansion and adoptive transfer.

Mycobacterial cell wall lipids bind the conserved CD1 family of antigen-presenting molecules and activate T cells via their T cell receptors (TCRs). Sulfoglycolipids (SGLs) are uniquely synthesized by Mycobacterium tuberculosis, but tools to study SGL-specific T cells in humans are lacking. We designed a novel hybrid synthesis of a naturally occurring SGL, generated CD1b tetramers loaded with natural or synthetic SGL analogs, and studied the molecular requirements for TCR binding and T cell activation. Two T cell lines derived using natural SGLs are activated by synthetic analogs independently of lipid chain length and hydroxylation, but differentially by saturation status. By contrast, two T cell lines derived using an unsaturated SGL synthetic analog were not activated by the natural antigen. Our data provide a bioequivalence hierarchy of synthetic SGL analogs and SGL-loaded CD1b tetramers. These reagents can now be applied to large-scale translational studies investigating the diagnostic potential of SGL-specific T cell responses or SGL-based vaccines. Sulfoglycolipids (SGLs) are uniquely synthesized by Mycobacterium tuberculosis (Mtb) and recognized by human T cells. James, Yu et al. describe a new hybrid synthesis for key antigenic determinants of SGLs and the development of SGL-specific tetramers that can now be applied to large-scale translational studies.

Mucosal‐associated invariant T (MAIT) cells represent up to 10% of circulating human T cells. They are usually defined using combinations of non‐lineage‐specific (surrogate) markers such as anti‐TRAV1‐2, CD161, IL‐18Rα and CD26. The development of MR1‐Ag tetramers now permits the specific identification of MAIT cells based on T‐cell receptor specificity. Here, we compare these approaches for identifying MAIT cells and show that surrogate markers are not always accurate in identifying these cells, particularly the CD4+ fraction. Moreover, while all MAIT cell subsets produced comparable levels of IFNγ, TNF and IL‐17A, the CD4+ population produced more IL‐2 than the other subsets. In a human ontogeny study, we show that the frequencies of most MR1 tetramer+ MAIT cells, with the exception of CD4+ MAIT cells, increased from birth to about 25 years of age and declined thereafter. We also demonstrate a positive association between the frequency of MAIT cells and other unconventional T cells including Natural Killer T (NKT) cells and Vδ2+ γδ T cells. Accordingly, this study demonstrates that MAIT cells are phenotypically and functionally diverse, that surrogate markers may not reliably identify all of these cells, and that their numbers are regulated in an age‐dependent manner and correlate with NKT and Vδ2+ γδ T cells.

Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells restricted by the major histocompatibility complex (MHC) class I-like molecule MHC-related protein 1 (MR1). MAIT cells are found throughout the body, especially in human blood and liver. Unlike conventional T cells, which are stimulated by peptide antigens presented by MHC molecules, MAIT cells recognize metabolite antigens derived from an intermediate in the microbial biosynthesis of riboflavin. MAIT cells mediate protective immunity to infections by riboflavin-producing microbes via the production of cytokines and cytotoxicity. The discovery of stimulating MAIT cell antigens allowed for the development of an analytical tool, the MR1 tetramer, that binds specifically to the MAIT T cell receptor (TCR) and is becoming the gold standard for identification of MAIT cells by flow cytometry. This article describes protocols to characterize the phenotype of human MAIT cells in blood and tissues by flow cytometry using fluorescently labeled human MR1 tetramers alongside antibodies specific for MAIT cell markers. © 2019 by John Wiley & Sons, Inc. The main protocols include: Basic Protocol 1: Determining the frequency and steady-state surface phenotype of human MAIT cells Basic Protocol 2: Determining the activation phenotype of human MAIT cells in blood Basic Protocol 3: Characterizing MAIT cell TCRs using TCR-positive reporter cell lines Alternate protocols are provided for determining the absolute number, transcription factor phenotype, and TCR usage of human MAIT cells; and determining activation phenotype by staining for intracellular markers, measuring secreted cytokines, and measuring fluorescent dye dilution due to proliferation. Additional methods are provided for determining the capacity of MAIT cells to produce cytokine independently of antigen using plate-bound or bead-immobilized CD3/CD28 stimulation; and determining the MR1-Ag dependence of MAIT cell activation using MR1-blocking antibody or competitive inhibition. For TCR-positive reporter cell lines, methods are also provided for evaluating the MAIT TCR-mediated MR1-Ag response, determining the capacity of the reporter lines to produce cytokine independently of antigen, determining the MR1-Ag dependence of the reporter lines, and evaluating the MR1-Ag response of the reporter lines using IL-2 secretion. Support Protocols describe the preparation of PBMCs from human blood, the preparation of single-cell suspensions from tissue, the isolation of MAIT cells by FACS and MACS, cloning MAIT TCRα and β chain genes and MR1 genes for transduction, generating stably and transiently transfected cells lines, generating a stable MR1 knockout antigen-presenting cell line, and generating monocyte-derived dendritic cells.

MR1-restricted mucosal-associated invariant T (MAIT) cells play a unique role in the immune system. These cells develop intrathymically through a three-stage process, but the events that regulate this are largely unknown. Here, using bulk and single-cell RNA sequencing-based transcriptomic analysis in mice and humans, we studied the changing transcriptional landscape that accompanies transition through each stage. Many transcripts were sharply modulated during MAIT cell development, including SLAM (signaling lymphocytic activation molecule) family members, chemokine receptors, and transcription factors. We also demonstrate that stage 3 "mature" MAIT cells comprise distinct subpopulations including newly arrived transitional stage 3 cells, interferon-γ-producing MAIT1 cells and interleukin-17-producing MAIT17 cells. Moreover, the validity and importance of several transcripts detected in this study are directly demonstrated using specific mutant mice. For example, MAIT cell intrathymic maturation was found to be halted in SLAM-associated protein (SAP)-deficient and CXCR6-deficient mouse models, providing clear evidence for their role in modulating MAIT cell development. These data underpin a model that maps the changing transcriptional landscape and identifies key factors that regulate the process of MAIT cell differentiation, with many parallels between mice and humans.

Mucosal-associated invariant T cells (MAIT cells) detect microbial vitamin B2 derivatives presented by the antigen-presenting molecule MR1. Here we defined three developmental stages and checkpoints for the MAIT cell lineage in humans and mice. Stage 1 and stage 2 MAIT cells predominated in thymus, while stage 3 cells progressively increased in abundance extrathymically. Transition through each checkpoint was regulated by MR1, whereas the final checkpoint that generated mature functional MAIT cells was controlled by multiple factors, including the transcription factor PLZF and microbial colonization. Furthermore, stage 3 MAIT cell populations were expanded in mice deficient in the antigen-presenting molecule CD1d, suggestive of a niche shared by MAIT cells and natural killer T cells (NKT cells). Accordingly, this study maps the developmental pathway and checkpoints that control the generation of functional MAIT cells.

Mucosal-associated invariant T (MAIT) cells express an invariant TRAV1/TRAJ33 TCR-α chain and are restricted to the MHC-I-like molecule, MR1. Whether MAIT cell development depends on this invariant TCR-α chain is unclear. Here we generate Traj33-deficient mice and show that they are highly depleted of MAIT cells; however, a residual population remains and can respond to exogenous antigen in vitro or pulmonary Legionella challenge in vivo. These residual cells include some that express Trav1+ TCRs with conservative Traj-gene substitutions, and others that express Trav1- TCRs with a broad range of Traj genes. We further report that human TRAV1-2- MR1-restricted T cells contain both MAIT-like and non-MAIT-like cells, as judged by their TCR repertoire, antigen reactivity and phenotypic features. These include a MAIT-like population that expresses a public, canonical TRAV36+ TRBV28+ TCR. Our findings highlight the TCR diversity and the resulting potential impact on antigen recognition by MR1-restricted T cells., Mucosal-associated invariant T (MAIT) cells express invariant TRAV1/TRAJ33 TCR-α gene segments and detect antigens presented by MR1. Here the authors show that atypical, MR1-restricted MAIT populations that include both Trav1+ and Trav1- cells are found in both Traj33-deficient mice and human peripheral blood.

A different way for γδ T cells to bind The ligands bound by γδ T cell receptors (TCRs) are less well characterized than those of their αβ TCR cousins, which are antigens presented by major histocompatibility complex (MHC) and related proteins. Le Nours et al. identified a phenotypically diverse γδ T cell subset in human tissues that reacts to MHC-related protein 1 (MR1), which presents vitamin B derivatives. A crystal structure of a γδ TCR–MR1–antigen complex revealed that some of these TCRs can bind underneath the MR1 antigen-binding cleft instead of recognizing the presented antigen. This work thus uncovers an additional ligand for γδ T cells and reconceptualizes the nature of T cell antigen recognition. Science , this issue p. 1522

Mucosal‐associated invariant T (MAIT) cells develop in the thymus and migrate into the periphery to become the largest antigen‐specific αβ T‐cell population in the human immune system. However, the frequency of MAIT cells varies widely between human individuals, and the basis for this is unclear. While MAIT cells are highly conserved through evolution and are phenotypically similar between humans and mice, they represent a much smaller proportion of total T cells in mice. In this review, we discuss how MAIT cells transition through a three‐stage development pathway in both mouse and human thymus, and continue to mature and expand after they leave the thymus. Moreover, we will explore and speculate on how specific factors regulate different stages of this process.

A central paradigm in αβ T cell–mediated immunity is the simultaneous co-recognition of antigens and antigen-presenting molecules by the αβ T cell antigen receptor (TCR). CD1a presents a broad repertoire of lipid-based antigens. We found that a prototypical autoreactive TCR bound CD1a when it was presenting a series of permissive endogenous ligands, while other lipid ligands were nonpermissive to TCR binding. The structures of two TCR-CD1a-lipid complexes showed that the TCR docked over the A′ roof of CD1a in a manner that precluded direct contact with permissive ligands. Nonpermissive ligands indirectly inhibited TCR binding by disrupting the TCR-CD1a contact zone. The exclusive recognition of CD1a by the TCR represents a previously unknown mechanism whereby αβ T cells indirectly sense self antigens that are bound to an antigen-presenting molecule.

Studies of lipid reactive CD1d-restricted T cells have focussed on α-galactosylceramide reactive semi-invariant Type I NKT cells, which influence a broad range of immune responses. Much less is known about other CD1d-restricted T cells with respect to TCR diversity, function, the types of antigens they recognize and how they specifically recognize antigens presented by CD1d. In this review, we reflect on recent literature that highlights unexpected complexity within the pool of CD1d-restricted T cells and emphasize how TCR diversity greatly broadens the scope of antigen recognition.

The T cell repertoire comprises αβ and γδ T cell lineages. Although it is established how αβ T cell antigen receptors (TCRs) interact with antigen presented by antigen-presenting molecules, this is unknown for γδ TCRs. We describe a population of human V δ 1 + γδ T cells that exhibit autoreactivity to CD1d and provide a molecular basis for how a γδ TCR binds CD1d-α- galactosylceramide (α-GalCer). The γδ TCR docked orthogonally, over the A′ pocket of CD1d, in which the V δ 1-chain, and in particular the germ line-encoded CDR1δ loop, dominated interactions with CD1d. The TCR γ-chain sat peripherally to the interface, with the CDR3γ loop representing the principal determinant for α-GalCer specificity. Accordingly, we provide insight into how a γδ TCR binds specifically to a lipid-loaded antigen-presenting molecule.

SummaryThe antigen receptor for natural killer T cells (NKT TCR) binds CD1d-restricted microbial and self-lipid antigens, although the molecular basis of self-CD1d recognition is unclear. Here, we have characterized NKT TCR recognition of CD1d molecules loaded with natural self-antigens (Ags) and report the 2.3 Å resolution structure of an autoreactive NKT TCR-phosphatidylinositol-CD1d complex. NKT TCR recognition of self- and foreign antigens was underpinned by a similar mode of germline-encoded recognition of CD1d. However, NKT TCR autoreactivity is mediated by unique sequences within the non-germline-encoded CDR3β loop encoding for a hydrophobic motif that promotes self-association with CD1d. Accordingly, NKT cell autoreactivity may arise from the inherent affinity of the interaction between CD1d and the NKT TCR, resulting in the recognition of a broad range of CD1d-restricted self-antigens. This demonstrates that multiple self-antigens can be recognized in a similar manner by autoreactive NKT TCRs.

SummaryNatural killer T (NKT) cells respond to a variety of CD1d-restricted antigens (Ags), although the basis for Ag discrimination by the NKT cell receptor (TCR) is unclear. Here we have described NKT TCR fine specificity against several closely related Ags, termed altered glycolipid ligands (AGLs), which differentially stimulate NKT cells. The structures of five ternary complexes all revealed similar docking. Acyl chain modifications did not affect the interaction, but reduced NKT cell proliferation, indicating an affect on Ag processing or presentation. Conversely, truncation of the phytosphingosine chain caused an induced fit mode of TCR binding that affected TCR affinity. Modifications in the glycosyl head group had a direct impact on the TCR interaction and associated cellular response, with ligand potency reflecting the t1/2 life of the interaction. Accordingly, we have provided a molecular basis for understanding how modifications in AGLs can result in striking alterations in the cellular response of NKT cells.

Introduction: Langerhans cell histiocytosis (LCH) is a rare disease involving inflammatory lesions that can occur in essentially any organ of the body. LCH lesions are defined by the presence of CD1a+/CD207+ myeloid lineage cells (LCH cells), which often have mutations within the RAS/RAF/MEK/ERK pathway and constitutive activation of ERK. A range of other immune cells are also present within lesions including an enrichment of Foxp3 regulatory T cells (Tregs). The immune suppressive cytokine transforming growth factor beta (TGF-β), which is commonly produced by Foxp3 Tregs has also been detected within lesions and blood from patients with active LCH disease. Groups have suggested that LCH cells are a source of TGF-β and that TGF-β is one of the drivers of the LCH cell phenotype. Given the enrichment of Foxp3 Tregs and the presence of TGF-β within LCH lesions it is conceivable that Foxp3 Tregs too are a source of TGF-β production. Historically the identification of Foxp3 Tregs has not allowed for functional studies to be conducted because staining for the Foxp3 transcription factor requires cell permeabilization. A newer surrogate gating strategy to detect CD3+CD4+CD25+CD127low lymphocytes allows for downstream functional assays on the Foxp3 Treg population. Our study aimed to better define the role of Tregs in LCH pathogenesis using this surrogate staining method. Results: A surrogate Treg identification method was employed to gate on CD3+CD4+CD25+CD127low lymphocytes in the blood from healthy donors and patients with LCH and in lesions from patients with LCH. Using this gating strategy we identified similarly to previous studies that the proportion of lesional Tregs (median = 12.85 %, IQR = 7.85-26.72 %, n = 6) was significantly (p < 0.0001) increased in the total T cell population when compared to those in the blood from healthy donors (median = 1.02 %, IQR = 0.84-1.20 %, n = 19). The proportion of Tregs in blood from patients with active LCH (median = 1.73 %, IQR = 1.58-3.80 %, n = 8) was also significantly (p = 0.232) increased when compared to those in the blood from healthy donors. We confirmed Foxp3 expression in Tregs in lesions from three independent LCH donors by staining for the transcription factor, and we confirmed that other lesional CD4+ T cells were mostly negative for Foxp3. Due to our interest in unconventional T cells we analysed specimens for CD56 expression in conjunction with investigating Tregs and we unexpectedly found CD56 expression on a considerable proportion of the Treg population in LCH lesions (median = 36.48 %, IQR = 24.94-55.14 %, n = 6). The proportion of Tregs that displayed CD56 expression on their cell surface was significantly higher in the lesions from patients with LCH compared to the blood from healthy donors (median = 2.29 %, IQR = 0.61-4.10 %, n = 15, p = 0.0092) and patients with active LCH (median = 0.57 %, IQR = 0.11-0.85 %, n = 8, p < 0.0001). Additionally, we found that the proportion of CD56+ Tregs in total T cells from LCH lesions was directly correlated to the proportion of total Tregs in overall lesional T cells (r = 1, p = 0.0028). Importantly, we confirmed Foxp3 expression by both the CD56+ and CD56- Treg populations in lesions from three independent LCH donors. We purified and then stimulated Tregs from healthy donors, and CD56+ and CD56- Treg populations from lesions from patients with LCH. Using supernatants from this assay we found that Tregs from lesions from patients with LCH produced active TGF-β when challenged with PMA and ionomycin for 16 hours (CD56+ Tregs n = 3, CD56- Tregs n = 3). Conclusion: We have confirmed that both CD56+ and CD56- Treg populations from the lesions from patients with LCH are able to produce TGF-β. Given their cytokine potential, the functional status of Tregs in LCH lesions is therefore most likely to be suppressive in nature. This means that Tregs are possibly responsible for a component of the well documented expression of TGF-β within lesions. Disclosures No relevant conflicts of interest to declare.

alphabeta T cell receptors (TCRs) have traditionally been viewed as receptors for peptide antigens presented by either Major Histocompatibility Complex (MHC) class I (for CD8 T cells) or MHC class II (for CD4 T cells) antigen-presenting molecules. However, it is now clear that some T cell lineages express TCRs that are specialized for recognition of lipid-based antigens presented by the MHC class I-like CD1 family. Recently, the molecular basis for the TCR recognition of glycolipid antigens presented by CD1d has revealed an evolutionarily conserved-docking mode that is distinct from that of peptide-based recognition. T cells carrying these receptors follow a unique developmental pathway that results not only in unconventional antigen specificity, but also seemingly exaggerated functional capabilities, which makes these cells and their antigens highly attractive targets for immunotherapeutic manipulation.

Mouse type I natural killer T cell receptors (iNKT TCRs) use a single V alpha 14-J alpha 18 sequence and V beta s that are almost always V beta 8.2, V beta 7, or V beta 2, although the basis of this differential usage is unclear. We showed that the V beta bias occurred as a consequence of the CDR2 beta loops determining the affinity of the iNKT TCR for CD1d-glycolipids, thus controlling positive selection. Within a conserved iNKT-TCR-CD1d docking framework, these inherent V beta-CD1d affinities are further modulated by the hypervariable CDR3 beta loop, thereby defining a functional interplay between the two iNKT TCR CDR beta loops. These V beta biases revealed a broadly hierarchical response in which V beta 8.2 > V beta 7 > V beta 2 in the recognition of diverse CD1d ligands. This restriction of the iNKT TCR repertoire during thymic selection paradoxically ensures that each peripheral iNKT cell recognizes a similar spectrum of antigens.

Invariant NKT cells play a critical role in controlling the strength and character of adaptive immune responses. We have previously reported deficiencies in the numbers and function of NKT cells in the NOD mouse strain, which is a well-validated model of type 1 diabetes and systemic lupus erythematosus. Genetic control of thymic NKT cell numbers was mapped to two linkage regions: Nkt1 on distal chromosome 1 and Nkt2 on chromosome 2. In this study, we report the production and characterization of a NOD.Nkrp1b.Nkt1b congenic mouse strain, apply microarray expression analyses to limit candidate genes within the 95% confidence region, identify Slamf1 (encoding signaling lymphocyte activation molecule) and Slamf6 (encoding Ly108) as potential candidates, and demonstrate retarded signaling lymphocyte activation molecule expression during T cell development of NOD mice, resulting in reduced expression at the CD4+CD8+ stage, which is consistent with decreased NKT cell production and deranged tolerance induction in NOD mice.

NKT cells recognize lipid-based Ags presented by CD1d. Type I NKT cells are often referred to as invariant owing to their mostly invariant TCR α-chain usage (Vα14-Jα18 in mice, Vα24-Jα18 in humans). However, these cells have diverse TCR β-chains, including Vβ8, Vβ7, and Vβ2 in mice and Vβ11 in humans, joined to a range of TCR Dβ and Jβ genes. In this study, we demonstrate that TCR β-chain composition can dramatically influence lipid Ag recognition in an Ag-dependent manner. Namely, the glycolipids α-glucosylceramide and isoglobotrihexosylceramide were preferentially recognized by Vβ7+ NKT cells from mice, whereas the α-galactosylceramide analog OCH, with a truncated sphingosine chain, was preferentially recognized by Vβ8+ NKT cells from mice. We show that the influence of the TCR β-chain is due to a combination of Vβ-, Jβ-, and CDR3β-encoded residues and that these TCRs can recapitulate the selective Ag reactivity in TCR-transduced cell lines. Similar observations were made with human NKT cells where different CDR3β-encoded residues determined Ag preference. These findings indicate that NKT TCR β-chain diversity results in differential and nonhierarchical Ag recognition by these cells, which implies that some Ags can preferentially activate type I NKT cell subsets.

Tissue-resident memory T (Trm) cells contribute to local immune protection in non-lymphoid tissues such as skin and mucosa, but little is known about their transcriptional regulation. Here we showed that CD8(+)CD103(+) Trm cells, independent of circulating memory T cells, were sufficient for protection against infection and described molecular elements that were crucial for their development in skin and lung. We demonstrated that the T-box transcription factors (TFs) Eomes and T-bet combined to control CD8(+)CD103(+) Trm cell formation, such that their coordinate downregulation was crucial for TGF-β cytokine signaling. TGF-β signaling, in turn, resulted in reciprocal T-box TF downregulation. However, whereas extinguishment of Eomes was necessary for CD8(+)CD103(+) Trm cell development, residual T-bet expression maintained cell surface interleukin-15 (IL-15) receptor β-chain (CD122) expression and thus IL-15 responsiveness. These findings indicate that the T-box TFs control the two cytokines, TGF-β and IL-15, which are pivotal for CD8(+)CD103(+) Trm cell development and survival.

A characteristic of mucosal-associated invariant T (MAIT) cells is the expression of TRAV1-2(+) T cell receptors (TCRs) that are activated by riboflavin metabolite-based antigens (Ag) presented by the MHC-I related molecule, MR1. Whether the MR1-restricted T cell repertoire and associated Ag responsiveness extends beyond these cells remains unclear. Here, we describe MR1 autoreactivity and folate-derivative reactivity in a discrete subset of TRAV1-2(+) MAIT cells. This recognition was attributable to CDR3β loop-mediated effects within a consensus TRAV1-2(+) TCR-MR1-Ag footprint. Furthermore, we have demonstrated differential folate- and riboflavin-derivative reactivity by a diverse population of "atypical" TRAV1-2(-) MR1-restricted T cells. We have shown that TRAV1-2(-) T cells are phenotypically heterogeneous and largely distinct from TRAV1-2(+) MAIT cells. A TRAV1-2(-) TCR docks more centrally on MR1, thereby adopting a markedly different molecular footprint to the TRAV1-2(+) TCR. Accordingly, diversity within the MR1-restricted T cell repertoire leads to differing MR1-restricted Ag specificity.

Glycolipid ligands for invariant natural killer T cells (iNKT cells) are loaded onto CD1d molecules in the late endosome/lysosome. Accumulation of glycosphingolipids (GSLs) in lysosomal storage diseases could potentially influence endogenous and exogenous lipid loading and/or presentation and, thus, affect iNKT cell selection or function. The percentages and frequency of iNKT cells were reduced in multiple mouse models of lysosomal GSL storage disease, irrespective of the specific genetic defect or lipid species stored. Reduced numbers of iNKT cells resulted in the absence of cytokine production in response to alpha-galactosylceramide (alpha-GalCer) and reduced iNKT cell-mediated lysis of wild-type targets loaded with alpha-GalCer. The reduction in iNKT cells did not result from defective expression of CD1d or a lack of antigen-presenting cells. Although H-2 restricted CD4(+) T cell responses were generally unaffected, processing of a lysosome-dependent analogue of alpha-GalCer was impaired in all the strains of mice tested. These data suggest that GSL storage may result in alterations in thymic selection of iNKT cells caused by impaired presentation of selecting ligands.

Little is known regarding the basis for selection of the semi-invariant alphabeta T cell receptor (TCR) expressed by natural killer T (NKT) cells or how this mediates recognition of CD1d-glycolipid complexes. We have determined the structures of two human NKT TCRs that differ in their CDR3beta composition and length. Both TCRs contain a conserved, positively charged pocket at the ligand interface that is lined by residues from the invariant TCR alpha- and semi-invariant beta-chains. The cavity is centrally located and ideally suited to interact with the exposed glycosyl head group of glycolipid antigens. Sequences common to mouse and human invariant NKT TCRs reveal a contiguous conserved "hot spot" that provides a basis for the reactivity of NKT cells across species. Structural and functional data suggest that the CDR3beta loop provides a plasticity mechanism that accommodates recognition of a variety of glycolipid antigens presented by CD1d. We propose a model of NKT TCR-CD1d-glycolipid interaction in which the invariant CDR3alpha loop is predicted to play a major role in determining the inherent bias toward CD1d. The findings define a structural basis for the selection of the semi-invariant alphabeta TCR and the unique antigen specificity of NKT cells.

NKT cells are typically defined as CD1d-dependent T cells that carry an invariant TCR α-chain and produce high levels of cytokines. Traditionally, these cells were defined as NK1.1+ T cells, although only a few mouse strains express the NK1.1 molecule. A popular alternative marker for NKT cells has been DX5, an Ab that detects the CD49b integrin, expressed by most NK cells and a subset of T cells that resemble NKT cells. Interpretation of studies using DX5 as an NKT cell marker depends on how well DX5 defines NKT cells. Using a range of DX5 and other anti-CD49b Abs, we reveal major differences in reactivity depending on which Ab and which fluorochrome are used. The brightest, PE-conjugated reagents revealed that while most CD1d-dependent NKT cells expressed CD49b, they represented only a minority of CD49b+ T cells. Furthermore, CD49b+ T cell numbers were near normal in CD1d−/− mice that are completely deficient for NKT cells. CD1d tetramer− CD49b+ T cells differ from NKT cells by their activation and memory marker expression, tissue distribution, and CD4/CD8 coreceptor profile. Interestingly, both NKT cells and CD1d tetramer− CD49b+ T cells produce cytokines, but the latter are clearly biased toward Th1-type cytokines, in contrast to NKT cells that produce both Th1 and Th2 cytokines. Finally, we demonstrate that expression of CD49b by NKT cells does not dramatically alter with age, contrasting with earlier reports proposing DX5 as a maturation marker for NKT cells. In summary, our data demonstrate that DX5/CD49b is a poor marker for identifying CD1d-dependent NKT cells.

After being positively selected on CD1d-expressing thymocytes, NKT cells undergo a series of developmental changes that can take place inside or outside the thymus. We asked whether CD1d continues to play a role in late-stage NKT cell development and, in particular, during the functionally significant acquisition of NK1.1 that is indicative of NKT cell maturity. We report that CD1d is indeed crucial for this step, because immature NK1.1− NKT cells fail to fully mature when transferred to a CD1d-deficient environment. Surprisingly, however, the lack of CD1d did not greatly affect the long-term survival of NKT cells, and they continued to express CD69 and slowly proliferate. This directly contradicts the currently held view that these phenomena are caused by autoreactivity directed against CD1d/TCR-restricted self-Ags. Our findings demonstrate an ongoing role for TCR-mediated signaling throughout NKT cell development, but the characteristic semiactivated basal state of NKT cells is controlled by CD1d-independent factors or is intrinsic to the cells themselves.

In the diabetes-prone NOD mouse, there is a proven association between a systemic deficiency of NKT cells and the onset of type 1 diabetes. Numerous reports of similar defects within the NKT cell compartment of human type 1 diabetes patients suggested NKT cell levels might be a valuable predictor of susceptibility and could provide a target for therapeutic intervention. Two recent studies, however, found no association between type 1 diabetes and blood NKT cell levels in humans and consequently rejected a link between the onset of diabetes and NKT cell deficiency. This cast considerable doubts on the potential for NKT cell-based clinical applications and challenged the validity of the NOD mouse as a model of human type 1 diabetes. We now report that NKT cell levels in blood are a poor representation of those in other organs. Strikingly, systemic NKT cell deficiencies were identified in NOD mice with normal, or even raised, blood levels. This re-establishes the correlation between NKT cell deficiency and type 1 diabetes and raises important questions regarding the assaying of NKT cell levels in humans.

The antimetastatic effect of the CD1d-binding glycolipid, alpha-galactosylceramide (alpha-GalCer), is mediated by NK1.1(+)T (NKT) cells; however, the mechanisms behind this process are poorly defined. Although it has been shown to involve NK cells and interferon-gamma (IFN-gamma) production, the way these factors collaborate to mediate effective tumor rejection and the importance of other factors characteristic of NKT cell and NK cell activation are unknown. Using gene-targeted mice and antibody treatments, the critical need for interleukin 12 (IL-12), IFN-gamma, and NK cells has been shown in the antimetastatic activity of alpha-GalCer in the lungs and the liver. By contrast, in lung and liver metastasis models, cytotoxic molecules expressed by NK cells and NKT cells (perforin, Fas ligand, and tumor necrosis factor-related apoptosis-inducing ligand) and an NKT cell-secreted cytokine, IL-4, were not necessary for the antitumor activity of alpha-GalCer. Like IL-12, IL-18 was required for optimal serum IFN-gamma induction and control of lung metastases by alpha-GalCer. IL-18 was unnecessary for alpha-GalCer-related suppression of liver metastases. Most importantly, after adoptive transfer of alpha-GalCer-reactive NKT cells or NK cells into NKT cell-deficient, IFN-gamma-deficient, or RAG-1-deficient mice, it was demonstrated that the sequential production of IFN-gamma by NKT cells and NK cells was absolutely required to reconstitute the antimetastatic activity of alpha-GalCer.

Natural killer T cells (NKT cells) are divided into type I and type II subsets on the basis of differences in their T cell antigen receptor (TCR) repertoire and CD1d-antigen specificity. Although the mode by which type I NKT cell TCRs recognize CD1d-antigen has been established, how type II NKT cell TCRs engage CD1d-antigen is unknown. Here we provide a basis for how a type II NKT cell TCR, XV19, recognized CD1d-sulfatide. The XV19 TCR bound orthogonally above the A' pocket of CD1d, in contrast to the parallel docking of type I NKT cell TCRs over the F' pocket of CD1d. At the XV19 TCR-CD1d-sulfatide interface, the TCRα and TCRβ chains sat centrally on CD1d, where the malleable CDR3 loops dominated interactions with CD1d-sulfatide. Accordingly, we highlight the diverse mechanisms by which NKT cell TCRs can bind CD1d and account for the distinct antigen specificity of type II NKT cells.

The most potent foreign antigens for natural killer T cells (NKT cells) are α-linked glycolipids, whereas NKT cell self-reactivity involves weaker recognition of structurally distinct β-linked glycolipid antigens. Here we provide the mechanism for the autoreactivity of T cell antigen receptors (TCRs) on NKT cells to the mono- and tri-glycosylated β-linked agonists β-galactosylceramide (β-GalCer) and isoglobotrihexosylceramide (iGb3), respectively. In binding these disparate antigens, the NKT cell TCRs docked onto CD1d similarly, achieving this by flattening the conformation of the β-linked ligands regardless of the size of the glycosyl head group. Unexpectedly, the antigenicity of iGb3 was attributable to its terminal sugar group making compensatory interactions with CD1d. Thus, the NKT cell TCR molds the β-linked self ligands to resemble the conformation of foreign α-linked ligands, which shows that induced-fit molecular mimicry can underpin the self-reactivity of NKT cell TCRs to β-linked antigens.

Type I natural killer T cells (NKT cells) are characterized by an invariant variable region 14-joining region 18 (V(α)14-J(α)18) T cell antigen receptor (TCR) α-chain and recognition of the glycolipid α-galactosylceramide (α-GalCer) restricted to the antigen-presenting molecule CD1d. Here we describe a population of α-GalCer-reactive NKT cells that expressed a canonical V(α)10-J(α)50 TCR α-chain, which showed a preference for α-glucosylceramide (α-GlcCer) and bacterial α-glucuronic acid-containing glycolipid antigens. Structurally, despite very limited TCRα sequence identity, the V(α)10 TCR-CD1d-α-GlcCer complex had a docking mode similar to that of type I TCR-CD1d-α-GalCer complexes, although differences at the antigen-binding interface accounted for the altered antigen specificity. Our findings provide new insight into the structural basis and evolution of glycolipid antigen recognition and have notable implications for the scope and immunological role of glycolipid-specific T cell responses.

CD1d presents lipid-based antigens (Ag) that are recognised by the semi-invariant T cell receptor (TCR) expressed on Natural Killer T (NKT) cells. While the TCR α-chain is typically invariant, the TCR β-chain expression is more diverse, particularly in mice where at least three different Vβ chains are commonly expressed. We report the structures of Vα14-Vβ8.2 and Vα14-Vβ7 NKT TCRs in complex with CD1d-α-galactosylceramide (α-GalCer), as well as a 2.5 Å structure of the human NKT TCR-CD1d-α-GalCer complex. Both Vβ8.2 and Vβ7 NKT TCRs, as well as the human NKT TCR, ligated CD1d-α-GalCer in a broadly similar manner, thereby highlighting the evolutionarily-conserved nature of this interaction. However, differences within the Vβ domains of the Vβ8.2 and Vβ7 NKT TCR-CD1d complexes not only resulted in altered TCR-β-CD1d-mediated contacts, but also surprisingly modulated recognition mediated by the invariant α-chain. Mutagenesis studies revealed the differing contributions of Vβ8.2 and Vβ7 residues within the CDR2β loop in mediating contacts with CD1d. Collectively we provide a structural basis for the differential NKT TCR Vβ usage in NKT cells.

Activation of NKT cells using the glycolipid α-galactosylceramide (α-GalCer4) has availed many investigations into their immunoregulatory and therapeutic potential. However, it remains unclear how NKT cells respond to stimulation in vivo, which co-stimulatory pathways are important, and what factors (eg. antigen availability and activation-induced cell death) limit their response. We have explored these questions in the context of anin vivo model of NKT cell dynamics spanning activation, population expansion and subsequent contraction. Neither the B7/CD28 nor the CD40/CD40-L co-stimulatory pathways were necessary for cytokine production by activated NKT cells, either early (2 hours) or late (3 days) following initial stimulation, but both pathways were necessary for normal proliferative expansion of NKT cells in vivo. The pro-apoptotic Bcl-2 family member Bim was necessary for normal contraction of the NKT cell population between days 3-9 after stimulation, suggesting the pool size is regulated by apoptotic cell death in a manner similar to that of conventional T cells. Antigen availability was not the limiting factor for NKT cell expansion in vivo, and a second injection of α-GalCer induced a very blunted response, whereby cytokine production was reduced and further expansion did not occur. This appeared to be a form of anergy that was intrinsic to the NKT cells and not associated with up-regulation of inhibitory NK cell receptors such as NKG2A or Ly49 family members. Furthermore, NKT cells from mice pre-challenged with α-GalCer in vivoshowed little cytokine production and reduced proliferation in vitro. In summary, this study significantly enhances our understanding of how NKT cells respond to α-GalCer in vivo, revealing that the full primary response depends on costimulation via the CD28 and CD40 pathways, with subsequent Bim-dependent contraction. After contraction, the NKT cells are hypo-responsive to further antigenic induced expansion.

NKT cells are emerging as an extremely influential regulatory subset of T lymphocytes that are functionally and developmentally distinct from their mainstream counterparts. Like other T cells, NKT cells are thymus-dependent but their apparently unique pathway of differentiation is poorly characterized. Given the strong association between NKT cell deficiency and increased incidences of autoimmunity and cancer it is imperative that the mechanisms by which NKT cells are generated becomes better understood. This review examines what is known about NKT cell development in the thymus and highlights elements of the pathway that differ significantly from mainstream T-cell development. It is here that NKT cell-specific disorders may originate and may best be addressed.

We analyzed the expression of granzyme H in human blood leukocytes, using a novel monoclonal antibody raised against recombinant granzyme H. 33-kDa granzyme H was easily detected in unfractionated peripheral blood mononuclear cells, due to its high constitutive expression in CD3(-)CD56(+) natural killer (NK) cells, whereas granzyme B was less abundant. The NK lymphoma cell lines, YT and Lopez, also expressed high granzyme H levels. Unstimulated CD4(+) and particularly CD8(+) T cells expressed far lower levels of granzyme H than NK cells, and various agents that classically induce T cell activation, proliferation, and enhanced granzyme B expression failed to induce granzyme H expression in T cells. Also, granzyme H was not detected in NK T cells, monocytes, or neutrophils. There was a good correlation between mRNA and protein expression in cells that synthesize both granzymes B and H, suggesting that gzmH gene transcription is regulated similarly to gzmB. Overall, our data indicate that although the gzmB and gzmH genes are tightly linked, expression of the proteins is quite discordant in T and NK cells. The finding that granzyme H is frequently more abundant than granzyme B in NK cells is consistent with a role for granzyme H in complementing the pro-apoptotic function of granzyme B in human NK cells.

NKT cell development takes place in the thymus, beginning when these cells branch away from CD4+CD8+ mainstream thymocytes upon expression of the Valpha14Jalpha18 T cell receptor (TCR) and recognition of the CD1d molecule. Although NKT cells express an invariant TCR alpha chain, the diverse TCR beta expression leaves open the possibility that the development of these cells is shaped by glycolipid antigen recognition in the context of CD1d. Here, we show that the presence of an agonist glycolipid ligand, alpha-galactosylceramide, while NKT cells are developing in vitro or in vivo, specifically ablates their development. In contrast, the delayed introduction of this compound in vitro or in vivo, after NKT cells have developed, does not deplete these cells. These data indicate that NKT cells pass through a developmental window where they are susceptible to TCR-mediated negative selection, and suggest that NKT cells with a potentially high level of self reactivity can be removed from the NKT cell repertoire before they exit the thymus.

The common γ-chain cytokine, IL-21, is produced by CD4+ T cells and mediates potent effects on a variety of immune cells including NK, T, and B cells. NKT cells express the receptor for IL-21; however, the effect of this cytokine on NKT cell function has not been studied. We show that IL-21 on its own enhances survival of NKT cells in vitro, and IL-21 increases the proliferation of NKT cells in combination with IL-2 or IL-15, and particularly with the CD1d-restricted glycosphingolipid Ag α-galactosylceramide. Similar to its effects on NK cells, IL-21 enhances NKT cell granular morphology, including granzyme B expression, and some inhibitory NK receptors, including Ly49C/I and CD94. IL-21 also enhanced NKT cell cytokine production in response to anti-CD3/CD28 in vitro. Furthermore, NKT cells may be subject to autocrine IL-21-mediated stimulation because they are potent producers of this cytokine following in vitro stimulation via CD3 and CD28, particularly in conjunction with IL-12 or following in vivo stimulation with α-galactosylceramide. Indeed, NKT cells produced much higher levels of IL-21 than conventional CD4 T cells in this assay. This study demonstrates that NKT cells are potentially a major source of IL-21, and that IL-21 may be an important factor in NKT cell-mediated immune regulation, both in its effects on NK, T, and B cells, as well as direct effects on NKT cells themselves. The influence of IL-21 in NKT cell-dependent models of tumor rejection, microbial clearance, autoimmunity, and allergy should be the subject of future investigations.