Mucosal-associated invariant T cells (MAIT cells) express a semi-invariant T cell receptor (TCR) ?-chain, TRAV1-2-TRAJ33, and are activated by vitamin B metabolites bound by the major histocompatibility complex (MHC)-related class I-like molecule, MR1. Understanding MAIT cell biology has been restrained by the lack of reagents to specifically identify and characterize these cells. Furthermore, the use of surrogate markers may misrepresent the MAIT cell population. We show that modified human MR1 tetramers loaded with the potent MAIT cell ligand, reduced 6-hydroxymethyl-8-D-ribityllumazine (rRL-6-CH?OH), specifically detect all human MAIT cells. Tetramer(+) MAIT subsets were predominantly CD8(+) or CD4(-)CD8(-), although a small subset of CD4(+) MAIT cells was also detected. Notably, most human CD8(+) MAIT cells were CD8?(+)CD8?(-/lo), implying predominant expression of CD8?? homodimers. Tetramer-sorted MAIT cells displayed a T(H)1 cytokine phenotype upon antigen-specific activation. Similarly, mouse MR1-rRL-6-CH?OH tetramers detected CD4(+), CD4(-)CD8(-) and CD8(+) MAIT cells in V?19 transgenic mice. Both human and mouse MAIT cells expressed a broad TCR-? repertoire, and although the majority of human MAIT cells expressed TRAV1-2-TRAJ33, some expressed TRAJ12 or TRAJ20 genes in conjunction with TRAV1-2. Accordingly, MR1 tetramers allow precise phenotypic characterization of human and mouse MAIT cells and revealed unanticipated TCR heterogeneity in this population.
The T cell repertoire comprises ?? and ?? T cell lineages. Although it is established how ?? T cell antigen receptors (TCRs) interact with antigen presented by antigen-presenting molecules, this is unknown for ?? TCRs. We describe a population of human V?1(+) ?? T cells that exhibit autoreactivity to CD1d and provide a molecular basis for how a ?? TCR binds CD1d-?-galactosylceramide (?-GalCer). The ?? TCR docked orthogonally, over the A pocket of CD1d, in which the V?1-chain, and in particular the germ line-encoded CDR1? loop, dominated interactions with CD1d. The TCR ?-chain sat peripherally to the interface, with the CDR3? loop representing the principal determinant for ?-GalCer specificity. Accordingly, we provide insight into how a ?? TCR binds specifically to a lipid-loaded antigen-presenting molecule.
Natural killer T cell antigen receptors (NKT TCRs) recognize lipid-based antigens (Ags) presented by CD1d. Although the TCR ?-chain is invariant, NKT TCR V? exhibits greater diversity, with one (V?11) and three (V?8, V?7, and V?2) V? chains in humans and mice, respectively. With the exception of the V?2 NKT TCR, NKT TCRs possess canonical tyrosine residues within complementarity determining region (CDR) 2? that are critical for CD1d binding. Thus, how V?2 NKT TCR docks with CD1d-Ag was unclear. Despite the absence of the CDR2?-encoded tyrosine residues, we show that the V?2 NKT TCR engaged CD1d-Ag in a similar manner and with a comparable affinity and energetic footprint to the manner observed for the V?8.2 and V?7 NKT TCRs. Accordingly, the germline-encoded regions of the TCR ?-chain do not exclusively dictate the innate NKT TCR-CD1d-Ag docking mode. Nevertheless, clear fine specificity differences for the CD1d-Ag existed between the V?2 NKT TCR and the V?8.2 and V?7 NKT TCRs, with the V?2 NKT TCR exhibiting greater sensitivity to modifications to the glycolipid Ag. Furthermore, within the V?2 NKT TCR-CD1d-?GalCer complex, the CDR2? loop mediated fewer contacts with CD1d, whereas the CDR1? and CDR3? loops contacted CD1d to a much greater extent compared with most V?11, V?8.2, and V?7 NKT TCRs. Accordingly, there is a greater interplay between the germline- and nongermline-encoded loops within the TCR ?-chain of the V?2 NKT TCR that enables CD1d-Ag ligation.
The most potent foreign antigens for natural killer T cells (NKT cells) are ?-linked glycolipids, whereas NKT cell self-reactivity involves weaker recognition of structurally distinct ?-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 ?-linked agonists ?-galactosylceramide (?-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 ?-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 ?-linked self ligands to resemble the conformation of foreign ?-linked ligands, which shows that induced-fit molecular mimicry can underpin the self-reactivity of NKT cell TCRs to ?-linked antigens.
Type I natural killer T cells (NKT cells) are characterized by an invariant variable region 14-joining region 18 (V(?)14-J(?)18) T cell antigen receptor (TCR) ?-chain and recognition of the glycolipid ?-galactosylceramide (?-GalCer) restricted to the antigen-presenting molecule CD1d. Here we describe a population of ?-GalCer-reactive NKT cells that expressed a canonical V(?)10-J(?)50 TCR ?-chain, which showed a preference for ?-glucosylceramide (?-GlcCer) and bacterial ?-glucuronic acid-containing glycolipid antigens. Structurally, despite very limited TCR? sequence identity, the V(?)10 TCR-CD1d-?-GlcCer complex had a docking mode similar to that of type I TCR-CD1d-?-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.
Natural 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 t(1/2) 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.
IL-21 has antitumor activity through actions on NK cells and CD8(+) T cells, and is currently in clinical development for the treatment of cancer. However, no studies have addressed the role of endogenous IL-21 in tumor immunity. In this study, we have studied both primary and secondary immune responses in IL-21(-/-) and IL-21R(-/-) mice against several experimental tumors. We found intact immune surveillance toward methylcholanthrene-induced sarcomas in IL-21(-/-) and IL-21R(-/-) mice compared with wild-type mice and B16 melanomas showed equal growth kinetics and development of lung metastases. IL-21R(-/-) mice showed competent NK cell-mediated rejection of NKG2D ligand (Rae1beta) expressing H-2b(-) RMAS lymphomas and sustained transition to CD8(+) T cell-dependent memory against H-2b(+) RMA lymphomas. alpha-Galactosylceramide stimulation showed equal expansion and activation of NKT and NK cells and mounted a powerful antitumor response in the absence of IL-21 signaling, despite reduced expression of granzyme B in NKT, NK, and CD8(+) T cells. Surprisingly, host IL-21 significantly restricted the expansion of Ag-specific CD8(+) T cells and inhibited primary CD8(+) T cell immunity against OVA-expressing EG7 lymphomas, as well as the secondary expansion of memory CD8(+) T cells. However, host IL-21 did not alter the growth of less immunogenic MC38 colon carcinomas with dim OVA expression. Overall, our results show that endogenous IL-21/IL-21R is not required for NK, NKT, and CD8(+) T cell-mediated tumor immunity, but restricts Ag-specific CD8(+) T cell expansion and rejection of immunogenic tumors, indicating novel immunosuppressive actions of this cytokine.
Suppressor of cytokine signaling (SOCS)-1 is a critical inhibitor of IFN-gamma signal transduction in vivo, but the precise biochemical mechanism of action of SOCS-1 is unclear. Studies in vitro have shown that SOCS-1 binds to Jaks and inhibits their catalytic activity, but recent studies indicate SOCS-1 may act in a similar manner to SOCS-3 by firstly interacting with cytokine receptors and then inhibiting Jak activity. Here, we have generated mice, termed Ifngr1(441F), in which a putative SOCS-1 binding site, tyrosine 441 (Y441), on the IFN-gamma receptor subunit 1 (IFNGR1) is mutated. We confirm that SOCS-1 binds to IFNGR1 in wild-type but not mutant cells. Mutation of Y441 results in impaired negative regulation of IFN-gamma signaling. IFN-gamma-induced STAT1 activation is prolonged in Ifngr1(441F) cells, but not to the extent seen in cells completely lacking SOCS-1, suggesting that SOCS-1 maintains activity to modulate IFN-gamma signaling via other mechanisms. Despite this, we show that hypersensitivity to IFN-gamma results in enhanced innate tumor protection in Ifngr1(441F) mice in vivo, and unregulated expression of an IFN-gamma-dependent chemokine, monokine-induced by IFN-gamma. Collectively, these data indicate that Y441 contributes to the regulation of signaling through IFNGR1 via the recruitment of SOCS-1 to the receptor.
CD1d-restricted T cells are considered to play a host protective effect in tumor immunity, yet the evidence for a role of natural killer T (NKT) cells in tumor immune surveillance has been weak and data from several tumor models has suggested that some (type II) CD1d-restricted T cells may also suppress some types of antitumor immune response. To substantiate an important role for CD1d-restricted T cells in host response to cancer, we have evaluated tumor development in p53(+/-) mice lacking either type I NKT cells (TCR Jalpha18(-/-)) or all CD1d-restricted T cells (CD1d(-/-)). Our findings support a key role for type I NKT cells in suppressing the onset of sarcomas and hematopoietic cancers caused by p53 loss but do not suggest that other CD1d-restricted T cells are critical in regulating the same tumor development.
CD1d-dependent NKT cells represent a heterogeneous family of effector T cells including CD4(+)CD8(-) and CD4(-)CD8(-) subsets that respond to glycolipid Ags with rapid and potent cytokine production. NKT cell development is regulated by a unique combination of factors, however very little is known about factors that control the development of NKT subsets. In this study, we analyze a novel mouse strain (helpless) with a mis-sense mutation in the BTB-POZ domain of ZBTB7B and demonstrate that this mutation has dramatic, intrinsic effects on development of NKT cell subsets. Although NKT cell numbers are similar in Zbtb7b mutant mice, these cells are hyperproliferative and most lack CD4 and instead express CD8. Moreover, the majority of ZBTB7B mutant NKT cells in the thymus are retinoic acid-related orphan receptor ?t positive, and a high frequency produce IL-17 while very few produce IFN-? or other cytokines, sharply contrasting the profile of normal NKT cells. Mice heterozygous for the helpless mutation also have reduced numbers of CD4(+) NKT cells and increased production of IL-17 without an increase in CD8(+) cells, suggesting that ZBTB7B acts at multiple stages of NKT cell development. These results reveal ZBTB7B as a critical factor genetically predetermining the balance of effector subsets within the NKT cell population.
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 TCR? and TCR? 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.
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