Thierry Mallevaey

Recognition of [beta]-linked self glycolipids mediated by natural killer T cell antigen receptors

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.

Recognition of CD1d-sulfatide mediated by a type II natural killer T cell antigen receptor

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.

T Cell Receptor CDR2β and CDR3β Loops Collaborate Functionally to Shape the iNKT Cell Repertoire

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 and Noninvariant Natural Killer T Cells Exert Opposite Regulatory Functions on the Immune Response during Murine Schistosomiasis

ABSTRACT CD1d-restricted natural killer T (NKT) cells represent a heterogeneous population of innate memory immune cells expressing both NK and T-cell markers distributed into two major subsets, i.e., invariant NKT (iNKT) cells, which express exclusively an invariant T-cell receptor (TCR) α chain (Vα14Jα18 in mice), and non-iNKT cells, which express more diverse TCRs. NKT cells quickly produce Th1- and/or Th2-type cytokines following stimulation with glycolipid antigen (Ag) and, through this property, play potent immunoregulatory roles in autoimmune diseases, cancer, and infection. No study has addressed the role of NKT cells in metazoan parasite infections so far. We show that during murine schistosomiasis, the apparent frequency of both iNKT cells and non-iNKT cells decreased in the spleen as early as 3 weeks postinfection (p.i.) and that both populations expressed a greater amount of the activation marker CD69 at 6 weeks p.i., suggesting an activated phenotype. Two different NKT-cell-deficient mouse models, namely, TCR Jα18−/− (exclusively deficient in iNKT cells) and CD1d−/− (deficient in both iNKT and non-iNKT cells) mice, were used to explore the implication of these subsets in infection. We show that whereas both iNKT and non-iNKT cells do not have a major impact on the immune response during the early phase (1 and 4 weeks) of infection, they exert important, although opposite, effects on the immune response during the acute phase of the disease (7 and 12 weeks), after schistosome egg production. Indeed, iNKT cells contribute to Th1 cell differentiation whereas non-iNKT cells might be mostly implicated in Th2 cell differentiation in response to parasite Ag. Our findings suggest, for the first time, that helminths activate both iNKT and non-iNKT cells in vivo, enabling them to differentially influence the Th1/Th2 balance of the immune response.

Vβ2 natural killer T cell antigen receptor-mediated recognition of CD1d-glycolipid antigen

Natural killer T cell antigen receptors (NKT TCRs) recognize lipid-based antigens (Ags) presented by CD1d. Although the TCR α-chain is invariant, NKT TCR Vβ exhibits greater diversity, with one (Vβ11) and three (Vβ8, Vβ7, and Vβ2) Vβ chains in humans and mice, respectively. With the exception of the Vβ2 NKT TCR, NKT TCRs possess canonical tyrosine residues within complementarity determining region (CDR) 2β that are critical for CD1d binding. Thus, how Vβ2 NKT TCR docks with CD1d-Ag was unclear. Despite the absence of the CDR2β-encoded tyrosine residues, we show that the Vβ2 NKT TCR engaged CD1d-Ag in a similar manner and with a comparable affinity and energetic footprint to the manner observed for the Vβ8.2 and Vβ7 NKT TCRs. Accordingly, the germline–encoded regions of the TCR β-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 Vβ2 NKT TCR and the Vβ8.2 and Vβ7 NKT TCRs, with the Vβ2 NKT TCR exhibiting greater sensitivity to modifications to the glycolipid Ag. Furthermore, within the Vβ2 NKT TCR-CD1d-αGalCer complex, the CDR2β loop mediated fewer contacts with CD1d, whereas the CDR1β and CDR3β loops contacted CD1d to a much greater extent compared with most Vβ11, Vβ8.2, and Vβ7 NKT TCRs. Accordingly, there is a greater interplay between the germline– and nongermline–encoded loops within the TCR β-chain of the Vβ2 NKT TCR that enables CD1d-Ag ligation.

CD3 bright signals on γδ T cells identify IL-17A-producing Vγ6Vδ1 + T cells

Interleukin‐17A (IL‐17A) is a pro‐inflammatory cytokine that has an important role at mucosal sites in a wide range of immune responses including infection, allergy and auto‐immunity. γδ T cells are recognized as IL‐17 producers, but based on the level of CD3 expression, we now define the remarkable ability of a CD3bright γδ T‐cell subset with an effector memory phenotype to rapidly produce IL‐17A, but not interferon‐γ. CD3bright γδ T cells uniformly express the canonical germline encoded Vγ6/Vδ1+ T‐cell receptor. They are widely distributed with a preferential representation in the lungs and skin are negatively impacted in the absence of retinoic acid receptor‐related orphan receptor gammat expression or endogenous flora. This population responded rapidly to various stimuli in a mechanism involving IL‐23 and NOD‐like receptor family, pyrin domain containing 3 (NLRP3)‐inflammasome‐dependent IL‐1β. Finally, we demonstrated that IL‐17‐producing CD3bright γδ T cells responded promptly and strongly to pneumococcal infection and during skin inflammation. Here, we propose a new way to specifically analyze IL‐17‐producing Vγ6/Vδ1+ T cells based on the level of CD3 signals. Using this gating strategy, our data reinforce the crucial role of this γδ T‐cell subset in respiratory and skin disorders.

MAIT Cell Recognition of MR1 on Bacterially Infected and Uninfected Cells

Mucosal-associated invariant T cells are a unique population of T cells that express a semi-invariant αβ TCR and are restricted by the MHC class I-related molecule MR1. MAIT cells recognize uncharacterized ligand(s) presented by MR1 through the cognate interaction between their TCR and MR1. To understand how the MAIT TCR recognizes MR1 at the surface of APCs cultured both with and without bacteria, we undertook extensive mutational analysis of both the MAIT TCR and MR1 molecule. We found differential contribution of particular amino acids to the MAIT TCR-MR1 interaction based upon the presence of bacteria, supporting the hypothesis that the structure of the MR1 molecules with the microbial-derived ligand(s) differs from the one with the endogenous ligand(s). Furthermore, we demonstrate that microbial-derived ligand(s) is resistant to proteinase K digestion and does not extract with common lipids, suggesting an unexpected class of antigen(s) might be recognized by this unique lymphocyte population.

Role of Invariant NK T Lymphocytes in Immune Responses to CpG Oligodeoxynucleotides

Unmethylated CpG oligodeoxynucleotides (ODNs), by activating cells of the innate immune system, such as dendritic cells and NK cells, are potent adjuvants for type 1 immune responses. In the present study, we aimed to investigate the role of invariant NKT (iNKT) cells, a subset of lipid-reactive innate lymphocytes, in CpG ODN-induced innate and acquired type 1 responses. Our data show that, in response to the CpG ODN type B 1826, splenic and hepatic iNKT cells become activated and produce IFN-γ, but not IL-4, both in vitro and in vivo. This Th1 bias is independent from the Ag-presenting molecule CD1d and strongly requires IL-12, at least in vitro. We also report that iNKT cell activation, in response to CpG ODN type B, results in the transactivation of NK cells. To address the potential role of iNKT cells in type 1 innate immunity induced by CpG ODN, a murine model of malignant melanoma was used. We show that CpG ODN type B protects mice against B16F10-induced lung metastasis in wild-type mice, but in a less efficient manner in iNKT cell-deficient animals. Finally, we report that immunization of wild-type mice with CpG ODN type B plus keyhole limpet hemocyanin biases the immune response toward a Th1 direction, an effect strongly mediated by iNKT cells. We conclude that iNKT cells amplify the innate and acquired response to CpG ODN type B, with potentially important consequences for the regulation of immune responses.

Nod1 and Nod2 Enhance TLR-Mediated Invariant NKT Cell Activation during Bacterial Infection

Invariant NKT (iNKT) cells act at the crossroad between innate and adaptive immunity and are important players in the defense against microbial pathogens. iNKT cells can detect pathogens that trigger innate receptors (e.g., TLRs, Rig-I, Dectin-1) within APCs, with the consequential induction of CD1d-mediated Ag presentation and release of proinflammatory cytokines. We show that the cytosolic peptidoglycan-sensing receptors Nod1 and Nod2 are necessary for optimal IFN-γ production by iNKT cells, as well as NK cells. In the absence of Nod1 and Nod2, iNKT cells had a blunted IFN-γ response following infection by Salmonella enterica serovar Typhimurium and Listeria monocytogenes. For Gram-negative bacteria, we reveal a synergy between Nod1/2 and TLR4 in dendritic cells that potentiates IL-12 production and, ultimately, activates iNKT cells. These findings suggest that multiple innate pathways can cooperate to regulate iNKT cell activation during bacterial infection.

Strategy of lipid recognition by invariant natural killer T cells: ‘one for all and all for one’

Invariant natural killer T (iNKT) cells are evolutionarily conserved lipid‐reactive T cells that bridge innate and adaptive immune responses. Despite a relatively restricted T‐cell receptor (TCR) diversity, these cells respond to a variety of structurally distinct foreign (i.e. microbial or synthetic) as well as host‐derived (self‐) lipid antigens presented by the CD1d molecule. These multi‐tasking lymphocytes are among the first responders in immunity, and produce an impressive array of cytokines and chemokines that can tailor the ensuing immune response. Accordingly, iNKT cells play important functions in autoimmune diseases, cancer, infection and inflammation. These properties make iNKT cells appealing targets in immune‐based therapies. Yet, much has to be learned on the mechanisms that allow iNKT cells to produce polarized responses. Responses of iNKT cells are influenced by the direct signals perceived by the cells through their TCRs, as well as by indirect co‐stimulatory (and potentially co‐inhibitory) cues that they receive from antigen‐presenting cells or the local milieu. A decade ago, biochemists and immunologists have started to describe synthetic lipid agonists with cytokine skewing potential, paving a new research avenue in the iNKT cell field. Yet how iNKT cells translate various antigenic signals into distinct functional responses has remained obscure. Recent findings have revealed a unique and innate mode of lipid recognition by iNKT cells, and suggest that both the lipid antigen presented and the diversity of the TCR modulate the strength of CD1d‐iNKT TCR interactions. In this review, we focus on novel discoveries on lipid recognition by iNKT cells, and how these findings may help us to design effective strategies to steer iNKT cell responses for immune intervention.