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

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

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 TCRß 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.

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

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

Recognition of ß-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 ß-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 ß-linked agonists ß-galactosylceramide (ß-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 ß-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 ß-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 ß-linked antigens.

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

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

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

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

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

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

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

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

Evolutionarily conserved features contribute to αß T cell receptor specificity.

αß T cell receptors (TCRs) bind specifically to foreign antigens presented by major histocompatibility complex proteins (MHC) or MHC-like molecules. Accumulating evidence indicates that the germline-encoded TCR segments have features that promote binding to MHC and MHC-like molecules, suggesting coevolution between TCR and MHC molecules. Here, we assess directly the evolutionary conservation of αß TCR specificity for MHC. Sequence comparisons showed that some Vßs from distantly related jawed vertebrates share amino acids in their complementarity determining region 2 (CDR2). Chimeric TCRs containing amphibian, bony fish, or cartilaginous fish Vßs can recognize antigens presented by mouse MHC class II and CD1d (an MHC-like protein), and this recognition is dependent upon the shared CDR2 amino acids. These results indicate that features of the TCR that control specificity for MHC and MHC-like molecules were selected early in evolution and maintained between species that last shared a common ancestor more than 400 million years ago.

A molecular basis for NKT cell recognition of CD1d-self-antigen.

The 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 CDR3ß 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.

A molecular basis for the exquisite CD1d-restricted antigen specificity and functional responses of natural killer T cells.

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.

Class II major histocompatibility complex mutant mice to study the germ-line bias of T-cell antigen receptors.

The interaction of αß 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.

CD1b-mycolic acid tetramers demonstrate T-cell fine specificity for mycobacterial lipid tails.

Mycobacterium tuberculosis synthesizes a thick cell wall comprised of mycolic acids (MA), which are foreign antigens for human T cells. T-cell clones from multiple donors were used to determine the fine specificity of MA recognition by human αß T cells. Most CD1-presented lipid antigens contain large hydrophilic head groups comprised of carbohydrates or peptides that dominate patterns of T-cell specificity. MA diverges from the consensus antigen motif in that it lacks a head group. Using multiple forms of natural and synthetic MA and MA-specific T-cells with different T-cell receptors, we found that, unlike antigens with larger head groups, lipid length strongly controlled T-cell responses to MA. In addition, the three forms of MA that naturally occur in M. tuberculosis that differ in modifications on the lipid tail, differ in their potency for activating MA-specific T-cell clones. Thus, naturally occurring MA forms should be considered as separate, partly cross-reactive antigens. Two of the three forms of MA could be loaded onto human CD1b proteins, creating working CD1b-MA tetramers. The creation of CD1b-MA tetramers represents a new tool for future studies that track the effector functions and kinetics of MA-specific T-cells ex vivo.

T cell receptor CDR2 beta and CDR3 beta 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.

Differential recognition of CD1d-alpha-galactosyl ceramide by the V beta 8.2 and V beta 7 semi-invariant NKT T cell receptors.

The semi-invariant natural killer T cell receptor (NKT TCR) recognizes CD1d-lipid antigens. Although the TCR alpha chain is typically invariant, the beta chain expression is more diverse, where three V beta chains are commonly expressed in mice. We report the structures of V alpha 14-V beta 8.2 and V alpha 14-V beta 7 NKT TCRs in complex with CD1d-alpha-galactosylceramide (alpha-GalCer) and the 2.5 A structure of the human NKT TCR-CD1d-alpha-GalCer complex. Both V beta 8.2 and V beta 7 NKT TCRs and the human NKT TCR ligated CD1d-alpha-GalCer in a similar manner, highlighting the evolutionarily conserved interaction. However, differences within the V beta domains of the V beta 8.2 and V beta 7 NKT TCR-CD1d complexes resulted in altered TCR beta-CD1d-mediated contacts and modulated recognition mediated by the invariant alpha chain. Mutagenesis studies revealed the differing contributions of V beta 8.2 and V beta 7 residues within the CDR2 beta loop in mediating contacts with CD1d. Collectively we provide a structural basis for the differential NKT TCR V beta usage in NKT cells.

It is time to beelieve the CD1a hype!

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.

IL-27 is required for shaping the magnitude, affinity distribution, and memory of T cells responding to subunit immunization.

An elusive goal of cellular immune vaccines is the generation of large numbers of antigen-specific T cells in response to subunit immunization. A broad spectrum of cytokines and cell-surface costimulatory molecules are known to shape the programming, magnitude, and repertoire of T cells responding to vaccination. We show here that the majority of innate immune receptor agonist-based vaccine adjuvants unexpectedly depend on IL-27 for eliciting CD4(+) and CD8(+) T-cell responses. This is in sharp contrast to infectious challenge, which generates T-cell responses that are IL-27-independent. Mixed bone marrow chimera experiments demonstrate that IL-27 dependency is T cell-intrinsic, requiring T-cell expression of IL-27Rα. Further, we show that IL-27 dependency not only dictates the magnitude of vaccine-elicited T-cell responses but also is critical for the programming and persistence of high-affinity T cells to subunit immunization. Collectively, our data highlight the unexpected central importance of IL-27 in the generation of robust, high-affinity cellular immune responses to subunit immunization.

Effective functional maturation of invariant natural killer T cells is constrained by negative selection and T-cell antigen receptor affinity.

The self-reactivity of their T-cell antigen receptor (TCR) is thought to contribute to the development of immune regulatory cells, such as invariant NK T cells (iNKT). In the mouse, iNKT cells express TCRs composed of a unique Vα14-Jα18 rearrangement and recognize lipid antigens presented by CD1d molecules. We created mice expressing a transgenic TCR-ß chain that confers high affinity for self-lipid/CD1d complexes when randomly paired with the mouse iNKT Vα14-Jα18 rearrangement to study their development. We show that although iNKT cells undergo agonist selection, their development is also shaped by negative selection in vivo. In addition, iNKT cells that avoid negative selection in these mice express natural sequence variants of the canonical TCR-α and decreased affinity for self/CD1d. However, limiting the affinity of the iNKT TCRs for "self" leads to inefficient Egr2 induction, poor expression of the iNKT lineage-specific zinc-finger transcription factor PLZF, inadequate proliferation of iNKT cell precursors, defects in trafficking, and impaired effector functions. Thus, proper development of fully functional iNKT cells is constrained by a limited range of TCR affinity that plays a key role in triggering the iNKT cell-differentiation pathway. These results provide a direct link between the affinity of the TCR expressed by T-cell precursors for self-antigens and the proper development of a unique population of lymphocytes essential to immune responses.