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Bacterial superantigens spur IgA secretion: Mucosal IgA is abundant and interacts with the gut microbiome. To examine microbial induction of IgA in humans, Bunker et al. screened microbiota from infants against mouse and human IgA. Unexpectedly, they saw a subset of samples bound IgA in a way that indicated the presence of superantigens, which bind T cell receptors or B cell receptors outside of the typical antigen-binding region, leading to nonspecific activation. Putative superantigens were identified in commensal members of Lachnospiraceae, which activated human VH3-positive B cells and induced IgA production in mice. These data identify commensal superantigens and suggest that they may be dominant forces behind IgA production in humans. IgA is prominently secreted at mucosal surfaces and coats a fraction of the commensal microbiota, a process that is critical for intestinal homeostasis. However, the mechanisms of IgA induction and the molecular targets of these antibodies remain poorly understood, particularly in humans. Here, we demonstrate that microbiota from a subset of human individuals encode two protein "superantigens" expressed on the surface of commensal bacteria of the family Lachnospiraceae such as Ruminococcus gnavus that bind IgA variable regions and stimulate potent IgA responses in mice. These superantigens stimulate B cells expressing human VH3 or murine VH5/6/7 variable regions and subsequently bind their antibodies, allowing these microbial organisms to become highly coated with IgA in vivo. These findings demonstrate a previously unappreciated role for commensal superantigens in host-microbiota interactions. Furthermore, as superantigen-expressing strains show an uneven distribution across human populations, they should be systematically considered in studies evaluating human B cell responses and microbiota during homeostasis and disease. [ABSTRACT FROM AUTHOR]

The molecular requirements for invariant Vα14-bearing natural killer T cells (iNKT) in the thymus are poorly understood. A minute population of ≈500 newly selected CD69+CD24+ stage 0 (ST0) iNKT cells gives rise to ≈100 times more CD44ne9/loCD24- stage 1 (ST1). cells, which then generate similar frequencies of CD44hiCD24- stage 2 (ST2) and mature iNKT cells. Although the increased number of 511 compared with STO cells indicates the initiation of a proliferation wave in the very early stages of iNKT cell development, details about the controlling mechanism are currently lacking. Here, we show that the transcription factor c-Myc is required for iNKT cell development. Conditional ablation of c-Myc in double-positive thymocytes specifically impacted iNKT but not conventional T cell development. Within the iNKT population, a progressive reduction of iNKT cells was observed starting at ST1 (≈50-fold) and ST2 (≈350-fold), with a complete lack of mature cells in thymus, spleen, and liver. ST0/ST1 c-Myc-deficient iNKT cells showed reduced proliferation. In contrast, annexin V staining did not reveal increased apoptosis, and transgenic overexpression of BCL-2 did not rescue iNKT cell development in c-Myc-deficient mice. Moreover, expression of known iNKT differentiation factors such as Plzf and Gata3 was not dramatically altered. These; findings provide compelling evidence that c-Myc mediates an intrathymic proliferation wave immediately after agonist selection of iNKT cells and illustrate the importance of this expansion for the generation of mature iNKT cells in vivo. [ABSTRACT FROM AUTHOR]

CD1d antigen presentation to natural killer T (NKT) cells expressing the semi-invariant T cell receptor V[sub α]14J[sub α]18 requires CD1d trafficking through endosomal compartments; however, the endosomal events remain undefined. We show that mice lacking the endosomal protease cathepsin L (catL) have greatly reduced numbers of V[sub α]14[sup +]NK1.1[sup +] T cells. In addition, catL expression in thymocytes is critical not only for selection of these cells in vivo but also for stimulation of V[sub α]14[sup +]NK1.1[sup +]T cells in vitro. CD1d cell-surface expression and intracellular localization appear normal in catL-deficient thymocytes, as does the lysosomal morphology; this implies a specific role for catL in regulating presentation of natural CD1d ligands mediating V[sub α]14[sup +]NK1.1[sup +]T cell selection. These data implicate lysosomal proteases as key regulators of not only classical major histocompatibility complex class II antigen presentation but also nonclassical CD1d presentation. [ABSTRACT FROM AUTHOR]

For some time, T cell receptor αβ (TCRαβ) signaling during thymocyte development was thought to induce one of three fates—negative selection to remove autoreactive cells, positive selection to enrich for host major histocompatibility complex (MHC)-restricted cells, and, in the absence of interactions with host MHC-peptides, death by neglect in experimental systems, negative selection is indeed found when thymocytes are presented with sufficient concentrations of their agonist antigen, that is, the same antigen that would activate them as mature T cells. In contrast, partial signaling mediated by variant peptides supports the positive selection of thymocytes, a step necessary for their lineage maturation into CD4 and CD8 T cells and exit from the thymus.

Abstract: The semi-invariant Vα14Jα18 T cell receptor (TCR) is expressed by regulatory NKT cells and has the unique ability to recognize chemically diverse ligands presented by CD1d. The crystal structure of CD1d complexed to a natural, endogenous ligand, isoglobotrihexosylceramide (iGb3), illustrates the extent of this diversity when compared to the binding of potent, exogenous ligands, such as α-galactosylceramide (α-GalCer). A single mode of recognition for these two classes of ligands would then appear problematic for a single T cell receptor. However, the Vα14 TCR adopts two different conformations in the crystal where, in one configuration, the presence of a larger cavity between the two CDR3 regions could accommodate iGb3 and, in the other, a smaller cavity fits α-GalCer more snugly. Alternatively, the extended iGb3 headgroup could be “squashed” upon docking of the TCR and accommodated between the CD1 and TCR surfaces. Thus, the same TCR may adopt alternative modes of recognition for these foreign and self-ligands for NKT cell activation. [Copyright &y& Elsevier]

Summary The precise impact of thymic positive and negative selection on the T cell receptor (TCR) repertoire remains controversial. Here, we used unbiased, high-throughput cloning and retroviral expression of individual pre-selection TCRs to provide a direct assessment of these processes at the clonal level in vivo. We found that 15% of random TCRs induced signaling and directed positive (7.5%) or negative (7.5%) selection, depending on strength of signal, whereas the remaining 85% failed to induce signaling or selection. Most negatively selected TCRs exhibited promiscuous crossreactivity toward multiple other major histocompatibility complex (MHC) haplotypes. In contrast, TCRs that were positively selected or non-selected were minimally crossreactive. Negative selection of crossreactive TCRs led to clonal deletion but also recycling into intestinal CD4 − CD8β − intraepithelial lymphocytes (iIELs). Thus, broadly crossreactive TCRs arise at low frequency in the pre-selection repertoire but constitute the primary drivers of thymic negative selection and iIEL lineage differentiation. [ABSTRACT FROM AUTHOR]

The developmental pathway of intestinal TCRαβ+CD4- CD8β- intraepithelial lymphocytes (unconventional iIELs) has remained elusive. Bendelac and colleagues demonstrate that expression of unconventional iIEL TCRs in thymocytes causes negative selection and that unconventional iIELs are derived from rare escapees of cortical negative selection. [ABSTRACT FROM AUTHOR]

Summary: The nature of the antigens recognized by γδ T cells and their potential recognition of major histocompatibility complex (MHC)-like molecules has remained unclear. Members of the CD1 family of lipid-presenting molecules are suggested ligands for Vδ1 TCR-expressing γδ T cells, the major γδ lymphocyte population in epithelial tissues. We crystallized a Vδ1 TCR in complex with CD1d and the self-lipid sulfatide, revealing the unusual recognition of CD1d by germline Vδ1 residues spanning all complementarity-determining region (CDR) loops, as well as sulfatide recognition separately encoded by nongermline CDR3δ residues. Binding and functional analysis showed that CD1d presenting self-lipids, including sulfatide, was widely recognized by gut Vδ1+ γδ T cells. These findings provide structural demonstration of MHC-like recognition of a self-lipid by γδ T cells and reveal the prevalence of lipid recognition by innate-like T cell populations. [Copyright &y& Elsevier]

Summary: Commitment to the T and natural killer T (NKT) cell lineages is determined during αβ T cell receptor (TCR)-mediated interactions of common precursors with ligand-expressing cells in the thymus. Whereas mainstream thymocyte precursors recognize major histocompatibility complex (MHC) ligands expressed by stromal cells, NKT cell precursors interact with CD1d ligands expressed by cortical thymocytes. Here, we demonstrated that such homotypic T-T interactions generated “second signals” mediated by the cooperative engagement of the homophilic receptors Slamf1 (SLAM) and Slamf6 (Ly108) and the downstream recruitment of the adaptor SLAM-associated protein (SAP) and the Src kinase Fyn, which are essential for the lineage expansion and differentiation of the NKT cell lineage. These receptor interactions were required during TCR engagement and therefore only occurred when selecting ligands were presented by thymocytes rather than epithelial cells, which do not express Slamf6 or Slamf1. Thus, the topography of NKT cell ligand recognition determines the availability of a cosignaling pathway that is essential for NKT cell lineage development. [Copyright &y& Elsevier]

Abstract: CD1d presentation of α-galactosyl ceramides to natural killer T cells has been a focal point of the study of regulatory T cells. KRN7000, an α-galactosyl ceramide originally generated from structure activity studies of antitumor properties of marine sponge glycolipids, is currently the most commonly used agonist ligand and is used to stain NKT cells. However, this glycolipid suffers from poor solubility and availability. We have developed an α-galactosyl ceramide with improved solubility over KRN7000 that effectively stains NKT cells, both mouse and human, and stimulates cytokine release at low concentrations. [Copyright &y& Elsevier]

NKT cells represent a distinct lineage of T cells that coexpress a conserved αβ T cell receptor (TCR) and natural killer (NK) receptors. Although the TCR of NKT cells is characteristically autoreactive to CD1d, a lipid-presenting molecule, endogenous ligands for these cells have not been identified. We show that a lysosomal glycosphingolipid of previously unknown function, isoglobotrihexosylceramide (iGb3), is recognized both by mouse and human NKT cells. Impaired generation of lysosomal iGb3 in mice lacking β-hexosaminidase b results in severe NKT cell deficiency, suggesting that this lipid also mediates development of NKT cells in the mouse. We suggest that expression of iGb3 in peripheral tissues may be involved in controlling NKT cell responses to infections and malignancy and in autoimmunity. [ABSTRACT FROM AUTHOR]