Direct recognition of an intact foreign protein by an αß T cell receptor.

αß T cell receptors (αßTCRs) co-recognise antigens when bound to Major Histocompatibility Complex (MHC) or MHC class I-like molecules. Additionally, some αßTCRs can bind non-MHC molecules, but how much intact antigen reactivities are achieved remains unknown. Here, we identify an αß T cell clone that directly recognises the intact foreign protein, R-phycoerythrin (PE), a multimeric (αß)6γ protein complex. This direct αßTCR-PE interaction occurs in an MHC-independent manner, yet triggers T cell activation and bound PE with an affinity comparable to αßTCR-peptide-MHC interactions. The crystal structure reveals how six αßTCR molecules simultaneously engage the PE hexamer, mediated by the complementarity-determining regions (CDRs) of the αßTCR. Here, the αßTCR mainly binds to two α-helices of the globin fold in the PE α-subunit, which is analogous to the antigen-binding platform of the MHC molecule. Using retrogenic mice expressing this TCR, we show that it supports intrathymic T cell development, maturation, and exit into the periphery as mature CD4/CD8 double negative (DN) T cells with TCR-mediated functional capacity. Accordingly, we show how an αßTCR can recognise an intact foreign protein in an antibody-like manner.

Lipidomic scanning of self-lipids identifies headless antigens for natural killer T cells.

To broadly measure the spectrum of cellular self-antigens for natural killer T cells (NKT), we developed a sensitive lipidomics system to analyze lipids trapped between CD1d and NKT T cell receptors (TCRs). We captured diverse antigen complexes formed in cells from natural endogenous lipids, with or without inducing endoplasmic reticulum (ER) stress. After separating protein complexes with no, low, or high CD1d-TCR interaction, we eluted lipids to establish the spectrum of self-lipids that facilitate this interaction. Although this unbiased approach identified fifteen molecules, they clustered into only two related groups: previously known phospholipid antigens and unexpected neutral lipid antigens. Mass spectrometry studies identified the neutral lipids as ceramides, deoxyceramides, and diacylglycerols, which can be considered headless lipids because they lack polar headgroups that usually form the TCR epitope. The crystal structure of the TCR-ceramide-CD1d complex showed how the missing headgroup allowed the TCR to predominantly contact CD1d, supporting a model of CD1d autoreactivity. Ceramide and related headless antigens mediated physiological TCR binding affinity, weak NKT cell responses, and tetramer binding to polyclonal human and mouse NKT cells. Ceramide and sphingomyelin are oppositely regulated components of the "sphingomyelin cycle" that are altered during apoptosis, transformation, and ER stress. Thus, the unique molecular link of ceramide to NKT cell response, along with the recent identification of sphingomyelin blockers of NKT cell activation, provide two mutually reinforcing links for NKT cell response to sterile cellular stress conditions.

History of tuberculosis disease is associated with genetic regulatory variation in Peruvians.

A quarter of humanity is estimated to have been exposed to Mycobacterium tuberculosis (Mtb) with a 5-10% risk of developing tuberculosis (TB) disease. Variability in responses to Mtb infection could be due to host or pathogen heterogeneity. Here, we focused on host genetic variation in a Peruvian population and its associations with gene regulation in monocyte-derived macrophages and dendritic cells (DCs). We recruited former household contacts of TB patients who previously progressed to TB (cases, n = 63) or did not progress to TB (controls, n = 63). Transcriptomic profiling of monocyte-derived DCs and macrophages measured the impact of genetic variants on gene expression by identifying expression quantitative trait loci (eQTL). We identified 330 and 257 eQTL genes in DCs and macrophages (False Discovery Rate (FDR) < 0.05), respectively. Four genes in DCs showed interaction between eQTL variants and TB progression status. The top eQTL interaction for a protein-coding gene was with FAH, the gene encoding fumarylacetoacetate hydrolase, which mediates the last step in mammalian tyrosine catabolism. FAH expression was associated with genetic regulatory variation in cases but not controls. Using public transcriptomic and epigenomic data of Mtb-infected monocyte-derived dendritic cells, we found that Mtb infection results in FAH downregulation and DNA methylation changes in the locus. Overall, this study demonstrates effects of genetic variation on gene expression levels that are dependent on history of infectious disease and highlights a candidate pathogenic mechanism through pathogen-response genes. Furthermore, our results point to tyrosine metabolism and related candidate TB progression pathways for further investigation.

Human Skin T Cells Express Conserved T-Cell Receptors that Cross-React with Staphylococcal Superantigens and CD1a.

Human Langerhans cells highly express CD1a antigen-presenting molecules. To understand the functions of CD1a in human skin, we used CD1a tetramers to capture T cells and determine their effector functions and TCR patterns. Skin T cells from all donors showed CD1a tetramer staining, which in three cases exceeded 10% of skin T cells. CD1a tetramer-positive T cells produced diverse cytokines, including IL-2, IL-4, IL-5, IL-9, IL-17, IL-22, and IFN-γ. Conserved TCRs often recognize nonpolymorphic antigen-presenting molecules, but no TCR motifs are known for CD1a. We detected highly conserved TCRs that used TRAV34 and TRBV28 variable genes, which is a known motif for recognition of staphylococcal enterotoxin B, a superantigen associated with atopic dermatitis. We found that these conserved TCRs did not respond to superantigen presented by CD1a, but instead showed a cross-reactive response with two targets: CD1a and staphylococcal enterotoxin B presented by classical major histocompatibility complex II. These studies identify a conserved human TCR motif for CD1a-reactive T cells. Furthermore, the demonstrated cross-reaction of T cells with two common skin-specific stimuli suggests a candidate mechanism by which CD1a and skin flora could synergize during natural immune response and in Staphylococcus-associated skin diseases.

Do antigen-presenting CD1a, CD1b, CD1c, and CD1d molecules bind different self-lipids?

Humans express four different lipid antigen-presenting molecules, CD1a, CD1b, CD1c, and CD1d, that are differentially expressed on antigen-presenting cells and which recycle through different endosomal compartments. Huang et al. now answer the question on whether the four CD1 isoforms selectively bind certain lipids.

History of tuberculosis disease is associated with genetic regulatory variation in Peruvians.

A quarter of humanity is estimated to be latently infected with Mycobacterium tuberculosis (Mtb) with a 5-10% risk of developing tuberculosis (TB) disease. Variability in responses to Mtb infection could be due to host or pathogen heterogeneity. Here, we focused on host genetic variation in a Peruvian population and its associations with gene regulation in monocyte-derived macrophages and dendritic cells (DCs). We recruited former household contacts of TB patients who previously progressed to TB (cases, n=63) or did not progress to TB (controls, n=63). Transcriptomic profiling of monocyte-derived dendritic cells (DCs) and macrophages measured the impact of genetic variants on gene expression by identifying expression quantitative trait loci (eQTL). We identified 330 and 257 eQTL genes in DCs and macrophages (False Discovery Rate (FDR) < 0.05), respectively. Five genes in DCs showed interaction between eQTL variants and TB progression status. The top eQTL interaction for a protein-coding gene was with FAH, the gene encoding fumarylacetoacetate hydrolase, which mediates the last step in mammalian tyrosine catabolism. FAH expression was associated with genetic regulatory variation in cases but not controls. Using public transcriptomic and epigenomic data of Mtb-infected monocyte-derived dendritic cells, we found that Mtb infection results in FAH downregulation and DNA methylation changes in the locus. Overall, this study demonstrates effects of genetic variation on gene expression levels that are dependent on history of infectious disease and highlights a candidate pathogenic mechanism through pathogen-response genes. Furthermore, our results point to tyrosine metabolism and related candidate TB progression pathways for further investigation.

Establishment of CD1b-restricted immunity to lipid antigens in the pulmonary response to Mycobacterium tuberculosis infection.

CD1 is an antigen presenting glycoprotein homologous to MHC I; however, CD1 proteins present lipid rather than peptide antigen. CD1 proteins are well established to present lipid antigens of Mycobacterium tuberculosis (Mtb) to T cells, but understanding the role of CD1-restricted immunity in vivo in response to Mtb infection has been limited by availability of animal models naturally expressing the CD1 proteins implicated in human response: CD1a, CD1b and CD1c. Guinea pigs, in contrast to other rodent models, express four CD1b orthologs, and here we utilize the guinea pig to establish the kinetics of gene and protein expression of CD1b orthologs, as well as the Mtb lipid-antigen and CD1b-restricted immune response at the tissue level over the course of Mtb infection. Our results indicate transient upregulation of CD1b expression during the effector phase of adaptive immunity that wanes with disease chronicity. Gene expression indicates that upregulation of CD1b is the result of transcriptional induction across all CD1b orthologs. We show high CD1b3 expression on B cells, and identify CD1b3 as the predominant CD1b ortholog in pulmonary granuloma lesions. We identify ex vivo cytotoxic activity directed against CD1b that closely paralleled the kinetic changes in CD1b expression in Mtb infected lung and spleen. This study confirms that CD1b expression is modulated by Mtb infection in lung and spleen, leading to pulmonary and extrapulmonary CD1b-restricted immunity as a component of the antigen-specific response to Mtb infection.

CD1 displays its own negative regulators.

After two decades of the study of lipid antigens that activate CD1-restricted T cells, new studies show how autoreactive αß T-cell receptors (TCRs) can directly recognize the outer surface of CD1 proteins in ways that are lipid-agnostic. Most recently, this lipid agnosticism has turned to negativity, with the discovery of natural CD1 ligands that dominantly negatively block autoreactive αß TCR binding to CD1a and CD1d. This review highlights the basic differences between positive and negative regulation of cellular systems. We outline strategies to discover lipid inhibitors of CD1-reactive T cells, whose roles in vivo are becoming clear, especially in CD1-mediated skin disease.

αß T-cell receptor recognition of self-phosphatidylinositol presented by CD1b.

CD1 glycoproteins present lipid-based antigens to T-cell receptors (TCRs). A role for CD1b in T-cell-mediated autoreactivity was proposed when it was established that CD1b can present self-phospholipids with short alkyl chains (∼C34) to T cells; however, the structural characteristics of this presentation and recognition are unclear. Here, we report the 1.9 Å resolution binary crystal structure of CD1b presenting a self-phosphatidylinositol-C34:1 and an endogenous scaffold lipid. Moreover, we also determined the 2.4 Å structure of CD1b-phosphatidylinositol complexed to an autoreactive αß TCR, BC8B. We show that the TCR docks above CD1b and directly contacts the presented antigen, selecting for both the phosphoinositol headgroup and glycerol neck region via antigen remodeling within CD1b and allowing lateral escape of the inositol moiety through a channel formed by the TCR α-chain. Furthermore, through alanine scanning mutagenesis and surface plasmon resonance, we identified key CD1b residues mediating this interaction, with Glu-80 abolishing TCR binding. We in addition define a role for both CD1b α1 and CD1b α2 molecular domains in modulating this interaction. These findings suggest that the BC8B TCR contacts both the presented phospholipid and the endogenous scaffold lipid via a dual mechanism of corecognition. Taken together, these data expand our understanding into the molecular mechanisms of CD1b-mediated T-cell autoreactivity.

Cell-Mediated Cytotoxicity in Lyme Arthritis.

OBJECTIVE: Obliterative microvascular lesions are found in the synovial tissue of ~50% of patients with post-antibiotic Lyme arthritis (LA) and correlate with autoantibodies to certain vascular antigens. In this study, we identified lymphocytes with cytotoxic potential that may also mediate this feature of synovial pathology. METHODS: The cytotoxic potential of lymphocytes and their T cell receptor (TCR) Vß gene usage were determined using samples of peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) from patients with antibiotic-responsive or post-antibiotic LA. Cell phenotypes were analyzed using flow cytometry and intracellular cytokine staining. Immunohistochemistry was performed on post-antibiotic synovial tissue samples. RESULTS: In SFMC and PBMC samples, the percentages of CD8+ T cells and double-negative T cells (primarily γδ T cells) were greater among 22 patients with post-antibiotic LA than in 14 patients with antibiotic-responsive LA. Moreover, CD8+ T cells and γδ T cells often expressed cytotoxic mediators, granzyme A/granzyme B, and perforin. The same 3 TCR Vß segments were over-represented in both CD4+ T cells and CD8+ T cells in SFMC samples from post-antibiotic LA patients. In synovial tissue samples from 3 patients with post-antibiotic LA, CD8+ T cells intermixed with CD4+ T cells were seen around blood vessels, and 2 patients with microvascular damage had autoantibodies to vascular-associated antigens. One of these 2 patients, the one in whom cytotoxicity appeared to be active, had complement (C5b-9) deposition on obliterated vessels. Very few natural killer cells or γδ T cells were seen. CONCLUSION: We propose that CD8+ T cell-mediated cytotoxicity, CD4+ T cell help, autoantibodies to vascular antigens, and complement deposition may each have a role in microvasculature damage in post-antibiotic LA.

Atypical sideways recognition of CD1a by autoreactive γδ T cell receptors.

CD1a is a monomorphic antigen-presenting molecule on dendritic cells that presents lipids to αß T cells. Whether CD1a represents a ligand for other immune receptors remains unknown. Here we use CD1a tetramers to show that CD1a is a ligand for Vδ1+ γδ T cells. Functional studies suggest that two γδ T cell receptors (TCRs) bound CD1a in a lipid-independent manner. The crystal structures of three Vγ4Vδ1 TCR-CD1a-lipid complexes reveal that the γδ TCR binds at the extreme far side and parallel to the long axis of the ß-sheet floor of CD1a's antigen-binding cleft. Here, the γδ TCR co-recognises the CD1a heavy chain and ß2 microglobulin in a manner that is distinct from all other previously observed γδ TCR docking modalities. The 'sideways' and lipid antigen independent mode of autoreactive CD1a recognition induces TCR clustering on the cell surface and proximal T cell signalling as measured by CD3ζ phosphorylation. In contrast with the 'end to end' binding of αß TCRs that typically contact carried antigens, autoreactive γδ TCRs support geometrically diverse approaches to CD1a, as well as antigen independent recognition.

Dual TCR-α Expression on Mucosal-Associated Invariant T Cells as a Potential Confounder of TCR Interpretation.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that are highly abundant in human blood and tissues. Most MAIT cells have an invariant TCRα-chain that uses T cell receptor α-variable 1-2 (TRAV1-2) joined to TRAJ33/20/12 and recognizes metabolites from bacterial riboflavin synthesis bound to the Ag-presenting molecule MHC class I related (MR1). Our attempts to identify alternative MR1-presented Ags led to the discovery of rare MR1-restricted T cells with non-TRAV1-2 TCRs. Because altered Ag specificity likely alters affinity for the most potent known Ag, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), we performed bulk TCRα- and TCRß-chain sequencing and single-cell-based paired TCR sequencing on T cells that bound the MR1-5-OP-RU tetramer with differing intensities. Bulk sequencing showed that use of V genes other than TRAV1-2 was enriched among MR1-5-OP-RU tetramerlow cells. Although we initially interpreted these as diverse MR1-restricted TCRs, single-cell TCR sequencing revealed that cells expressing atypical TCRα-chains also coexpressed an invariant MAIT TCRα-chain. Transfection of each non-TRAV1-2 TCRα-chain with the TCRß-chain from the same cell demonstrated that the non-TRAV1-2 TCR did not bind the MR1-5-OP-RU tetramer. Thus, dual TCRα-chain expression in human T cells and competition for the endogenous ß-chain explains the existence of some MR1-5-OP-RU tetramerlow T cells. The discovery of simultaneous expression of canonical and noncanonical TCRs on the same T cell means that claims of roles for non-TRAV1-2 TCR in MR1 response must be validated by TCR transfer-based confirmation of Ag specificity.

Rational design of a hydrolysis-resistant mycobacterial phosphoglycolipid antigen presented by CD1c to T cells.

Whereas proteolytic cleavage is crucial for peptide presentation by classical major histocompatibility complex (MHC) proteins to T cells, glycolipids presented by CD1 molecules are typically presented in an unmodified form. However, the mycobacterial lipid antigen mannosyl-ß1-phosphomycoketide (MPM) may be processed through hydrolysis in antigen presenting cells, forming mannose and phosphomycoketide (PM). To further test the hypothesis that some lipid antigens are processed, and to generate antigens that lead to defined epitopes for future tuberculosis vaccines or diagnostic tests, we aimed to create hydrolysis-resistant MPM variants that retain their antigenicity. Here, we designed and tested three different, versatile synthetic strategies to chemically stabilize MPM analogs. Crystallographic studies of CD1c complexes with these three new MPM analogs showed anchoring of the lipid tail and phosphate group that is highly comparable to nature-identical MPM, with considerable conformational flexibility for the mannose head group. MPM-3, a difluoromethylene-modified version of MPM that is resistant to hydrolysis, showed altered recognition by cells, but not by CD1c proteins, supporting the cellular antigen processing hypothesis. Furthermore, the synthetic analogs elicited T cell responses that were cross-reactive with nature-identical MPM, fulfilling important requirements for future clinical use.

Benzofuran sulfonates and small self-lipid antigens activate type II NKT cells via CD1d.

Natural killer T (NKT) cells detect lipids presented by CD1d. Most studies focus on type I NKT cells that express semi-invariant αß 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 αßTCRs 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.

CD1a selectively captures endogenous cellular lipids that broadly block T cell response.

We optimized lipidomics methods to broadly detect endogenous lipids bound to cellular CD1a proteins. Whereas membrane phospholipids dominate in cells, CD1a preferentially captured sphingolipids, especially a C42, doubly unsaturated sphingomyelin (42:2 SM). The natural 42:2 SM but not the more common 34:1 SM blocked CD1a tetramer binding to T cells in all human subjects tested. Thus, cellular CD1a selectively captures a particular endogenous lipid that broadly blocks its binding to TCRs. Crystal structures show that the short cellular SMs stabilized a triad of surface residues to remain flush with CD1a, but the longer lipids forced the phosphocholine group to ride above the display platform to hinder TCR approach. Whereas nearly all models emphasize antigen-mediated T cell activation, we propose that the CD1a system has intrinsic autoreactivity and is negatively regulated by natural endogenous inhibitors selectively bound in its cleft. Further, the detailed chemical structures of natural blockers could guide future design of therapeutic blockers of CD1a response.

Multimodally profiling memory T cells from a tuberculosis cohort identifies cell state associations with demographics, environment and disease.

Multimodal T cell profiling can enable more precise characterization of elusive cell states underlying disease. Here, we integrated single-cell RNA and surface protein data from 500,089 memory T cells to define 31 cell states from 259 individuals in a Peruvian tuberculosis (TB) progression cohort. At immune steady state >4 years after infection and disease resolution, we found that, after accounting for significant effects of age, sex, season and genetic ancestry on T cell composition, a polyfunctional type 17 helper T (TH17) cell-like effector state was reduced in abundance and function in individuals who previously progressed from Mycobacterium tuberculosis (M.tb) infection to active TB disease. These cells are capable of responding to M.tb peptides. Deconvoluting this state-uniquely identifiable with multimodal analysis-from public data demonstrated that its depletion may precede and persist beyond active disease. Our study demonstrates the power of integrative multimodal single-cell profiling to define cell states relevant to disease and other traits.

CD1 and MR1 recognition by human γδ T cells.

The two main T cell lineages, αß and γδ T cells, play a central role in immunity. Unlike αß T cells that recognize antigens bound to the Major Histocompatibility Complex (MHC) or MHC class I-like antigen-presenting molecules, the ligands for γδ T cell receptors (TCRs) are much more diverse. However, it is now clear that γδ TCRs can also recognize MHC class I-like molecules, including CD1b, CD1c, CD1d and the MHC class I-related protein 1 (MR1). Yet, our understanding at the molecular level of γδ T cell immunity to CD1 and MR1 is still very limited. Here, we discuss new molecular paradigms underpinning γδ TCRs recognition of antigens, antigen-presenting molecules or both. The recent discovery of recognition of MR1 by a γδ TCR at a position located underneath the antigen display platform reinforces the view that γδ TCRs can approach their ligands from many directions, unlike αß TCRs that bind MHC, CD1 and MR1 targets in an aligned, end to end fashion.

Human skin is colonized by T cells that recognize CD1a independently of lipid.

CD1a-autoreactive T cells contribute to skin disease, but the identity of immunodominant self-lipid antigens and their mode of recognition are not yet solved. In most models, MHC and CD1 proteins serve as display platforms for smaller antigens. Here, we showed that CD1a tetramers without added antigen stained large T cell pools in every subject tested, accounting for approximately 1% of skin T cells. The mechanism of tetramer binding to T cells did not require any defined antigen. Binding occurred with approximately 100 lipid ligands carried by CD1a proteins, but could be tuned upward or downward with certain natural self-lipids. TCR recognition mapped to the outer A' roof of CD1a at sites remote from the antigen exit portal, explaining how TCRs can bind CD1a rather than carried lipids. Thus, a major antigenic target of CD1a T cell autoreactivity in vivo is CD1a itself. Based on their high frequency and prevalence among donors, we conclude that CD1a-specific, lipid-independent T cells are a normal component of the human skin T cell repertoire. Bypassing the need to select antigens and effector molecules, CD1a tetramers represent a simple method to track such CD1a-specific T cells from tissues and in any clinical disease.

Synthetic mycobacterial diacyl trehaloses reveal differential recognition by human T cell receptors and the C-type lectin Mincle.

The cell wall of Mycobacterium tuberculosis is composed of diverse glycolipids which potentially interact with the human immune system. To overcome difficulties in obtaining pure compounds from bacterial extracts, we recently synthesized three forms of mycobacterial diacyltrehalose (DAT) that differ in their fatty acid composition, DAT1, DAT2, and DAT3. To study the potential recognition of DATs by human T cells, we treated the lipid-binding antigen presenting molecule CD1b with synthetic DATs and looked for T cells that bound the complex. DAT1- and DAT2-treated CD1b tetramers were recognized by T cells, but DAT3-treated CD1b tetramers were not. A T cell line derived using CD1b-DAT2 tetramers showed that there is no cross-reactivity between DATs in an IFN-γ release assay, suggesting that the chemical structure of the fatty acid at the 3-position determines recognition by T cells. In contrast with the lack of recognition of DAT3 by human T cells, DAT3, but not DAT1 or DAT2, activates Mincle. Thus, we show that the mycobacterial lipid DAT can be both an antigen for T cells and an agonist for the innate Mincle receptor, and that small chemical differences determine recognition by different parts of the immune system.

Human γδ T cells recognize CD1b by two distinct mechanisms.

γδ T cells form an abundant part of the human cellular immune system, where they respond to tissue damage, infection, and cancer. The spectrum of known molecular targets recognized by Vδ1-expressing γδ T cells is becoming increasingly diverse. Here we describe human γδ T cells that recognize CD1b, a lipid antigen-presenting molecule, which is inducibly expressed on monocytes and dendritic cells. Using CD1b tetramers to study multiple donors, we found that many CD1b-specific γδ T cells use Vδ1. Despite their common use of Vδ1, three CD1b-specific γδ T cell receptors (TCRs) showed clear differences in the surface of CD1b recognized, the requirement for lipid antigens, and corecognition of butryophilin-like proteins. Several Vγ segments were present among the CD1b-specific TCRs, but chain swap experiments demonstrated that CD1b specificity was mediated by the Vδ1 chain. One of the CD1b-specific Vδ1+ TCRs paired with Vγ4 and shows dual reactivity to CD1b and butyrophilin-like proteins. αß TCRs typically recognize the peptide display platform of MHC proteins. In contrast, our results demonstrate the use of rearranged receptors to mediate diverse modes of recognition across the surface of CD1b in ways that do and do not require carried lipids.