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CD1a molecules capture lipid classes that can prevent the binding of autoreactive T cell antigen receptors., CD1a-autoreactive T cells represent a significant proportion of circulating αβ T cells in humans and appear to be enriched in the skin. How their autoreactivity is regulated remains unclear. In this issue of JEM, Cotton et al. (2021. J. Exp. Med. https://doi.org/10.1084/jem.20202699) show that CD1a molecules do not randomly survey cellular lipids but instead capture certain lipid classes that broadly interfere with the binding of autoreactive T cell antigen receptors to the target CD1a. These findings provide new potential therapeutic avenues for manipulating CD1a autoreactive T cell responses.

Recent advances in single-cell technologies and integration algorithms make it possible to construct large, comprehensive reference atlases from multiple datasets encompassing many donors, studies, disease states, and sequencing platforms. Much like mapping sequencing reads to a reference genome, it is essential to be able to map new query cells onto complex, multimillion-cell reference atlases to rapidly identify relevant cell states and phenotypes. We present Symphony, a novel algorithm for building compressed, integrated reference atlases of ≥106 cells and enabling efficient query mapping within seconds. Based on a linear mixture model framework, Symphony precisely localizes query cells within a low-dimensional reference embedding without the need to reintegrate the reference cells, facilitating the downstream transfer of many types of reference-defined annotations to the query cells. We demonstrate the power of Symphony by (1) mapping a query containing multiple levels of experimental design to predict pancreatic cell types in human and mouse, (2) localizing query cells along a smooth developmental trajectory of human fetal liver hematopoiesis, and (3) harnessing a multimodal CITE-seq reference atlas to infer query surface protein expression in memory T cells. Symphony will enable the sharing of comprehensive integrated reference atlases in a convenient, portable format that powers fast, reproducible querying and downstream analyses.

This work sheds light on the role of the lipid 1-tuberculosinyladenosine in the evolution of an environmental ancestor to M. tuberculosis. On a larger scale, it reinforces the importance of horizontal gene transfer in bacterial evolution and examines novel models and methods to provide a better understanding of the subtle effects of individual M. tuberculosis-specific virulence factors in infection settings that are relevant to the pathogen., Mycobacterium kansasii is an environmental nontuberculous mycobacterium that causes opportunistic tuberculosis-like disease. It is one of the most closely related species to the Mycobacterium tuberculosis complex. Using M. kansasii as a proxy for the M. kansasii-M. tuberculosis common ancestor, we asked whether introducing the M. tuberculosis-specific gene pair Rv3377c-Rv3378c into M. kansasii affects the course of experimental infection. Expression of these genes resulted in the production of an adenosine-linked lipid species, known as 1-tuberculosinyladenosine (1-TbAd), but did not alter growth in vitro under standard conditions. Production of 1-TbAd enhanced growth of M. kansasii under acidic conditions through a bacterial cell-intrinsic mechanism independent of controlling pH in the bulk extracellular and intracellular spaces. Production of 1-TbAd led to greater burden of M. kansasii in the lungs of C57BL/6 mice during the first 24 h after infection, and ex vivo infections of alveolar macrophages recapitulated this phenotype within the same time frame. However, in long-term infections, production of 1-TbAd resulted in impaired bacterial survival in both C57BL/6 mice and Ccr2−/− mice. We have demonstrated that M. kansasii is a valid surrogate of M. tuberculosis to study virulence factors acquired by the latter organism, yet shown the challenge inherent to studying the complex evolution of mycobacterial pathogenicity with isolated gene complementation.

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.

CD1d‐restricted invariant natural killer T (iNKT) cells constitute a common glycolipid‐reactive innate‐like T‐cell subset with a broad impact on innate and adaptive immunity. While several microbial glycolipids are known to activate iNKT cells, the cellular mechanisms leading to endogenous CD1d‐dependent glycolipid responses remain largely unclear. Here, we show that endoplasmic reticulum (ER) stress in APCs is a potent inducer of CD1d‐dependent iNKT cell autoreactivity. This pathway relies on the presence of two transducers of the unfolded protein response: inositol‐requiring enzyme‐1a (IRE1α) and protein kinase R‐like ER kinase (PERK). Surprisingly, the neutral but not the polar lipids generated within APCs undergoing ER stress are capable of activating iNKT cells. These data reveal that ER stress is an important mechanism to elicit endogenous CD1d‐restricted iNKT cell responses through induction of distinct classes of neutral lipids., Upon ER stress immunogenic lipids are produced that activate NKT cells. Defining the conditions required to generate endogenous high immunogenic lipid antigens paves the way for the design of novel lipids with immunomodulatory function.

This work describes the discovery, biosynthesis, and chemical synthesis of a previously unknown class of lipids, trehalose phospholipids, in pathogenic Salmonella species. Trehalose phospholipids stimulate the innate immune receptor Mincle, the receptor for the strong adjuvant mycobacterial cord factor., Salmonella species are among the world’s most prevalent pathogens. Because the cell wall interfaces with the host, we designed a lipidomics approach to reveal pathogen-specific cell wall compounds. Among the molecules differentially expressed between Salmonella Paratyphi and S. Typhi, we focused on lipids that are enriched in S. Typhi, because it causes typhoid fever. We discovered a previously unknown family of trehalose phospholipids, 6,6′-diphosphatidyltrehalose (diPT) and 6-phosphatidyltrehalose (PT). Cardiolipin synthase B (ClsB) is essential for PT and diPT but not for cardiolipin biosynthesis. Chemotyping outperformed clsB homology analysis in evaluating synthesis of diPT. DiPT is restricted to a subset of Gram-negative bacteria: large amounts are produced by S. Typhi, lower amounts by other pathogens, and variable amounts by Escherichia coli strains. DiPT activates Mincle, a macrophage activating receptor that also recognizes mycobacterial cord factor (6,6′-trehalose dimycolate). Thus, Gram-negative bacteria show convergent function with mycobacteria. Overall, we discovered a previously unknown immunostimulant that is selectively expressed among medically important bacterial species.

Mycobacterium tuberculosis (M.tb) results in 10 million active tuberculosis (TB) cases and 1.5 million deaths each year1, making it the world’s leading infectious cause of death2. Infection leads to either an asymptomatic latent state or TB disease. Memory T cells have been implicated in TB disease progression, but the specific cell states involved have not yet been delineated because of the limited scope of traditional profiling strategies. Furthermore, immune activation during infection confounds underlying differences in T cell state distributions that influence risk of progression. Here, we used a multimodal single-cell approach to integrate measurements of transcripts and 30 functionally relevant surface proteins to comprehensively define the memory T cell landscape at steady state (i.e., outside of active infection). We profiled 500,000 memory T cells from 259 Peruvians > 4.7 years after they had either latent M.tb infection or active disease and defined 31 distinct memory T cell states, including a CD4+CD26+CD161+CCR6+ effector memory state that was significantly reduced in patients who had developed active TB (OR = 0.80, 95% CI: 0.73–0.87, p = 1.21 × 10−6). This state was also polyfunctional; in ex vivo stimulation, it was enriched for IL-17 and IL-22 production, consistent with a Th17-skewed phenotype, but also had more capacity to produce IFNγ than other CD161+CCR6+ Th17 cells. Additionally, in progressors, IL-17 and IL-22 production in this cell state was significantly lower than in non-progressors. Reduced abundance and function of this state may be an important factor in failure to control M.tb infection.

The Mycobacterium tuberculosis Ser/Thr protein kinases PknA and PknB are essential for growth and have been proposed as possible drug targets. We used a titratable conditional depletion system to investigate the functions of these kinases. Depletion of PknA or PknB or both kinases resulted in growth arrest, shortening of cells, and time-dependent loss of acid-fast staining with a concomitant decrease in mycolate synthesis and accumulation of trehalose monomycolate. Depletion of PknA and/or PknB resulted in markedly increased susceptibility to β-lactam antibiotics, and to the key tuberculosis drug rifampin. Phosphoproteomic analysis showed extensive changes in protein phosphorylation in response to PknA depletion and comparatively fewer changes with PknB depletion. These results identify candidate substrates of each kinase and suggest specific and coordinate roles for PknA and PknB in regulating multiple essential physiologies. These findings support these kinases as targets for new antituberculosis drugs and provide a valuable resource for targeted investigation of mechanisms by which protein phosphorylation regulates pathways required for growth and virulence in M. tuberculosis., Author summary Tuberculosis is a major global health threat, with approximately 10 million new cases per year and 1.5 million deaths. Although tuberculosis is treatable, cure requires prolonged antibiotic courses and strains that are resistant to current treatments are increasingly common. Understanding how Mycobacterium tuberculosis adapts to the human host during infection can lead to new approaches to treat tuberculosis. A major adaptive mechanism is reversible protein phosphorylation mediated by protein kinases, which detect signals in the extracellular environment and transmit this information to regulate M. tuberculosis physiology. In this study we analyzed two M. tuberculosis kinases, PknA and PknB, by comparing strains in which these kinases are present to strains in which one or both of these kinases has been depleted. We identified features that differ between the kinase replete and kinase depleted strains including bacterial growth and viability, cell shape, the bacterial cell envelope and gene regulation. We also identified proteins that show decreased phosphorylation in the kinase depleted strains, which can result from direct or indirect effects of kinase depletion. These findings provide insight into how these kinases are important for M. tuberculosis pathogenesis and indicate that these kinases may be valuable targets for development of new TB drugs.

Peptide and lipid antigens are presented to T cells when bound to MHC or CD1 proteins, respectively. The general paradigm of T cell antigen recognition is that T cell receptors (TCRs) co-recognize an epitope comprised of the antigen and antigen presenting molecule. Here we review the latest studies in which T cells operate outside the co-recognition paradigm: TCRs can broadly contact CD1 itself, but not the carried lipid. The essential structural feature in these new mechanisms is a large 'antigen free' zone on the outer surface of certain antigen presenting molecules. Whereas peptides dominate the exposed surface of MHC-peptide complexes, all human CD1 proteins have a closed, antigen-free surface, which is known as the A' roof. These new structural models help to interpret recent biological studies of CD1 autoreactive T cells in vivo, which have now been broadly observed in studies on TCR-transgenic mice, healthy humans and patients with autoimmune disease.

Mycobacterial cell wall lipids bind the conserved CD1 family of antigen-presenting molecules and activate T cells via their T cell receptors (TCRs). Sulfoglycolipids (SGLs) are uniquely synthesized by Mycobacterium tuberculosis, but tools to study SGL-specific T cells in humans are lacking. We designed a novel hybrid synthesis of a naturally occurring SGL, generated CD1b tetramers loaded with natural or synthetic SGL analogs, and studied the molecular requirements for TCR binding and T cell activation. Two T cell lines derived using natural SGLs are activated by synthetic analogs independently of lipid chain length and hydroxylation, but differentially by saturation status. By contrast, two T cell lines derived using an unsaturated SGL synthetic analog were not activated by the natural antigen. Our data provide a bioequivalence hierarchy of synthetic SGL analogs and SGL-loaded CD1b tetramers. These reagents can now be applied to large-scale translational studies investigating the diagnostic potential of SGL-specific T cell responses or SGL-based vaccines. Sulfoglycolipids (SGLs) are uniquely synthesized by Mycobacterium tuberculosis (Mtb) and recognized by human T cells. James, Yu et al. describe a new hybrid synthesis for key antigenic determinants of SGLs and the development of SGL-specific tetramers that can now be applied to large-scale translational studies.

Glycolipid antigens recognized by αβ T-cell receptors (TCRs) drive the activation of invariant natural killer T (iNKT) cells, a specialized subset of innate T lymphocytes. Glycolipids with α-linked anomeric carbohydrates have been identified as potent microbial lipid antigens for iNKT cells, and their unusual α-anomeric linkage has been thought to define a "foreign" lipid antigen motif. However, mammals use endogenous lipids to select iNKT cells, and there is compelling evidence for iNKT cell responses in various types of sterile inflammation. The nature of endogenous or environmental lipid antigens encountered by iNKT cells is not well defined. Here, we sought to identify lipid antigens in cow's milk, a prominent part of the human diet. We developed a method to directly capture lipid antigens within CD1d-lipid-TCR complexes, while excluding CD1d bound to nonantigenic lipids, followed by direct biochemical analysis of the lipid antigens trapped at the TCR-CD1d interface. The specific antigens captured by this "TCR trap" method were identified as α-linked monohexosylceramides by mass spectrometry fragmentation patterns that distinguished α- from β-anomeric monohexosylceramides. These data provide direct biochemical evidence for α-linked lipid antigens from a common dietary source.

CD1a, CD1b, CD1c and CD1d proteins migrate through distinct subcellular compartments of antigen presenting cells and so can be considered to take four separate pathways leading to display of lipid antigens to T cell receptors. This review discusses the intersection of CD1 trafficking and lipid antigen loading mechanisms in cells, highlighting key controversies relating to CD1 gene expression, size mismatches between antigens and CD1 binding clefts and unexpected mechanisms of T cell receptor-based recognition.

A large proportion of human T cells are autoreactive to group 1 CD1 proteins, which include CD1a, CD1b, and CD1c. However, the physiological role of the CD1 proteins remains poorly defined. Here, we have generated a double-transgenic mouse model that expresses human CD1b and CD1c molecules (hCD1Tg) as well as a CD1b-autoreactive TCR (HJ1Tg) in the ApoE-deficient background (hCD1Tg HJ1Tg Apoe–/– mice) to determine the role of CD1-autoreactive T cells in hyperlipidemia-associated inflammatory diseases. We found that hCD1Tg HJ1Tg Apoe–/– mice spontaneously developed psoriasiform skin inflammation characterized by T cell and neutrophil infiltration and a Th17-biased cytokine response. Anti–IL-17A treatment ameliorated skin inflammation in vivo. Additionally, phospholipids and cholesterol preferentially accumulated in diseased skin and these autoantigens directly activated CD1b-autoreactive HJ1 T cells. Furthermore, hyperlipidemic serum enhanced IL-6 secretion by CD1b+ DCs and increased IL-17A production by HJ1 T cells. In psoriatic patients, the frequency of CD1b-autoreactive T cells was increased compared with that in healthy controls. Thus, this study has demonstrated the pathogenic role of CD1b-autoreactive T cells under hyperlipidemic conditions in a mouse model of spontaneous skin inflammation. As a large proportion of psoriatic patients are dyslipidemic, this finding is of clinical significance and indicates that self-lipid–reactive T cells might serve as a possible link between hyperlipidemia and psoriasis.

During industrialization, humans have been exposed to increasing numbers of foreign chemicals. Failure of the immune system to tolerate drugs, cosmetics, and other skin products causes allergic contact dermatitis, a T cell-mediated disease with rising prevalence. Models of αβ T cell response emphasize T cell receptor (TCR) contact with peptide-MHC complexes, but this model cannot readily explain activation by most contact dermatitis allergens, which are nonpeptidic molecules. We tested whether CD1a, an abundant MHC I-like protein in human skin, mediates contact allergen recognition. Using CD1a-autoreactive human αβ T cell clones to screen clinically important allergens present in skin patch testing kits, we identified responses to balsam of Peru, a tree oil widely used in cosmetics and toothpaste. Additional purification identified benzyl benzoate and benzyl cinnamate as antigenic compounds within balsam of Peru. Screening of structurally related compounds revealed additional stimulants of CD1a-restricted T cells, including farnesol and coenzyme Q2. Certain general chemical features controlled response: small size, extreme hydrophobicity, and chemical constraint from rings and unsaturations. Unlike lipid antigens that protrude to form epitopes and contact TCRs, the small size of farnesol allows sequestration deeply within CD1a, where it displaces self-lipids and unmasks the CD1a surface. These studies identify molecular connections between CD1a and hypersensitivity to consumer products, defining a mechanism that could plausibly explain the many known T cell responses to oily substances.

High-throughput TCR sequencing allows interrogation of the human TCR repertoire, potentially connecting TCR sequences to antigenic targets. Unlike the highly polymorphic MHC proteins, monomorphic Ag-presenting molecules such as MR1, CD1d, and CD1b present Ags to T cells with species-wide TCR motifs. CD1b tetramer studies and a survey of the 27 published CD1b-restricted TCRs demonstrated a TCR motif in humans defined by the TCR β-chain variable gene 4-1 (TRBV4-1) region. Unexpectedly, TRBV4-1 was involved in recognition of CD1b regardless of the chemical class of the carried lipid. Crystal structures of two CD1b-specific TRBV4-1+ TCRs show that germline-encoded residues in CDR1 and CDR3 regions of TRBV4-1–encoded sequences interact with each other and consolidate the surface of the TCR. Mutational studies identified a key positively charged residue in TRBV4-1 and a key negatively charged residue in CD1b that is shared with CD1c, which is also recognized by TRBV4-1 TCRs. These data show that one TCR V region can mediate a mechanism of recognition of two related monomorphic Ag-presenting molecules that does not rely on a defined lipid Ag.

Improved tuberculosis diagnostics and tools for monitoring treatment response are urgently needed. We developed a robust and simple, PCR-based host-blood transcriptomic signature, RISK6, for multiple applications: identifying individuals at risk of incident disease, as a screening test for subclinical or clinical tuberculosis, and for monitoring tuberculosis treatment. RISK6 utility was validated by blind prediction using quantitative real-time (qRT) PCR in seven independent cohorts.Prognostic performance significantly exceeded that of previous signatures discovered in the same cohort. Performance for diagnosing subclinical and clinical disease in HIV-uninfected and HIV-infected persons, assessed by area under the receiver-operating characteristic curve, exceeded 85%. As a screening test for tuberculosis, the sensitivity at 90% specificity met or approached the benchmarks set out in World Health Organization target product profiles for non-sputum-based tests. RISK6 scores correlated with lung immunopathology activity, measured by positron emission tomography, and tracked treatment response, demonstrating utility as treatment response biomarker, while predicting treatment failure prior to treatment initiation. Performance of the test in capillary blood samples collected by finger-prick was noninferior to venous blood collected in PAXgene tubes. These results support incorporation of RISK6 into rapid, capillary blood-based point-of-care PCR devices for prospective assessment in field studies.

The CD1 and MHC systems are specialized for lipid and peptide display, respectively. Here, we review evidence showing how cellular CD1a, CD1b, CD1c, and CD1d proteins capture and display many cellular lipids to T cell receptors (TCRs). Increasing evidence shows that CD1-reactive T cells operate outside two classical immunogenetic concepts derived from the MHC paradigm. First, because CD1 proteins are non-polymorphic in human populations, T cell responses are not restricted to the donor's genetic background. Second, the simplified population genetics of CD1 antigen-presenting molecules can lead to simplified patterns of TCR usage. As contrasted with donor-restricted patterns of MHC-TCR interaction, the donor-unrestricted nature of CD1-TCR interactions raises the prospect that lipid agonists and antagonists of T cells could be developed.

Global control of tuberculosis has become increasingly complicated with the emergence of multidrug-resistant strains of Mycobacterium tuberculosis. First-line treatments are anchored by two antibiotics, rifampin and isoniazid. Most rifampin resistance occurs through the acquisition of missense mutations in the rifampin resistance-determining region, an 81-base pair region encoding the rifampin binding site on the β subunit of RNA polymerase (rpoB). Although these mutations confer a survival advantage in the presence of rifampin, they may alter the normal process of transcription, thereby imposing significant fitness costs. Because the downstream biochemical consequences of the rpoB mutations are unknown, we used an organism-wide screen to identify the number and types of lipids changed after rpoB mutation. A new mass spectrometry-based profiling platform systematically compared ∼10,000 cell wall lipids in a panel of rifampin-resistant mutants within two genetically distinct strains, CDC1551and W-Beijing. This unbiased lipidomic survey detected quantitative alterations (>2-fold, p < 0.05) in more than 100 lipids in each mutant. By focusing on molecular events that change among most mutants and in both genetic backgrounds, we found that rifampin resistance mutations lead to altered concentrations of mycobactin siderophores and acylated sulfoglycolipids. These findings validate a new organism-wide lipidomic analysis platform for drug-resistant mycobacteria and provide direct evidence for characteristic remodeling of cell wall lipids in rifampin-resistant strains of M. tuberculosis. The specific links between rifampin resistance and named lipid factors provide diagnostic and therapeutic targets that may be exploited to address the problem of drug resistance.

CD1 proteins are expressed on dendritic cells, where they display lipid antigens to T-cell receptors (TCRs). Here we describe T-cell autoreactivity towards ubiquitous human membrane phospholipids presented by CD1b. These T-cells discriminate between two major types of lipids, sphingolipids and phospholipids, but were broadly cross-reactive towards diverse phospholipids including phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine. The crystal structure of a representative TCR bound to CD1b-phosphatidylcholine provides a molecular mechanism for this promiscuous recognition. We observe a lateral escape channel in the TCR, which shunted phospholipid head groups sideways along the CD1b-TCR interface, without contacting the TCR. Instead the TCR recognition site involved the neck region phosphate that is common to all major self-phospholipids but absent in sphingolipids. Whereas prior studies have focused on foreign lipids or rare self-lipids, we define a new molecular mechanism of promiscuous recognition of common self-phospholipids including those that are known targets in human autoimmune disease., CD1 proteins present lipid antigens to T cells via the T cell receptor. Here the authors describe T cell reactivity to human membrane lipid moieties and provide structural data to define a molecular mechanism of promiscuous recognition of self-derived phospholipids.

Lyme disease is a common multi-system disease caused by infection with a tick-transmitted spirochete, Borrelia burgdorferi and related Borrelia species. The monoglycosylated diacylglycerol known as B. burgdorferi glycolipid II (BbGL-II) is a major target of antibodies in sera from infected individuals. Here we show that CD1b presents BbGL-II to human T cells and that the T cell receptor (TCR) mediates the recognition. However, we did not detect increased frequency of CD1b-BbGL-II binding T cells in the peripheral blood of Lyme disease patients compared to controls. Unexpectedly, mapping the T cell specificity for BbGL-II-like molecules using tetramers and activation assays revealed a concomitant response to CD1b-expressing antigen presenting cells in absence of BbGL-II. Further, among all major classes of self-lipid tested, BbGL-II responsive TCRs show strong cross-reactivity to diacylglycerol, a self-lipid antigen with structural similarities to BbGL-II. Extending prior work on MHC and CD1b, CD1c, and CD1d proteins, this study provides evidence for cross-reactive CD1b-restricted T cell responses to bacterial and self-antigens, and identifies chemically defined targets for future discovery of self and foreign antigen cross-reactive T cells. This article is protected by copyright. All rights reserved.

Mycobacterium tuberculosis (Mtb) is the world's most deadly pathogen. Unlike less virulent mycobacteria, Mtb produces 1-tuberculosinyladenosine (1-TbAd), an unusual terpene nucleoside of unknown function. In the present study 1-TbAd has been shown to be a naturally evolved phagolysosome disruptor. 1-TbAd is highly prevalent among patient-derived Mtb strains, where it is among the most abundant lipids produced. Synthesis of TbAd analogs and their testing in cells demonstrate that their biological action is dependent on lipid linkage to the 1-position of adenosine, which creates a strong conjugate base. Furthermore, C2O lipid moieties confer passage through membranes. 1-TbAd selectively accumulates in acidic compartments, where it neutralizes the pH and swells lysosomes, obliterating their multilamellar structure. During macrophage infection, a 1-TbAd biosynthesis gene (Rv3378c) confers marked phagosomal swelling and intraphagosomal inclusions, demonstrating an essential role in regulating the Mtb cellular microenvironment. Although macrophages kill intracellular bacteria through phagosome acidification, Mtb coats itself abundantly with antacid.

The role of CD1a‐reactive T cells in human allergic disease is unknown. We have previously shown that circulating CD1a‐reactive T cells recognize neolipid antigens generated by bee and wasp venom phospholipase, and here tested the hypothesis that venom‐responsive CD1a‐reactive T cells associate with venom allergy. Circulating T cells from bee and wasp venom allergic individuals, before and during immunotherapy, were exposed to CD1a‐transfected K562 cells in the presence of wasp or bee venom. T‐cell response was evaluated based on IFNγ, GM‐CSF, and IL‐13 cytokine production. Venom allergic individuals showed significantly higher frequencies of IFN‐γ, GM‐CSF, and IL‐13 producing CD1a‐reactive T cells responsive to venom and venom‐derived phospholipase than healthy individuals. Venom‐responsive CD1a‐reactive T cells were cross‐responsive between wasp and bee suggesting shared pathways of allergenicity. Frequencies of CD1a‐reactive T cells were initially induced during subcutaneous immunotherapy, peaking by weeks 5, but then reduced despite escalation of antigen dose. Our current understanding of venom allergy and immunotherapy is largely based on peptide and protein‐specific T cell and antibody responses. Here, we show that lipid antigens and CD1a‐reactive T cells associate with the allergic response. These data have implications for mechanisms of allergy and approaches to immunotherapy.

Human T cells are activated by both peptide and nonpeptide Ags produced by Mycobacterium tuberculosis. T cells recognize cell wall lipids bound to CD1 molecules, but effector functions of CD1-reactive T cells have not been systematically assessed in M. tuberculosis–infected humans. It is also not known how these features correlate with T cell responses to secreted protein Ags. We developed a flow cytometric assay to profile CD1-restricted T cells ex vivo and assessed T cell responses to five cell wall lipid Ags in a cross-sectional study of 19 M. tuberculosis–infected and 22 M. tuberculosis–uninfected South African adolescents. We analyzed six T cell functions using a recently developed computational approach for flow cytometry data in high dimensions. We compared these data with T cell responses to five protein Ags in the same cohort. We show that CD1b-restricted T cells producing antimycobacterial cytokines IFN-γ and TNF-α are detectable ex vivo in CD4+, CD8+, and CD4−CD8− T cell subsets. Glucose monomycolate was immunodominant among lipid Ags tested, and polyfunctional CD4 T cells specific for this lipid simultaneously expressed CD40L, IFN-γ, IL-2, and TNF-α. Lipid-reactive CD4+ T cells were detectable at frequencies of 0.001–0.01%, and this did not differ by M. tuberculosis infection status. Finally, CD4 T cell responses to lipids were poorly correlated with CD4 T cell responses to proteins (Spearman rank correlation −0.01; p = 0.95). These results highlight the functional diversity of CD1-restricted T cells circulating in peripheral blood as well as the complementary nature of T cell responses to mycobacterial lipids and proteins. Our approach enables further population-based studies of lipid-specific T cell responses during natural infection and vaccination.

While most studies of T lymphocytes have focused on T cells reactive to complexes of peptide and major histocompatibility complex (MHC) proteins, many other types of T cells do not fit this paradigm. These include CD1-restricted T cells, MR1-restricted mucosal associated invariant T cells (MAIT cells), MHC class Ib-reactive T cells, and γδ T cells. Collectively, these T cells are considered 'unconventional', in part because they can recognize lipids, small-molecule metabolites and specially modified peptides. Unlike MHC-reactive T cells, these apparently disparate T cell types generally show simplified patterns of T cell antigen receptor (TCR) expression, rapid effector responses and 'public' antigen specificities. Here we review evidence showing that unconventional T cells are an abundant component of the human immune system and discuss the immunotherapeutic potential of these cells and their antigenic targets.

Improved biomarkers are needed for tuberculosis. To develop tests based on products secreted by tubercle bacilli that are strictly associated with viability, we evaluated 3 bacterial-derived, species-specific, small molecules as biomarkers: 2 mycobactin siderophores and tuberculosinyladenosine. Using liquid chromatography–tandem mass spectrometry, we demonstrated the presence of 1 or both mycobactins and/or tuberculosinyladenosine in serum and whole lung tissues from infected mice and sputum, cerebrospinal fluid (CSF), or lymph nodes from infected patients but not uninfected controls. Detection of the target molecules distinguished host infection status in 100% of mice with both serum and lung as the target sample. In human subjects, we evaluated detection of the bacterial small molecules (BSMs) in multiple body compartments in 3 patient cohorts corresponding to different forms of tuberculosis. We detected at least 1 of the 3 molecules in 90%, 71%, and 40% of tuberculosis patients' sputum, CSF, and lymph node samples, respectively. In paucibacillary forms of human tuberculosis, which are difficult to diagnose even with culture, detection of 1 or more BSM was rapid and compared favorably to polymerase chain reaction–based detection. Secreted BSMs, detectable in serum, warrant further investigation as a means for diagnosis and therapeutic monitoring in patients with tuberculosis.

Although small molecules shed from pathogens are widely used to diagnose infection, such tests have not been widely implemented for tuberculosis. Here we show that the recently identified compound, 1-tuberculosinyladenosine (1-TbAd), accumulates to comprise >1% of all Mycobacterium tuberculosis lipid. Invitro and invivo, two isomers of TbAd weredetected that might serve as infection markers. Using mass spectrometry and nuclear magnetic resonance, we established the structure of the previously unknown molecule, N(6)-tuberculosinyladenosine (N(6)-TbAd). Its biosynthesis involves enzymatic production of 1-TbAd by Rv3378c followed by conversion to N(6)-TbAd via the Dimroth rearrangement. Intact biosynthetic genes are observed only within M.tuberculosis complex bacteria, and TbAd was not detected among other medically important pathogens, environmental bacteria, and vaccine strains. With no substantially similar known molecules in nature, the discovery and invivo detection of two abundant terpene nucleosides support their development as specific diagnostic markers of tuberculosis.

Problem CD11cHI human decidual macrophages express several isoforms of CD1 molecules. Their expression pattern and function required investigation. Method of Study CD11cHI macrophages were isolated from decidua. Expression of CD1 isoforms and their ability to present lipid antigens to T cells was studied. Results CD1a, CD1c, and CD1d were all expressed on CD11cHI dMϕ, a pattern differing from those previously observed. Exposure of peripheral monocytes and dendritic cells to lipid isolates from decidua led to increased surface CD1a levels only. The CD1a and CD1c on dMϕ were able to present the appropriate lipid antigens to lipid antigen-specific T cells. Finally, autoreactivity of decidual T cells to CD1a was observed. Conclusion The unique pattern of expression of CD1 isoforms on CD11cHI dMϕ is consistent with organ-specific roles of CD1 in human T-cell responses. dMϕ are able to present lipid antigens to both peripheral and decidual T cells and are major antigen-presenting cells in human decidua.

For decades, proteins were thought to be the sole or at least the dominant source of antigens for T cells. Studies in the 1990s demonstrated that CD1 proteins and mycobacterial lipids form specific targets of human αβ T cells. The molecular basis by which T-cell receptors (TCRs) recognize CD1-lipid complexes is now well understood. Many types of mycobacterial lipids function as antigens in the CD1 system, and new studies done with CD1 tetramers identify T-cell populations in the blood of tuberculosis patients. In human populations, a fundamental difference between the CD1 and major histocompatibility complex systems is that all humans express nearly identical CD1 proteins. Correspondingly, human CD1 responsive T cells show evidence of conserved TCRs. In addition to natural killer T cells and mucosal-associated invariant T (MAIT cells), conserved TCRs define other subsets of human T cells, including germline-encoded mycolyl-reactive (GEM) T cells. The simple immunogenetics of the CD1 system and new investigative tools to measure T-cell responses in humans now creates a situation in which known lipid antigens can be developed as immunodiagnostic and immunotherapeutic reagents for tuberculosis disease.

A central paradigm in αβ T cell–mediated immunity is the simultaneous co-recognition of antigens and antigen-presenting molecules by the αβ T cell antigen receptor (TCR). CD1a presents a broad repertoire of lipid-based antigens. We found that a prototypical autoreactive TCR bound CD1a when it was presenting a series of permissive endogenous ligands, while other lipid ligands were nonpermissive to TCR binding. The structures of two TCR-CD1a-lipid complexes showed that the TCR docked over the A′ roof of CD1a in a manner that precluded direct contact with permissive ligands. Nonpermissive ligands indirectly inhibited TCR binding by disrupting the TCR-CD1a contact zone. The exclusive recognition of CD1a by the TCR represents a previously unknown mechanism whereby αβ T cells indirectly sense self antigens that are bound to an antigen-presenting molecule.

The hallmark function of αβ T cell antigen receptors (TCRs) involves the highly specific co-recognition of a major histocompatibility complex molecule and its carried peptide. However, the molecular basis of the interactions of TCRs with the lipid antigen-presenting molecule CD1c is unknown. We identified frequent staining of human T cells with CD1c tetramers across numerous subjects. Whereas TCRs typically show high specificity for antigen, both tetramer binding and autoreactivity occurred with CD1c in complex with numerous, chemically diverse self lipids. Such extreme polyspecificity was attributable to binding of the TCR over the closed surface of CD1c, with the TCR covering the portal where lipids normally protrude. The TCR essentially failed to contact lipids because they were fully seated within CD1c. These data demonstrate the sequestration of lipids within CD1c as a mechanism of autoreactivity and point to small lipid size as a determinant of autoreactive T cell responses.

Solving the mechanism by which MHC I and II proteins capture protein fragments represented a triumph for T cell biologists. This discovery highlighted the peptidic nature of antigens for T cells. Investigation of MHC-peptide–T cell receptor (TCR) interactions provides the basis for epitope mapping, which is the process of identifying the particular chemical elements of an antigen that contact the TCR and determine immune response. Accordingly, for decades, nearly every aspect of T cell-related technology development in autoimmunity, infection, antitumor response, and vaccination has focused on peptide epitopes. However, the known universe of antigens for human T cells widened considerably based on work in the early 1990s by Porcelli, Brenner, and colleagues (1, 2). They identified lipid antigens for T cells, including mycolic acid, a membrane lipid from Mycobacterium tuberculosis (Fig. 1). This discovery forced a collision of two previously unrelated fields: lipid chemistry and antigen presentation. Mycolic acids and other lipids are now well established to bind within CD1b antigen-presenting molecules, and the resulting CD1b–lipid complexes are recognized by TCRs (3). In PNAS, Salah Mansour’s laboratory returns to the question of epitope mapping of mycobacterial mycolic acid antigens (4). Their work not only solves certain questions, but also highlights new controversies regarding how clonotypic TCRs can specifically grasp and recognize lipids. Fig. 1. Mycolic acid and glucose monomycolate antigens possess the same kinds of mycolyl groups, yet function differently in activation of T cells. T cell response to GMM absolutely requires the glucose head group, including the indicated hydroxyl groups at positions C2 and C4. T cell response to GMM proceeds normally when the lipid is truncated from C80 to C32 removing functional groups from its meromycolate chain (5). In contrast, the functional groups on mycolic acid lipid moiety strongly control T cell response (4, 12, 13). The … [↵][1]1Email: bmoody{at}bwh.harvard.edu. [1]: #xref-corresp-1-1

The spread of antibiotic resistance is a major challenge for treatment of Mycobacterium tuberculosis infection. In addition, efficacy of drugs is often limited by the restricted permeability of the mycomembrane. Frontline antibiotics inhibit mycomembrane biosynthesis leading to rapid cell death. Inspired by this mechanism we exploit β-lactones as putative mycolic acid mimics to block serine hydrolases involved in their biosynthesis. Among a collection of β-lactones we found one hit with potent anti-mycobacterial and bactericidal activity. Chemical proteomics using an alkynylated probe identified Pks13 and Ag85 serine hydrolases as major targets. Validation via enzyme assays and customized 13C metabolite profiling showed that both targets are functionally impaired by the β-lactone. Co-administration with front-line antibiotics enhanced the potency against M. tuberculosis by more than 100-fold demonstrating a therapeutic potential of targeting mycomembrane biosynthesis serine hydrolases.

SUMMARYMycobacterium lepraecauses leprosy, and is unique among mycobacterial diseases in producing peripheral neuropathy. This debilitating morbidity is attributed to axon demyelination resulting from direct interactions of theM. leprae-specific phenolic glycolipid 1 (PGL-1) with myelinating glia, and their subsequent infection. Here, we use transparent zebrafish larvae to visualize the earliest events ofM. leprae-induced nerve damage. We find that demyelination and axonal damage are not directly initiated byM. lepraebut by infected macrophages that patrol axons; demyelination occurs in areas of intimate contact. PGL-1 confers this neurotoxic response on macrophages: macrophages infected withM. marinumexpressing PGL-1 also damage axons. PGL-1 induces nitric oxide synthase in infected macrophages, and the resultant increase in reactive nitrogen species damages axons by injuring their mitochondria and inducing demyelination. Our findings implicate the response of innate macrophages toM. lepraePGL-1 in initiating nerve damage in leprosy.

To identify lipids with roles in tuberculosis disease, we systematically compared the lipid content of virulent Mycobacterium tuberculosis with the attenuated vaccine strain Mycobacterium bovis bacillus Calmette-Guerin. Comparative lipidomics analysis identified more than 1,000 molecular differences, including a previously unknown, Mycobacterium tuberculosis-specific lipid that is composed of a diterpene unit linked to adenosine. We established the complete structure of the natural product as 1-tuberculosiny-ladenosine (1-TbAd) using mass spectrometry and NMR spectroscopy. A screen for 1-TbAd mutants, complementation studies, and gene transfer identified Rv3378c as necessary for 1-TbAd biosynthesis. Whereas Rv3378c was previously thought to function as a phosphatase, these studies establish its role as a tuberculosinyl transferase and suggest a revised biosynthetic pathway for the sequential action of Rv3377c-Rv3378c. In agreement with this model, recombinant Rv3378c protein produced 1-TbAd, and its crystal structure revealed a cis-prenyl transferase fold with hydrophobic residues for isoprenoid binding and a second binding pocket suitable for the nucleoside substrate. The dual-substrate pocket distinguishes Rv3378c from classical cis-prenyl transferases, providing a unique model for the prenylation of diverse metabolites. Terpene nucleosides are rare in nature, and 1-TbAd is known only in Mycobacterium tuberculosis. Thus, this intersection of nucleoside and terpene pathways likely arose late in the evolution of the Mycobacterium tuberculosis complex; 1-TbAd serves as an abundant chemical marker of Mycobacterium tuberculosis, and the extracellular export of this amphipathic molecule likely accounts for the known virulence-promoting effects of the Rv3378c enzyme.

Siderophores are small iron-binding molecules secreted by bacteria to scavenge iron. Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis, produces the siderophores mycobactin and carboxymycobactin. Complexes of the mycobacterial membrane proteins MmpS4 and MmpS5 with the transporters MmpL4 and MmpL5 are required for siderophore export and virulence in Mtb. Here we show that, surprisingly, mycobactin or carboxymycobactin did not rescue the low-iron growth defect of the export mutant but severely impaired growth. Exogenous siderophores were taken up by the export mutant, and siderophore-delivered iron was used, but the deferrated siderophores accumulated intracellularly, indicating a blockade of siderophore recycling. This hypothesis was confirmed by the observation that radiolabeled carboxymycobactin was taken up and secreted again by Mtb. Addition of iron salts to an Mtb siderophore biosynthesis mutant stimulated more growth in the presence of a limiting amount of siderophores than iron-loaded siderophores alone. Thus, recycling enables Mtb to acquire iron at lower metabolic cost because Mtb cannot use iron salts without siderophores. Exogenous siderophores were bactericidal for the export mutant in submicromolar quantities. High-resolution mass spectrometry revealed that endogenous carboxymycobactin also accumulated in the export mutant. Toxic siderophore accumulation is prevented by a drug that inhibits siderophore biosynthesis. Intracellular accumulation of siderophores was toxic despite the use of an alternative iron source such as hemin, suggesting an additional inhibitory mechanism independent of iron availability. This study indicates that targeting siderophore export/recycling would deliver a one-two punch to Mtb: restricting access to iron and causing toxic intracellular siderophore accumulation.

Human CD1a mediates foreign Ag recognition by a T cell clone, but the nature of possible TCR interactions with CD1a/lipid are unknown. After incubating CD1a with a mycobacterial lipopeptide Ag, dideoxymycobactin (DDM), we identified and measured binding to a recombinant TCR (TRAV3/ TRBV3-1, KD of ≈100 μM). Detection of ternary CD1a/lipid/TCR interactions enabled development of CD1a tetramers and CD1a multimers with carbohydrate backbones (dextramers), which specifically stained T cells using a mechanism that was dependent on the precise stereochemistry of the peptide backbone and was blocked with a soluble TCR. Furthermore, sorting of human T cells from unrelated tuberculosis patients for bright DDM-dextramer staining allowed recovery of T cells that were activated by CD1a and DDM. These studies demonstrate that the mechanism of T cell activation by lipopeptides occurs via ternary interactions of CD1a/Ag/TCR. Furthermore, these studies demonstrate the existence of lipopeptide-specific T cells in humans ex vivo.

CD1 proteins evolved to present diverse lipid Ags to T cells. In comparison with MHC proteins, CD1 proteins exhibit minimal allelic diversity as a result of nonsynonymous single nucleotide polymorphisms (SNPs). However, it is unknown if common SNPs in gene regulatory regions affect CD1 expression and function. We report surprising diversity in patterns of inducible CD1a expression on human dendritic cells (DCs), spanning the full range from undetectable to high density, a finding not seen with other CD1 isoforms. CD1a-deficient DCs failed to present mycobacterial lipopeptide to T cells but had no defects in endocytosis, cytokine secretion, or expression of costimulatory molecules after LPS treatment. We identified an SNP in the 5′ untranslated region (rs366316) that was common and strongly associated with low CD1a surface expression and mRNA levels (p = 0.03 and p = 0.001, respectively). Using a CD1a promoter-luciferase system in combination with mutagenesis studies, we found that the polymorphic allele reduced luciferase expression by 44% compared with the wild-type variant (p < 0.001). Genetic regulation of lipid Ag presentation by varying expression on human DCs provides a mechanism for achieving population level differences in immune responses despite limited structural variation in CD1a proteins.

Human T cell autoreactivity toward lipid antigens presented by CD1 proteins can manifest in numerous diseases, including psoriasis, contact hypersensitivities, and allergies. However, the molecular mechanisms for regulating T cell autoreactivity toward lipid antigens remain unclear. We determined the basis for T cell receptor (TCR) autoreactivity toward CD1b bound to self-phospholipids. The spectrum of self-antigens captured by CD1b skews toward abundant membrane phospholipids such as phosphatidylcholine and phosphatidylethanolamine. However, TCRs can specifically recognize rare phospholipids, including phosphatidylglycerol (PG). The structure of an autoreactive TCR bound to CD1b-PG shows that discrimination occurs through a marked induced fit movement of PG so that its polar head group fits snugly into the cationic cup of the TCR. Conversely, TCR binding toward ubiquitous self-phospholipids was sterically or electrostatically repelled. Accordingly, we describe a mechanism of TCR autoreactivity toward rare phospholipids and avoidance of autoreactivity to the most abundant self-phospholipids.

Mycobacterium tuberculosis produces dideoxymycobactin-838 (DDM-838), a lipopeptide that potently activates T cells upon binding to the MHC-like antigen-presenting molecule CD1a. M. tuberculosis produces DDM-838 in only trace amounts and a previous solid-phase synthesis provided sub-milligram quantities. We describe a high yielding solution-phase synthesis of DDM-838 that features a Mitsunobu substitution that avoids yield-limiting epimerization at lysine during esterification, and amidation conditions that prevent double-bond isomerization of the Z-C20:1 acyl chain, and provides material with equivalent antigenicity to natural DDM-838. Isomers of DDM-838 that varied in stereochemistry at the central lysine and the C20:1 acyl chain were compared for their ability to be recognised by CD1a-restricted T cell receptors (TCRs). These TCRs, derived from unrelated human donors, exhibited a similar spectrum of reactivity towards the panel of DDM-838 isomers, highlighting the exquisite sensitivity of lipopeptide-reactive T cells for the natural DDM stereochemistry.

CD1 proteins present microbial lipids to T cells. Germline-encoded mycolyl lipid-reactive (GEM) T cells with conserved αβ T cell receptors (TCRs) recognize CD1b presenting mycobacterial mycolates. As the molecular basis underpinning TCR recognition of CD1b remains unknown, here we determine the structure of a GEM TCR bound to CD1b presenting glucose-6-O-monomycolate (GMM). The GEM TCR docks centrally above CD1b, whereby the conserved TCR α-chain extensively contacts CD1b and GMM. Through mutagenesis and study of T cells from tuberculosis patients, we identify a consensus CD1b footprint of TCRs present among GEM T cells. Using both the TCR α- and β-chains as tweezers to surround and grip the glucose moiety of GMM, GEM TCRs create a highly specific mechanism for recognizing this mycobacterial glycolipid., Germline-encoded mycolyl lipid-reactive (GEM) T cells recognize CD1b proteins presenting mycobacterial mycolates via their T-cell receptors (TCRs). Here, the authors present the structure of this interaction and provide a molecular basis for the co-recognition of CD1b and a mycobacterial glycolipid.

SummaryCD1a is expressed on Langerhans cells (LCs) and dendritic cells (DCs), where it mediates T cell recognition of glycolipid and lipopeptide antigens that contain either one or two alkyl chains. We demonstrate here that CD1a-restricted T cells can discriminate the peptide component of didehydroxymycobactin lipopeptides. Structure analysis of CD1a cocrystallized with a synthetic mycobactin lipopeptide at 2.8 Å resolution further reveals that the single alkyl chain is inserted deep within the A′ pocket of the groove, whereas its two peptidic branches protrude along the F′ pocket to the outer, α-helical surface of CD1a for recognition by the TCR. Remarkably, the cyclized lysine branch of the peptide moiety lies in the shallow F′ pocket in a conformation that closely mimics that of the alkyl chain in the CD1a-sulfatide structure. Thus, this structural study illustrates how a single chain lipid can be presented by CD1 and that the peptide moiety of the lipopeptide is recognized by the TCR.

Protected from host immune attack and antibiotic penetration by their unique cell envelope, mycobacterial pathogens cause devastating human diseases such as tuberculosis. Seamless coordination of cell growth with cell envelope elongation at the pole maintains this barrier. Unraveling this spatiotemporal regulation is a potential strategy for controlling mycobacterial infections. Our biochemical analysis previously revealed two functionally distinct membrane fractions in Mycobacterium smegmatis cell lysates: plasma membrane tightly associated with the cell wall (PM-CW) and a distinct fraction of pure membrane free of cell wall components (PMf). To provide further insight into the functions of these membrane fractions, we took the approach of comparative proteomics and identified more than 300 proteins specifically associated with the PMf, including essential enzymes involved in cell envelope synthesis such as a mannosyltransferase, Ppm1, and a galactosyltransferase, GlfT2. Furthermore, comparative lipidomics revealed the distinct lipid composition of the PMf, with specific association of key cell envelope biosynthetic precursors. Live-imaging fluorescence microscopy visualized the PMf as patches of membrane spatially distinct from the PM-CW and notably enriched in the pole of the growing cells. Taken together, our study provides the basis for assigning the PMf as a spatiotemporally distinct and metabolically active membrane domain involved in cell envelope biogenesis.

The mechanisms that lead to phenotypic antibacterial tolerance in bacteria remain poorly understood. We investigate whether changes in NaCl concentration toward physiologically higher values affect antibacterial efficacy against Mycobacterium tuberculosis (Mtb), the causal agent of human tuberculosis. Indeed, multiclass phenotypic antibacterial tolerance is observed during Mtb growth in physiologic saline. This includes changes in sensitivity to ethionamide, ethambutol, d-cycloserine, several aminoglycosides, and quinolones. By employing organism-wide metabolomic and lipidomic approaches combined with phenotypic tests, we identified a time-dependent biphasic adaptive response after exposure of Mtb to physiological levels of NaCl. A first rapid, extensive, and reversible phase was associated with changes in core and amino acid metabolism. In a second phase, Mtb responded with a substantial remodelling of plasma membrane and outer lipid membrane composition. We demonstrate that phenotypic tolerance at physiological concentrations of NaCl is the result of changes in plasma and outer membrane lipid remodeling and not changes in core metabolism. Altogether, these results indicate that physiologic saline-induced antibacterial tolerance is kinetically coupled to cell envelope changes and demonstrate that metabolic changes and growth arrest are not the cause of phenotypic tolerance observed in Mtb exposed to physiologic concentrations of NaCl. Importantly, this work uncovers a role for bacterial cell envelope remodeling in antibacterial tolerance, alongside well-documented allterations in respiration, metabolism, and growth rate.

When it comes to lipid diversity, no bacterial genus approaches Mycobacterium. In this issue of Cell Chemical Biology, Burbaud et al. (2016) provide a multi-genic working model for the biosynthesis of trehalose polyphleate (TPP), one of the largest known lipids in mycobacteria. They demonstrate that this lipid is made by diverse mycobacterial species, including those of medical importance.

In most adult humans, hepatitis B is a self-limiting disease leading to life-long protective immunity, which is the consequence of a robust adaptive immune response occurring weeks after hepatitis B virus (HBV) infection. Notably, HBV-specific T cells can be detected shortly after infection, but the mechanisms underlying this early immune priming and its consequences for subsequent control of viral replication are poorly understood. Using primary human and mouse hepatocytes and mouse models of transgenic and adenoviral HBV expression, we show that HBV-expressing hepatocytes produce endoplasmic reticulum (ER)-associated endogenous antigenic lipids including lysophospholipids that are generated by HBV-induced secretory phospholipases and that lead to activation of natural killer T (NKT) cells. The absence of NKT cells or CD1d or a defect in ER-associated transfer of lipids onto CD1d results in diminished HBV-specific T and B cell responses and delayed viral control in mice. NKT cells may therefore contribute to control of HBV infection through sensing of HBV-induced modified self-lipids.

Infections caused by biofilms are abundant and highly persistent, displaying phenotypic resistance to high concentrations of antimicrobials and modulating host immune systems. Tuberculosis (TB), caused by Mycobacterium tuberculosis , shares these qualities with biofilm infections. To identify genetic determinants of biofilm formation in M. tuberculosis , we performed a small-scale transposon screen using an in vitro pellicle biofilm assay. We identified five M. tuberculosis mutants that were reproducibly attenuated for biofilm production relative to that of the parent strain H37Rv. One of the most attenuated mutants is interrupted in pks1 , a polyketide synthase gene. When fused with pks15 , as in some M. tuberculosis isolates, pks1 contributes to synthesis of the immunomodulatory phenolic glycolipids (PGLs). However, in strains such as H37Rv with split pks15 and pks1 loci, PGL is not produced and pks1 has no previously defined role. We showed that pks1 complementation restores biofilm production independently of the known role of pks1 in PGL synthesis. We also assessed the relationship among biofilm formation, the pks15/1 genotype, and M. tuberculosis phylogeography. A global survey of M. tuberculosis clinical isolates revealed surprising sequence variability in the pks15/1 locus and substantial variation in biofilm phenotypes. Our studies identify novel M. tuberculosis genes that contribute to biofilm production, including pks1 . In addition, we find that the ability to make pellicle biofilms is common among M. tuberculosis isolates from throughout the world, suggesting that this trait is relevant to TB propagation or persistence.

Glucose monomycolate–loaded CD1b tetramers identify a subset of CD4+ T cells in patients with Mycobacterium tuberculosis infection., Microbial lipids activate T cells by binding directly to CD1 and T cell receptors (TCRs) or by indirect effects on antigen-presenting cells involving induction of lipid autoantigens, CD1 transcription, or cytokine release. To distinguish among direct and indirect mechanisms, we developed fluorescent human CD1b tetramers and measured T cell staining. CD1b tetramer staining of T cells requires glucose monomycolate (GMM) antigens, is specific for TCR structure, and is blocked by a recombinant clonotypic TCR comprised of TRAV17 and TRBV4-1, proving that CD1b–glycolipid complexes bind the TCR. GMM-loaded tetramers brightly stain a small subpopulation of blood-derived cells from humans infected with Mycobacterium tuberculosis, providing direct detection of a CD1b-reactive T cell repertoire. Polyclonal T cells from patients sorted with tetramers are activated by GMM antigens presented by CD1b. Whereas prior studies emphasized CD8+ and CD4−CD8− CD1b-restricted clones, CD1b tetramer-based studies show that nearly all cells express the CD4 co-receptor. These findings prove a cognate mechanism whereby CD1b–glycolipid complexes bind to TCRs. CD1b tetramers detect a natural CD1b-restricted T cell repertoire ex vivo with unexpected features, opening a new investigative path to study the human CD1 system.