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After two decades of the study of lipid antigens that activate CD1-restricted T cells, new studies show how autoreactive αβ 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 αβ 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., Competing Interests: Declaration of Competing Interest The authors declare possible perceived conflicts based on consultation for Pfizer, EnaraBio, and based on Brigham and Women's pending patents., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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 αβ 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., Competing Interests: Conflict of interest D. B. M. consults for Enara and Pfizer. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

CD1a is a monomorphic antigen-presenting molecule on dendritic cells that presents lipids to αβ 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 β-sheet floor of CD1a's antigen-binding cleft. Here, the γδ TCR co-recognises the CD1a heavy chain and β2 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 αβ TCRs that typically contact carried antigens, autoreactive γδ TCRs support geometrically diverse approaches to CD1a, as well as antigen independent recognition., (© 2022. The Author(s).)

As single-cell datasets grow in sample size, there is a critical need to characterize cell states that vary across samples and associate with sample attributes, such as clinical phenotypes. Current statistical approaches typically map cells to clusters and then assess differences in cluster abundance. Here we present co-varying neighborhood analysis (CNA), an unbiased method to identify associated cell populations with greater flexibility than cluster-based approaches. CNA characterizes dominant axes of variation across samples by identifying groups of small regions in transcriptional space-termed neighborhoods-that co-vary in abundance across samples, suggesting shared function or regulation. CNA performs statistical testing for associations between any sample-level attribute and the abundances of these co-varying neighborhood groups. Simulations show that CNA enables more sensitive and accurate identification of disease-associated cell states than a cluster-based approach. When applied to published datasets, CNA captures a Notch activation signature in rheumatoid arthritis, identifies monocyte populations expanded in sepsis and identifies a novel T cell population associated with progression to active tuberculosis., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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-β1-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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Disclosures: R.N. Cotton reported personal fees from MPM Capital outside the submitted work. G. Ogg reported grants from UCB outside the submitted work. No other disclosures were reported., (© 2021 Cotton et al.)

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.

The non-polymorphic nature of CD1 proteins creates a situation in which T cells with invariant T cell receptors (TCRs), like CD1d-specific NKT cells, are present in all humans. CD1b is an abundant protein on human dendritic cells that presents M. tuberculosis ( Mtb ) lipid antigens to T cells. Analysis of T cell clones suggested that semi-invariant TCRs exist in the CD1b system, but their prevalence in humans is not known. Here we used CD1b tetramers loaded with mycolic acid or glucose monomycolate to study polyclonal T cells from 150 Peruvian subjects. We found that CD1b tetramers loaded with mycolic acid or glucose monomycolate antigens stained TRAV1-2 + GEM T cells or TRBV4-1 + LDN5-like T cells in the majority of subjects tested, at rates ~10-fold lower than NKT cells. Thus, GEM T cells and LDN5-like T cells are a normal part of the human immune system. Unlike prior studies measuring MHC- or CD1b-mediated activation, this large-scale tetramer study found no significant differences in rates of CD1b tetramer-mycobacterial lipid staining of T cells among subjects with Mtb exposure, latent Mtb infection or active tuberculosis (TB) disease. In all subjects, including "uninfected" subjects, CD1b tetramer + T cells expressed memory markers at high levels. However, among controls with lower mycobacterial antigen exposure in Boston, we found significantly lower frequencies of T cells staining with CD1b tetramers loaded with mycobacterial lipids. These data link CD1b-specific T cell detection to mycobacterial exposure, but not TB disease status, which potentially explains differences in outcomes among CD1-based clinical studies, which used control subjects with low Mtb exposure., (Copyright © 2020 Lopez, Iwany, Suliman, Reijneveld, Ocampo, Jimenez, Calderon, Lecca, Murray, Moody and Van Rhijn.)

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., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Lyme disease is a common multisystem 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 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 APCs 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., (© 2019 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.

Competing Interests: The author declares no conflict of interest.

Mycobacterium tuberculosis synthesizes a thick cell wall comprised of mycolic acids (MA), which are foreign antigens for human T cells. T-cell clones from multiple donors were used to determine the fine specificity of MA recognition by human αβ T cells. Most CD1-presented lipid antigens contain large hydrophilic head groups comprised of carbohydrates or peptides that dominate patterns of T-cell specificity. MA diverges from the consensus antigen motif in that it lacks a head group. Using multiple forms of natural and synthetic MA and MA-specific T-cells with different T-cell receptors, we found that, unlike antigens with larger head groups, lipid length strongly controlled T-cell responses to MA. In addition, the three forms of MA that naturally occur in M. tuberculosis that differ in modifications on the lipid tail, differ in their potency for activating MA-specific T-cell clones. Thus, naturally occurring MA forms should be considered as separate, partly cross-reactive antigens. Two of the three forms of MA could be loaded onto human CD1b proteins, creating working CD1b-MA tetramers. The creation of CD1b-MA tetramers represents a new tool for future studies that track the effector functions and kinetics of MA-specific T-cells ex vivo., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

Single-cell analysis captures the heterogeneity of T-cell populations that target defined antigens. Human immunodeficiency virus (HIV) infection results in defects of antimycobacterial immunity, which remain poorly defined. We therefore recruited a small number of subjects, including those with latent and active M. tuberculosis infection, with or without concomitant HIV infection, and tracked the mycobacterial glycolipid-reactive T-cell repertoire by using CD1b tetramers. Glycolipid-reactive T cells expressed memory markers and the HIV coreceptors CD4 and CCR5; they were not detected in subjects with HIV-associated active M. tuberculosis infection. HIV infection may affect T cells that recognize mycobacterial glycolipids and influence immunity., (© The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

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., (© 2015 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The now-famous term "restriction" derived from experiments in which T cells from Donor A failed to recognize Ags presented by cells from Donor B. Restriction results from interdonor variation in MHC genes. Donor restriction dominates immunologists' thinking about the T cell response because it governs organ transplantation and hinders the discovery of disease-associated Ags. However, other T cells can be considered "donor unrestricted" because their targets, CD1a, CD1b, CD1c, CD1d, or MR1, are expressed in a similar form among all humans. A striking feature of donor unrestricted T cells is the expression of invariant TCRs with nearly species-wide distribution. In this article, we review new evidence that donor unrestricted T cells are common in humans. NKT cells, mucosa-associated invariant T cells, and germline-encoded mycolyl-reactive T cells operate outside of the familiar principles of the MHC system, providing a broader picture of T cell function and new opportunities for therapy., (Copyright © 2015 by The American Association of Immunologists, Inc.)

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.

For decades immunologists thought that T cells solely recognize peptides bound to Major Histocompatibility Complex (MHC) proteins. Therefore, nearly all medical technology that seeks to measure and manipulate human T cells during immunization, infection, allergy and autoimmune diseases relies on peptide antigens. Newer insights into αβ and γδ T cell activation by CD1 or MR1 proteins greatly expand the biochemical range of T cell antigens to include lipids and non-peptidic small molecules. Moving beyond in vitro studies, the recent development of human CD1a, CD1b, CD1c and MR1 tetramers allows direct and specific enumeration of lipid-reactive and small molecule-reactive T cells, providing a new approach to study of T cell-mediated diseases., (Copyright © 2014. Published by Elsevier Ltd.)

Lipids from mycobacteria can be presented to human T cells by group 1 CD1 Ag-presenting molecules (CD1a, CD1b, and CD1c). Group 1 CD1-restricted T cells are activated by lipid Ags presented by myeloid dendritic cells (DCs), after which they generate antibacterial effector functions, including IFN-γ secretion and cytolysis. Thus, mycobacterial lipids are being investigated as components of novel vaccines for mycobacterial infections. In this study we show that the mycobacterial lipid Ag C80 glucose-6-monomycolate can be delivered to human CD1b(+) DCs via targeted liposomal nanoparticles, leading to robust group 1 CD1-restricted activation of T cells. Targeting was achieved by decorating the liposomes with a high-affinity glycan ligand of sialic acid-binding Ig-like lectin (Siglec)-7, a siglec receptor expressed on DCs that mediates rapid endocytosis and transport of its cargo to lysosomes. An Ab to Siglec-7 completely blocked the binding of targeted liposomes to human monocyte-derived DCs (Mo-DCs), demonstrating their targeting specificity. Mo-DCs pulsed with targeted liposomes containing C80 glucose-6-monomycolate more potently activated a CD1b-restricted T cell line relative to Mo-DCs pulsed with free lipid Ag or antigenic liposomes without Siglec-7 ligand. These data suggest that the endocytic function of Siglec-7 can be exploited to deliver glycolipid Ags to their target cell and increase the efficiency of display to T cells., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Whereas research on CD1d has emphasized a few glycosyl ceramides, the broader family of four human CD1 antigen-presenting molecules binds hundreds of distinct self-lipids. Individual lipid types bind within CD1 grooves in different ways, such that they partially fill the groove, match the groove volume, or protrude substantially from the groove. These differing modes of binding can now be connected to differing immunological functions, as individual lipids can act as stimulatory antigens, inhibitory ligands, or space-filling scaffolds. Because each type of CD1 protein folds to produce antigen-binding grooves with differing sizes and shapes, CD1a, CD1b, CD1c, CD1d, and CD1e have distinct mechanisms of capturing self-lipids and exchanging them for foreign lipids. The size discrepancy between endogeneous lipids and groove volume is most pronounced for CD1b. Recent studies show that the large CD1b cavity can simultaneously bind two self-lipids, the antigen, and its scaffold lipid, which can be exchanged for one large bacterial lipid. In this review, we will highlight recent studies showing how cells regulate lipid antigen loading and the roles CD1 groove structures have in control of the presentation of chemically diverse lipids to T cells.

T cells autoreactive to the antigen-presenting molecule CD1a are common in human blood and skin, but the search for natural autoantigens has been confounded by background T cell responses to CD1 proteins and self lipids. After capturing CD1a-lipid complexes, we gently eluted ligands while preserving non-ligand-bound CD1a for testing lipids from tissues. CD1a released hundreds of ligands of two types. Inhibitory ligands were ubiquitous membrane lipids with polar head groups, whereas stimulatory compounds were apolar oils. We identified squalene and wax esters, which naturally accumulate in epidermis and sebum, as autoantigens presented by CD1a. The activation of T cells by skin oils suggested that headless mini-antigens nest within CD1a and displace non-antigenic resident lipids with large head groups. Oily autoantigens naturally coat the surface of the skin; thus, this points to a previously unknown mechanism of barrier immunity.

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.

Human T cell antigen receptors (TCRs) pair in millions of combinations to create complex and unique T cell repertoires for each person. Through the use of tetramers to analyze TCRs reactive to the antigen-presenting molecule CD1b, we detected T cells with highly stereotyped TCR α-chains present among genetically unrelated patients with tuberculosis. The germline-encoded, mycolyl lipid-reactive (GEM) TCRs had an α-chain bearing the variable (V) region TRAV1-2 rearranged to the joining (J) region TRAJ9 with few nontemplated (N)-region additions. Analysis of TCRs by high-throughput sequencing, binding and crystallography showed linkage of TCRα sequence motifs to high-affinity recognition of antigen. Thus, the CD1-reactive TCR repertoire is composed of at least two compartments: high-affinity GEM TCRs, and more-diverse TCRs with low affinity for CD1b-lipid complexes. We found high interdonor conservation of TCRs that probably resulted from selection by a nonpolymorphic antigen-presenting molecule and an immunodominant antigen.

CD1c is expressed with high density on human dendritic cells (DCs) and B cells, yet its antigen presentation functions are the least well understood among CD1 family members. Using a CD1c-reactive T cell line (DN6) to complete an organism-wide survey of M. tuberculosis lipids, we identified C32 phosphomycoketide (PM) as a previously unknown molecule and a CD1c-presented antigen. CD1c binding and presentation of mycoketide antigens absolutely required the unusual, mycobacteria-specific lipid branching patterns introduced by polyketide synthase 12 (pks12). Unexpectedly, one TCR responded to diversely glycosylated and unglycosylated forms of mycoketide when presented by DCs and B cells. Yet cell-free systems showed that recognition was mediated only by the deglycosylated phosphoantigen. These studies identify antigen processing of a natural bacterial antigen in the human CD1c system, indicating that cells act on glycolipids to generate a highly simplified neoepitope composed of a sugar-free phosphate anion. Using knowledge of this processed antigen, we generated human CD1c tetramers, and demonstrate that CD1c-PM complexes stain T cell receptors (TCRs), providing direct evidence for a ternary interaction among CD1c-lipid-TCR. Furthermore, PM-loaded CD1c tetramers detect fresh human T cells from peripheral blood, demonstrating a polyclonal response to PM antigens in humans ex vivo.

Transferring lipid antigens from membranes into CD1 antigen-presenting proteins represents a major molecular hurdle necessary for T-cell recognition. Saposins facilitate this process, but the mechanisms used are not well understood. We found that saposin B forms soluble saposin protein-lipid complexes detected by native gel electrophoresis that can directly load CD1 proteins. Because saposin B must bind lipids directly to function, we found it could not accommodate long acyl chain containing lipids. In contrast, saposin C facilitates CD1 lipid loading in a different way. It uses a stable, membrane-associated topology and was capable of loading lipid antigens without forming soluble saposin-lipid antigen complexes. These findings reveal how saposins use different strategies to facilitate transfer of structurally diverse lipid antigens.

Although CD1 proteins are known to present mycobacterial lipid antigens to T cells, there is little understanding of the in vivo behavior of T cells restricted by CD1a, CD1b and CD1c, and the relative immunogenicity and immunodominance of individual lipids within the total array of lipids that comprise a bacterium. Because bovines express multiple CD1 proteins and are natural hosts of Mycobacterium bovis and Mycobacterium avium paratuberculosis (MAP), we used them as a new animal model of CD1 function. Here, we report the surprisingly divergent responses against lipids produced by these two pathogens during infection. Despite considerable overlap in lipid content, only three out of 69 animals cross-react with M. bovis and MAP total lipid preparations. The unidentified immunodominant compound of M. bovis is a hydrophilic compound, whereas the immunodominant lipid of MAP is presented by CD1b and was identified as glucose monomycolate (GMM). The preferential recognition of GMM antigen by MAP-infected cattle may be explained by the higher expression of GMM by MAP than by M. bovis. The bacterial species-specific nature of the CD1-restricted, adaptive T-cell response affects the approach to development of lipid based immunodiagnostic tests.

Mycobacterium tuberculosis survival in cells requires mycobactin siderophores. Recently, the search for lipid antigens presented by the CD1a antigen-presenting protein led to the discovery of a mycobactin-like compound, dideoxymycobactin (DDM). Here we synthesize DDMs using solution phase and solid phase peptide synthesis chemistry. Comparison of synthetic standards to natural mycobacterial mycobactins by nuclear magnetic resonance and mass spectrometry allowed identification of an unexpected alpha-methyl serine unit in natural DDM. This finding further distinguishes these pre-siderophores as foreign compounds distinct from conventional peptides, and we provide evidence that this chemical variation influences the T cell response. One synthetic DDM recapitulated natural structures and potently stimulated T cells, making it suitable for patient studies of CD1a in infectious disease. DDM analogs differing in the stereochemistry of their butyrate or oxazoline moieties were not recognized by human T cells. Therefore, we conclude that T cells show precise specificity for both arms of the peptide, which are predicted to lie at the CD1a-T cell receptor interface.

CD1-restricted T cells can be activated by diverse lipids derived from mammals, bacteria and protozoa. Certain lipids function as antigens, which bind to CD1 proteins and contact T cell antigen receptors. Other lipids activate CD1-restricted T cells by functioning as adjuvants. By stimulating Toll-like receptors on antigen-presenting cells, these adjuvants alter cytokine secretion, lipid antigen synthesis and CD1 protein translation. Delineation of the separate mechanisms by which adjuvants and antigens activate CD1-restricted T cells is leading to new hypotheses about the functions of individual CD1 proteins during the transition from innate to acquired immune responses.

The most well-known molecular paradigm of antigen recognition by T cells involves partial digestion of proteins to generate small peptides, which bind to major histocompatibility complex (MHC) proteins. Recent studies of CD1, an MHC class I homolog encoded outside the MHC, have revealed that it presents diverse glycolipids to T cells. The molecular mechanism for lipid antigen recognition involves insertion of the lipid portion of antigens into a hydrophobic groove to form CD1-lipid complexes, which contact T-cell receptors (TCRs). Here, we examine the known antigen structures presented by CD1, the majority of which have sugar moieties that are capable of interacting with TCRs. Recognition of carbohydrate epitopes is precise, and lipid-reactive T cells alter systemic immune responses in models of infectious and autoimmune disease. These findings provide a previously unrecognized mechanism by which the cellular immune system can recognize alterations in many types of carbohydrate structures.

The discovery of the CD1 antigen-presenting system reveals that T cells survey the lipid content of target cells via T-cell receptor (TCR) contact with CD1 bound to lipids, glycolipids and small molecules. Recently, CD1 proteins have been found to present mycobacterial lipopeptides that are involved in scavenging iron from infected cells. The mechanism of lipopeptide antigen presentation by CD1 involves the anchoring of antigens in the hydrophobic binding groove, resulting in exposure of the peptide moiety for TCR contact. These findings expand the range of known antigens for T cells and raise the intriguing possibility that CD1, similar to MHC class I and II molecules, enables T cells to discriminate among peptide sequences.

Molecular studies have shown that CD1 proteins present self and foreign lipid Ags to T cells, but the possible roles of CD1 in human autoimmune diseases in vivo are not known, especially for the group 1 CD1 isoforms (CD1a, CD1b, and CD1c). To investigate the hypothesis that CD1-restricted T cells might be activated and home to target tissues involved in Hashimoto's thyroiditis and Graves' disease, we performed ex vivo analysis of lymphocytes from peripheral blood and autoinflammatory lesions of thyroid tissue. Immunofluorescence analysis identified two types of CD1-expressing APCs in inflamed thyroid tissues. CD1a, CD1b, and CD1c were expressed on CD83+ dendritic cells, and CD1c was expressed on an abundant population of CD20+ IgD+ CD23- CD38- B cells that selectively localized to the mantle zone of lymphoid follicles within the thyroid gland. CD1c-restricted, glycolipid-specific T cells could not be detected in the peripheral blood, but were present in polyclonal lymphocyte populations isolated from affected thyroid glands. In addition, polyclonal thyroid-derived lymphocytes and short-term T cell lines were found to recognize and lyse targets in a CD1a- or CD1c-dependent manner. The targeting of CD1-restricted T cells and large numbers of CD1-expressing APCs to the thyroid gland during the early stages of autoimmune thyroiditis suggests a possible effector function of CD1-restricted T cells in tissue destruction and point to a new model of organ-specific autoimmune disease involving lipid Ag presentation.

CD1c-mediated T cells are activated by a mycobacterial phospholipid antigen whose carbohydrate structure precisely corresponds to mammalian mannosyl beta-1-phosphodolichol (MPD), but contains an unusual lipid moiety. Here, we show that this T cell antigen is a member of a family of branched, alkane lipids that vary in length (C30-34) and are produced by medically important mycobacteria such as M. tuberculosis and M. bovis Bacille-Calmette-Guerin. The alkane moiety distinguished these mycobacterial lipid antigens from mammalian MPDs and was necessary for activation of CD1c-restricted T cells, but could not be accounted for by any known lipid biosynthetic pathway. Metabolic labeling and mass spectrometric analyses suggested a mechanism for elongating lipids using alternating C2 and C3 units, rather than C5 isopentenyl pyrophosphate. Inspection of the M. tuberculosis genome identified one candidate gene, pks12, which was predicted to encode the largest protein in M. tuberculosis, consisting of 12 catalytic domains that correspond to key steps in the proposed pathway. Genetic deletion and complementation showed that Pks12 was necessary for antigen production, but did not affect synthesis of true isoprenols. These studies establish the genetic and enzymatic basis for a previously unknown type of polyketide, designated mycoketide, which contains a lipidic pathogen-associated molecular pattern.

In addition to NK T cells expressing invariant Valpha14 or Valpha24 T cell receptors (TCRs), the CD1d-restricted T cell repertoire is comprised of T cells with diverse TCRs that mediate inflammation during autoimmune and infectious disease. Here we describe the isolation of human Valpha24(-) T cells that are activated by antigen and CD1d. Mass spectrometric and NMR studies revealed that the stimulatory compounds were neither peptidic nor lipidic but instead were composed of sulfur and aromatic hydrocarbon rings, corresponding to the general structure of phenyl pentamethyldihydrobenzofuran sulfonates. Studies of the molecular mechanism of T cell activation showed that a clonotypic Valpha2/Vbeta21 TCR transmitted activating signals, which were highly specific for hydroxylation and methylation patterns at the terminal structures of stimulatory compounds. These studies provide evidence for noninvariant CD1d-restricted T cells in humans and identify the complete molecular structure of a nonlipidic small molecule that activates T cells through an alphabeta TCR.

Unlike major histocompatibility proteins, which bind peptides, CD1 proteins display lipid antigens to T cells. Here, we report that CD1a presents a family of previously unknown lipopeptides from Mycobacterium tuberculosis, named didehydroxymycobactins because of their structural relation to mycobactin siderophores. T cell activation was mediated by the alphabeta T cell receptors and was specific for structure of the acyl and peptidic components of these antigens. These studies identify a means of intracellular pathogen detection and identify lipopeptides as a biochemical class of antigens for T cells, which, like conventional peptides, have a potential for marked structural diversity.

Little is known about the antigen specificity of CD1d-restricted T cells, except that they frequently recognize CD1d-expressing antigen-presenting cells in the absence of exogenous antigen. We previously demonstrated that the 24.8.A iNKT cell hybridoma was broadly reactive with CD1d-transfected cell lines and recognized the polar lipid fraction of a tumor cell extract. In the present study, the antigen recognized by the 24.8.A iNKT cell hybridoma was purified to homogeneity and identified as palmitoyl-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1 PE). The 24.8.A iNKT cell hybridoma recognized synthetic 16:0-18:1[cis] PE, confirming that this phospholipid is antigenic. Recognition correlated with the degree of unsaturation of the acyl chains. Using a panel of synthetic PEs, the 24.8.A iNKT cell hybridoma was shown to be activated by PEs that contained at least one unsaturated acyl chain. The configuration of the double bonds was important, as the 24.8.A iNKT cell hybridoma recognized unsaturated acyl chains in the cis, but not the trans, configuration. PEs with multiple double bonds were recognized better than those with a single double bond, and increasing acyl chain unsaturation correlated with increased binding of PE to CD1d. These data illustrate the potential importance of the acyl chain structure for phospholipid antigen binding to CD1d.

CD1 proteins mediate T cell activation in response to self and foreign lipids, including lipid antigens from the intracellular pathogen Mycobacterium tuberculosis. During natural infections, myeloid cells migrate to sites of infection and use microbial pattern recognition receptors to internalize live bacteria and lipid antigens into the endosomal network. New studies show that certain CD1 proteins are particularly receptive to binding lipid antigens in the low pH environment of endosomes. Therefore, the endosomal network may represent a depot for concentrating and then selectively presenting exogenous foreign lipid antigens to T cells.

CD1-restricted presentation of lipid or glycolipid antigens derived from Mycobacterium tuberculosis has been demonstrated by in vitro experiments using cultured T-cell lines. In the present work, the frequency of T-cell responses to natural mycobacterial lipids was analyzed in ex vivo studies of peripheral blood lymphocytes from human patients with pulmonary tuberculosis, from asymptomatic individuals with known contact with M. tuberculosis documented by conversion of their tuberculin skin tests, and from healthy tuberculin skin test-negative individuals or individuals vaccinated with Mycobacterium bovis BCG. Proliferation and gamma interferon enzyme-linked immunospot assays using peripheral blood lymphocytes and autologous CD1(+) immature dendritic cells revealed that T cells from asymptomatic M. tuberculosis-infected donors responded with significantly greater magnitude and frequency to mycobacterial lipid antigen preparations than lymphocytes from uninfected healthy donors. By use of these methods, lipid-antigen-specific proliferative responses were minimally detectable or absent in blood samples from patients with active tuberculosis prior to chemotherapy but became detectable in blood samples drawn 2 weeks after the start of treatment. Lipid antigen-reactive T cells were detected predominantly in the CD4-enriched T-cell fractions of circulating lymphocytes, and anti-CD1 antibody blocking experiments confirmed the CD1 restriction of these T-cell responses. Our results provide further support for the hypothesis that lipid antigens serve as targets of the recall response to M. tuberculosis, and they indicate that CD1-restricted T cells responding to these antigens comprise a significant portion of the circulating pool of M. tuberculosis-reactive T cells in healthy individuals with previous exposure to M. tuberculosis.

T cells are well known to recognize peptide antigens presented by major histocompatibility (MHC) class I or class II molecules. More recently, the CD1 family of antigen-presenting molecules has been shown to present both mammalian and microbial glycolipid antigens for specific recognition by T cells. Human CD1c proteins mediate T cell recognition of polyisoprenyl glycolipids, evolutionarily conserved phosphoglycolipids, which function in glycan synthesis pathways. This family of antigenic molecules is particularly attractive for the study of the molecular features that control T cell recognition of self and foreign glycolipids because natural polyisoprenols from mammals, fungi, protozoa, mycobacteria and eubacteria differ in structure. Moreover, these naturally occurring structural differences can influence their recognition by CDlc-restricted T cells. This review of the structural diversity and evolutionary relationships of polyisoprenoid glycolipids emphasizes those features of polyisoprenyl glycolipid biosynthesis that are relevant to their functions as targets of CD1-mediated T cell responses.

Four human CD1 isoforms (CD1a, -b,-c and -d) are now known to be antigen presenting molecules with the unique ability to present lipid antigens to T cells. CD1b and CD1d are found in acidic, late endocytic compartments, whereas CD1a and CD1c molecules accumulate at the plasma membrane and in early endosomes. Consistent with their differences in intracellular localization, most studies show antigen presentation by CD1b/CD1d to be dependent on endosomal acidification while CD1a/CD1c mediated antigen presentation is not. Taken together, recent advances in the analysis of CD1 molecules reinforce the hypothesis that the different CD1 isoforms are specialized to survey the lipid content of distinct intracellular compartments. This may help to explain the duplication and diversification of CD1 genes in humans and other mammalian species., (Copyright 2000 Academic Press.)

T cells recognize microbial glycolipids presented by CD1 proteins, but there is no information regarding the generation of natural glycolipid antigens within infected tissues. Therefore, we determined the molecular basis of CD1b-restricted T cell recognition of mycobacterial glycosylated mycolates, including those produced during tissue infection in vivo. Transfection of the T cell receptor (TCR) alpha and beta chains from a glucose monomycolate (GMM)-specific T cell line reconstituted GMM recognition in TCR-deficient T lymphoblastoma cells. This TCR-mediated response was highly specific for natural mycobacterial glucose-6-O-(2R, 3R) monomycolate, including the precise structure of the glucose moiety, the stereochemistry of the mycolate lipid, and the linkage between the carbohydrate and the lipid. Mycobacterial production of antigenic GMM absolutely required a nonmycobacterial source of glucose that could be supplied by adding glucose to media at concentrations found in mammalian tissues or by infecting tissue in vivo. These results indicate that mycobacteria synthesized antigenic GMM by coupling mycobacterial mycolates to host-derived glucose. Specific T cell recognition of an epitope formed by interaction of host and pathogen biosynthetic pathways provides a mechanism for immune response to those pathogenic mycobacteria that have productively infected tissues, as distinguished from ubiquitous, but innocuous, environmental mycobacteria.

The discovery of the CD1 antigen presentation pathway has expanded the spectrum of T-cell antigens to include lipids, but the range of natural lipid antigens and functions of CD1-restricted T cells in vivo remain poorly understood. Here we show that the T-cell antigen receptor and the CD1c protein mediate recognition of an evolutionarily conserved family of isoprenoid glycolipids whose members include essential components of protein glycosylation and cell-wall synthesis pathways. A CD1c-restricted, mycobacteria-specific T-cell line recognized two previously unknown mycobacterial hexosyl-1-phosphoisoprenoids and structurally related mannosyl-beta1-phosphodolichols. Responses to mannosyl-beta1-phosphodolichols were common among CD1c-restricted T-cell lines and peripheral blood T lymphocytes of human subjects recently infected with M. tuberculosis, but were not seen in naive control subjects. These results define a new class of broadly distributed lipid antigens presented by the CD1 system during infection in vivo and suggest an immune mechanism for recognition of senescent or transformed cells that are known to have altered dolichol lipids.

CD1 is a family of cell surface glycoproteins that are related in structure and evolutionary origin to the major histocompatibility complex (MHC)-encoded antigen-presenting molecules. In contrast to MHC-encoded antigen-presenting molecules, CD1 binds and presents lipid and glycolipid antigens for specific recognition by T cell antigen receptors. Recent work shows that several CD1 family members colocalize with MHC class II proteins within the endocytic system of antigen-presenting cells. Detailed studies of the intracellular trafficking of CD1 proteins reveal new mechanisms controlling delivery of antigen-presenting molecules to particular compartments within cells. The combination of overlapping yet distinct trafficking routes for the various CD1 family members, combined with emerging information on the heterogeneity of CD1-presented lipid antigens, suggest a model whereby different members of the CD1 family could present antigens that occur in various cellular compartments. Furthermore, the CD1 family as a group may present antigens from pathogens that are not normally accessible to or efficiently surveyed by the MHC Class I or II systems. The discovery of this third pathway for antigen presentation, together with the appreciation of a previously unrecognized universe of nonpeptide lipid antigens for T cell responses, are likely to have broad implications for our understanding of the cell-mediated immune response and its role in health and disease.