RESUMEN
The CD1 system binds lipid antigens for display to T cells. Here, we solved lipidomes for the four human CD1 antigen-presenting molecules, providing a map of self-lipid display. Answering a basic question, the detection of >2,000 CD1-lipid complexes demonstrates broad presentation of self-sphingolipids and phospholipids. Whereas peptide antigens are chemically processed, many lipids are presented in an unaltered form. However, each type of CD1 protein differentially edits the self-lipidome to show distinct capture motifs based on lipid length and chemical composition, suggesting general antigen display mechanisms. For CD1a and CD1d, lipid size matches the CD1 cleft volume. CD1c cleft size is more variable, and CD1b is the outlier, where ligands and clefts show an extreme size mismatch that is explained by uniformly seating two small lipids in one cleft. Furthermore, the list of compounds that comprise the integrated CD1 lipidome supports the ongoing discovery of lipid blockers and antigens for T cells.
Asunto(s)
Antígenos CD1 , Lípidos , Humanos , Presentación de Antígeno , Antígenos CD1/química , Antígenos CD1/metabolismo , Lipidómica , Lípidos/química , Linfocitos T , Secuencias de AminoácidosRESUMEN
Expressed on epidermal Langerhans cells, CD1a presents a range of self-lipid antigens found within the skin; however, the extent to which CD1a presents microbial ligands from bacteria colonizing the skin is unclear. Here we identified CD1a-dependent T cell responses to phosphatidylglycerol (PG), a ubiquitous bacterial membrane phospholipid, as well as to lysylPG, a modified PG, present in several Gram-positive bacteria and highly abundant in Staphylococcus aureus. The crystal structure of the CD1a-PG complex showed that the acyl chains were buried within the A'- and F'-pockets of CD1a, while the phosphoglycerol headgroup remained solvent exposed in the F'-portal and was available for T cell receptor contact. Using lysylPG and PG-loaded CD1a tetramers, we identified T cells in peripheral blood and in skin that respond to these lipids in a dose-dependent manner. Tetramer+CD4+ T cell lines secreted type 2 helper T cell cytokines in response to phosphatidylglycerols as well as to co-cultures of CD1a+ dendritic cells and Staphylococcus bacteria. The expansion in patients with atopic dermatitis of CD4+ CD1a-(lysyl)PG tetramer+ T cells suggests a response to lipids made by bacteria associated with atopic dermatitis and provides a link supporting involvement of PG-based lipid-activated T cells in atopic dermatitis pathogenesis.
Asunto(s)
Dermatitis Atópica , Humanos , Piel , Células de Langerhans , Antígenos CD1 , Autoantígenos/metabolismo , Staphylococcus/metabolismo , FosfatidilglicerolesRESUMEN
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.
Asunto(s)
Memoria Inmunológica , Mycobacterium tuberculosis/inmunología , Células Th17/inmunología , Tuberculosis Pulmonar/inmunología , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Niño , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Predisposición Genética a la Enfermedad , Técnicas de Genotipaje , Humanos , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis/aislamiento & purificación , Perú , RNA-Seq , Factores Sexuales , Análisis de la Célula Individual , Factores Socioeconómicos , Tuberculosis Pulmonar/sangre , Tuberculosis Pulmonar/genética , Tuberculosis Pulmonar/microbiología , Adulto JovenRESUMEN
Mycobacterium leprae causes leprosy and is unique among mycobacterial diseases in producing peripheral neuropathy. This debilitating morbidity is attributed to axon demyelination resulting from direct interaction of the M. 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 of M. leprae-induced nerve damage. We find that demyelination and axonal damage are not directly initiated by M. leprae but by infected macrophages that patrol axons; demyelination occurs in areas of intimate contact. PGL-1 confers this neurotoxic response on macrophages: macrophages infected with M. marinum-expressing 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 to M. leprae PGL-1 in initiating nerve damage in leprosy.
Asunto(s)
Antígenos Bacterianos/metabolismo , Modelos Animales de Enfermedad , Glucolípidos/metabolismo , Lepra/microbiología , Lepra/patología , Macrófagos/inmunología , Mycobacterium leprae/fisiología , Animales , Axones/metabolismo , Axones/patología , Enfermedades Desmielinizantes , Larva/crecimiento & desarrollo , Lepra/inmunología , Mycobacterium marinum/metabolismo , Vaina de Mielina/química , Vaina de Mielina/metabolismo , Vaina de Mielina/ultraestructura , Neuroglía/metabolismo , Neuroglía/patología , Óxido Nítrico/metabolismo , Pez CebraRESUMEN
The hallmark function of αß 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.
Asunto(s)
Antígenos CD1/inmunología , Autoantígenos/inmunología , Autoinmunidad/inmunología , Glicoproteínas/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Presentación de Antígeno/inmunología , Humanos , Lípidos/inmunología , Activación de Linfocitos/inmunologíaRESUMEN
Non-coding genetic variants may cause disease by modulating gene expression. However, identifying these expression quantitative trait loci (eQTLs) is complicated by differences in gene regulation across fluid functional cell states within cell types. These states-for example, neurotransmitter-driven programs in astrocytes or perivascular fibroblast differentiation-are obscured in eQTL studies that aggregate cells1,2. Here we modelled eQTLs at single-cell resolution in one complex cell type: memory T cells. Using more than 500,000 unstimulated memory T cells from 259 Peruvian individuals, we show that around one-third of 6,511 cis-eQTLs had effects that were mediated by continuous multimodally defined cell states, such as cytotoxicity and regulatory capacity. In some loci, independent eQTL variants had opposing cell-state relationships. Autoimmune variants were enriched in cell-state-dependent eQTLs, including risk variants for rheumatoid arthritis near ORMDL3 and CTLA4; this indicates that cell-state context is crucial to understanding potential eQTL pathogenicity. Moreover, continuous cell states explained more variation in eQTLs than did conventional discrete categories, such as CD4+ versus CD8+, suggesting that modelling eQTLs and cell states at single-cell resolution can expand insight into gene regulation in functionally heterogeneous cell types.
Asunto(s)
Predisposición Genética a la Enfermedad , Células T de Memoria , Sitios de Carácter Cuantitativo , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad/genética , Humanos , Células T de Memoria/inmunología , Células T de Memoria/metabolismo , Perú , Sitios de Carácter Cuantitativo/genéticaRESUMEN
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.
Asunto(s)
Subgrupos de Linfocitos T/inmunología , Animales , Presentación de Antígeno , Antígenos CD1/química , Antígenos CD1/metabolismo , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Ratones , Modelos Inmunológicos , Estructura Molecular , Células T Asesinas Naturales/inmunología , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
A central paradigm in αß T cell-mediated immunity is the simultaneous co-recognition of antigens and antigen-presenting molecules by the αß 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 αß T cells indirectly sense self antigens that are bound to an antigen-presenting molecule.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos CD1/inmunología , Autoantígenos/inmunología , Lípidos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Línea Celular , Línea Celular Tumoral , Células HEK293 , Humanos , Células Jurkat , Ligandos , Unión ProteicaRESUMEN
To broadly measure the spectrum of cellular self-antigens for natural killer T cells (NKT), we developed a sensitive lipidomics system to analyze lipids trapped between CD1d and NKT T cell receptors (TCRs). We captured diverse antigen complexes formed in cells from natural endogenous lipids, with or without inducing endoplasmic reticulum (ER) stress. After separating protein complexes with no, low, or high CD1d-TCR interaction, we eluted lipids to establish the spectrum of self-lipids that facilitate this interaction. Although this unbiased approach identified fifteen molecules, they clustered into only two related groups: previously known phospholipid antigens and unexpected neutral lipid antigens. Mass spectrometry studies identified the neutral lipids as ceramides, deoxyceramides, and diacylglycerols, which can be considered headless lipids because they lack polar headgroups that usually form the TCR epitope. The crystal structure of the TCR-ceramide-CD1d complex showed how the missing headgroup allowed the TCR to predominantly contact CD1d, supporting a model of CD1d autoreactivity. Ceramide and related headless antigens mediated physiological TCR binding affinity, weak NKT cell responses, and tetramer binding to polyclonal human and mouse NKT cells. Ceramide and sphingomyelin are oppositely regulated components of the "sphingomyelin cycle" that are altered during apoptosis, transformation, and ER stress. Thus, the unique molecular link of ceramide to NKT cell response, along with the recent identification of sphingomyelin blockers of NKT cell activation, provide two mutually reinforcing links for NKT cell response to sterile cellular stress conditions.
Asunto(s)
Antígenos CD1d , Lipidómica , Células T Asesinas Naturales , Receptores de Antígenos de Linfocitos T , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/metabolismo , Antígenos CD1d/inmunología , Antígenos CD1d/metabolismo , Animales , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Ratones , Lipidómica/métodos , Humanos , Autoantígenos/inmunología , Autoantígenos/metabolismo , Ceramidas/metabolismo , Ceramidas/inmunología , Lípidos/química , Lípidos/inmunología , Estrés del Retículo Endoplásmico/inmunologíaRESUMEN
A quarter of humanity is estimated to have been exposed to Mycobacterium tuberculosis (Mtb) with a 5-10% risk of developing tuberculosis (TB) disease. Variability in responses to Mtb infection could be due to host or pathogen heterogeneity. Here, we focused on host genetic variation in a Peruvian population and its associations with gene regulation in monocyte-derived macrophages and dendritic cells (DCs). We recruited former household contacts of TB patients who previously progressed to TB (cases, n = 63) or did not progress to TB (controls, n = 63). Transcriptomic profiling of monocyte-derived DCs and macrophages measured the impact of genetic variants on gene expression by identifying expression quantitative trait loci (eQTL). We identified 330 and 257 eQTL genes in DCs and macrophages (False Discovery Rate (FDR) < 0.05), respectively. Four genes in DCs showed interaction between eQTL variants and TB progression status. The top eQTL interaction for a protein-coding gene was with FAH, the gene encoding fumarylacetoacetate hydrolase, which mediates the last step in mammalian tyrosine catabolism. FAH expression was associated with genetic regulatory variation in cases but not controls. Using public transcriptomic and epigenomic data of Mtb-infected monocyte-derived dendritic cells, we found that Mtb infection results in FAH downregulation and DNA methylation changes in the locus. Overall, this study demonstrates effects of genetic variation on gene expression levels that are dependent on history of infectious disease and highlights a candidate pathogenic mechanism through pathogen-response genes. Furthermore, our results point to tyrosine metabolism and related candidate TB progression pathways for further investigation.
Asunto(s)
Células Dendríticas , Macrófagos , Mycobacterium tuberculosis , Sitios de Carácter Cuantitativo , Tuberculosis , Humanos , Perú , Tuberculosis/genética , Tuberculosis/microbiología , Macrófagos/metabolismo , Macrófagos/microbiología , Mycobacterium tuberculosis/patogenicidad , Mycobacterium tuberculosis/genética , Femenino , Células Dendríticas/metabolismo , Masculino , Adulto , Predisposición Genética a la Enfermedad , Variación Genética , Regulación de la Expresión Génica , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Perfilación de la Expresión GénicaRESUMEN
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.
Asunto(s)
Antígenos CD1/inmunología , Autoantígenos/inmunología , Lípidos/inmunología , Piel/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Presentación de Antígeno , Antígenos CD1/genética , Autoantígenos/química , Autoantígenos/aislamiento & purificación , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Lípidos/química , Lípidos/aislamiento & purificación , Activación de Linfocitos , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes/genética , Relación Estructura-ActividadRESUMEN
The CD1 family of antigen-presenting molecules adopt a major histocompatibility complex class I (MHC-I) fold. Whereas MHC molecules present peptides, the CD1 family has evolved to bind self- and foreign-lipids. The CD1 family of antigen-presenting molecules comprises four members-CD1a, CD1b, CD1c, and CD1d-that differ in their architecture around the lipid-binding cleft, thereby enabling diverse lipids to be accommodated. These CD1-lipid complexes are recognized by T cell receptors (TCRs) expressed on T cells, either through dual recognition of CD1 and lipid or in a new model whereby the TCR directly contacts CD1, thereby triggering an immune response. Chemical syntheses of lipid antigens, and analogs thereof, have been crucial in understanding the underlying specificity of T cell-mediated lipid immunity. This review will focus on our current understanding of how TCRs interact with CD1-lipid complexes, highlighting how it can be fundamentally different from TCR-MHC-peptide corecognition.
RESUMEN
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.
Asunto(s)
Antígenos CD1/inmunología , Infecciones por Mycobacterium/inmunología , Mycobacterium/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Secuencia de Bases , Células Clonales , Cristalografía por Rayos X , Citometría de Flujo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Infecciones por Mycobacterium/microbiología , ARN/química , ARN/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Subgrupos de Linfocitos T/citología , Linfocitos T/citologíaRESUMEN
Mycobacterial plasma membrane, together with the peptidoglycan-arabinogalactan cell wall and waxy outer membrane, creates a robust permeability barrier against xenobiotics. The fact that several antituberculosis drugs target plasma membrane-embedded enzymes underscores the importance of the plasma membrane in bacterial physiology and pathogenesis. Nevertheless, its accurate phospholipid composition remains undefined, with conflicting reports on the abundance of phosphatidylinositol mannosides (PIMs), physiologically important glycolipids evolutionarily conserved among mycobacteria and related bacteria. Some studies indicate cardiolipin, phosphatidylethanolamine, and phosphatidylinositol as dominant structural phospholipids. Conversely, some suggest PIMs dominate the plasma membrane. A striking example of the latter is the use of reverse micelle extraction, showing diacyl phosphatidylinositol dimannoside (Ac2PIM2) as the most abundant phospholipid in a model organism, Mycobacterium smegmatis. Our recent work reveals a rapid response mechanism to membrane-fluidizing stress in mycobacterial plasma membrane: monoacyl phosphatidylinositol dimannoside and hexamannoside (AcPIM2 and AcPIM6) are converted to diacyl forms (Ac2PIM2 and Ac2PIM6). Given the dynamic nature of PIMs, we aimed to resolve the conflicting data in the literature. We show that unstressed M. smegmatis lacks an Ac2PIM2-dominated plasma membrane. Ac2PIM2 accumulation is induced by experimental conditions involving sodium docusate, a component of the reverse micellar solution. Using chemically synthesized PIMs as standards, we accurately quantified phospholipid ratio in M. smegmatis through liquid chromatography-mass spectrometry, revealing that mycobacterial plasma membrane is dominated by cardiolipin, phosphatidylethanolamine, and phosphatidylinositol. PIMs are quantitatively minor but responsive to environmental stresses in M. smegmatis. Our study paves the way for accurate modeling of mycobacterial plasma membrane.
Asunto(s)
Mycobacterium smegmatis , Fosfatidilinositoles , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/efectos de los fármacos , Fosfatidilinositoles/metabolismo , Fosfatidilinositoles/química , Detergentes/química , Detergentes/farmacología , Membrana Celular/metabolismoRESUMEN
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 (â¼C34) to T cells; however, the structural characteristics of this presentation and recognition are unclear. Here, we report the 1.9 Å resolution binary crystal structure of CD1b presenting a self-phosphatidylinositol-C34:1 and an endogenous scaffold lipid. Moreover, we also determined the 2.4 Å structure of CD1b-phosphatidylinositol complexed to an autoreactive αß TCR, BC8B. We show that the TCR docks above CD1b and directly contacts the presented antigen, selecting for both the phosphoinositol headgroup and glycerol neck region via antigen remodeling within CD1b and allowing lateral escape of the inositol moiety through a channel formed by the TCR α-chain. Furthermore, through alanine scanning mutagenesis and surface plasmon resonance, we identified key CD1b residues mediating this interaction, with Glu-80 abolishing TCR binding. We in addition define a role for both CD1b α1 and CD1b α2 molecular domains in modulating this interaction. These findings suggest that the BC8B TCR contacts both the presented phospholipid and the endogenous scaffold lipid via a dual mechanism of corecognition. Taken together, these data expand our understanding into the molecular mechanisms of CD1b-mediated T-cell autoreactivity.
Asunto(s)
Presentación de Antígeno , Antígenos CD1 , Fosfatidilinositoles , Receptores de Antígenos de Linfocitos T , Linfocitos T , Antígenos CD1/metabolismo , Fosfolípidos/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismoRESUMEN
In addition to serving as the main physical barrier with the outside world, human skin is abundantly infiltrated with resident αß T cells that respond differently to self, infectious, microbiome, and noxious stimuli. To study skin T cells during infection and inflammation, experimental biologists track T-cell surface phenotypes and effector functions, which are often interpreted with the untested assumption that MHC proteins and peptide antigens drive measured responses. However, a broader perspective is that CD1 proteins also activate human T cells, and in skin, Langerhans cells (LCs) are abundant antigen presenting cells that express extremely high levels of CD1a. The emergence of new experimental tools, including CD1a tetramers carrying endogenous lipids, now show that CD1a-reactive T cells comprise a large population of resident T cells in human skin. Here, we review studies showing that skin-derived αß T cells directly recognize CD1a proteins, and certain bound lipids, such as contact dermatitis allergens, trigger T-cell responses. Other natural skin lipids inhibit CD1a-mediated T-cell responses, providing an entry point for the development of therapeutic lipids that block T-cell responses. Increasing evidence points to a distinct role of CD1a in type 2 and 22 T-cell responses, providing new insights into psoriasis, contact dermatitis, and other T-cell-mediated skin diseases.
Asunto(s)
Dermatitis por Contacto , Enfermedades de la Piel , Humanos , Linfocitos T , Piel , Lípidos , Antígenos CD1/metabolismoRESUMEN
Mucosal-associated invariant T (MAIT) cells are innate-like T cells that are highly abundant in human blood and tissues. Most MAIT cells have an invariant TCRα-chain that uses T cell receptor α-variable 1-2 (TRAV1-2) joined to TRAJ33/20/12 and recognizes metabolites from bacterial riboflavin synthesis bound to the Ag-presenting molecule MHC class I related (MR1). Our attempts to identify alternative MR1-presented Ags led to the discovery of rare MR1-restricted T cells with non-TRAV1-2 TCRs. Because altered Ag specificity likely alters affinity for the most potent known Ag, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), we performed bulk TCRα- and TCRß-chain sequencing and single-cell-based paired TCR sequencing on T cells that bound the MR1-5-OP-RU tetramer with differing intensities. Bulk sequencing showed that use of V genes other than TRAV1-2 was enriched among MR1-5-OP-RU tetramerlow cells. Although we initially interpreted these as diverse MR1-restricted TCRs, single-cell TCR sequencing revealed that cells expressing atypical TCRα-chains also coexpressed an invariant MAIT TCRα-chain. Transfection of each non-TRAV1-2 TCRα-chain with the TCRß-chain from the same cell demonstrated that the non-TRAV1-2 TCR did not bind the MR1-5-OP-RU tetramer. Thus, dual TCRα-chain expression in human T cells and competition for the endogenous ß-chain explains the existence of some MR1-5-OP-RU tetramerlow T cells. The discovery of simultaneous expression of canonical and noncanonical TCRs on the same T cell means that claims of roles for non-TRAV1-2 TCR in MR1 response must be validated by TCR transfer-based confirmation of Ag specificity.
Asunto(s)
Células T Invariantes Asociadas a Mucosa , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/metabolismo , Membrana Mucosa , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
Natural killer T (NKT) cells detect lipids presented by CD1d. Most studies focus on type I NKT cells that express semi-invariant αß T cell receptors (TCR) and recognize α-galactosylceramides. However, CD1d also presents structurally distinct lipids to NKT cells expressing diverse TCRs (type II NKT cells), but our knowledge of the antigens for type II NKT cells is limited. An early study identified a nonlipidic NKT cell agonist, phenyl pentamethyldihydrobenzofuransulfonate (PPBF), which is notable for its similarity to common sulfa drugs, but its mechanism of NKT cell activation remained unknown. Here, we demonstrate that a range of pentamethylbenzofuransulfonates (PBFs), including PPBF, activate polyclonal type II NKT cells from human donors. Whereas these sulfa drug-like molecules might have acted pharmacologically on cells, here we demonstrate direct contact between TCRs and PBF-treated CD1d complexes. Further, PBF-treated CD1d tetramers identified type II NKT cell populations expressing αßTCRs and γδTCRs, including those with variable and joining region gene usage (TRAV12-1-TRAJ6) that was conserved across donors. By trapping a CD1d-type II NKT TCR complex for direct mass-spectrometric analysis, we detected molecules that allow the binding of CD1d to TCRs, finding that both selected PBF family members and short-chain sphingomyelin lipids are present in these complexes. Furthermore, the combination of PPBF and short-chain sphingomyelin enhances CD1d tetramer staining of PPBF-reactive T cell lines over either molecule alone. This study demonstrates that nonlipidic small molecules, which resemble sulfa drugs implicated in systemic hypersensitivity and drug allergy reactions, are targeted by a polyclonal population of type II NKT cells in a CD1d-restricted manner.