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

ABSTRACT: 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 TCRβ-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 tetramer^low 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 TCRβ-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 β-chain explains the existence of some MR1-5-OP-RU tetramer^low 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.

SUBMITTER: Suliman S

PROVIDER: S-EPMC9359468 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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

Suliman Sara S Kjer-Nielsen Lars L Iwany Sarah K SK Lopez Tamara Kattya K Loh Liyen L Grzelak Ludivine L Kedzierska Katherine K Ocampo Tonatiuh A TA Corbett Alexandra J AJ McCluskey James J Rossjohn Jamie J León Segundo R SR Calderon Roger R Lecca-Garcia Leonid L Murray Megan B MB Moody D Branch DB Van Rhijn Ildiko I

Journal of immunology (Baltimore, Md. : 1950) 20220304 6

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. Beca ...[more]

PMID: 35246495

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Project description:Over the past decade, we have contributed to the chemistry of microbial natural products and synthetic ligands, related to riboflavin and uracils, that modulate immune cells called mucosal associated invariant T cells (MAIT cells). These highly abundant T lymphocytes were only discovered in 2003 and have become recognized for their importance in mammalian immunology. Unlike other T cells, MAIT cells are not activated by peptide or lipid antigens. In collaboration with immunology and structural biology research groups, we discovered that they are instead activated by unstable nitrogen-containing heterocycles synthesized by bacteria. The most potent naturally occurring activating compound (antigen) is 5-(2-oxopropylideneamino)-d-ribitylaminouracil (5-OP-RU). This compound is an imine (Schiff base) formed through condensation between an intermediate in the biosynthesis of riboflavin (vitamin B2) and a metabolic byproduct of mammalian and microbial glycolysis. Although it is very unstable in water due to intramolecular ring closure or hydrolysis, we were able to develop a non-enzymatic synthesis that yields a pure kinetically stable compound in a nonaqueous solvent. This compound has revolutionized the study of MAIT cell immunology due to its potent activation (EC50 = 2 pM) of MAIT cells and its development into immunological reagents for detecting and characterizing MAIT cells in tissues. MAIT cells are now linked to key physiological processes and disease, including antibacterial defense, tissue repair, regulation of graft-vs-host disease, gastritis, inflammatory bowel diseases, and cancer. 5-OP-RU activates MAIT cells and, like a vaccine, has been shown to protect mice from bacterial infections and cancers. Mechanistic studies on the binding of 5-OP-RU to its dual protein targets, the major histocompatibility complex class I related protein (MR1) and the MAIT cell receptor (MAIT TCR), have involved synthetic chemistry, 2D 1H NMR spectroscopy, mass spectrometry, computer modeling and molecular dynamics simulations, biochemical, cellular, and immunological assays, and protein structural biology. These combined studies have revealed structural influences for 5-OP-RU in solution on protein binding and antigen presentation and potency; informed the development of potent (EC50 = 2 nM) and water stable analogues; led to fluorescent analogues for detecting and tracking binding proteins in and on cells; and enabled discovery of drugs and drug-like molecules that bind MR1 and modulate MAIT cell function. MAIT cells offer new opportunities for chemical synthesis to enhance the stability, potency, selectivity, and bioavailability of small molecule ligands for MR1 or MAIT TCR proteins, and to contribute to the understanding of T cell immunity and the development of prospective new immunomodulating medicines.

Project description:BackgroundFibromyalgia (FM) is defined as a widely distributed pain. While many rheumatologists and pain physicians have considered it to be a pain disorder, psychiatry, psychology, and general medicine have deemed it to be a syndrome (FMS) or psychosomatic disorder. The lack of concrete structural and/or pathological evidence has made patients suffer prejudice that FMS is a medically unexplained symptom, implying inauthenticity. Furthermore, FMS often exhibits comorbidity with rheumatoid arthritis (RA) or spondyloarthritis (SpA), both of which show similar indications. In this study, disease specific biomarkers were sought in blood samples from patients to facilitate objective diagnoses of FMS, and distinguish it from RA and SpA.MethodsPeripheral blood mononuclear cells (PBMCs) from patients and healthy donors (HD) were subjected to multicolor flow cytometric analysis. The percentage of mucosal-associated invariant T (MAIT) cells in PBMCs and the mean fluorescent intensity (MFI) of cell surface antigen expression in MAIT cells were analyzed.ResultsThere was a decrease in the MAIT cell population in FMS, RA, and SpA compared with HD. Among the cell surface antigens in MAIT cells, three chemokine receptors, CCR4, CCR7, and CXCR1, a natural killer (NK) receptor, NKp80, a signaling lymphocyte associated molecule (SLAM) family, CD150, a degrunulation marker, CD107a, and a coreceptor, CD8β emerged as potential biomarkers for FMS to distinguish from HD. Additionally, a memory marker, CD44 and an inflammatory chemokine receptor, CXCR1 appeared possible markers for RA, while a homeostatic chemokine receptor, CXCR4 deserved for SpA to differentiate from FMS. Furthermore, the drug treatment interruption resulted in alternation of the expression of CCR4, CCR5, CXCR4, CD27, CD28, inducible costimulatory molecule (ICOS), CD127 (IL-7 receptor α), CD94, NKp80, an activation marker, CD69, an integrin family member, CD49d, and a dipeptidase, CD26, in FMS.ConclusionsCombined with the currently available diagnostic procedures and criteria, analysis of MAIT cells offers a more objective standard for the diagnosis of FMS, RA, and SpA, which exhibit multifaceted and confusingly similar clinical manifestations.

Project description:A recently identified unconventional T cell population known as mucosal-associated invariant T (MAIT) cells are characterized by the expression of semi-invariant T cell receptor (TCR) with a canonical TRAV1-2/TRAJ33 (Vα7.2/Jα33). These evolutionary conserved, innate-like T cells recognize vitamin B metabolites, derived from some bacteria and fungi. Due to their presence not only in the T cell repertoire of mucosal surfaces but also in peripheral blood and liver, and their significant involvement in a wide range of diseases, in-depth characterization of human MAIT cells is a timely requirement. Studies that examined the transcriptome, immunoproteome, and whole-cell proteome characterized the role of cytotoxic molecules and cytokines in effector functions of MAIT cells and their relationship with some other immune cell subsets. As MAIT cells are classified under the CD3+ T cell compartment and the majority express surface receptor CD8, identifying their proteomic relationship with CD3+ and CD8+ T cells is pivotal. Thus, a high-resolution dataset was generated using the cell populations sorted from peripheral blood mononuclear cells of three healthy volunteers to describe the whole cell proteomes of MAIT, CD3+, and CD8+ T cells. Trypsin-digested peptide samples obtained from the methanol co-precipitation method were analyzed using an Orbitrap FusionTM TribridTM mass spectrometer (Thermo Fisher Scientific, USA) inline coupled to nanoACQUITY ultra-performance liquid chromatography system (Waters, USA) to acquire data-dependent shotgun proteomic data (DDA-MS) for label-free quantification. Analysis of raw DDA-MS data using MaxQuant software and maxLFQ identified and quantified 4,442 protein groups at a 1% false discovery rate. Further analysis identified 3,680 proteins which were detected with a single UniProt accession and a minimum of 2 unique or razor peptides. Thus this proteomic dataset can be used as a reference proteome for future studies on human MAIT cells.

Project description:IntroductionMucosal Associated Invariant T (MAIT) cells are innate-like T cells that respond to conserved pathogen-derived vitamin B metabolites presented by the MHC class I related-1 molecule (MR1) antigen presentation pathway. Whilst viruses do not synthesize these metabolites, we have reported that varicella zoster virus (VZV) profoundly suppresses MR1 expression, implicating this virus in manipulation of the MR1:MAIT cell axis. During primary infection, the lymphotropism of VZV is likely to be instrumental in hematogenous dissemination of virus to gain access to cutaneous sites where it clinically manifests as varicella (chickenpox). However, MAIT cells, which are found in the blood and at mucosal and other organ sites, have yet to be examined in the context of VZV infection. The goal of this study was to examine any direct impact of VZV on MAIT cells.MethodsUsing flow cytometry, we interrogated whether primary blood derived MAIT cells are permissive to infection by VZV whilst further analysing differential levels of infection between various MAIT cell subpopulations. Changes in cell surface extravasation, skin homing, activation and proliferation markers after VZV infection of MAIT cells was also assessed via flow cytometry. Finally the capacity of MAIT cells to transfer infectious virus was tested through an infectious center assay and imaged via fluorescence microscopy.ResultsWe identify primary blood-derived MAIT cells as being permissive to VZV infection. A consequence of VZV infection of MAIT cells was their capacity to transfer infectious virus to other permissive cells, consistent with MAIT cells supporting productive infection. When subgrouping MAIT cells by their co- expression of a variety cell surface markers, there was a higher proportion of VZV infected MAIT cells co-expressing CD4+ and CD4+/CD8+ MAIT cells compared to the more phenotypically dominant CD8+ MAIT cells, whereas infection was not associated with differences in co-expression of CD56 (MAIT cell subset with enhanced responsiveness to innate cytokine stimulation), CD27 (co-stimulatory) or PD-1 (immune checkpoint). Infected MAIT cells retained high expression of CCR2, CCR5, CCR6, CLA and CCR4, indicating a potentially intact capacity for transendothelial migration, extravasation and trafficking to skin sites. Infected MAIT cells also displayed increased expression of CD69 (early activation) and CD71 (proliferation) markers.DiscussionThese data identify MAIT cells as being permissive to VZV infection and identify impacts of such infection on co- expressed functional markers.

Dataset Information

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

Publications

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

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