Innate-like activation of mucosal-associated invariant T cells in mycobacterial infection
ABSTRACT: Gene expression of Va7.2+CD161+CD4-CD8+MAIT cells from two subsets (CD69+CD26++ and CD69-CD26+/-). Overall design: Human blood mononuclear cells were magnetically sorted to enrich MAIT cells, which were stimulated with M. bovis-incubated MR1-expressing cells or anti-CD3 antibody and further sorted to CD69+CD26++ and CD69-CD26+/- subsets from Va7.2+CD161+CD4-CD8+ -gated MAIT cells.
Project description:Conventional T cells exhibit a delayed response to the initial priming of peptide antigens presented by major histocompatibility complex (MHC) proteins. Unlike conventional T cells, mucosal-associated invariant T (MAIT) cells quickly respond to non-peptidic metabolite antigens presented by MHC-related protein 1 (MR1). To elucidate the MR1-dependent activation program of MAIT cells in response to mycobacterial infections, we determined the surface markers, transcriptomic profiles, and effector responses of activated human MAIT cells. Results revealed that mycobacterial-incubated antigen-presenting cells stimulated abundant human CD8+ MAIT cells to upregulate the co-expression of CD69 and CD26, as a combinatorial activation marker. Further transcriptomic analyses demonstrated that CD69+CD26++ CD8+MAIT cells highly expressed numerous genes for mediating anti-mycobacterial immune responses, including pro-inflammatory cytokines, cytolytic molecules, NK cell receptors, and transcription factors, in contrast to inactivated counterparts CD69+/-CD26+/- CD8+MAIT cells. Gene co-expression, enrichment, and pathway analyses yielded high statistical significance to strongly support that activated CD8+ MAIT cells shared gene expression and numerous pathways with NK and CD8+ T cells in activation, cytokine production, cytokine signaling, and effector functions. Flow cytometry detected that activated CD8+MAIT cells produced TNF?, IFN?, and granulysin to inhibit mycobacterial growth and fight mycobacterial infection. Together, results strongly support that the combinatorial activation marker CD69+CD26++ labels the activated CD8+MAIT cells that develop an innate-like activation program in anti-mycobacterial immune responses. We speculate that the rapid production of anti-mycobacterial effector molecules facilitates MAIT cells to fight early mycobacterial infection in humans.
Project description:BACKGROUND:Mucosal-associated invariant T (MAIT) cells are innate-like T cells that recognise bacterial metabolites presented by MHC class I-related protein 1 (MR1). Bacterial dysbiosis has been implicated in auto-inflammatory disease development. We investigated MAIT cells in early, untreated rheumatoid arthritis (RA) and spondyloarthritis (SpA) patients. METHODS:Blood and synovial fluid mononuclear cells obtained from patients (SpA/RA) and controls were stimulated with fixed Escherichia coli to provide MAIT ligand. Cells were analysed by flow cytometry and MAIT cells were identified by MR1-5-OP-RU tetramers. Synovial biopsies were studied by confocal microscopy. RESULTS:Peripheral and synovial CD3+ MR1-tet+ MAIT cell frequencies were comparable in all groups. MAIT cells were detected in RA and SpA synovium based on CD3, CD161 and V?7.2 expression. Peripheral RA MAIT cells were mostly CD4+ (controls 8.3%, SpA 12.3%, RA 52.6%; p?<?0.001) and CD161 expression was strongly reduced (control mean fluorescence intensity (MFI)?=?2348, SpA MFI?=?2219, RA MFI?=?226; p?<?0.001). MAIT cells were hyporesponsive, shown by minimal upregulation of CD25 and CD69 to E. coli stimulation (control, CD25 MFI =?177, CD69 MFI?=?1307; SpA, CD25 MFI?=?95, CD69 MFI =?1257; RA, CD25 MFI?=?0, CD69 MFI =?467; p?<?0.001 and p?=?0.01 respectively). CONCLUSION:In early untreated RA patients, the peripheral MAIT cell composition was altered, with reduced levels of CD161 expression, and cells were hyporesponsive to stimulation. MAIT cell dysfunction may provide a link between the microbiome and development of RA.
Project description:Human mucosal-associated invariant T (MAIT) cells are an important T cell subset that are enriched in tissues and possess potent effector functions. Typically such cells are marked by their expression of V?7.2-J?33/J?20/J?12 T cell receptors, and functionally they are major histocompatibility complex class I-related protein 1 (MR1)-restricted, responding to bacterially derived riboflavin synthesis intermediates. MAIT cells are contained within the CD161++ V?7.2+ T cell population, the majority of which express the CD8 receptor (CD8+), while a smaller fraction expresses neither CD8 or CD4 coreceptor (double negative; DN) and a further minority are CD4+. Whether these cells have distinct homing patterns, phenotype and functions have not been examined in detail. We used a combination of phenotypic staining and functional assays to address the similarities and differences between these CD161++ V?7.2+ T cell subsets. We find that most features are shared between CD8+ and DN CD161++ V?7.2+ T cells, with a small but detectable role evident for CD8 binding in tuning functional responsiveness. By contrast, the CD4+ CD161++ V?7.2+ T cell population, although showing MR1-dependent responsiveness to bacterial stimuli, display reduced T helper 1 effector functions, including cytolytic machinery, while retaining the capacity to secrete interleukin-4 (IL-4) and IL-13. This was consistent with underlying changes in transcription factor (TF) expression. Although we found that only a proportion of CD4+ CD161++ V?7.2+ T cells stained for the MR1-tetramer, explaining some of the heterogeneity of CD4+ CD161++ V?7.2+ T cells, these differences in TF expression were shared with CD4+ CD161++ MR1-tetramer+ cells. These data reveal the functional diversity of human CD161++ V?7.2+ T cells and indicate potentially distinct roles for the different subsets <i>in vivo</i>.
Project description:Mucosal-associated invariant T (MAIT) cells represent up to 10% of circulating human T cells. They are usually defined using combinations of non-lineage-specific (surrogate) markers such as anti-TRAV1-2, CD161, IL-18Rα and CD26. The development of MR1-Ag tetramers now permits the specific identification of MAIT cells based on T-cell receptor specificity. Here, we compare these approaches for identifying MAIT cells and show that surrogate markers are not always accurate in identifying these cells, particularly the CD4+ fraction. Moreover, while all MAIT cell subsets produced comparable levels of IFNγ, TNF and IL-17A, the CD4+ population produced more IL-2 than the other subsets. In a human ontogeny study, we show that the frequencies of most MR1 tetramer+ MAIT cells, with the exception of CD4+ MAIT cells, increased from birth to about 25 years of age and declined thereafter. We also demonstrate a positive association between the frequency of MAIT cells and other unconventional T cells including Natural Killer T (NKT) cells and Vδ2+ γδ T cells. Accordingly, this study demonstrates that MAIT cells are phenotypically and functionally diverse, that surrogate markers may not reliably identify all of these cells, and that their numbers are regulated in an age-dependent manner and correlate with NKT and Vδ2+ γδ T cells.
Project description:Mucosal-associated invariant T (MAIT) cells have a semi-invariant TCR V?-chain, and their optimal development is dependent upon commensal flora and expression of the nonpolymorphic MHC class I-like molecule MR1. MAIT cells are activated in an MR1-restricted manner by diverse strains of bacteria and yeast, suggesting a widely shared Ag. Recently, human and mouse MR1 were found to bind bacterial riboflavin metabolites (ribityllumazine [RL] Ags) capable of activating MAIT cells. In this study, we used MR1/RL tetramers to study MR1 dependency, subset heterogeneity, and protective effector functions important for tuberculosis immunity. Although tetramer(+) cells were detected in both MR1(+/+) and MR1(-/-) TCR V?19i-transgenic (Tg) mice, MR1 expression resulted in significantly increased tetramer(+) cells coexpressing TCR V?6/8, NK1.1, CD44, and CD69 that displayed more robust in vitro responses to IL-12 plus IL-18 and RL Ag, indicating that MR1 is necessary for the optimal development of the classic murine MAIT cell memory/effector subset. In addition, tetramer(+) MAIT cells expressing CD4, CD8, or neither developing in MR1(+/+) V?19i-Tg mice had disparate cytokine profiles in response to RL Ag. Therefore, murine MAIT cells are considerably more heterogeneous than previously thought. Most notably, after mycobacterial pulmonary infection, heterogeneous subsets of tetramer(+) V?19i-Tg MAIT cells expressing CXCR3 and ?4?1 were recruited into the lungs and afforded early protection. In addition, V?19iC?(-/-)MR(+/+) mice were significantly better protected than were V?19iC?(-/-)MR1(-/-), wild-type, and MR1(-/-) non-Tg mice. Overall, we demonstrate considerable functional diversity of MAIT cell responses, as well as that MR1-restricted MAIT cells are important for tuberculosis protective immunity.
Project description:Mucosal-associated invariant T cells (MAIT cells) express a semi-invariant T cell receptor (TCR) ?-chain, TRAV1-2-TRAJ33, and are activated by vitamin B metabolites bound by the major histocompatibility complex (MHC)-related class I-like molecule, MR1. Understanding MAIT cell biology has been restrained by the lack of reagents to specifically identify and characterize these cells. Furthermore, the use of surrogate markers may misrepresent the MAIT cell population. We show that modified human MR1 tetramers loaded with the potent MAIT cell ligand, reduced 6-hydroxymethyl-8-D-ribityllumazine (rRL-6-CH?OH), specifically detect all human MAIT cells. Tetramer(+) MAIT subsets were predominantly CD8(+) or CD4(-)CD8(-), although a small subset of CD4(+) MAIT cells was also detected. Notably, most human CD8(+) MAIT cells were CD8?(+)CD8?(-/lo), implying predominant expression of CD8?? homodimers. Tetramer-sorted MAIT cells displayed a T(H)1 cytokine phenotype upon antigen-specific activation. Similarly, mouse MR1-rRL-6-CH?OH tetramers detected CD4(+), CD4(-)CD8(-) and CD8(+) MAIT cells in V?19 transgenic mice. Both human and mouse MAIT cells expressed a broad TCR-? repertoire, and although the majority of human MAIT cells expressed TRAV1-2-TRAJ33, some expressed TRAJ12 or TRAJ20 genes in conjunction with TRAV1-2. Accordingly, MR1 tetramers allow precise phenotypic characterization of human and mouse MAIT cells and revealed unanticipated TCR heterogeneity in this population.
Project description:MR1-restricted T (MR1T) cells are defined by their recognition of metabolite antigens presented by the monomorphic MHC class 1-related molecule, MR1, the most highly conserved MHC class I related molecule in mammalian species. Mucosal-associated invariant T (MAIT) cells are the predominant subset of MR1T cells expressing an invariant TCR ?-chain, TRAV1-2. These cells comprise a T cell subset that recognizes and mediates host immune responses to a broad array of microbial pathogens, including Mycobacterium tuberculosis. Here, we sought to characterize development of circulating human MR1T cells as defined by MR1-5-OP-RU tetramer labeling and of the TRAV1-2+ MAIT cells defined by expression of TRAV1-2 and high expression of CD26 and CD161 (TRAV1-2+CD161++CD26++ cells). We analyzed postnatal expansion, maturation, and functionality of peripheral blood MR1-5-OP-RU tetramer+ MR1T cells in cohorts from three different geographic settings with different tuberculosis (TB) vaccination practices, levels of exposure to and infection with M. tuberculosis. Early after birth, frequencies of MR1-5-OP-RU tetramer+ MR1T cells increased rapidly by several fold. This coincided with the transition from a predominantly CD4+ and TRAV1-2- population in neonates, to a predominantly TRAV1-2+CD161++CD26++ CD8+ population. We also observed that tetramer+ MR1T cells that expressed TNF upon mycobacterial stimulation were very low in neonates, but increased ~10-fold in the first year of life. These functional MR1T cells in all age groups were MR1-5-OP-RU tetramer+TRAV1-2+ and highly expressed CD161 and CD26, markers that appeared to signal phenotypic and functional maturation of this cell subset. This age-associated maturation was also marked by the loss of naïve T cell markers on tetramer+ TRAV1-2+ MR1T cells more rapidly than tetramer+TRAV1-2- MR1T cells and non-MR1T cells. These data suggest that neonates have infrequent populations of MR1T cells with diverse phenotypic attributes; and that exposure to the environment rapidly and preferentially expands the MR1-5-OP-RU tetramer+TRAV1-2+ population of MR1T cells, which becomes the predominant population of functional MR1T cells early during childhood.
Project description:BACKGROUND:Immune dysregulation is one of the mechanisms to promote endometriosis (EMS). Various T cell subpopulations have been reported to play different roles in the development of EMS. The mucosa-associated invariant T cell (MAIT) is an important T cell subset in the pathogenesis of various autoimmune diseases. Evidence has indicated that there are three functionally distinct MAIT subsets: CD4+, CD8+ and CD4/CD8-/- (double negative, DN) MAIT cells. Till now, the associations between endometriosis and MAIT have not been studied. Our research investigates different MAIT subpopulations in peripheral blood (PB) and peritoneal fluid (PF) from EMS patients. METHODS:Thirty-two EMS patients and eighteen controls were included. PB and PF were collected. Tests of cytokines in plasma and PF were performed by ELISA kit. Characterisations of MAIT were done by flow cytometry. MAIT cells have been defined as CD3?+?CD161?+?V?7.2+ cells. Based on CD4 and CD8 expression, they were divided into CD8+MAIT, CD4+MAIT and DN MAIT. RESULTS:Enrichments of MAIT cells, especially CD4 and CD8 MAIT subsets were found. Moreover, CD8 MAIT cells had a high activation in the EMS group. EMS patients produced higher level of IL-8/12/17 as compared to these from controls. On the contrary, control patients exhibited an impressive upregulation of DN MAIT cells, however, these DN MAIT cells from controls showed a higher expression of PD-1. Lastly, we performed the relevance analysis, and discovered that the accumulation of PB MAIT cells positively correlated with an elevated level of serum CA125 production in EMS group. CONCLUSION:These results suggest that different MAIT subsets play distinct roles in the progression of endometriosis.
Project description:Studies on the biology of mucosal-associated invariant T cells (MAIT cells) in mice have been hampered by a lack of specific reagents. Using MR1-antigen (Ag) tetramers that specifically bind to the MR1-restricted MAIT T cell receptors (TCRs), we demonstrate that MAIT cells are detectable in a broad range of tissues in C57BL/6 and BALB/c mice. These cells include CD4(-)CD8(-), CD4(-)CD8(+), and CD4(+)CD8(-) subsets, and their frequency varies in a tissue- and strain-specific manner. Mouse MAIT cells have a CD44(hi)CD62L(lo) memory phenotype and produce high levels of IL-17A, whereas other cytokines, including IFN-?, IL-4, IL-10, IL-13, and GM-CSF, are produced at low to moderate levels. Consistent with high IL-17A production, most MAIT cells express high levels of retinoic acid-related orphan receptor ?t (ROR?t), whereas ROR?t(lo) MAIT cells predominantly express T-bet and produce IFN-?. Most MAIT cells express the promyelocytic leukemia zinc finger (PLZF) transcription factor, and their development is largely PLZF dependent. These observations contrast with previous reports that MAIT cells from V?19 TCR transgenic mice are PLZF(-) and express a naive CD44(lo) phenotype. Accordingly, MAIT cells from normal mice more closely resemble human MAIT cells than previously appreciated, and this provides the foundation for further investigations of these cells in health and disease.
Project description:Mucosal-associated invariant T (MAIT) cells help protect against certain infections and are related to some autoimmune diseases. Immune thrombocytopenia (ITP) is a relatively rare hematological autoimmune disease associated with low platelet count. We designed a cross-sectional study wherein we examined peripheral blood samples of patients with ITP for the number of MAIT cells (CD3+TCR-V?7.2+CD161+IL-18R?+ lymphocytes) and their CD4/8 subsets (by flow cytometry) and levels of cytokines (by multiplex assays). The study cohort included 18 patients with ITP and 20 healthy controls (HCs). We first compared the number of MAIT cells between HCs and patients with ITP and then performed subgroup analysis in patients with ITP. The number of total MAIT cells in patients with ITP was significantly lower than that in HCs (p < 0.0001), and the CD4-CD8+ subset of MAIT cells showed the same trend. Moreover, patients with ITP refractory to prednisolone exhibited a significantly lower number of total MAIT and CD4-CD8+ MAIT cells than patients sensitive to prednisolone. The number of total MAIT and CD4-CD8+ MAIT cells was not correlated with the response to thrombopoietin receptor agonist treatment or with Helicobacter pylori infection. We found no relation between cytokine levels and response to prednisolone treatment, although the levels of IP-10 and RANTES showed a correlation with the number of total MAIT and CD4-CD8+ MAIT cells. In conclusion, total MAIT and CD4-CD8+ MAIT cells in peripheral blood were decreased in patients with ITP, correlating with their response to prednisolone.