RESUMO
Mucosal-associated invariant T (MAIT) cells are activated by microbial riboflavin-based metabolite antigens when presented by MR1. How modifications to the potent antigen 5-OP-RU affect presentation by MR1 and MAIT cell activation remains unclear. Here we design 20 derivatives, termed altered metabolite ligands (AMLs), to dissect the impact of different antigen components on the human MAIT-MR1 axis. Analysis of 11 crystal structures of MAIT T cell antigen receptor (TCR)-MR1-AML ternary complexes, along with biochemical and functional assays, shows that MR1 cell-surface upregulation is influenced by ribityl and non-ribityl components of the ligand and the hydrophobicity of the MR1-AML interface. The polar ribityl chain of the AML strongly influences MAIT cell activation potency through dynamic compensatory interactions within a MAIT TCR-MR1-AML interaction triad. We define the basis by which the MAIT TCR can differentially recognize AMLs, thereby providing insight into MAIT cell antigen specificity and potency.
Assuntos
Antígenos/imunologia , Células T Invariantes Associadas à Mucosa/imunologia , Linhagem Celular Tumoral , Humanos , Células Jurkat , Ligantes , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Riboflavina/imunologiaRESUMO
The major-histocompatibility-complex-(MHC)-class-I-related molecule MR1 can present activating and non-activating vitamin-B-based ligands to mucosal-associated invariant T cells (MAIT cells). Whether MR1 binds other ligands is unknown. Here we identified a range of small organic molecules, drugs, drug metabolites and drug-like molecules, including salicylates and diclofenac, as MR1-binding ligands. Some of these ligands inhibited MAIT cells ex vivo and in vivo, while others, including diclofenac metabolites, were agonists. Crystal structures of a T cell antigen receptor (TCR) from a MAIT cell in complex with MR1 bound to the non-stimulatory and stimulatory compounds showed distinct ligand orientations and contacts within MR1, which highlighted the versatility of the MR1 binding pocket. The findings demonstrated that MR1 was able to capture chemically diverse structures, spanning mono- and bicyclic compounds, that either inhibited or activated MAIT cells. This indicated that drugs and drug-like molecules can modulate MAIT cell function in mammals.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Células T Invariantes Associadas à Mucosa/efeitos dos fármacos , Células T Invariantes Associadas à Mucosa/metabolismo , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Descoberta de Drogas , Antígenos de Histocompatibilidade Classe I/química , Humanos , Ligação de Hidrogênio , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Antígenos de Histocompatibilidade Menor/química , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Células T Invariantes Associadas à Mucosa/imunologia , Ligação Proteica , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo , Relação Estrutura-AtividadeRESUMO
Mucosal-associated invariant T cells (MAIT cells) detect microbial vitamin B2 derivatives presented by the antigen-presenting molecule MR1. Here we defined three developmental stages and checkpoints for the MAIT cell lineage in humans and mice. Stage 1 and stage 2 MAIT cells predominated in thymus, while stage 3 cells progressively increased in abundance extrathymically. Transition through each checkpoint was regulated by MR1, whereas the final checkpoint that generated mature functional MAIT cells was controlled by multiple factors, including the transcription factor PLZF and microbial colonization. Furthermore, stage 3 MAIT cell populations were expanded in mice deficient in the antigen-presenting molecule CD1d, suggestive of a niche shared by MAIT cells and natural killer T cells (NKT cells). Accordingly, this study maps the developmental pathway and checkpoints that control the generation of functional MAIT cells.
Assuntos
Diferenciação Celular/imunologia , Células T Invariantes Associadas à Mucosa/citologia , Células T Invariantes Associadas à Mucosa/fisiologia , Timo/imunologia , Timo/metabolismo , Animais , Antígenos CD1d/genética , Biomarcadores , Diferenciação Celular/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Imunofenotipagem , Células Progenitoras Linfoides/imunologia , Células Progenitoras Linfoides/metabolismo , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/genéticaRESUMO
Mucosal-associated invariant T (MAIT) cells are unconventional T cells that respond to riboflavin biosynthesis and cytokines through TCR-dependent and -independent pathways, respectively. MAIT cell activation plays an immunoprotective role against several pathogens, however the functional capacity of MAIT cells following direct infection or exposure to infectious agents remains poorly defined. We investigated the impact of Varicella Zoster Virus (VZV) on blood-derived MAIT cells and report virus-mediated impairment of activation, cytokine production, and altered transcription factor expression by VZV infected (antigen+) and VZV exposed (antigen-) MAIT cells in response to TCR-dependent and -independent stimulation. Furthermore, we reveal that suppression of VZV exposed (antigen-) MAIT cells is not mediated by a soluble factor from neighbouring VZV infected (antigen+) MAIT cells. Finally, we demonstrate that VZV impairs the cytolytic potential of MAIT cells in response to riboflavin synthesising bacteria. In summary, we report a virus-mediated immune-evasion strategy that disarms MAIT cell responses.
Assuntos
Herpesvirus Humano 3 , Células T Invariantes Associadas à Mucosa , Humanos , Células T Invariantes Associadas à Mucosa/imunologia , Herpesvirus Humano 3/imunologia , Ativação Linfocitária/imunologia , Citocinas/metabolismo , Citocinas/imunologia , Riboflavina/imunologia , Infecção pelo Vírus da Varicela-Zoster/imunologia , Infecção pelo Vírus da Varicela-Zoster/virologia , Evasão da Resposta Imune/imunologia , Herpes Zoster/imunologia , Herpes Zoster/virologiaRESUMO
Mucosal-associated invariant T (MAIT) cells can elicit immune responses against riboflavin-based antigens presented by the evolutionary conserved MHC class I related protein, MR1. While we have an understanding of the structural basis of human MAIT cell receptor (TCR) recognition of human MR1 presenting a variety of ligands, how the semi-invariant mouse MAIT TCR binds mouse MR1-ligand remains unknown. Here, we determine the crystal structures of 2 mouse TRAV1-TRBV13-2+ MAIT TCR-MR1-5-OP-RU ternary complexes, whose TCRs differ only in the composition of their CDR3ß loops. These mouse MAIT TCRs mediate high affinity interactions with mouse MR1-5-OP-RU and cross-recognize human MR1-5-OP-RU. Similarly, a human MAIT TCR could bind mouse MR1-5-OP-RU with high affinity. This cross-species recognition indicates the evolutionary conserved nature of this MAIT TCR-MR1 axis. Comparing crystal structures of the mouse versus human MAIT TCR-MR1-5-OP-RU complexes provides structural insight into the conserved nature of this MAIT TCR-MR1 interaction and conserved specificity for the microbial antigens, whereby key germline-encoded interactions required for MAIT activation are maintained. This is an important consideration for the development of MAIT cell-based therapeutics that will rely on preclinical mouse models of disease.
Assuntos
Antígenos de Histocompatibilidade Classe I , Antígenos de Histocompatibilidade Menor , Células T Invariantes Associadas à Mucosa , Ribitol , Animais , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Menor/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/imunologia , Antígenos de Histocompatibilidade Menor/química , Camundongos , Células T Invariantes Associadas à Mucosa/imunologia , Células T Invariantes Associadas à Mucosa/metabolismo , Humanos , Ribitol/análogos & derivados , Ribitol/metabolismo , Ribitol/química , Uracila/análogos & derivados , Uracila/metabolismo , Uracila/química , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Cristalografia por Raios XRESUMO
Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells that recognize small molecule metabolites presented by major histocompatibility complex class I related protein 1 (MR1), via an αß T cell receptor (TCR). MAIT TCRs feature an essentially invariant TCR α-chain, which is highly conserved between mammals. Similarly, MR1 is the most highly conserved major histocompatibility complex-I-like molecule. This extreme conservation, including the mode of interaction between the MAIT TCR and MR1, has been shown to allow for species-mismatched reactivities unique in T cell biology, thereby allowing the use of selected species-mismatched MR1-antigen (MR1-Ag) tetramers in comparative immunology studies. However, the pattern of cross-reactivity of species-mismatched MR1-Ag tetramers in identifying MAIT cells in diverse species has not been formally assessed. We developed novel cattle and pig MR1-Ag tetramers and utilized these alongside previously developed human, mouse, and pig-tailed macaque MR1-Ag tetramers to characterize cross-species tetramer reactivities. MR1-Ag tetramers from each species identified T cell populations in distantly related species with specificity that was comparable to species-matched MR1-Ag tetramers. However, there were subtle differences in staining characteristics with practical implications for the accurate identification of MAIT cells. Pig MR1 is sufficiently conserved across species that pig MR1-Ag tetramers identified MAIT cells from the other species. However, MAIT cells in pigs were at the limits of phenotypic detection. In the absence of sheep MR1-Ag tetramers, a MAIT cell population in sheep blood was identified phenotypically, utilizing species-mismatched MR1-Ag tetramers. Collectively, our results validate the use and define the limitations of species-mismatched MR1-Ag tetramers in comparative immunology studies.
Assuntos
Antígenos de Histocompatibilidade Classe I , Antígenos de Histocompatibilidade Menor , Células T Invariantes Associadas à Mucosa , Especificidade da Espécie , Animais , Células T Invariantes Associadas à Mucosa/imunologia , Células T Invariantes Associadas à Mucosa/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Camundongos , Bovinos , Antígenos de Histocompatibilidade Menor/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/imunologia , Antígenos de Histocompatibilidade Menor/química , Suínos , Macaca , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
Activating and inhibitory receptors on natural killer (NK) cells have a crucial role in innate immunity, although the basis of the engagement of activating NK cell receptors is unclear. The activating receptor Ly49H confers resistance to infection with murine cytomegalovirus by binding to the 'immunoevasin' m157. We found that m157 bound to the helical stalk of Ly49H, whereby two m157 monomers engaged the Ly49H dimer. The helical stalks of Ly49H lay centrally across the m157 platform, whereas its lectin domain was not required for recognition. Instead, m157 targeted an 'aromatic peg motif' present in stalks of both activating and inhibitory receptors of the Ly49 family, and substitution of this motif abrogated binding. Furthermore, ligation of m157 to Ly49H or Ly49C resulted in intracellular signaling. Accordingly, m157 has evolved to 'tackle the legs' of a family of NK cell receptors.
Assuntos
Infecções por Herpesviridae/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Imunidade Inata/imunologia , Células Matadoras Naturais/imunologia , Muromegalovirus/imunologia , Subfamília A de Receptores Semelhantes a Lectina de Células NK/imunologia , Motivos de Aminoácidos/imunologia , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência Molecular , Transdução de Sinais/imunologia , Organismos Livres de Patógenos Específicos , Ressonância de Plasmônio de SuperfícieRESUMO
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.
Assuntos
Células T Invariantes Associadas à Mucosa , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Mucosa , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
Bacterial synthesis of vitamin B2 generates a by-product, 5-(2-oxopropylideneamino)-d-ribityl-aminouracil (5-OP-RU), with potent immunological properties in mammals, but it is rapidly degraded in water. This natural product covalently bonds to the key immunological protein MR1 in the endoplasmic reticulum of antigen presenting cells (APCs), enabling MR1 refolding and trafficking to the cell surface, where it interacts with T cell receptors (TCRs) on mucosal associated invariant T lymphocytes (MAIT cells), activating their immunological and antimicrobial properties. Here, we strategically modify this natural product to understand the molecular basis of its recognition by MR1. This culminated in the discovery of new water-stable compounds with extremely powerful and distinctive immunological functions. We report their capacity to bind MR1 inside APCs, triggering its expression on the cell surface (EC50 17â nM), and their potent activation (EC50 56â pM) or inhibition (IC50 80â nM) of interacting MAIT cells. We further derivatize compounds with diazirine-alkyne, biotin, or fluorophore (Cy5 or AF647) labels for detecting, monitoring, and studying cellular MR1. Computer modeling casts new light on the molecular mechanism of activation, revealing that potent activators are first captured in a tyrosine- and serine-lined cleft in MR1 via specific pi-interactions and H-bonds, before more tightly attaching via a covalent bond to Lys43 in MR1. This chemical study advances our molecular understanding of how bacterial metabolites are captured by MR1, influence cell surface expression of MR1, interact with T cells to induce immunity, and offers novel clues for developing new vaccine adjuvants, immunotherapeutics, and anticancer drugs.
Assuntos
Riboflavina , Humanos , Riboflavina/metabolismo , Riboflavina/química , Riboflavina/farmacologia , Riboflavina/biossíntese , Riboflavina/análogos & derivados , Agentes de Imunomodulação/química , Agentes de Imunomodulação/farmacologia , Agentes de Imunomodulação/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Menor/metabolismo , Células T Invariantes Associadas à Mucosa/metabolismo , Células T Invariantes Associadas à Mucosa/imunologia , Fatores Imunológicos/farmacologia , Fatores Imunológicos/química , Fatores Imunológicos/metabolismo , Células Apresentadoras de Antígenos/metabolismo , Células Apresentadoras de Antígenos/imunologia , Ribitol/análogos & derivados , Uracila/análogos & derivadosRESUMO
T-cell receptor+ CD4- CD8- double-negative (DN) T cells are a population of T cells present in low abundance in blood and lymphoid organs, but enriched in various organs including the kidney. Despite burgeoning interest in these cells, studies examining their abundance in the kidney have reported conflicting results. Here we developed a flow cytometry strategy to clearly segregate DN T cells from other immune cells in the mouse kidney and used it to characterize their phenotype and response in renal ischemia-reperfusion injury (IRI). These experiments revealed that in the healthy kidney, most DN T cells are located within the renal parenchyma and exhibit an effector memory phenotype. In response to IRI, the number of renal DN T cells is unaltered after 24 h, but significantly increased by 72 h. This increase is not related to alterations in proliferation or apoptosis. By contrast, adoptive transfer studies indicate that circulating DN T cells undergo preferential recruitment to the postischemic kidney. Furthermore, DN T cells show the capacity to upregulate CD8, both in vivo following adoptive transfer and in response to ex vivo activation. Together, these findings provide novel insights regarding the phenotype of DN T cells in the kidney, including their predominant extravascular location, and show that increases in their abundance in the kidney following IRI occur in part as a result of increased recruitment from the circulation. Furthermore, the observation that DN T cells can upregulate CD8 in vivo has important implications for detection and characterization of DN T cells in future studies.
Assuntos
Traumatismo por Reperfusão , Linfócitos T , Camundongos , Animais , Rim , Receptores de Antígenos de Linfócitos T alfa-beta , Receptores de Antígenos de Linfócitos TRESUMO
The antigen-presenting molecule MR1 (MHC class I-related protein 1) presents metabolite antigens derived from microbial vitamin B2 synthesis to activate mucosal-associated invariant T (MAIT) cells. Key aspects of this evolutionarily conserved pathway remain uncharacterized, including where MR1 acquires ligands and what accessory proteins assist ligand binding. We answer these questions by using a fluorophore-labeled stable MR1 antigen analog, a conformation-specific MR1 mAb, proteomic analysis, and a genome-wide CRISPR/Cas9 library screen. We show that the endoplasmic reticulum (ER) contains a pool of two unliganded MR1 conformers stabilized via interactions with chaperones tapasin and tapasin-related protein. This pool is the primary source of MR1 molecules for the presentation of exogenous metabolite antigens to MAIT cells. Deletion of these chaperones reduces the ER-resident MR1 pool and hampers antigen presentation and MAIT cell activation. The MR1 antigen-presentation pathway thus co-opts ER chaperones to fulfill its unique ability to present exogenous metabolite antigens captured within the ER.
Assuntos
Retículo Endoplasmático/genética , Antígenos de Histocompatibilidade Classe I/genética , Metaboloma/genética , Antígenos de Histocompatibilidade Menor/genética , Proteômica , Apresentação de Antígeno/genética , Antígenos/genética , Antígenos/imunologia , Sistemas CRISPR-Cas/genética , Humanos , Ligantes , Ativação Linfocitária/genética , Proteínas de Membrana Transportadoras/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/imunologia , Células T Invariantes Associadas à Mucosa/imunologia , Riboflavina/genéticaRESUMO
The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers.
Assuntos
Doenças Autoimunes/imunologia , Citometria de Fluxo , Infecções/imunologia , Neoplasias/imunologia , Animais , Doença Crônica , Humanos , Camundongos , Guias de Prática Clínica como AssuntoRESUMO
Mucosal-associated invariant T (MAIT) cells are a major subset of innate-like T cells mediating protection against bacterial infection through recognition of microbial metabolites derived from riboflavin biosynthesis. Mouse MAIT cells egress from the thymus as two main subpopulations with distinct functions, namely, T-bet-expressing MAIT1 and RORγt-expressing MAIT17 cells. Previously, we reported that inducible T-cell costimulator and interleukin (IL)-23 provide essential signals for optimal MHC-related protein 1 (MR1)-dependent activation and expansion of MAIT17 cells in vivo. Here, in a model of tularemia, in which MAIT1 responses predominate, we demonstrate that IL-12 and IL-23 promote MAIT1 cell expansion during acute infection and that IL-12 is indispensable for MAIT1 phenotype and function. Furthermore, we showed that the bias toward MAIT1 or MAIT17 responses we observed during different bacterial infections was determined and modulated by the balance between IL-12 and IL-23 and that these responses could be recapitulated by cytokine coadministration with antigen. Our results indicate a potential for tailored immunotherapeutic interventions via MAIT cell manipulation.
Assuntos
Infecções Bacterianas , Células T Invariantes Associadas à Mucosa , Animais , Citocinas , Antígenos de Histocompatibilidade Classe I/metabolismo , Interleucina-12 , Interleucina-23 , CamundongosRESUMO
BACKGROUND: In human blood, mucosal-associated invariant T (MAIT) cells are abundant T cells that recognize antigens presented on non-polymorphic major histocompatibility complex-related 1 (MR1) molecules. The MAIT cells are activated by mycobacteria, and prior human studies indicate that blood frequencies of MAIT cells, defined by cell surface markers, decline during tuberculosis (TB) disease, consistent with redistribution to the lungs. METHODS: We tested whether frequencies of blood MAIT cells were altered in patients with TB disease relative to healthy Mycobacterium tuberculosis-exposed controls from Peru and South Africa. We quantified their frequencies using MR1 tetramers loaded with 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil. RESULTS: Unlike findings from prior studies, frequencies of blood MAIT cells were similar among patients with TB disease and latent and uninfected controls. In both cohorts, frequencies of MAIT cells defined by MR1-tetramer staining and coexpression of CD161 and the T-cell receptor alpha variable gene TRAV1-2 were strongly correlated. Disease severity captured by body mass index or TB disease transcriptional signatures did not correlate with MAIT cell frequencies in patients with TB. CONCLUSIONS: Major histocompatibility complex (MHC)-related 1-restrictied MAIT cells are detected at similar levels with tetramers or surface markers. Unlike MHC-restricted T cells, blood frequencies of MAIT cells are poor correlates of TB disease but may play a role in pathophysiology.
Assuntos
Células T Invariantes Associadas à Mucosa/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose/epidemiologia , Tuberculose/imunologia , Adulto , Biomarcadores , Estudos de Casos e Controles , Feminino , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Células T Invariantes Associadas à Mucosa/metabolismo , Prevalência , Vigilância em Saúde Pública , Medição de Risco , Fatores de Risco , Tuberculose/microbiologiaRESUMO
This article discusses mucosal-associated invariant T-cell prophylactic and therapeutic vaccination schemes tested by Sakai et al. in mouse models of Mycobacterium tuberculosis infection together with the outcomes and observations.
Assuntos
Células T Invariantes Associadas à Mucosa , Tuberculose , Animais , Camundongos , VacinaçãoRESUMO
Superantigens (SAgs) are potent exotoxins secreted by Staphylococcus aureus and Streptococcus pyogenes. They target a large fraction of T cell pools to set in motion a "cytokine storm" with severe and sometimes life-threatening consequences typically encountered in toxic shock syndrome (TSS). Given the rapidity with which TSS develops, designing timely and truly targeted therapies for this syndrome requires identification of key mediators of the cytokine storm's initial wave. Equally important, early host responses to SAgs can be accompanied or followed by a state of immunosuppression, which in turn jeopardizes the host's ability to combat and clear infections. Unlike in mouse models, the mechanisms underlying SAg-associated immunosuppression in humans are ill-defined. In this work, we have identified a population of innate-like T cells, called mucosa-associated invariant T (MAIT) cells, as the most powerful source of pro-inflammatory cytokines after exposure to SAgs. We have utilized primary human peripheral blood and hepatic mononuclear cells, mouse MAIT hybridoma lines, HLA-DR4-transgenic mice, MAIThighHLA-DR4+ bone marrow chimeras, and humanized NOD-scid IL-2Rγnull mice to demonstrate for the first time that: i) mouse and human MAIT cells are hyperresponsive to SAgs, typified by staphylococcal enterotoxin B (SEB); ii) the human MAIT cell response to SEB is rapid and far greater in magnitude than that launched by unfractionated conventional T, invariant natural killer T (iNKT) or γδ T cells, and is characterized by production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2, but not IL-17A; iii) high-affinity MHC class II interaction with SAgs, but not MHC-related protein 1 (MR1) participation, is required for MAIT cell activation; iv) MAIT cell responses to SEB can occur in a T cell receptor (TCR) Vß-specific manner but are largely contributed by IL-12 and IL-18; v) as MAIT cells are primed by SAgs, they also begin to develop a molecular signature consistent with exhaustion and failure to participate in antimicrobial defense. Accordingly, they upregulate lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin-3 (TIM-3), and/or programmed cell death-1 (PD-1), and acquire an anergic phenotype that interferes with their cognate function against Klebsiella pneumoniae and Escherichia coli; vi) MAIT cell hyperactivation and anergy co-utilize a signaling pathway that is governed by p38 and MEK1/2. Collectively, our findings demonstrate a pathogenic, rather than protective, role for MAIT cells during infection. Furthermore, we propose a novel mechanism of SAg-associated immunosuppression in humans. MAIT cells may therefore provide an attractive therapeutic target for the management of both early and late phases of severe SAg-mediated illnesses.
Assuntos
Antígenos de Bactérias/toxicidade , Anergia Clonal , Modelos Imunológicos , Células T Invariantes Associadas à Mucosa/imunologia , Staphylococcus aureus/imunologia , Streptococcus pyogenes/imunologia , Superantígenos/toxicidade , Animais , Antígenos de Bactérias/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linhagem Celular , Células Cultivadas , Anergia Clonal/efeitos dos fármacos , Cruzamentos Genéticos , Enterotoxinas/metabolismo , Enterotoxinas/toxicidade , Feminino , Humanos , Hibridomas , Imunidade Inata , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Camundongos Transgênicos , Células T Invariantes Associadas à Mucosa/citologia , Células T Invariantes Associadas à Mucosa/efeitos dos fármacos , Células T Invariantes Associadas à Mucosa/metabolismo , Organismos Livres de Patógenos Específicos , Staphylococcus aureus/metabolismo , Streptococcus pyogenes/metabolismo , Superantígenos/metabolismo , Quimeras de Transplante/sangue , Quimeras de Transplante/imunologia , Quimeras de Transplante/metabolismoRESUMO
Mucosal-associated invariant T (MAIT) cells produce inflammatory cytokines and cytotoxic granzymes in response to by-products of microbial riboflavin synthesis. Although MAIT cells are protective against some pathogens, we reasoned that they might contribute to pathology in chronic bacterial infection. We observed MAIT cells in proximity to Helicobacter pylori bacteria in human gastric tissue, and so, using MR1-tetramers, we examined whether MAIT cells contribute to chronic gastritis in a mouse H. pylori SS1 infection model. Following infection, MAIT cells accumulated to high numbers in the gastric mucosa of wild-type C57BL/6 mice, and this was even more pronounced in MAIT TCR transgenic mice or in C57BL/6 mice where MAIT cells were preprimed by Ag exposure or prior infection. Gastric MAIT cells possessed an effector memory Tc1/Tc17 phenotype, and were associated with accelerated gastritis characterized by augmented recruitment of neutrophils, macrophages, dendritic cells, eosinophils, and non-MAIT T cells and by marked gastric atrophy. Similarly treated MR1-/- mice, which lack MAIT cells, showed significantly less gastric pathology. Thus, we demonstrate the pathogenic potential of MAIT cells in Helicobacter-associated immunopathology, with implications for other chronic bacterial infections.
Assuntos
Gastrite/imunologia , Infecções por Helicobacter/imunologia , Helicobacter pylori/imunologia , Células T Invariantes Associadas à Mucosa/imunologia , Adulto , Animais , Linhagem Celular Tumoral , Feminino , Mucosa Gástrica/imunologia , Humanos , Memória Imunológica/imunologia , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Linfócitos T Citotóxicos/imunologiaRESUMO
T cells discriminate between foreign and host molecules by recognizing distinct microbial molecules, predominantly peptides and lipids. Riboflavin precursors found in many bacteria and yeast also selectively activate mucosal-associated invariant T (MAIT) cells, an abundant population of innate-like T cells in humans. However, the genesis of these small organic molecules and their mode of presentation to MAIT cells by the major histocompatibility complex (MHC)-related protein MR1 (ref. 8) are not well understood. Here we show that MAIT-cell activation requires key genes encoding enzymes that form 5-amino-6-d-ribitylaminouracil (5-A-RU), an early intermediate in bacterial riboflavin synthesis. Although 5-A-RU does not bind MR1 or activate MAIT cells directly, it does form potent MAIT-activating antigens via non-enzymatic reactions with small molecules, such as glyoxal and methylglyoxal, which are derived from other metabolic pathways. The MAIT antigens formed by the reactions between 5-A-RU and glyoxal/methylglyoxal were simple adducts, 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil (5-OE-RU) and 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU), respectively, which bound to MR1 as shown by crystal structures of MAIT TCR ternary complexes. Although 5-OP-RU and 5-OE-RU are unstable intermediates, they became trapped by MR1 as reversible covalent Schiff base complexes. Mass spectra supported the capture by MR1 of 5-OP-RU and 5-OE-RU from bacterial cultures that activate MAIT cells, but not from non-activating bacteria, indicating that these MAIT antigens are present in a range of microbes. Thus, MR1 is able to capture, stabilize and present chemically unstable pyrimidine intermediates, which otherwise convert to lumazines, as potent antigens to MAIT cells. These pyrimidine adducts are microbial signatures for MAIT-cell immunosurveillance.
Assuntos
Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Ativação Linfocitária/imunologia , Redes e Vias Metabólicas , Pirimidinas/metabolismo , Riboflavina/metabolismo , Subpopulações de Linfócitos T/imunologia , Amino Açúcares/química , Amino Açúcares/imunologia , Amino Açúcares/metabolismo , Apresentação de Antígeno/imunologia , Antígenos de Bactérias/química , Glioxal/química , Glioxal/metabolismo , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunidade Inata/imunologia , Imunidade nas Mucosas/imunologia , Ligantes , Antígenos de Histocompatibilidade Menor , Modelos Moleculares , Conformação Molecular , Mucosa/imunologia , Pirimidinas/química , Pirimidinas/imunologia , Aldeído Pirúvico/química , Aldeído Pirúvico/metabolismo , Riboflavina/biossíntese , Riboflavina/imunologia , Bases de Schiff/química , Subpopulações de Linfócitos T/citologia , Uracila/análogos & derivados , Uracila/química , Uracila/imunologia , Uracila/metabolismo , Complexo Vitamínico B/imunologia , Complexo Vitamínico B/metabolismoRESUMO
Mucosal-associated invariant T (MAIT) cells are unconventional T cells that recognize antigens derived from riboflavin biosynthesis. In addition to anti-microbial functions, human MAIT cells are associated with cancers, autoimmunity, allergies and inflammatory disorders, although their role is poorly understood. Activated MAIT cells are well known for their rapid release of Th1 and Th17 cytokines, but we have discovered that chronic stimulation can also lead to potent interleukin (IL)-13 expression. We used RNA-seq and qRT-PCR to demonstrate high expression of the IL-13 gene in chronically stimulated MAIT cells, and directly identify IL-13 using intracellular flow cytometry and multiplex bead analysis of MAIT cell cultures. This unexpected finding has important implications for IL-13-dependent diseases, such as colorectal cancer (CRC), that occur in mucosal areas where MAIT cells are abundant. We identify MAIT cells near CRC tumors and show that these areas and precancerous polyps express high levels of the IL-13 receptor, which promotes tumor progression and metastasis. Our data suggest that MAIT cells have a more complicated role in CRC than currently realized and that they represent a promising new target for immunotherapies where IL-13 can be a critical factor.
Assuntos
Neoplasias Colorretais/imunologia , Interleucina-13/metabolismo , Células T Invariantes Associadas à Mucosa/imunologia , Lesões Pré-Cancerosas/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Colo/citologia , Colo/imunologia , Colo/patologia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Humanos , Imunoterapia/métodos , Interleucina-13/imunologia , Subunidade alfa1 de Receptor de Interleucina-13 , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Ativação Linfocitária/imunologia , Pessoa de Meia-Idade , Células T Invariantes Associadas à Mucosa/metabolismo , Lesões Pré-Cancerosas/patologia , Lesões Pré-Cancerosas/terapia , RNA-Seq , Receptores de Interleucina-13/metabolismo , Reto/citologia , Reto/imunologia , Reto/patologiaRESUMO
Mucosal-associated invariant T (MAIT) cells are innate-like T lymphocytes known to elicit potent immunity to a broad range of bacteria, mainly via the rapid production of inflammatory cytokines. Whether MAIT cells contribute to antiviral immunity is less clear. Here we asked whether MAIT cells produce cytokines/chemokines during severe human influenza virus infection. Our analysis in patients hospitalized with avian H7N9 influenza pneumonia showed that individuals who recovered had higher numbers of CD161(+)Vα7.2(+) MAIT cells in peripheral blood compared with those who succumbed, suggesting a possible protective role for this lymphocyte population. To understand the mechanism underlying MAIT cell activation during influenza, we cocultured influenza A virus (IAV)-infected human lung epithelial cells (A549) and human peripheral blood mononuclear cells in vitro, then assayed them by intracellular cytokine staining. Comparison of influenza-induced MAIT cell activation with the profile for natural killer cells (CD56(+)CD3(-)) showed robust up-regulation of IFNγ for both cell populations and granzyme B in MAIT cells, although the individual responses varied among healthy donors. However, in contrast to the requirement for cell-associated factors to promote NK cell activation, the induction of MAIT cell cytokine production was dependent on IL-18 (but not IL-12) production by IAV-exposed CD14(+) monocytes. Overall, this evidence for IAV activation via an indirect, IL-18-dependent mechanism indicates that MAIT cells are protective in influenza, and also possibly in any human disease process in which inflammation and IL-18 production occur.