RESUMO
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 (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
Pre-treatment screening of individuals for human leukocyte antigens (HLA) HLA-B*57:01 is recommended for the prevention of life-threatening hypersensitivity reactions to abacavir, a drug widely prescribed for HIV treatment. However, the implementation of screening in clinical practice is hindered by the slow turnaround time and high cost of conventional HLA genotyping methods. We have developed a biosensor platform using interdigitated electrode (IDE) functionalized with a monoclonal antibody to detect cells expressing HLA-B*57:01. This platform was evaluated using cell lines and peripheral blood mononuclear cells expressing different HLA-B alleles. The functionalized IDE sensor was able to specifically capture HLA-B*57:01 cells, resulting in a significant change in the impedance magnitude in 20 min. This IDE platform has the potential to be further developed to enable point-of-care HLA-B*57:01 screening.
Assuntos
Técnicas Biossensoriais/métodos , Didesoxinucleosídeos/efeitos adversos , Hipersensibilidade a Drogas/prevenção & controle , Antígenos HLA-B/análise , Leucócitos Mononucleares/metabolismo , Alelos , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Didesoxinucleosídeos/uso terapêutico , Hipersensibilidade a Drogas/etiologia , Técnicas Eletroquímicas , Eletrodos , Infecções por HIV/tratamento farmacológico , Antígenos HLA-B/genética , Antígenos HLA-B/imunologia , HumanosRESUMO
Human leukocyte antigens (HLAs) are highly polymorphic proteins that initiate immunity by presenting pathogen-derived peptides to T cells. HLA polymorphisms mostly map to the antigen-binding cleft, thereby diversifying the repertoire of self-derived and pathogen-derived peptide antigens selected by different HLA allotypes. A growing number of immunologically based drug reactions, including abacavir hypersensitivity syndrome (AHS) and carbamazepine-induced Stevens-Johnson syndrome (SJS), are associated with specific HLA alleles. However, little is known about the underlying mechanisms of these associations, including AHS, a prototypical HLA-associated drug reaction occurring exclusively in individuals with the common histocompatibility allele HLA-B*57:01, and with a relative risk of more than 1,000 (refs 6, 7). We show that unmodified abacavir binds non-covalently to HLA-B*57:01, lying across the bottom of the antigen-binding cleft and reaching into the F-pocket, where a carboxy-terminal tryptophan typically anchors peptides bound to HLA-B*57:01. Abacavir binds with exquisite specificity to HLA-B*57:01, changing the shape and chemistry of the antigen-binding cleft, thereby altering the repertoire of endogenous peptides that can bind HLA-B*57:01. In this way, abacavir guides the selection of new endogenous peptides, inducing a marked alteration in 'immunological self'. The resultant peptide-centric 'altered self' activates abacavir-specific T-cells, thereby driving polyclonal CD8 T-cell activation and a systemic reaction manifesting as AHS. We also show that carbamazepine, a widely used anti-epileptic drug associated with hypersensitivity reactions in HLA-B*15:02 individuals, binds to this allotype, producing alterations in the repertoire of presented self peptides. Our findings simultaneously highlight the importance of HLA polymorphism in the evolution of pharmacogenomics and provide a general mechanism for some of the growing number of HLA-linked hypersensitivities that involve small-molecule drugs.
Assuntos
Apresentação de Antígeno/efeitos dos fármacos , Autoimunidade/efeitos dos fármacos , Autoimunidade/imunologia , Didesoxinucleosídeos/farmacologia , Antígenos HLA-B/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Sítios de Ligação , Doadores de Sangue , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Carbamazepina/farmacologia , Hipersensibilidade a Drogas , Antígenos HLA-B/química , Humanos , Modelos Moleculares , Conformação Proteica , SíndromeRESUMO
Antigen-presenting molecules, encoded by the major histocompatibility complex (MHC) and CD1 family, bind peptide- and lipid-based antigens, respectively, for recognition by T cells. Mucosal-associated invariant T (MAIT) cells are an abundant population of innate-like T cells in humans that are activated by an antigen(s) bound to the MHC class I-like molecule MR1. Although the identity of MR1-restricted antigen(s) is unknown, it is present in numerous bacteria and yeast. Here we show that the structure and chemistry within the antigen-binding cleft of MR1 is distinct from the MHC and CD1 families. MR1 is ideally suited to bind ligands originating from vitamin metabolites. The structure of MR1 in complex with 6-formyl pterin, a folic acid (vitamin B9) metabolite, shows the pterin ring sequestered within MR1. Furthermore, we characterize related MR1-restricted vitamin derivatives, originating from the bacterial riboflavin (vitamin B2) biosynthetic pathway, which specifically and potently activate MAIT cells. Accordingly, we show that metabolites of vitamin B represent a class of antigen that are presented by MR1 for MAIT-cell immunosurveillance. As many vitamin biosynthetic pathways are unique to bacteria and yeast, our data suggest that MAIT cells use these metabolites to detect microbial infection.
Assuntos
Ácido Fólico/metabolismo , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/imunologia , Pterinas/química , Pterinas/imunologia , Linfócitos T/imunologia , Apresentação de Antígeno , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Ácido Fólico/química , Ácido Fólico/imunologia , Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Vigilância Imunológica/imunologia , Células Jurkat , Ligantes , Ativação Linfocitária , Antígenos de Histocompatibilidade Menor , Modelos Moleculares , Redobramento de Proteína/efeitos dos fármacos , Pterinas/metabolismo , Pterinas/farmacologia , Salmonella/imunologia , Salmonella/metabolismo , Infecções por Salmonella/imunologia , Infecções por Salmonella/microbiologia , Eletricidade Estática , Microglobulina beta-2/imunologia , Microglobulina beta-2/metabolismoRESUMO
The basis for strong immunogenetic associations between particular human leukocyte antigen (HLA) class I allotypes and inflammatory conditions like Behçet's disease (HLA-B51) and ankylosing spondylitis (HLA-B27) remain mysterious. Recently, however, even stronger HLA associations are reported in drug hypersensitivities to the reverse-transcriptase inhibitor abacavir (HLA-B57), the gout prophylactic allopurinol (HLA-B58), and the antiepileptic carbamazepine (HLA-B*1502), providing a defined disease trigger and suggesting a general mechanism for these associations. We show that systemic reactions to abacavir were driven by drug-specific activation of cytokine-producing, cytotoxic CD8+ T cells. Recognition of abacavir required the transporter associated with antigen presentation and tapasin, was fixation sensitive, and was uniquely restricted by HLA-B*5701 and not closely related HLA allotypes with polymorphisms in the antigen-binding cleft. Hence, the strong association of HLA-B*5701 with abacavir hypersensitivity reflects specificity through creation of a unique ligand as well as HLA-restricted antigen presentation, suggesting a basis for the strong HLA class I-association with certain inflammatory disorders.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Didesoxinucleosídeos/efeitos adversos , Hipersensibilidade a Drogas/imunologia , Antígenos HLA-B/imunologia , Ativação Linfocitária , Inibidores da Transcriptase Reversa/efeitos adversos , Fármacos Anti-HIV/efeitos adversos , Fármacos Anti-HIV/imunologia , Fármacos Anti-HIV/metabolismo , Apresentação de Antígeno , Didesoxinucleosídeos/imunologia , Didesoxinucleosídeos/metabolismo , Hipersensibilidade a Drogas/metabolismo , Antígenos HLA-B/química , Antígenos HLA-B/metabolismo , Humanos , Inibidores da Transcriptase Reversa/imunologia , Inibidores da Transcriptase Reversa/metabolismoRESUMO
BACKGROUND/OBJECTIVES: Mucosal-associated invariant T (MAIT) cells are a novel subset of innate-like T-cells that are enriched in mucosal tissues. Their presence in human skin has only recently been recognised. We describe the expression of skin-tropic molecules on human skin MAIT cells at steady state and investigate their contribution to various dermatoses with known T-cell involvement. METHODS: To examine the expression of skin-tropic molecules by MAIT cells at steady state, we performed a flow cytometric analysis of blood and skin samples from healthy donors. To investigate any potential wider contribution of MAIT cells to skin disease, we examined psoriasis, alopecia areata and dermatitis herpetiformis biopsies using immunofluorescent staining to identify the proportion of T-cells expressing MAIT cell surface markers. RESULTS: We found that MAIT cells constituted a small population of T-cells in normal human skin, similar to the percentage found in peripheral blood. Like other skin T-cells, skin MAIT cells expressed high levels of the skin-associated markers, cutaneous lymphocyte antigen and CD103. In psoriasis and alopecia areata the proportion of MAIT cells was similar to that found in normal skin, but in dermatitis herpetiformis it was significantly elevated. CONCLUSIONS: The expression of skin-tropic molecules by skin MAIT cells is consistent with their resident status in normal human skin. Our results suggest that MAIT cells may play a role in the pathogenesis of dermatitis herpetiformis.
Assuntos
Dermatite Herpetiforme/imunologia , Células T Invariantes Associadas à Mucosa/metabolismo , Pele/imunologia , Alopecia em Áreas/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Humanos , Cadeias alfa de Integrinas/metabolismo , Contagem de Linfócitos , Glicoproteínas de Membrana/metabolismo , Psoríase/imunologiaRESUMO
Current views emphasize TCR diversity as a key feature that differentiates the group 1 (CD1a, CD1b, CD1c) and group 2 (CD1d) CD1 systems. Whereas TCR sequence motifs define CD1d-reactive NKT cells, the available data do not allow a TCR-based organization of the group 1 CD1 repertoire. The observed TCR diversity might result from donor-to-donor differences in TCR repertoire, as seen for MHC-restricted T cells. Alternatively, diversity might result from differing CD1 isoforms, Ags, and methods used to identify TCRs. Using CD1b tetramers to isolate clones recognizing the same glycolipid, we identified a previously unknown pattern of V gene usage (TRAV17, TRBV4-1) among unrelated human subjects. These TCRs are distinct from those present on NKT cells and germline-encoded mycolyl lipid-reactive T cells. Instead, they resemble the TCR of LDN5, one of the first known CD1b-reactive clones that was previously thought to illustrate the diversity of the TCR repertoire. Interdonor TCR conservation was observed in vitro and ex vivo, identifying LDN5-like T cells as a distinct T cell type. These data support TCR-based organization of the CD1b repertoire, which consists of at least two compartments that differ in TCR sequence motifs, affinity, and coreceptor expression.
Assuntos
Motivos de Aminoácidos/imunologia , Receptores de Antígenos de Linfócitos T/química , Subpopulações de Linfócitos T/química , Antígenos CD1/imunologia , Sequência de Bases , Sequência Conservada/imunologia , Citometria de Fluxo , Glicolipídeos/imunologia , Humanos , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Subpopulações de Linfócitos T/imunologiaRESUMO
alphabeta T cell receptors (TCRs) are genetically restricted to corecognize peptide antigens bound to self-major histocompatibility complex (pMHC) molecules; however, the basis for this MHC specificity remains unclear. Despite the current dogma, evaluation of the TCR-pMHC-I structural database shows that the nongermline-encoded complementarity-determining region (CDR)-3 loops often contact the MHC-I, and the germline-encoded CDR1 and -2 loops frequently participate in peptide-mediated interactions. Nevertheless, different TCRs adopt a roughly conserved docking mode over the pMHC-I, in which three MHC-I residues (65, 69, and 155) are invariably contacted by the TCR in one way or another. Nonetheless, the impact of mutations at these three positions, either individually or together, was not uniformly detrimental to TCR recognition of pHLA-B*0801 or pHLA-B*3508. Moreover, when TCR-pMHC-I recognition was impaired, this could be partially restored by expression of the CD8 coreceptor. The structure of a TCR-pMHC-I complex in which these three (65, 69, and 155) MHC-I positions were all mutated resulted in shifting of the TCR footprint relative to the cognate complex and formation of compensatory interactions. Collectively, our findings reveal the inherent adaptability of the TCR in maintaining peptide recognition while accommodating changes to the central docking site on the pMHC-I.
Assuntos
Imunidade Adaptativa , Complexo Principal de Histocompatibilidade , Receptores de Antígenos de Linfócitos T/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Bases de Dados Genéticas , Humanos , Ativação Linfocitária , Modelos Moleculares , Mutação , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
Little is known regarding the basis for selection of the semi-invariant alphabeta T cell receptor (TCR) expressed by natural killer T (NKT) cells or how this mediates recognition of CD1d-glycolipid complexes. We have determined the structures of two human NKT TCRs that differ in their CDR3beta composition and length. Both TCRs contain a conserved, positively charged pocket at the ligand interface that is lined by residues from the invariant TCR alpha- and semi-invariant beta-chains. The cavity is centrally located and ideally suited to interact with the exposed glycosyl head group of glycolipid antigens. Sequences common to mouse and human invariant NKT TCRs reveal a contiguous conserved "hot spot" that provides a basis for the reactivity of NKT cells across species. Structural and functional data suggest that the CDR3beta loop provides a plasticity mechanism that accommodates recognition of a variety of glycolipid antigens presented by CD1d. We propose a model of NKT TCR-CD1d-glycolipid interaction in which the invariant CDR3alpha loop is predicted to play a major role in determining the inherent bias toward CD1d. The findings define a structural basis for the selection of the semi-invariant alphabeta TCR and the unique antigen specificity of NKT cells.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos CD1/imunologia , Glicolipídeos/imunologia , Células Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T/imunologia , Animais , Apresentação de Antígeno/genética , Antígenos CD1/genética , Genes Codificadores da Cadeia alfa de Receptores de Linfócitos T/genética , Genes Codificadores da Cadeia alfa de Receptores de Linfócitos T/imunologia , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/genética , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/imunologia , Humanos , Camundongos , Ligação Proteica/genética , Ligação Proteica/imunologia , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína/fisiologia , Especificidade da Espécie , Homologia Estrutural de Proteína , Relação Estrutura-AtividadeRESUMO
Mucosal-associated invariant T (MAIT) cells detect microbial infection via recognition of riboflavin-based antigens presented by the major histocompatibility complex class I (MHC-I)-related protein 1 (MR1). Most MAIT cells in human peripheral blood express CD8αα or CD8αß coreceptors, and the binding site for CD8 on MHC-I molecules is relatively conserved in MR1. Yet, there is no direct evidence of CD8 interacting with MR1 or the functional consequences thereof. Similarly, the role of CD8αα in lymphocyte function remains ill-defined. Here, using newly developed MR1 tetramers, mutated at the CD8 binding site, and by determining the crystal structure of MR1-CD8αα, we show that CD8 engaged MR1, analogous to how it engages MHC-I molecules. CD8αα and CD8αß enhanced MR1 binding and cytokine production by MAIT cells. Moreover, the CD8-MR1 interaction was critical for the recognition of folate-derived antigens by other MR1-reactive T cells. Together, our findings suggest that both CD8αα and CD8αß act as functional coreceptors for MAIT and other MR1-reactive T cells.
Assuntos
Células T Invariantes Associadas à Mucosa , Receptores de Antígenos de Linfócitos T alfa-beta , Antígenos , Antígenos CD8 , Linfócitos T CD8-Positivos , Antígenos de Histocompatibilidade Classe I , Humanos , Antígenos de Histocompatibilidade MenorRESUMO
Abacavir hypersensitivity syndrome can occur in individuals expressing the HLA-B*57:01 major histocompatibility complex class I allotype when utilising the drug abacavir as a part of their anti-retroviral regimen. The drug is known to bind within the HLA-B*57:01 antigen binding cleft, leading to the selection of novel self-peptide ligands, thus provoking life-threatening immune responses. However, the sub-cellular location of abacavir binding and the mechanics of altered peptide selection are not well understood. Here, we probed the impact of abacavir on the assembly of HLA-B*57:01 peptide complexes. We show that whilst abacavir had minimal impact on the maturation or average stability of HLA-B*57:01 molecules, abacavir was able to differentially enhance the formation, selectively decrease the dissociation, and alter tapasin loading dependency of certain HLA-B*57:01-peptide complexes. Our data reveals a spectrum of abacavir mediated effects on the immunopeptidome which reconciles the heterogeneous functional T cell data reported in the literature.
Assuntos
Fármacos Anti-HIV/imunologia , Didesoxinucleosídeos/imunologia , Hipersensibilidade a Drogas/imunologia , Antígenos HLA-B/imunologia , Linfócitos T/imunologia , Fármacos Anti-HIV/efeitos adversos , Linhagem Celular , Didesoxinucleosídeos/efeitos adversos , Antígenos HLA-B/metabolismo , Humanos , Cinética , Ativação Linfocitária/imunologiaRESUMO
Mucosal-associated invariant T (MAIT) cells are MR1-restricted innate-like T cells conserved across mammalian species, including mice and humans. By sequencing RNA from sorted MR1-5-OP-RU tetramer+ cells derived from either human blood or murine lungs, we define the basic transcriptome of an activated MAIT cell in both species and demonstrate how this profile changes during the resolution of infection and during reinfection. We observe strong similarities between MAIT cells in humans and mice. In both species, activation leads to strong expression of pro-inflammatory cytokines and chemokines as well as a strong tissue repair signature, recently described in murine commensal-specific H2-M3-restricted T cells. Transcriptomes of MAIT cells and H2-M3-specific CD8+ T cells displayed the most similarities to invariant natural killer T (iNKT) cells when activated, but to γδ T cells after the resolution of infection. These data define the requirements for and consequences of MAIT cell activation, revealing a tissue repair phenotype expressed upon MAIT cell activation in both species.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Ativação Linfocitária , Células T Invariantes Associadas à Mucosa/imunologia , Células T Matadoras Naturais/imunologia , Transcriptoma/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Humanos , Camundongos , Células T Invariantes Associadas à Mucosa/citologia , Células T Matadoras Naturais/citologiaRESUMO
Mucosal-associated invariant T (MAIT) cells are activated in a TCR-dependent manner by antigens derived from the riboflavin synthesis pathway, including 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), bound to MHC-related protein-1 (MR1). However, MAIT cell activation in vivo has not been studied in detail. Here, we have found and characterized additional molecular signals required for optimal activation and expansion of MAIT cells after pulmonary Legionella or Salmonella infection in mice. We show that either bone marrow-derived APCs or non-bone marrow-derived cells can activate MAIT cells in vivo, depending on the pathogen. Optimal MAIT cell activation in vivo requires signaling through the inducible T cell costimulator (ICOS), which is highly expressed on MAIT cells. Subsequent expansion and maintenance of MAIT-17/1-type responses are dependent on IL-23. Vaccination with IL-23 plus 5-OP-RU augments MAIT cell-mediated control of pulmonary Legionella infection. These findings reveal cellular and molecular targets for manipulating MAIT cell function under physiological conditions.
Assuntos
Antígenos de Bactérias/imunologia , Interleucina-23/imunologia , Legionella/imunologia , Doença dos Legionários/imunologia , Células T Invariantes Associadas à Mucosa/imunologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , VacinaçãoRESUMO
Mucosal associated invariant T (MAIT) cells recognise conserved microbial metabolites from riboflavin synthesis. Striking evolutionary conservation and pulmonary abundance implicate them in antibacterial host defence, yet their functions in protection against clinically important pathogens are unknown. Here we show that mouse Legionella longbeachae infection induces MR1-dependent MAIT cell activation and rapid pulmonary accumulation of MAIT cells associated with immune protection detectable in immunocompetent host animals. MAIT cell protection is more evident in mice lacking CD4+ cells, and adoptive transfer of MAIT cells rescues immunodeficient Rag2-/-γC-/- mice from lethal Legionella infection. Protection is dependent on MR1, IFN-γ and GM-CSF, but not IL-17A, TNF or perforin, and enhanced protection is detected earlier after infection of mice antigen-primed to boost MAIT cell numbers before infection. Our findings define a function for MAIT cells in protection against a major human pathogen and indicate a potential role for vaccination to enhance MAIT cell immunity.
Assuntos
Legionella longbeachae/patogenicidade , Pulmão/microbiologia , Células T Invariantes Associadas à Mucosa/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Interleucina-17/metabolismo , Legionella longbeachae/imunologia , Legionelose/imunologia , Legionelose/microbiologia , Pulmão/metabolismo , Masculino , Camundongos , Células T Invariantes Associadas à Mucosa/metabolismo , Perforina/metabolismoRESUMO
Mucosal associated invariant T (MAIT) cells are evolutionarily-conserved, innate-like lymphocytes which are abundant in human lungs and can contribute to protection against pulmonary bacterial infection. MAIT cells are also activated during human viral infections, yet it remains unknown whether MAIT cells play a significant protective or even detrimental role during viral infections in vivo. Using murine experimental challenge with two strains of influenza A virus, we show that MAIT cells accumulate and are activated early in infection, with upregulation of CD25, CD69 and Granzyme B, peaking at 5 days post-infection. Activation is modulated via cytokines independently of MR1. MAIT cell-deficient MR1-/- mice show enhanced weight loss and mortality to severe (H1N1) influenza. This is ameliorated by prior adoptive transfer of pulmonary MAIT cells in both immunocompetent and immunodeficient RAG2-/-γC-/- mice. Thus, MAIT cells contribute to protection during respiratory viral infections, and constitute a potential target for therapeutic manipulation.
Assuntos
Influenza Humana/patologia , Influenza Humana/virologia , Células T Invariantes Associadas à Mucosa/virologia , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Transferência Adotiva , Animais , Citocinas/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Pulmão/patologia , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Menor/metabolismoRESUMO
HLA-G is a nonclassical class I major histocompatibility complex (MHC) molecule that is primarily expressed at the foetal-maternal interface. Although the role of HLA-G has not been fully elucidated, current evidence suggests it protects the foetus from the maternal immune response. In this report, HLA-G (44 kDa) is characterized by expression in Escherichia coli. The inclusion bodies were refolded in complex with a peptide derived from histone H2A (RIIPRHLQL), purified and subsequently crystallized. Correct refolding was determined using two conformation-dependent antibodies. Cobalt ions were shown to be an essential ingredient for obtaining diffraction-quality crystals. The crystals, which diffracted to 1.9 A resolution, belonged to space group P3(2)2(1), with unit-cell parameters a = b = 77.15, c = 151.72 A.
Assuntos
Cobalto/química , Antígenos HLA/química , Antígenos HLA/isolamento & purificação , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Substituição de Aminoácidos/genética , Cátions Bivalentes/química , Linhagem Celular Tumoral , Cisteína/genética , Antígenos HLA/genética , Antígenos HLA-G , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Serina/genética , SolubilidadeRESUMO
Mucosal-associated invariant T (MAIT) cells express an invariant T cell receptor (TCR) α-chain (TRAV1-2 joined to TRAJ33, TRAJ20, or TRAJ12 in humans), which pairs with an array of TCR ß-chains. MAIT TCRs can bind folate- and riboflavin-based metabolites restricted by the major histocompatibility complex (MHC)-related class I-like molecule, MR1. However, the impact of MAIT TCR and MR1-ligand heterogeneity on MAIT cell biology is unclear. We show how a previously uncharacterized MR1 ligand, acetyl-6-formylpterin (Ac-6-FP), markedly stabilized MR1, potently up-regulated MR1 cell surface expression, and inhibited MAIT cell activation. These enhanced properties of Ac-6-FP were attributable to structural alterations in MR1 that subsequently affected MAIT TCR recognition via conformational changes within the complementarity-determining region (CDR) 3ß loop. Analysis of seven TRBV6-1(+) MAIT TCRs demonstrated how CDR3ß hypervariability impacted on MAIT TCR recognition by altering TCR flexibility and contacts with MR1 and the Ag itself. Ternary structures of TRBV6-1, TRBV6-4, and TRBV20(+) MAIT TCRs in complex with MR1 bound to a potent riboflavin-based antigen (Ag) showed how variations in TRBV gene usage exclusively impacted on MR1 contacts within a consensus MAIT TCR-MR1 footprint. Moreover, differential TRAJ gene usage was readily accommodated within a conserved MAIT TCR-MR1-Ag docking mode. Collectively, MAIT TCR heterogeneity can fine-tune MR1 recognition in an Ag-dependent manner, thereby modulating MAIT cell recognition.
Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Mucosa/citologia , Mucosa/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Antígenos/metabolismo , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/metabolismo , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Antígenos de Histocompatibilidade Menor , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Pterinas/química , Pterinas/farmacologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Coloração e Rotulagem , Ressonância de Plasmônio de Superfície , Linfócitos T/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Mucosal-associated invariant T cells (MAIT cells) express a semi-invariant T cell receptor (TCR) α-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-CH2OH), 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 CD8α(+)CD8ß(-/lo), implying predominant expression of CD8αα homodimers. Tetramer-sorted MAIT cells displayed a T(H)1 cytokine phenotype upon antigen-specific activation. Similarly, mouse MR1-rRL-6-CH2OH tetramers detected CD4(+), CD4(-)CD8(-) and CD8(+) MAIT cells in Vα19 transgenic mice. Both human and mouse MAIT cells expressed a broad TCR-ß 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.
Assuntos
Antígenos/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Células T Matadoras Naturais/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Animais , Células Cultivadas , Cristalografia por Raios X , Antígenos de Histocompatibilidade Classe I/química , Humanos , Camundongos , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor , Proteínas Mutantes/metabolismo , Redobramento de Proteína , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismoRESUMO
Mucosal-associated invariant T (MAIT) cells express a semiinvariant αß T cell receptor (TCR) that binds MHC class I-like molecule (MR1). However, the molecular basis for MAIT TCR recognition by MR1 is unknown. In this study, we present the crystal structure of a human Vα7.2Jα33-Vß2 MAIT TCR. Mutagenesis revealed highly conserved requirements for the MAIT TCR-MR1 interaction across different human MAIT TCRs stimulated by distinct microbial sources. Individual residues within the MAIT TCR ß chain were dispensable for the interaction with MR1, whereas the invariant MAIT TCR α chain controlled specificity through a small number of residues, which are conserved across species and located within the Vα-Jα regions. Mutagenesis of MR1 showed that only two residues, which were centrally positioned and on opposing sides of the antigen-binding cleft of MR1, were essential for MAIT cell activation. The mutagenesis data are consistent with a centrally located MAIT TCR-MR1 docking that was dominated by the α chain of the MAIT TCR. This candidate docking mode contrasts with that of the NKT TCR-CD1d-antigen interaction, in which both the α and ß chain of the NKT TCR is required for ligation above the F'-pocket of CD1d.