Expression Of MR1 Research Articles

How MAIT cells control the growth of intracellular mycobacteria (P4381)

Abstract Mucosal associated invariant T cells or MAIT cells have a semi-invariant TCR Vα chain and limited Vβ chains. In addition MAIT cell development is dependent upon the commensal microbiota and the expression of MR1, a highly conserved class Ib molecule of mammals. Mouse and human MAIT cells are activated in an MR1-restricted manner by cells infected with diverse strains for bacteria suggesting a widely shared antigen. Remarkably, human MR1 was recently shown to bind select bacterial vitamin B metabolites capable of activating MAIT cells in an MR1-restricted manner. This finding raised the interesting question of the importance of MR1 presentation of vitamin B metabolites for controlling bacterial infection. To address this question we have developed a mouse model whereby mycobacteria infected macrophages are co-cultured with purified MAIT cells, and control of intracellular bacterial growth is monitored. Using this assay, MAIT cell control of mycobacterial growth was found to depend upon IL-12 secretion by infected macrophages and IFN-γ secretion by MAIT cells. Interestingly, in the absence of infection, MAIT cells secreted IFN-γ in response to recombinant IL-12 alone or antigenic vitamin B metabolites. These findings demonstrate that MAIT cells can be activated by either TCR or non-TCR interactions. The contribution of these two pathways of MAIT cell activation in the context of mycobacterial infection is currently being investigated.

Purification and Characterization of the [NiFe]-Hydrogenase of Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 possesses a periplasmic [NiFe]-hydrogenase (MR-1 [NiFe]-H(2)ase) that has been implicated in H(2) production and oxidation as well as technetium [Tc(VII)] reduction. To characterize the roles of MR-1 [NiFe]-H(2)ase in these proposed reactions, the genes encoding both subunits of MR-1 [NiFe]-H(2)ase were cloned and then expressed in an MR-1 mutant without hyaB and hydA genes. Expression of recombinant MR-1 [NiFe]-H(2)ase in trans restored the mutant's ability to produce H(2) at 37% of that for the wild type. Following purification, MR-1 [NiFe]-H(2)ase coupled H(2) oxidation to reduction of Tc(VII)O(4)(-) and methyl viologen. Change of the buffers used affected MR-1 [NiFe]-H(2)ase-mediated reduction of Tc(VII)O(4)(-) but not methyl viologen. Under the conditions tested, all Tc(VII)O(4)(-) used was reduced in Tris buffer, while in HEPES buffer, only 20% of Tc(VII)O(4)(-) was reduced. The reduced products were soluble in Tris buffer but insoluble in HEPES buffer. Transmission electron microscopy analysis revealed that Tc precipitates reduced in HEPES buffer were aggregates of crystallites with diameters of ∼5 nm. Measurements with X-ray absorption near-edge spectroscopy revealed that the reduction products were a mixture of Tc(IV) and Tc(V) in Tris buffer but only Tc(IV) in HEPES buffer. Measurements with extended X-ray adsorption fine structure showed that while the Tc bonding environment in Tris buffer could not be determined, the Tc(IV) product in HEPES buffer was very similar to Tc(IV)O(2)·nH(2)O, which was also the product of Tc(VII)O(4)(-) reduction by MR-1 cells. These results shows for the first time that MR-1 [NiFe]-H(2)ase catalyzes Tc(VII)O(4)(-) reduction directly by coupling to H(2) oxidation.

Bacteria, mucosal‐associated invariant T cells and MR1

Certain subpopulations of T lymphoctyes show innate-like properties that distinguish them from conventional T cells and thus have been collectively termed innate T cells. Two extensively studied subsets of innate T cells implicated in pathogen detection are invariant natural killer T (iNKT) cells and γδ T cells. Conventional T cells detect pathogens in a manner that is exquisitely antigen specific, based on discrimination of peptides bound to classical major histocompatibility complex (MHC) molecules. By contrast, innate T cells have only limited antigen discrimination and when antigen presentation has been implicated the antigen is not an unmodified peptide. For example, iNKT cells are activated by lipids presented by CD1d molecules,1 whereas certain γδ T cells are directly activated by ‘danger signals’ such as the small microbial product isopentenyl pyrophosphate.2 Their limited ligand discrimination and relatively larger clonal size allow innate T cells to mount earlier responses than conventional T cells. In comparison with conventional T cells, innate T-cell populations develop by a distinct ontogenic pathway and preferentially home to specific tissues. On the basis of these combined properties of innate T cells and the fact that their receptors and ligands have been highly conserved in evolution, it is attractive to propose that they represent ancient innate pathways of pathogen protection. Mucosal-associated invariant T (MAIT) cells were previously shown to have several properties in common with innate T cells, yet their physiological function remained unknown. Two new articles, Le Bourhis et al. published in Nature Immunology3 (reviewed here) and Gold et al. published in PLoS Biology4 implicate MAIT cells in the detection of bacterial infection in a manner restricted by the novel MHC class I-related molecule, MR1.

Endogenous MR1 is transiently expressed on the cell surface and can be stabilized with a unique mAb for activation of mucosal-associated invariant T cells (130.19)

Abstract MHC-related protein 1 (MR1) is highly conserved among mammals and restricts the development of mucosal-associated invariant T (MAIT) cells. MAIT cells bear an invariant TCRα chain preferentially paired with limited β chains. MAIT cells reside predominantly in the mucosal tissues but are absent in mice lacking commensal bacteria or B cells. Upon activation, MAIT cells rapidly secret cytokines including IFN-γ, IL-4, and IL-10. Surprisingly, endogenous MR1 has not been detected at the cell surface; but, surface MR1 is detected on cells overexpressing MR1 by transfection or transduction. To better characterize endogenous MR1 expression, we generated mAbs to mouse MR1. Using these mAbs, we show that select B cell lines express low levels of endogenous MR1 at the cell surface. Interestingly, a unique mAb to MR1 was found to enhance MAIT cell activation, whereas other mAbs to MR1 blocked MAIT cell activation. The enhancing and the blocking mAbs recognize different MR1 epitopes mapped by a panel of MR1 mutants and by mAb competition assays. Mechanistically, the enhancing mAb stabilizes MR1 at the cell surface suggesting it either promotes assembly of the MR1 complex or allosterically induces a more favorable conformation. These findings demonstrate that endogenous MR1 transiently visits the cell surface of APCs, and that induced levels of MR1/ligand complexes are required for MAIT cell activation.

MR1, a novel class Ib molecule, utilizes the endocytic pathway to activate mucosal associated invariant T cells (93.1)

Abstract MR1 is a non-MHC linked class Ib molecule that is highly conserved among mammals and its expression is required for the in vivo development of mucosal associated invariant T (MAIT) cells. MAIT cells, with a memory phenotype, express a canonical TCR α chain and preferentially reside in the gut in a manner dependent upon B cells and commensal flora. Although the nature of the ligand for MR1 is unknown, our previous structure-function data strongly suggested MAIT cell activation by MR1 is ligand dependent. Here we show that, unlike other class I molecules, MR1 surface expression and activation of MAIT cells are independent of TAP, tapasin, calreticulin and proteasome function. Alternatively, using fibroblast cells overexpressing MR1 and the class II chaperone Ii, we showed MR1/Ii were physically associated, which significantly enhanced the MAIT cell activation. The association with Ii suggested that MR1 might traffic to endocytic compartments, a hypothesis we tested in a B cell line since B cells express endogenous Ii and are though to be the physiologic APC for MAIT cells. Confocal staining of the B cells displayed colocalization of MR1 and Lamp1, and treatment with lysosome inhibitors was found to inhibit MR1 expression and MAIT cell activation. Thus the endocytic pathway used by MR1 mimics that of class II and certain CD1 isoforms, thereby providing a potential to sample both endocytosed and endogenous antigens.

Evidence for MR1 Antigen Presentation to Mucosal-associated Invariant T Cells

The novel class Ib molecule MR1 is highly conserved in mammals, particularly in its alpha1/alpha2 domains. Recent studies demonstrated that MR1 expression is required for development and expansion of a small population of T cells expressing an invariant T cell receptor (TCR) alpha chain called mucosal-associated invariant T (MAIT) cells. Despite these intriguing properties it has been difficult to determine whether MR1 expression and MAIT cell recognition is ligand-dependent. To address these outstanding questions, monoclonal antibodies were produced in MR1 knock-out mice immunized with recombinant MR1 protein, and a series of MR1 mutations were generated at sites previously shown to disrupt the ability of class Ia molecules to bind peptide or TCR. Here we show that 1) MR1 molecules are detected by monoclonal antibodies in either an open or folded conformation that correlates precisely with peptide-induced conformational changes in class Ia molecules, 2) only the folded MR1 conformer activated 2/2 MAIT hybridoma cells tested, 3) the pattern of MAIT cell activation by the MR1 mutants implies the MR1/TCR orientation is strikingly similar to published major histocompatibility complex/alphabetaTCR engagements, 4) all the MR1 mutations tested and found to severely reduce surface expression of folded molecules were located in the putative ligand binding groove, and 5) certain groove mutants of MR1 that are highly expressed on the cell surface disrupt MAIT cell activation. These combined data strongly support the conclusion that MR1 has an antigen presentation function.

Characterization of MR-1, a novel myofibrillogenesis regulator in human muscle.

The actin-myosin contractile apparatus consists of several thick filament and thin filament proteins. Specific regulatory mechanisms are involved in this highly ordered process. In this paper, we reported the identification and characterization of a novel myofibrillogenesis regulator, MR-1. The MR-1 gene was cloned from human skeletal muscle cDNA library by using a strategy that involves EST data base searching, PCR and RACE. The MR-1 gene is located on human chromosome 2q35 and encodes a 142 aa protein. Northern blot revealed that the mRNA level of MR-1 was highest in the skeletal muscle and certain level of MR-1 expression was also observed in heart, liver and kidney. Immunohistochemical assay confirmed that the MR-1 protein existed in human myocardial myofibrils. It was found by yeast two-hybrid screening and confirmed by in vitro binding assay that MR-1 could interact with sarcomeric proteins, such as myosin regulatory light chain, myomesin 1 and beta-enolase. These studies suggested that MR-1 might play a regulatory role in the muscle cell and it was worth investigating further.

Biochemical features of the MHC-related protein 1 consistent with an immunological function.

MHC-related protein (MR)1 is an MHC class I-related molecule encoded on chromosome 1 that is highly conserved among mammals and is more closely related to classical class I molecules than are other nonclassical class I family members. In this report, we show for the first time that both mouse and human MR1 molecules can associate with the peptide-loading complex and can be detected at low levels at the surface of transfected cells. We also report the production of recombinant human MR1 molecules in insect cells using highly supplemented media and provide evidence that the MR1 H chain can assume a folded conformation and is stoichiometrically associated with beta(2)-microglobulin, similar to class I molecules. Cumulatively, these findings demonstrate that surface expression of MR1 is possible but may be limited by a specific ligand or associated molecule.

Posttranscriptional Regulation of Cellular Gene Expression by the c-myc Oncogene

The c-myc oncogene has been implicated in the development of many different cancers, yet the mechanism by which the c-myc protein alters cellular growth control has proven elusive. We used a cDNA hybridization difference assay to isolate two genes, mr1 and mr2, that were constitutively expressed (i.e., deregulated) in rodent fibroblast cell lines immortalized by transfection of a viral promoter-linked c-myc gene. Both cDNAs were serum inducible in quiescent G0 fibroblasts, suggesting that they are functionally related to cellular proliferative processes. Although there were significant differences in cytoplasmic mRNA levels between myc-immortalized and control cells, the rates of transcription and mRNA turnover of both genes were similar, suggesting that c-myc regulates mr1 and mr2 expression by some nuclear posttranscriptional mechanism. mr1 was also rapidly (within 2 h) and specifically induced by dexamethasone in BALB/c cell lines expressing a mouse mammary tumor virus long terminal repeat-driven myc gene, under conditions where other growth factor-inducible genes were unaffected. A frameshift mutation in the mouse mammary tumor virus myc gene destroyed the dexamethasone stimulation of mr1, indicating that c-myc protein is required for the effect. As in the myc-immortalized cells, the induction of mr1 by c-myc occurred without detectable changes in mr1 transcription or cytoplasmic mRNA stability, implicating regulation, either direct or indirect, through a nuclear posttranscriptional mechanism. These results provide evidence that c-myc can rapidly modulate cellular gene expression and suggest that c-myc may function in gene regulation at the level of RNA export, splicing, or nuclear RNA turnover.

Posttranscriptional regulation of cellular gene expression by the c-myc oncogene.

The c-myc oncogene has been implicated in the development of many different cancers, yet the mechanism by which the c-myc protein alters cellular growth control has proven elusive. We used a cDNA hybridization difference assay to isolate two genes, mr1 and mr2, that were constitutively expressed (i.e., deregulated) in rodent fibroblast cell lines immortalized by transfection of a viral promoter-linked c-myc gene. Both cDNAs were serum inducible in quiescent G0 fibroblasts, suggesting that they are functionally related to cellular proliferative processes. Although there were significant differences in cytoplasmic mRNA levels between myc-immortalized and control cells, the rates of transcription and mRNA turnover of both genes were similar, suggesting that c-myc regulates mr1 and mr2 expression by some nuclear posttranscriptional mechanism. mr1 was also rapidly (within 2 h) and specifically induced by dexamethasone in BALB/c cell lines expressing a mouse mammary tumor virus long terminal repeat-driven myc gene, under conditions where other growth factor-inducible genes were unaffected. A frameshift mutation in the mouse mammary tumor virus myc gene destroyed the dexamethasone stimulation of mr1, indicating that c-myc protein is required for the effect. As in the myc-immortalized cells, the induction of mr1 by c-myc occurred without detectable changes in mr1 transcription or cytoplasmic mRNA stability, implicating regulation, either direct or indirect, through a nuclear posttranscriptional mechanism. These results provide evidence that c-myc can rapidly modulate cellular gene expression and suggest that c-myc may function in gene regulation at the level of RNA export, splicing, or nuclear RNA turnover.