The MHC-Ib molecule MR1 presents microbial metabolites to MR1-restricted T cells (MR1Ts). Given the ubiquitous expression of MR1 and the high prevalence of human MR1Ts, it is important to understand the mechanisms of MR1-dependent antigen presentation. Here, we show that MR1-dependent antigen presentation can be distinguished between intracellular Mycobacterium tuberculosis (Mtb) infection and exogenously added antigens. Although both Mtb infection and exogenously added antigens are presented by preformed MR1, only exogenously added antigens are capable of reusing MR1 that had been bound to the folic acid metabolite 6-formylpterin (6-FP). In addition, we identify an endosomal trafficking protein, Syntaxin 4, which is specifically involved in the presentation of exogenously delivered antigens but not Mtb-dependent antigen presentation. These data reveal there are multiple ways that MR1 can sample antigens and that MR1-mediated sampling of intracellular Mtb infection is distinguishable from the sampling of exogenously added antigens.

Mucosal Associated Invariant T (MAIT) cells can sense intracellular infection by a broad array of pathogens. These cells are activated upon encountering microbial antigen(s) displayed by MR1 on the surface of an infected cell. Human MR1 undergoes alternative splicing. The full-length isoform, MR1A, can activate MAIT cells, while the function of the isoforms, MR1B and MR1C, are incompletely understood. In this report, we sought to characterize the expression and function of these splice variants. Using a transcriptomic analysis in conjunction with qPCR, we find that that MR1A and MR1B transcripts are widely expressed. However only MR1A can present mycobacterial antigen to MAIT cells. Coexpression of MR1B with MR1A decreases MAIT cell activation following bacterial infection. Additionally, expression of MR1B prior to MR1A lowers total MR1A abundance, suggesting competition between MR1A and MR1B for either ligands or chaperones required for folding and/or trafficking. Finally, we evaluated CD4/CD8 double positive thymocytes expressing surface MR1. Here, we find that relative expression of MR1A/MR1B transcript is associated with the prevalence of MR1 + CD4/CD8 cells in the thymus. Our results suggest alternative splicing of MR1 represents a means of regulating MAIT activation in response to microbial ligand(s).

Mucosal-associated invariant T (MAIT) cells are key players in the immune response against microbial infection. The MAIT T-cell receptor (TCR) recognizes a diverse array of microbial ligands, and recent reports have highlighted the variability in the MAIT TCR that could further contribute to discrimination of ligand. The MAIT TCR complementarity determining region (CDR)3b sequence displays a high level of diversity across individuals, and clonotype usage appears to be dependent on antigenic exposure. To address the relationship between the MAIT TCR and microbial ligand, we utilized a previously defined panel of MAIT cell clones that demonstrated variability in responses against different microbial infections. Sequencing of these clones revealed four pairs, each with shared (identical) CDR3a and different CDR3b sequences. These pairs demonstrated varied responses against microbially infected dendritic cells as well as against 5-(2-oxopropylideneamino)-6-Dribitylaminouracil, a ligand abundant in Salmonella enterica serovar Typhimurium, suggesting that the CDR3b contributes to differences in ligand discrimination. Taken together, these results highlight a key role for the MAIT CDR3b region in distinguishing between MR1-bound antigens and ligands.

Modulation of aryl hydrocarbon receptor (AHR) activity by a class of ligands termed selective AHR modulators (SAhRMs) has been demonstrated to attenuate proinflammatory gene expression and signaling, including repression of cytokine-mediated induction of acute-phase genes (e.g., Saa1). These effects are observed to occur through an AHR-dependent mechanism that does not require canonical signaling through dioxin response elements. Previously, we have demonstrated that the SAhRM 3Ј,4Ј-dimethoxy-␣-naphthoflavone (DiMNF) can repress the cytokine-mediated induction of complement factor genes. Here, we report that the activation of the AHR with DiMNF can suppress cytokine-mediated induction of the membrane complement regulatory protein CD55. When CD55 is expressed on host cells, it facilitates the decay of the complement component 3 (C3) convertase, thereby protecting the cell from complement-mediated lysis. Tumor cells often exhibit elevated CD55 expression on the cell surface in the inflammatory microenvironment of the tumor, and such enhanced expression could represent a means of escaping immune surveillance. DiMNF can repress the cytokine-mediated induction of CD55 mRNA and protein. Luciferase reporter analysis has identified possible response elements on the CD55 promoter, which may be targets for this repression. A C3 deposition assay with [ 125 I]C3 revealed that repression of cytokine-mediated CD55 expression by DiMNF led to an increase of C3 deposition on the surface of Huh7 cells, which would likely stimulate the formation of the membrane attack complex. These results suggest that SAhRMs such as DiMNF have therapeutic potential in regulating the immune response to tumor formation.

Mucosal Associated Invariant T (MAIT) cells can sense intracellular infection by a broad array of pathogens. These cells are activated upon encountering microbial antigen(s) displayed by MR1 on the surface of an infected cell. Human MR1 undergoes alternative splicing. The full length isoform, MR1A, can activate MAIT cells, while the function of the isoforms, MR1B and MR1C, are not well characterized.In this report, we sought to characterize these splice variants. Using a transcriptomic analysis in conjunction with qPCR, we find that that MR1A and MR1B transcripts are widely expressed. Despite the widespread expression of MR1A and MR1B, only MR1A can present mycobacterial antigen to MAIT cells. Coexpression of MR1B with MR1A serves to decrease MAIT cell activation following bacterial infection.However, expression of MR1B prior to MR1A lowers total MR1A abundance, suggesting competition between MR1A and MR1B for either ligands or chaperones required for folding and/or trafficking. Finally, we evaluated CD4/CD8 double positive thymocytes expressing surface MR1. Relative MR1A/MR1B expression in MR1-expressing thymocytes is associated with their prevalence.Our results suggest alternative splicing of MR1 represents a means of regulating MAIT activation in response to microbial ligand.