Abstract
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.
Highlights
While the regulatory mechanisms governing antigen presentation have been rigorously defined for MHC-I and MHC-II1, the molecular mechanisms of antigen presentation by the non-classical class I molecule MR1 are still being elucidated
Explanations for this finding could include the possibility that Mycobacterium tuberculosis (Mtb) produces a lower quantity of MR1 antigens, and optimal loading occurs intracellularly where more MR1 molecules are located, or it is possible that Mtb does not secrete MR1 antigens or that they are secreted in the phagosome
We found that when doxycycline is present, the cells were able to present mycobacterial antigen to MR1-restricted T cells (MR1Ts) compared to transfected cells that were never treated with doxycycline (Fig. 3A)
Summary
While the regulatory mechanisms governing antigen presentation have been rigorously defined for MHC-I and MHC-II1, the molecular mechanisms of antigen presentation by the non-classical class I molecule MR1 are still being elucidated. The addition of 6-FP shortly before the addition of exogenous MR1 antigens resulted in a complete loss of MR1-dependent antigen presentation, whereas Mtb-dependent antigen presentation was intact in the presence of 6-FP15 These data suggested distinct MR1 trafficking pathways between exogenously added antigens versus antigen derived from an intracellular infection. To clearly establish that there are distinct trafficking pathways of MR1, we knocked down endosomal trafficking proteins and found that Syntaxin 4 knockdown affected the presentation of exogenously delivered antigens but has no effect on Mtb-dependent antigen presentation These results demonstrate three important findings: (1) preformed MR1 can present antigens from both Mtb infection and exogenously delivered mycobacterial antigens, (2) 6-FP bound MR1 can be efficiently recycled to present exogenously delivered mycobacterial antigens and (3) the mechanisms of MR1-dependent antigen presentation can differ between exogenously added antigens versus intracellular Mtb infection
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