Kulicke Corinna A, Lemon Chance, Krawic Jason R, Ramirez Luisa Maria Nieto, Kim Se-Jin, Narayanan Gitanjali, Tafesse Fikadu G, Hildebrand William H, Dobos Karen M, Lewinsohn David M
Division of Pulmonary, Allergy, and Critical Care Medicine, Oregon Health & Science University, Portland, OR 97239, USA.
Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
bioRxiv. 2025 May 19:2025.05.14.654109. doi: 10.1101/2025.05.14.654109.
The antigen presenting molecule MHC class I-related protein 1 (MR1) binds small molecule metabolites derived from microbial riboflavin biosynthetic pathways and presents them at the cell surface for surveillance by MR1-restricted mucosal-associated invariant T cells (MAIT cells). MR1 ligands can originate in the extracellular space or in endosomal compartments that contain microbial pathogens. Distinct, complementary antigen processing and presentation pathways enable MR1 to survey diverse intracellular locations and present both exogenous and intracellular antigens. Here, we generated a panel of BEAS-2B MR1 KO cells reconstituted with MR1 proteins mutated at amino acids 9 - 16. The mutated MR1 molecules differentially translocated to the cell surface in response to 6-formylpterin and differed in their ability to present mycobacterial antigens to MAIT cell clones. While they barely presented supernatant and other exogenous MAIT cell antigens, their ability to present antigens derived from mycobacterial infection and a 5-A-RU prodrug requiring endosomal processing remained largely intact. Protein co-immunoprecipitation and mass spectrometry-based proteomic analysis showed that mutated MR1 differentially associated with calnexin and β-microglobulin (B2M). Knock-down of B2M in cells over-expressing MR1 phenocopied the loss of exogenous antigen presentation but did not impact presentation of intracellular antigens. Thus, the MR1-mediated presentation of exogenous antigen appears to be limited by binding to B2M whereas the lower sensitivity to B2M deficiency implies that MAIT cell activation via the endosomal antigen presentation pathway may be limited by the availability of MR1 itself.
抗原呈递分子MHC I类相关蛋白1（MR1）结合源自微生物核黄素生物合成途径的小分子代谢产物，并将它们呈递至细胞表面，以供受MR1限制的黏膜相关恒定T细胞（MAIT细胞）进行监测。MR1配体可源自细胞外空间或含有微生物病原体的内体区室。不同的、互补的抗原加工和呈递途径使MR1能够监测不同的细胞内位置，并呈递外源性和内源性抗原。在此，我们构建了一组用在第9至16位氨基酸处发生突变的MR1蛋白重建的BEAS-2B MR1基因敲除细胞。突变的MR1分子对6-甲酰蝶呤的反应不同，向细胞表面的转运也不同，并且它们向MAIT细胞克隆呈递分枝杆菌抗原的能力也有所不同。虽然它们几乎不呈递上清液和其他外源性MAIT细胞抗原，但它们呈递源自分枝杆菌感染的抗原以及需要内体加工的5-A-RU前药的能力在很大程度上保持完整。蛋白质免疫共沉淀和基于质谱的蛋白质组学分析表明，突变的MR1与钙连蛋白和β2微球蛋白（B₂M）的结合存在差异。在过表达MR1的细胞中敲低B₂M模拟了外源性抗原呈递的丧失，但不影响内源性抗原的呈递。因此，MR1介导的外源性抗原呈递似乎受到与B₂M结合的限制，而对B₂M缺陷较低的敏感性意味着通过内体抗原呈递途径激活MAIT细胞可能受到MR1自身可用性的限制。
