Study On The Presence Of MR1-MAIT Cell Axis In Pigs

MR1-MAIT cell axis which is involved in innate and adaptive immunity consists of major histocompatibility complex(MHC)class I-related protein 1(MR1)and mucosal-associated invariant T(MAIT)cells in mammals.MR1 that is a non-classical MHC-?b molecule and has the similar structure with classical MHC-?a molecule was first discovered in 1995.MR1 is the most conserved MHC molecule across species,implying the important role of MR1 in the process of antigen recognition and immune activation.It is known that MR1 is the restricting molecule for MAIT cells,and can present microbial metabolites from riboflavin synthesis,such as 5-OP-RU,to MAIT cells,which are activated and then exert immune function.MAIT cells are the hottest population of innate-like T cells that have been studied for nearly two decades,and express an evolutionarily conserved invariant TCR?chain paired with a limited diversity of TCR V?chains.MAIT cells also express some cytokine receptors,chemokine receptors and transcription factors.MAIT cells are abundant in humans,especially in non-lymphoid tissues.Currently,the function of MAIT cells is closely related to the pathogenesis,immune regulation and immune prevention of various types of diseases.researches on MAIT cell function have been involved in all disease types.Studies have shown that MR1 and MAIT TCR?have a co-evolutionary feature in mammals.Besides humans and mice,MR1-MAIT cell axis has been identified in animals such as Pongo pygnaeus,cows,and sheep.Because pigs are the main source of food protein for people,the model animals that are the closest to humans in physiology and immunology,and the potential donors for human organ transplantation,study on their immune cells has an economic and comparative immunological significance that cannot be underestimated.Genomic data analysis revealed that pigs also have encoding genes of MR1 and MAIT TCR?;to date,however,there are no studies on the distribution and function of the porcine MR1-MAIT axis.Therefore,our project is the first to study the tissue distribution of MR1-MAIT axis,the binding characteristics of MR1 and ligand,the diversity of TCR?chain and the use preference of TCR?chain in porcine MAIT cells,and the expression of cluster of differentiation antigens and surface markers on porcine MAIT cells using RT-PCR,RACE,qPCR and single-cell PCR assays.Firstly,six MR1 transcripts were obtained using RT-PCR and RACE assays,and then their sequence characteristics and the expression of them in tissues were analyzed.Our data showed that these six transcripts have distinct features.Furthermore,we found the expression of MR1 transcripts was highest in spleen,thymus,mesenteric lymph node and PBMC,but lower in mucosal tissues.Secondly,the expression of pig MR1 was analyzed in primary and passage cells,the 3D structure of pig MR1 was predicted,and then the docking mode of MR1 with 5-OP-RU was simulated.Our data showed that the expression of MR1 could be detected in all cell types through intracellular staining,but not through surface staining.Furthermore,we found the binding of the 5-OP-RU ligand to pig MR1 was similar to that observed with human MR1;specifically,5-OP-RU was bound within the A'-pocket of pig MR1,as it is in human MR1,and residues of the A'-pocket of MR1 that were implicated in the interaction with 5-OP-RU between human and pig were also similar.Thirdly,porcine MAIT TCR?sequences were obtained using RT-PCR and RACE assays,and the usage of MAIT TCR?chains was analyzed using single-cell PCR.Our data showed that pig MAIT TCR?sequence has a high similarity with human and mouse MAIT TCR?,and the usage of MAIT TCR V?segments has a preference,as their human and mouse counterparts do,but bias to use TRBV20S and TRBV29S segments.Finally,the tissue distribution of porcine MAIT cells and the expression of cell surface markers and transcription factors by them were analyzed using qPCR and single-cell PCR assays,respectively.Our data showed that porcine MAIT cells are present in all tested tissues,but prefer to locate in peripheral non-lymphoid mucosal tissues and are less frequent in spleen,thymus,and lymph node.Porcine MAIT cells consist of three subsets(CD4~-CD8~-,CD8~+CD4~-,and CD4~+CD8~-),and the frequencies of the three subsets are different in different tissues.Porcine MAIT cells can express cell surface markers IL-7R?,IL-18R?,CCR9,CCR5 and/or CXCR6,and also express transcription factors PLZF,T-bet and/or RORC.In summary,we have first uncovered that pig possesses the MAIT cell system which has similar molecular phenotype to human and/or mouse MR1-MAIT cell axis.These results will provide the foundation for the deep analysis of pig MAIT cell functions in anti-infective immunity and vaccine development,especially for the study of human MAIT cell functions using pig models.