Establishment And Application Of MAIT Cell Expansion System Based On MR1 Dimer

Aim:Mucosal-associated invariant T（MAIT）cells are unconventional,intrinsic immune T lymphocytes that are mainly enriched in mucosal and liver tissues,MAIT cells have antibacterial,antiviral and tissue repair functions,MAIT cells mainly recognise antigens presented by the MHC class I-related molecule 1（MR1）.Although MAIT cells are known to recognise bacterial-derived riboflavin pathway vitamin B-like metabolites,the antigens they can recognise remain largely unknown.The aim of this study was to construct an artificial antigen-presenting cell（a APC）based on MR1 capable of presenting antigens to MAIT cells in vitro,with the aim of investigating the substances that influence MAIT cell activation.Previous laboratory studies have found that direct bilirubin（DBIL）can affect the function of MAIT cells and that this effect is DBIL concentration dependent.This study will investigate whether DBIL controls disease progression in liver diseases by influencing the function of immune cells and the specific mechanism of influence,which will provide a reference for immune-oriented therapy in the treatment of clinical liver diseases.Content:We constructed a dimer based on MR1（MR1 dimer）that can probe the antigenic substances recognized by MAIT cells,and MR1-dependent artificial antigen-presenting cells（MR1 a APCs）were made to carry out the next step of research on the effects of different antigenic substances on activation of MAIT cells;regarding the mechanism of DBIL affecting MAIT cells,two main directions of research were conducted:1.The effect of DBIL on the MAIT cell pathway whether TCR-dependent;2.The mechanism of DBIL affecting MAIT cells was investigated at the gene expression level.Methods:1.Using DNA splicing technology,the human MR1 extracellular segment gene was spliced with the Fc gene of Ig G1 to form a complete gene sequence MR1-Fc,and then MR1-Fc was inserted with the humanβ2m gene into the vector p Fast Bac ^TMDual to express MR1 dimer using the Bac-to-Bac system.MR1 dimers were made into MR1-based artificial antigen-presenting cells（MR1 a APCs）.MR1 a APC was used to investigate whether E.coli-derived metabolites have the function of activating MAIT cell activation.2.Whether the effect of DBIL on MAIT cells is TCR-dependent:Peripheral blood mononuclear cells（PBMCs）from healthy donor were co-cultured in TCR-dependent or TCR-independent stimulation systems for 7 days to detect whether the proliferation of MAIT cells was differentially affected by DBIL.DBIL was co-incubated with the constructed MR1 a APC to detect whether DBIL could bind to MR1.3.To investigate the mechanism of DBIL affecting MAIT cells at the gene expression level:MAIT cells from PBMC of healthy donor were expanded,and the expanded MAIT cells were co-cultured in TCR-dependent stimulation system（CD3/CD28,5-OP-RU/MR1 a APC）and TCR-independent stimulation system（IL-12/IL-18）for 24 h.RNA was extracted from MAIT cells for transcriptome sequencing to investigate the alteration of gene levels in MAIT cells affected by DBIL in the three different stimulation systems.Results:MR1 dimer was successfully constructed in vitro,and the yield,molecular size,etc.of MR1 dimer were examined.MR1 a APC was successfully constructed,and MR1 a APC loaded with E.coli metabolites was co-cultured with PBMC in vitro.1-day culture results showed that E.coli metabolites could activate MAIT cells,although weaker than5-OP-RU/MR1 a APC.7-day culture results showed that E.coli metabolites could proliferate MAIT cells,also weaker than 5-OP-RU/MR1 a APC.Next,mass spectrometric assays were performed to screen for antigenic substances recognizable by MAIT cells after elution of bound metabolites on MR1 a APC with weak hydrochloric acid.In addition,transcriptomic data after co-culture of DBIL with MAIT cells showed that DBIL affected antigen presentation and ferroptosis pathways in MAIT cells.Conclusion:MR1 dimer was successfully constructed in vitro and MR1 a APC was made.MR1 a APC can be used to probe the antigenic substances that activate MAIT cells,and DBIL may play its regulatory role by affecting the antigen presentation and ferroptosis-related pathways in MAIT cells.