Study On The Differentiation Mechanisms Of T Cell-dependent IgA~+B Cell In Peyer's Patches Induced By Porcine Epidemic Diarrhea Virus

Secretory IgA(sIgA)is a dominant antibody in the mucosal surface,which can bind to pathogens and defend their invasion.Peyer's patches(PPs)are the leading inducing site of intestinal sIgA.Studies have shown that porcine epidemic diarrhea virus(PEDV)induced IgA response in PPs.However,the detailed mechanisms remain unclear.In this study,we aimed to investigate the primary factors and mechanisms involved in intestinal immune response in porcine PPs during PEDV infection.To verify if PEDV induce the intestinal immune response in pigs,an attenuated PEDV strain was used to inoculate one-month-old pigs through oral combined with Houhai point administration.The immune response was investigated.The results showed that PEDV increased the production of PEDV-specific IgG and IgA antibodies in the serum and promoted PEDV-specific IgA production in the intestinal contents,indicating that PEDV boosted the intestinal mucosal immune response in pigs.To evaluate porcine IgA class switch recombination accurately,PCR methods were established for the amplification of three critical molecular markers(GLT??PST?and CT?)produced during antibody class recombination in IgA~+B cells,and their sequences were obtained.Then,the amplifying conditions were optimized.Finally,the PCR method with excellent repeatability was established for the detection of these three critical molecular markers,which laid the solid foundation for the study on IgA production in pigs.To investigate the vital cytokines promoting IgA production in porcine PPs,IgM~+B cells were isolated and purified from porcine PPs and cultured with T cell-independent cytokines(BAFF and APRIL)or T cell-dependent cytokines(CD40L,TGF?-1,IL-6,IL-10,IL-17A,and IL-21)in vitro.The IgA levels in the supernatant were measured.The results showed that IL-21 actively promoted IgA production in B cells.Then,further investigation revealed that IL-21 promoted the proliferation and inhibited the apoptosis of porcine B cells,thus improving B cell viability.IL-21 also promoted plasma cell differentiation by up-regulating the expression of IRF4 and BLIMP1 and down-regulating the expression of PAX5 and BCL6.Further,this study indicated that IL-21 promoted IgA class switch recombination through the JAK-STAT signaling pathway,leading to the generation of IgA~+B cells,which ultimately contributed to IgA production.To investigate whether follicular T helper(Tfh)cells were the primary source of IL-21 in porcine PPs,porcine CXCR5 were cloned,polyclonal antibodies against 54 extracellular amino acids in the N-terminal were prepared.The anti-pig CXCR5 polyclonal antibody showed good reactivity and specificity.Then,peripheral blood lymphocytes were labeled with the CXCR5 antibody.The quantitative PCR and flow cytometry results showed that pig CXCR5 was mainly expressed in B cells.Subsequently,the CXCR5 polyclonal antibody was used to identify and isolate the naive Th cells,Tfh cells,and other activated non-Tfh cells in the porcine PPs.Quantitative PCR results showed that porcine Tfh cells were the primary Th cell type producing IL-21 in the porcine PPs.In conclusion,our results showed that PEDV inoculation induced IgA production in the porcine intestine;Besides,PCR methods were established for the amplification of three critical molecular markers(GLT?,PST?and CT?)produced during porcine IgA class recombination;The IL-21,mainly produced by Tfh cells in porcine PPs,promoted the differentiation of IgA-producing plasma cells in porcine PPs through the JAK-STAT signaling pathway,thus improving the production of IgA in the gut.The findings from this study enriched the knowledge of porcine mucosal immunology and provided insights and theoretical basis for the design and development of the swine mucosal vaccine.