| Objective:Previous studies confirmed that interferon-?(IFN-?)can promote the differentiation of follicular helper T cells(Tfh)induced by foot-and-mouth disease recombinant adenovirus vaccine and promote the formation of germinal centers and enhance the humoral immune response in mice.Studies have confirmed that long-lived memory Tfh cells are present in CD4~+T cells and are primarily responsible for activating IgG1 memory B cells,initiating an adaptive immune response.However,it is unclear whether IFN-?can promote the production of memory Tfh cells.Therefore,in this study,the effects of IFN-?on the differentiation and function of FMD recombinant adenovirus vaccine induced memory Tfh cells were determined by in vivo experiments,and the mechanism of IFN-?promoting FMD recombinant adenovirus vaccine-induced memory Tfh cell differentiation was investigated.Further research on the new mechanism of IFN-?regulation of vaccine immunity provides theoretical data.Method:1.6-8 weeks female BALB/c mice were randomly divided into 6 groups,20 rats in each group,respectively.Each group was immunized respectively with adenoviral empty vector(rAd),recombinant adenovirus expressing FMDV VP1(rAd5VP1),recombinant adenovirus expressing porcine IFN-?(rAd5poIFN-?),recombinant adenovirus co-expressing FMDV VP1 and porcine IFN-?(rAd5VP1-2A-poIFN-?),combined with rAd5VP1 and rAd5poIFN-?and porcine O-type foot-and-mouth disease inactivated vaccine(Inactivited vaccine).immunized once every two weeks,a total of 2 immunizations.The spleens of mice were taken 30 days,60 days,and 90 days after the initial immunization.Mononuclear cells were harvested by density gradient centrifugation.The ratio of CD4~+CD44~+activated T cells,CD4~+CD44~+CD62L~-memory T cells and CD4~+CXCR5~+memory Tfh cells were detected by flow cytometry.The ratio of CD4~+CXCR5~+memory Tfh cells.The ratio of CD38~+B220~+memory B cells,CD38~+B220~+GL-7~-IgG1~+memory B cells were detected by flow cytometry.The expression of chemokines CXCR3 and CCR6 in Tfh cells was detected by flow cytometry 90 days after the initial immunization.2.After 90 days of initial immunization,the mice were re-immunized,and the following studies were performed before immunization and 3 days after immunization:2.1 Take the mouse spleen,harvest mononuclear cells by density gradient centrifugation,and the ratio of CD4~+CD44~+activated T cells,CD4~+CD44~+CD62L~-memory T cells,CD4~+CXCR5~+memory Tfh cells,the expression of Tfh cell-specific transcription factor Bcl-6,the expression level of cytokines IL-21,IL-4 and IFN-?secreted by Tfh cells,and the expression level of synergistic costimulatory molecule CD86 in memory B cells by flow cytometry.2.2 Take the mouse spleen,harvest mononuclear cells by density gradient centrifugation,and the expression levels of Tfh cell-associated transcription factors STAT1 and Bcl-6 were detected by real-time PCR.2.3 Mouse eye blood was taken,serum was separated,and the secretion levels of total IgG,IgG1,IgG2a and IgG2b were detected by ELISA.Result:1.IFN-a promotes foot-and-mouth disease recombinant adenovirus vaccine to induce the production of memory T cells and memory Tfh cells.1.1 After 30 days of mouse immunization with recombinant adenovirus,the results of flow cytometry showed that the ratio of CD4~+CD44~+cells in rAd5VP1+rAd5poIFN-?group and rAd5VP1-2A-poIFN-?group was significantly higher than that in rAd group,The difference was statistically significant.The proportion of CD4~+CD44~+cells in rAd5VP1-2A-poIFN-?group was significantly higher than that in rAd5VP1 group,The difference was statistically significant.The number of CD4~+CD44~+cells persisted in 60 days and decreased slightly after 90 days.1.2 After 30 days of immunization with recombinant adenovirus,the ratio of CD4~+CD44~+CD62L~-cells in rAd5VP1+rAd5poIFN-?group and rAd5VP1-2A-poIFN-?group was significantly higher than that in rAd group,the difference was statistically significant.The number of CD4~+CD44~+CD62L~-cells persisted in 60 days and decreased slightly after 90 days.1.3 After 30 days of mouse immunization with recombinant adenovirus,the ratio of CD4~+CXCR5~+cells in rAd5VP1+rAd5poIFN-?group and rAd5VP1-2A-poIFN-?group was significantly higher than that in rAd group,rAd5VP1 group and rAd5poIFN-?group,The difference was statistically significant.The proportion of CD4~+CXCR5~+cells in the rAd5VP1-2A-poIFN-?group was significantly higher than that in the inactivited vaccine group,The difference was statistically significant.The number of CD4~+CXCR5~+cells persisted at 60 days and decreased slightly after 90 days.1.4 After 90 days of mouse immunization with recombinant adenovirus,the results of flow cytometry showed that the ratio of Th1 type Tfh cells(CXCR3~+CCR6~-)in rAd5VP1-2A-poIFN-?group was significantly higher than that in rAd group,rAd5VP1 group,rAd5poIFN-?group and inactivited vaccine group,The difference was statistically significant.The proportion of Th1 type Tfh cells(CXCR3~+CCR6~-)in the rAd5VP1+rAd5poIFN-?group was significantly higher than that in the rAd group,and the difference was statistically significant.The proportion of Th2 Tfh cells(CXCR3~-CCR6~-)in rAd5VP1+rAd5poIFN-?group was significantly higher than that in rAd group,rAd5VP1 group and rAd5poIFN-?group,The difference was statistically significant.The proportion of Th2 Tfh cells(CXCR3~-CCR6~-)in rAd5VP1-2A-poIFN-?group was significantly higher than that in rAd group and rAd5poIFN-?group,The difference was statistically significant.There was no significant difference in the number of Th17 Tfh cells(CXCR3~-CCR6~+)between the groups.2.IFN-a promotes differentiation of memory-bearing B cells induced by foot-and-mouth disease recombinant adenovirus vaccine2.1 After 30 days of mouse immunization with recombinant adenovirus,the results of flow cytometry showed that the ratio of CD38~+B220~+cells in rAd5VP1+rAd5poIFN-?group,rAd5VP1-2A-poIFN-?group and inactivited vaccine group was significantly higher than that in rAd group and rAd5poIFN-?group,the difference was statistically significant.The proportion of CD38~+B220~+cells in rAd5VP1 group was significantly higher than that in rAd group,the difference was statistically significant.The number of CD38~+B220~+cells persisted in 60 days and decreased slightly after 90 days.2.2 After 30 days of immunization with recombinant adenovirus,the proportion of CD38~+B220~+GL-7~-IgG1~+cells in rAd5VP1-2A-poIFN-?group was significantly higher than that in rAd group,rAd5VP1 group and rAd5poIFN-?group,the difference was statistically significant.The ratio of CD38~+B220~+GL-7~-IgG1~+cells in rAd5VP1+rAd5poIFN-?group was significantly higher than that in rAd5VP1 group,the difference was statistically significant.The number of CD38~+B220~+GL-7~-IgG1~+cells persisted in 60 days and decreased slightly after90 days.3.Effect of re-immunized recombinant adenovirus on memory Tfh cells and humoral immune response in mice3.1 The recombinant adenovirus was re-immunized 90 days after the first immunization.After 3 days,the results of flow cytometry showed that the proportion of CD4~+CD44~+cells,CD4~+CD44~+CD62L~-cells and CD4~+CXCR5~+cells in each group was significantly higher than that before reimmunization,the difference was statistically significant.3.2 The recombinant adenovirus was re-immunized 90 days after the first immunization of the mice.After 3 days,the results of flow cytometry showed that the expression of IL-21 in Tfh cells in rAd5VP1 group,rAd5VP1+rAd5poIFN-?group,rAd5VP1-2A-poIFN-?group and inactivited vaccine group was significantly higher than that before reimmunization,and the difference was statistically significant.The other groups had an increasing trend,but there was no statistical difference.The expression of IFN-?in rAd5poIFN-?group,rAd5VP1+rAd5poIFN-?group and rAd5VP1-2A-poIFN-?group was significantly higher than that before re-immunization,and the difference was statistically significant.The other groups had an increasing trend,but there was no statistical difference.The expression of IL-4in each group was significantly higher than that before re-immunization,and the difference was statistically significant.3.3 The recombinant adenovirus was re-immunized 90 days after the first immunization of the mice.After 3 days,the results of flow cytometry showed that the expression of co-stimulatory molecule CD86 on the surface of memory B cells in rAd5VP1+rAd5poIFN-?group and rAd5VP1-2A-poIFN-?group was significantly higher than that before re-immunization,the difference was statistically significant.The other groups had an increasing trend,but there was no statistical difference.3.4 The recombinant adenovirus was re-immunized 90 days after the first immunization of the mice.After 3 days,the ELISA results showed that the secretion levels of total IgG and IgG1 antibodies in the serum of each group were significantly higher than those before re-immunization,the difference was statistically significant.The secretion level of IgG2a antibody in serum of inactivited vaccine group was significantly higher than that before re-immunization,and the difference was statistically significant.the other groups were not statistically different.The secretion levels of IgG2b antibody in serum of rAd5VP1+rAd5poIFN-?group,rAd5VP1-2A-poIFN-?group and inactivited vaccine group were significantly higher than those before re-immunization,and the differences were statistically significant.4.IFN-a promotes the expression of transcription factors Bcl-6 and STAT1 by foot-and-mouth disease recombinant adenovirus vaccine.4.1 After 90 days of mouse immunization with recombinant adenovirus,the results of flow cytometry showed that the expression of transcription factor Bcl-6 in CXCR5~+T cells was significantly higher than that in CXCR5~-T cells,and the difference was statistically significant.4.2 The recombinant adenovirus was re-immunized 90 days after the first immunization.After 3 days,the results of flow cytometry showed that the proportion of Tfh cells(CXCR5~+Bcl-6~+)inrAdgroup,rAd5VP1group,rAd5poIFN-?group,rAd5VP1+rAd5poIFN-?group and rAd5VP1-2A-poIFN-?was significantly higher than that before re-immunization,and the difference was statistically significant.The proportion of Tfh cells(CXCR5~+Bcl-6~+)in inactivited vaccine group had an increasing trend,but there was no statistical difference.4.3 The recombinant adenovirus was re-immunized 90 days after the first immunization.After 3 days,the real-time PCR results showed that the expression level of STAT1 mRNA in each group was significantly higher than that before re-immunization,the difference was statistically significant.The expression levels of Bcl-6 mRNA in rAd group,rAd5VP1 group,rAd5poIFN-?group,rAd5VP1+rAd5poIFN-?group and rAd5VP1-2A-poIFN-?group were significantly higher than those before re-immunization,and the difference was statistically significant.The expression of Bcl-6 mRNA in inactivited vaccine group had an increasing trend,but there was no statistical difference.Conclusion:IFN-?is an immunomodulator of foot-and-mouth disease recombinant adenovirus vaccine,which can enhance the differentiation of memory T cells,memory Tfh cells and memory B cells induced by recombinant adenovirus vaccine,and enhance the expression of Tfh cell chemokines(CXCR3,CCR6),promotes the secretion of cytokines(IL-21,IL-4,IFN-?)and antibodies.After re-immunization,IFN-?promotes the immune function of Tfh cells induced by foot-and-mouth disease recombinant adenovirus vaccine.IFN-?may promote the expression of Bcl-6 by increasing the expression of the transcription factor STAT1,thereby promoting the differentiation of Tfh cells.It can provide theoretical data for further study of new mechanisms by which IFN-?regulates vaccine immunity. |
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