| Objective During the early phase of HCV infection,the rapid development of protective neutralizing antibodies responses is associated with viral control and HCV spontaneous clearance.Follicular helper T cells(Tfh)play a pivotal role in the differentiation of B cells and are essential for the generation of long-live,high affinity neutralizing antibodies.The role of circulating follicular helper T cells(c Tfh)in neutralizing antibodies response during the HCV infection was still remains unclear.This study aims to investigate the correlation between c Tfh cells differentiation and neutralizing antibody immune responses in HCV-infected patients,will providing a novel strategy for rational HCV vaccine designs.Method(1)From December 2016 to September 2018,blood samples were collected from the injection drug users in Chen Zhou City,Hu Nan Province.Excluded the individuals whose serum HBs Ag,anti-HIV Ig G,anti-HDV Ig G or anti-TP Ig G were positive.According to the results of HCV RNA and anti-HCV Ig G,the individuals were divided into healthy control(n=28)and hepatitis C virus infection(n=43).All Inclusive patients were followed up at least 6 months to ensure without seroconversion.(2)The phenotype frequency of c Tfh cells and their subsets were measured by flow cytometry,intracellular cytokine levels of IL-21,IFN-γ,IL-10 were detected after PMA and Ionomycin stimulation for 6 hours in vitro.The expression of transcription factors Bcl-6,T-bet were detected by flow cytometry after nuclear antibody staining.(3)The strength and breadth of neutralizing antibody were detected by Luciferase reporter system,The correlation of c Tfh cells and their subsets with neutralizing antibody were analyzed.(4)HCV viral load was detected by real-time fluorescent quantitative PCR;HCV genome-E1 and NS5B sequences were amplified by PCR,according to HCV gene sequencing analysis the HCV viral genotypes.Analyzed the correlation of HCV RNA with neutralizing antibody or c Tfh cells and their subsets.(5)The function of c Tfh cells supporting B cell differention and antibody generation were detected by co-culture system in vitro.Results(1)Compared with healthy control,the frequency of c Tfh cells in HCV-infected patients increased(P=0.024),the expression of CXCR3~+c Tfh cells was significantly increased(P=0.001),meanwhile the ratio of CXCR3~+c Tfh/CXCR3~-c Tfh cells was also significantly increased(P=0.001).(2)The frequency of CXCR3~+c Tfh cells was positive correlations with the neutralizing antibody strength,breadth and each HCV genotype,and the ratio of CXCR3~+c Tfh/CXCR3~–c Tfh cell was also positively correlated with the neutralizing antibody titer(R=0.317,P=0.038).There was no relationship between CXCR3~–c Tfh cells and the neutralizing antibodies.(3)There was no correlation between HCV RNA and neutralizing antibody or c Tfh cells and their subsets.(4)Compared with CXCR3~–c Tfh cells,the expression of CD40L,PD-1,Ki-67,HLA-DR,ICOS and CD127 in CXCR3~+c Tfh cells were highly expressed(P<0.001).Functionally,the secretion of IL-21,IFN-γ,IL-10 higher in CXCR3~+c Tfh cells(P<0.001).The level of transcription factor Bcl-6,T-bet were also significantly higher in CXCR3~+c Tfh cells(P<0.001).(5)CXCR3~+c Tfh cells or CXCR3~-c Tfh cells was co-cultured with homologous B cells for 7 days,found that there was no significant difference in level of supernatant Ig A,Ig G and Ig M between CXCR3~+c Tfh cells and CXCR3~-c Tfh cells.While the frequency of HCV E2c specific B cell was significantly enhanced in CXCR3~+c Tfh cells(P=0.046).Conclusion:Our results suggested that CXCR3~+c Tfh cells may supporting B cell differentiation and participated in neutralizing antibody response during HCV infection.This found predicted that targeting CXCR3~+c Tfh cells differentiation may effectively facilitated the neutralizing antibody response,and promoting HCV clearance and control of viral infection.This finding will hopefully provide some direct implication for vaccine optimization. |
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