The Relationship Of LAG-3 On CD4~+T Cells And HCV Neutralizing Antibody Response During HCV Infection

Objective Lymphocyte activation gene-3(LAG-3)is an immunosuppressive receptor which could negatively regulate the function of T cells and make CD4 and CD8 T cells exhaustion.The CD4~+T cells subsets which express LAG-3,such as type I regulatory T cells(Tr1)is involved in the negative regulate of humoral immunity.However,whether LAG-3 participates in the negative regulation of HCV neutralizing antibody response during hepatitis C virus(HCV)infection has not been reported.Therefore,in this study we aim to investigate the relationship between CD4~+LAG-3~+T cells and HCV neutralizing antibodies.Methods1.Samples inclusion and exclusion criteria:Individuals with a history of intravenous drug abuse were recruited from Chenzhou city,Hunan province.HBs Ag,anti-HDV Ig G,anti-HIV Ig G and anti-TP Ig G in serum were detected by using enzyme-linked immunosorbent assay(ELISA).Inclusion criteria:According to the results of HCV RNA and anti-HCV antibody(HCV-Ig G),HCV infection individuals were divided into chronic hepatitis C infection(CHC)(n=40),Self-limited HCV infection(SL)(n=30)and Healthy control group(HC)(n=36).All individuals were followed up for at least 6 months to be sure no seroconversion.Exclusion criteria:HBV surface antigen-positive?anti HDV-Ig G positive?anti HIV-Ig G positive and anti-TP-Ig G positive individuals2.HCV viral load and genotype analysis:HCV viral load was detected by real-time fluoreslence quantitative PCR;HCV Genome-E1and NS5B sequences were amplified by using PCR,then HCV viral genotypes was confirmed by using HCV genotype sequencing detection.3.Neutralizing antibody detection:1:100-fold diluted serum sample co-incubation with different types genotype HCV membrane proteins pseudovirions(genotype:1a,1b,2a,3a,4a,5a),then infected Huh7.5 cells.Serum neutralizing activity indicated as the relative inhibition of serum on viral infection.4.Detection and analysis of immune cells:the peripheral blood of LAG-3 on CD4+T cells,type I regulatory T cells(Trl),regulatory T cells(Tregs)and T follicular helper cell(Tfh)frequency were analyzed by flow cytometry.5.Correlation analysis:(1)analysis the relationship of LAG-3~+CD4~+T,Tr1,Treg and LAG-3~+Treg with Tfh and neutralizing antibody;(2)analysis correlation of Tfh and neutralizing antibodyResults1.LAG-3 on CD4~+T cells was up-regulated during HCV infection:we found LAG-3~+CD4~+T cells level in HCV chronic infection and HCV self-limited infection group were significantly higher than healthy control group(P=0.020,P=0.035,respectively).We further analyzed LAG-3~+-related CD4 regulatory T cells:LAG-3~+CD4~+T cells:Tr1(CD4~+CD45RA~-CD49b~+LAG-3~+T cells)and Treg(CD4~+CD25~+Foxp3~+LAG-3~+T cells)frequency changed during HCV infection,and found Tr1 and LAG-3~+Treg cells in HCV self-limited infection group were significantly higher than healthy control group(P=0.034,P=0.011 respectively),While there was no significant difference between chronic HCV infection group and the other two groups.These results suggested that HCV infection drives increasing LAG-3~+in regulatory T cells.2.LAG-3~+CD4~+T cells negatively correlate with HCV neutralizing antibody:LAG-3 is a negative regulatory factor which could inhibit cell?Lar immunity during HCV infection.We further analyze the relationship between LAG-3 and neutralizing antibody:there was a significant negative correlation between LAG-3~+CD4~+T cell frequency and neutralizing antibody response during HCV chronic infection and HCV self-limiting infection(R=-0.446,P=0.007,R=-0.693,P<0.001 respectively).Further analysis found LAG-3~+Treg but not Treg showed a negative correlation with neutralizing antibodies(R=-0.418,P=0.038,R=-0.615,P=0.004).This result suggested that LAG-3~+CD4~+T cells may be involved in the negatively regulate neutralizing antibody responses during HCV infection.3.The relationship between follic?Lar helper T cells and neutralizing antibodies in HCV infection:Tfh cells is a type of helper CD4~+T cells which is necessary for produce long-term and broad-spectrum neutralizing antibody.We analyzed the correlation between Tfh cells and neutralizing antibodies and found:the frequency of CXCR3~+Tfh cells was positively correlated with HCV neutralizing antibody(R=0.646,P=0.004).This result indicated that CXCR3~+Tfh cells may the major Tfh cells participate in the production of neutralizing antibodies.4.Analysis the correlation of elevated LAG-3~+CD4~+T cells and CXCR3~+Tfh cells during HCV infection:LAG-3 was upregulated in CD4~+T cells,which was negatively correlated with neutralizing antibodies during HCV infection.While CXCR3~+Tfh cells showed a positive correlation with neutralizing antibodies.Therefore,we further analyzed the association between CD4~+LAG-3~+T cells and CXCR3~+Tfh cells.The results showed that CD4~+LAG-3~+T cells were negatively correlated with CXCR3~+Tfh cells in HCV self-limited infection(P<0.05).It is suggested that CD4~+LAG-3~+T cells may affect CXCR3~+Tfh cells and affect HCV neutralizing antibody response.Conclusion From our study results,we found elevated LAG-3expression on CD4~+T cells and biased CXCR3~+Tfh cells differentiation due to HCV infection.The frequency of CD4~+LAG-3~+T cells in Self-limited HCV-infected individual is negatively correlated with the frequency of CXCR3~+Tfh cells and the level of HCV neutralizing antibody,While the frequency of CXCR3~+Tfh cells was positively correlated with the level of HCV neutralizing antibody.All results implied that immunosuppressive receptors LAG-3 may negatively affect CXCR3~+Tfh cells thereby affect the immune response of HCV neutralizing antibodies.