The Relationship Between The Antiviral Treatment Effect And NK And Specific CD8~+T Cells Activity In Chronic Hepatitis C

ObjectsHepatitis C virus (HCV) infection is easy to chronic and can progress to cirrhosis and liver cancer. Interferon and ribavirin antiviral therapy can significantly improve the prognosis of the disease, but the efficacy is affected by a variety of viral and host factors. This study mainly investigated the host immune relevant factors affecting the hepatitis C antiviral efficacy, the main contents include:1. To observe the effect of peg-IFNa or ordinary IFNa and ribavirin antiviral therapy in patients with chronic hepatitis C (CHC), and analyze influence the common clinical factors.2. To explore the relationship between the functional activity of natural killer cells (NK cells) and antiviral treatment effect of CHC patients.3. To explore the relationship between specific CD8+T cell responses and antiviral treatment effect of CHC patients.Methods 1.70patients with chronic hepatitis C were treated with peg-IFNa or ordinary IFNa and ribavirin according to genotype therapy24or48weeks,60patients with follow-up for24weeks after treatment at least,10patients were lost. According to the results of virological response in24weeks after treatment evaluated the treatment effect (SVR and non-SVR). We collected the blood samples in before antiviral treatment (Ow), treatment for4w,12w,24w,48w and after the end of treatment24w,48w, respectively, and separated peripheral blood mononuclear cells (PBMC) and serum (plasma).2. Using the flow cytometry detected the frequency of NK cells and their subtypes, the expression levels of surface receptors, cytotoxic activity and cytokine secretion capacity.3. Using type-specific primers detected the HLA-A genotype in CHC patients, after specific HCV antigen peptide stimulated PBMC, using the flow cytometry detected specific CD8+T cell cytotoxic activity and cytokine secretion capacity.4. GraphPad Prism software was used for mapping, and SPSS13.0software was used for statistical analysis, to explore the relationship between hepatitis C antiviral efficacy and NK cells and specific CD8+T cell activity.Results一、 The antiviral efficacy in patients with chronic hepatitis C and related factors of common clinical:60patients were followed up for24weeks after treatment at least in patients with CHC,49patients with HCV genotype1,11patients with genotype2or3, other genotypes were not detected.50patients used peg-IFNa and ribavirin treatment,10patients used ordinary IFNa and ribavirin treatment.50patients achieved SVR (83.33%),10patients with non-SVR (16.67%). Compared to non-SVR patients, the EVR and ETVR were higher than SVR patients (P＜0.05). The SVR rate of patients with dose adjustment was lower than those without dose adjustments (37.50%vs90.38%), the difference was statistically significant (P＜0.05). The SVR rates of the elderly was lower than young patients (57.14%vs86.79%), the patients with high viral load lower than low viral load (81.08%vs86.96%), the patients with cirrhosis lower than without cirrhosis (62.50%vs86.54%), the patients with complication lower than without complication(66.67%vs 86.27%), the patients with peg-IFNa/ribavirin treatment lower than ordinary IFNa/ribavirin treatment (60.00%vs88.00%), but the difference was not statistically significant (P>0.05), then patients with HCV genotype1and non-genotype1were no significant difference.二、 NK cells functional activity analysis in CHC patients1. The changes of NK cells activity in CHC patients with antiviral therapy before and after treatment:①Compared to healthy controls, the frequency of NK cells, the expression levels of activating receptors NKp30, NKp46, NKG2D and cytotoxic activity decreased (P＜0.05) in CHC patients before treatment, and the expression level of inhibitory receptor NKG2A increased (P＜0.05), NK cells secreted cytokines IFN-y and TNF-a levels also decreased, but the difference was not statistically significant (P>0.05);②NK cells cytotoxic activity and secret cytokine IFN-y level with viral load exist negative correlation, in addition the expression levels of NK cells activating receptors NKG2C and NKG2D with ALT levels exist negative correlation;③ompared to CHC patients before antiviral treatment, the frequency of NK cells and the expression level of activating receptor NKp30were increased (P＜0.05) after treatment, then the expression levels of NKG2C, granzyme B and perforin were decreased (P＜0.05), while changes in the levels of cytokine secretion were no significant difference.2. Compare the NK cells activity in CHC patients with different genotypes:①NK cells activity was no significant difference in genotype1and non-genotype1patients before treatment; Compared to non-genotype1patients, the expression levels of NK cells activating receptor NKG2C and granzyme B were lower than genotype1patients in the end of the treatment (P＜0.05);②NK cells activity was no significant difference in genotype1patients with high viral load and low viral load before treatment; Compared to patients with high viral load, the frequency of NK cells was lower than patients with low viral load in the treatment for4w (P＜0.05);③Compare to non-SVR patients with viral rebound or recurrent positive, the frequency of CD56dim was higher than SVR patients after treatment, then the frequency of CD56bright was lower (P<0.05); Compare to non-SVR patients, the expression level of inhibitory receptor NKG2A was lower than SVR patients before treatment, during treatment and after treatment, then NK cells cytotoxic activity was higher, but the difference were not statistically significant (P>0.05); NK cells secreted cytokine levels were no significant difference in two groups of patients.3. The relationship between the dynamic changes of NK cells activity and response to therapy in antiviral treatment:①Compared to CHC patients before antiviral treatment, the frequencies of NK cells and their subtypes CD56dim, NK cells cytotoxic activity and secreted cytokines IFN-y and TNF-a levels decreased during treatment, then CD56bright frequency, the expression levels of activating receptors NKp30, NKp46and NKG2A were increased during treatment (P＜0.05); Compared to the end of treatment, the frequency of CD56dim and secreted cytokines IFN-y levels increased after treatment, then the frequency of CD56bright and the expression level of inhibitory receptor NKG2A decreased (P＜0.05);②According to the treatment effect to compare, SVR patients compared to before treatment, the frequency of NK cells decreased during treatment, and increased after treatment, the expression level of activating receptor NKp46increased during treatment, the expression levels of activating receptor NKG2C and cytokines TNF-a decreased during treatment (P<0.05); SVR patients compared to the end of treatment, the frequency of CD56dim and expression level of CD107a increased after treatment, the frequency of CD56bright decreased (P<0.05). But non-SVR patients showed no significant change.三、 HCV-specific CD8+T cells functional activity analysis in CHC patients1. The analysis of HLA-A specific CD8+T cells activity in different treatment effects: Compared to non-SVR group and chronic hepatitis C group, specific CD8+T cells secreted cytokines IFN-y and TNF-a levels in SVR group were higher in HLA-A2, HLA-A11and HLA-A24patients (P<0.05), the expression level of granzyme B in SVR group was lower in HLA-A2patients (P＜0.05), but the expression levels of granzyme B and perforin between SVR group and non-SVR group showed no significant difference in HLA-A11and HLA-A24patients.2. The dynamic change of specific CD8+T cells activity in antiviral treatment: Compared to CHC patients before antiviral treatment, specific CD8+T cells secreted cytokines IFN-y and TNF-a levels increased after treatment in HLA-A2, HLA-A11and HLA-A24patients (P<0.05), then the expression level of granzyme B decreased (P<0.05).3. The relationship between specific CD8+T cells activity and viral load, ALT levels: The cytokine TNF-a expression level of HLA-A2restricted Core131-140antigen peptide-specific CD8+T cells and the perforin expression level of HLA-A24-restricted Core35-44peptide-specific CD8+T cells with viral load were negatively correlated, but the specific CD8+T cell functions and ALT levels were no relationship.Conclusions1. Common clinical factors of influencing antiviral efficacy include:dose adjustment, the patient’s age, cirrhosis, complication, options of treatment programs.2. Altered NK cell phenotype and functional may affect the treatment effect in chronic hepatitis C patients during antiviral treatment, and there are correlation with the phenotype and function of NK cells and viral load or ALT levels.3. Altered different epitope peptide-specific CD8+T cell function are not exactly the same in chronic hepatitis C patients during antiviral treatment; there is correlation between specific CD8+T cell cytokine secretion function and treatment effect, and non-cytotoxic function plays a leading role in antiviral; there is related to specific CD8+T cell immune dysfunction and the level of virus, regardless of ALT levels.