Effects Of IL-21on CD8~+T Cell Function Of Chronic HBV Infection Patients In Vitro

Hepatitis B virus (HBV) infection is a global public health problem, and isharmfulness of the most serious diseases which is in our country. According to the data bythe Ministry of Health, there are30million chronic HBV patients and approximately300,000people who died from chronic HBV infection caused by the end-stage liverdiseases, such as hepatic failure, liver cirrhosis, and hepatocellular carcinoma. Yet, thepathogenic mechanism of chronic HBV infection remains unclear. Interleukin (IL)-21, apleiotropic cytokine that is produced mainly by activated CD4+T cells play a key role ofpromotes the proliferation and differatiation of CD8+T cells, and enhances the CD8+T cellresponse to various chronic infections.Aims: To observe the differences and relationship of expression lever of PD-1, IFN-γ,IL-21on HBV-specific CD8+T cells of HBV-infected patients with different clinicalmanifestation stimulated with IL-21in vitro, as well as their correlations to serological,virological, and biochemical markers. Simultaneously，to provide research data forelucidating the mechanism of chronic HBV infection in T cell function exhausted.Method:1. Thirty-eight of HBV-infected patients, including18of chronic hepatitis B (CHB),18of acute hepatitis B (AHB) were recruited for this study and10of age and sex matchedpeople for normal controls. The level of HBsAg, HBeAg, HBeAb were quantified. Theviral load was determined in all patients. ALT, AST, ultrasonic examination concerningliver and any other clinical data were examined.2. Peripheral blood mononuclear cells(PBMCs) were extracted from whole blood by density gradient centrifugation.3. PBMCswere suspended in RPMI1640+10%FCS in present of IL-2and anti-CD28andstimulated with HBV whole core protein T cell epitope peptides, HBV whole core proteinT cell epitope peptides+IL-21, IL-21alone, or medium for4days.4. Flow cytometrywere used to detect expression of INF-γ, PD-1in the CD8+T cells and IL-21in the CD4+T cells.5. To analyze the differences and relationship of expression lever of PD-1+CD8+T,IFN-γ+CD8+T and IL-21+CD4+T cells in the HBV infection patients.Results:1.The percentage of CD3+CD8+IFN-γ+T cells in CHBs were increased successivelyin the HBV whole core protein T cell epitope peptides stimulation（2.93±1.22%）, HBVwhole core protein T cell epitope peptides and IL-21co-stimulation（3.36±1.01%）,non-stimulation（4.16±1.57%）, IL-21stimulation alone（4.80±1.91%）. Among theproportion of CD3+CD8+IFN-γ+T cells in the IL-21stimulation alone higher than in theHBV core protein antigen peptide stimulation, the difference is statistically significant. Itis also elevated than in the whole core protein T cell epitope peptides and IL-21co-stimulation, the difference is statistically significant. 2. The percentage of CD3+CD8+IFN-γ+T cells in CHBs with four kinds of stimulationwas notably increased than in AHB. The percentage of CD3+CD8+IFN-γ+T cells in thenon-stimulation in CHBs（4.16±1.57%） was elevated than in AHB（2.13±1.53%）(P<0.001). It is also higher in the whole core protein T cell epitope peptides stimulation inCHBs（2.93±1.22%） than in AHB（1.34±0.38%）(P<0.001). As well as it is higher in thewhole core protein T cell epitope peptides stimulation and IL-21co-stimulation in CHBs（3.36±1.01%）than than inAHBs（1.86±0.90%）(P<0.001). In addition, the proportionof CD3+CD8+IFN-γ+T cells in the IL-21stimulation alone is significantly elevated inCHBs（4.80±1.91%）than in AHBs（1.54±0.53%）(P<0.001).3. The percentage of CD3+CD8+PD-1+T cells in CHBs with four kinds of stimulationwas notably increased than in AHBs. The percentage of CD3+CD8+PD-1+T cells in thenon-stimulation in CHBs（5.44±3.65%） was elevated than in AHBs（2.68±1.72%）(P<0.01). It is also higher in the HBV whole core protein T cell epitope peptidesstimulation in CHBs（5.26±3.51%）than in AHBs（2.34±1.82%）(P<0.01). As well as itis higher in the whole core protein T cell epitope peptides stimulation and IL-21co-stimulation in CHBs（5.08±3.36%）than than in AHBs（2.96±2.23%）(P<0.05). Inaddition, the proportion of CD3+CD8+PD-1+T cells in the IL-21stimulation alone ishigher in CHBs（7.32±5.09%）than in AHBs（4.03±2.08%）(P<0.05).4. Correlation analysis of each factors. The percentages of CD3+CD4+IL-21+T celland CD3+CD8+PD-1+T cell achieve positive correlations in AHBs with HBV whole coreprotein T cell epitope peptides stimulation（r=0.774，p=0.0001）, HBV whole core proteinT cell epitope peptides stimulation and IL-21co-stimulation（r=0.737，p=0.0003）,but notin non-stimulation（r=0.527，p=0.019）and IL-21stimulation alone（r=0.313，p=0.207）.The percentages of CD3+CD4+IL-21+T cell and CD3+CD8+PD-1+T cell achieve positivecorrelations in CHBs with non-stimulation（r=0.753，p=0.0003）, HBV whole core proteinT cell epitope peptides stimulation（r=0.930，p＜0.0001）, HBV whole core protein T cell epitope peptides stimulation and IL-21co-stimulation（r=0.82，p＜0.0001）, but not inIL-21stimulation alone（r=0.533，p=0.027）.5. The proportions of CD3+CD4+IL-21+T cell, CD3+CD8+IFN-γ+T cell andCD3+CD8+PD-1+T cell in AHBs and CHBs did not show significant correlations withALT, AST, HBsAg, HBeAg and HBV DNA.Conclusion:1. The percentages of CD8+IFN-γ+T cell and CD8+PD-1+T cell in CHBs was elevatedthan in AHBs, which suggested that there existed persistent the HBV-specific CD8+T cellresponses in CHB patients, and these cell responses could be controlled by PD-1.2. IL-21augments the hepatitis B virus-specific CD8+T cell response and secret ofIFN–γ in vitro in patients of CHB.3. The expression of PD-1on CD8+T cell was not directly inhibited by IL-21in CHB,the expression of PD-1on CD8+T cell increase in a passive state when up-regulation theexpression of IL-21on CD4+T cell.4. T cell function may be inhibited by HBV whole core protein antigen T cell epitopepeptides.