| OBJECTIVE:To investigate the T-cell subsets,Foxp3 mRNA and cytokine levels secreted by peripheral blood mononuclear cells (PBMC) with or without stimulation of PHA and HBsAg in the cases with and without responses to HBsAg vaccination, and to explore the relationship between immune respone to HBsAg and activation of T-cell subsets,the expression of Foxp3 mRNA and cytokines.METHODS:After examination of serological markers of hepatitis B of 1024 students of short course in institution, we selected students with no serological markers of hepatitis B to be immunized with hepatitis B vaccine.45 days after the end of vaccination, we examined the serological markers of hepatitis B of the students again. Peripheral blood from 18 responders to HBsAg vaccination(responders),22 nonresponders and 10 naive nonimmunized healthy controls (controls) were analyzed for the expression of CD6,CD25,CD4,CD8 in peripheral blood mononuclear cell, and CD4+CD25+regulatory T cell frequency was also analyzed by Flow cytometry; the expression of Foxp3 mRNA of peripheral blood mononuclear cells with or without stimulation of PHA and HBsAg were analysed by real-time quantitative PCR;IL-4,IL-12,IL-18IFN-γsecreted by PBMC after stimulation of PHA and HBsAg were analysed by ELISA. RESULTS:1. Of 1024 students of short course in institution 405 were negative for any serological marker of hepatitis B virus infection. There were 1.7% cases with HBsAg positive. After vaccination of HBsAg, only 22 cases (5.5%) were negative for antibody to HBsAg (HBsAb), as nonresponders. Response rate to HBsAg vaccination was as high as 94.5% in the present study. The rates of nonresponders and hyporesponders was no significantly different between male and females. The titres of HBsAb in the most students belonged to hyperresponders' group.2. The percentage of CD3+CD4+T cells in CD3- T cells from responders,nonresponders and controls were 50.84±6.36%,53.67±6.68%,and 51.02±9.30%, respectively. There was no difference statistically between responders and controls. The same result could be found between responders and nonresponders(p>0.05). CD8 expression on CD3+T cells in responders,nonresponders and controls were 41.02±4.41%,42.69±7.35%,and 42.36±8.34%, respectively. There were no difference statistically between responders and controls as well as nonresponders (p>0.05).3. The percentage of CD69+CD4+T cells in CD4+T cells from responders,nonresponders and controls were 1.81±1.33%,0.64±0.48%,and 0.56±0.39%, respectively. CD69 expression on CD4+T cells in responders was increased as compared to controls as well as nonresponders(p<0.01), but no difference between nonresponders and controls(p>0.05). The percentage of CD69+CD8+T cells in CD8+T cells from responders was significantly lower than that of nonresponders(0.43±0.25% vs.3.05±1.69%,p<0.01) and controls (0.43±0.25% vs.3.11±2.41%,p<0.01).4. The percentage of CD4+CD25+regulatory T cell in CD4+cells from responders,nonresponders and controls were 0.59±0.46%,0.77±0.40%,and 1.30±1.44%, respectively. There were no significant statistical difference between them (p>0.05).5. Expression of Foxp3 mRNA of peripheral blood monoclear cells from responders,nonresponders and controls were analysed by real-time quantitative PCR. Eachβ-actin had clear amplification curve which implied that DNA of all samples extracted were good. The melting curve of Foxp3 andβ-actin was a high single peak which showed that specificity of primers was good. Before stimulation, there were significant difference statistically between responders and controls as well as nonresponders (p<0.05).After stimulation, the difference between responders and nonresponders was not clear, but compared with controls,there was significant difference statistically (p<0.05)6. There were significant difference statistically the concentration of IFN-γproduced by PBMCs after stimulation by PHA and HBsAg between the responders and controls as well as nonresponders (p<0.05). However, there were no significant difference statistically the concentration of IL-18,IL-4,IL-12 produced by PBMCs after stimulation by PHA and HBsAg between responders and controls as well as nonresponders (p>0.05).CONCLUSION:1. The rates of nonresponse to hepatitis B vaccine were 5.5%; Among which had response to hepatitis B vaccine,the rates of hyporesponse,mid-response,hyperresponse were 13%,22%,65%, respectively.2. Insufficient early activation of Th and the increasing activation of Ts were concerned with nonresponse to HBsAg vaccination.3. The CD4+CD25+Foxp3+regulatory T cell might play an important role in the negativity regulation of response to HBsAg vaccination.4. Nonresponse to hepatitis B vaccine was related to down-regulated IFN-y production.
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