Effect On Regulatory Immune Function Of Chronic Hepatitis B Patients After Anti-hbv Treatment

The pathogenesis of Chronic Hepatitis B ( CHB) has been one of the hot points in the field of infectious diseases for a long time. So far, the detailed mechanism of CHB persistent infection hasn't been clarified. Since the mid-90s, anti-viral treatment of CHB patients has made great progress, but the existing antiviral drugs can not completely terminate the duplication of hepatitis B virus in vivo. It is important and essential to clarify the mechanism of chronic HBV infection. Some reports show that the immunity of CHB patients improved after these patients take anti-virus medince.Few study has revealed precise mechanism.In the study,we completed the following two parts:一:Circulating CD4+CD25high regulatory T cells and expression of PD-1 and BTLA on CD4+ T cells in patients with chronic hepatitis B virus infectionThe roles of regulatory T cells (Tregs) and PD-1 in hepatitis B have not been clearly described. Also, the role of B and T lymphocyte attenuator (BTLA), which serves as a negative regulator of T cell activation, is still unknown in hepatitis B. In this study, we analyzed the frequency of circulating CD4+CD25high Tregs in patients with chronic hepatitis B (CHB) infection as well as the expression of PD-1 and BTLA on CD4+ T cells and their relationships with HBV-related clinical markers. Flow cytometry was performed to assess the expression of PD-1 and BTLA on the CD4+Tcells and circulating CD4+CD25high Tregs of 39 patients with chronic hepatitis B (CHB) and 19 healthy controls (NC). In addition ,the expression of BTLA were further analyzed by real-time PCR.Result:①In CHB patients, the CD4+CD25high population represented 4.48 to 13.89% of CD4+ T cells, These data indicate that the frequency of CD4+CD25high Tregs in CHB patients (mean±SD: 7.38±2.02%) was significantly higher than in normal controls (mean±SD: 4.99±1.15%) (P <0.0001). CHB patients with serum HBV DNA load≥108 copies/ml had a higher mean percentage of circulating CD4+CD25high Tregs than patients with serum HBV DNA load<108 copies/ml (7.75±2.18% vs 6.55±2.34%, respectively, P=0.0043). There were also significant differences in CD4+ CD25high Treg populations between the two groups and normal controls.②The percentage of CD4+ PD-1+ T cells in CHB patients (mean±SD: 58.73±11.11%) was significantly higher than normal controls (mean±SD: 47.75±12.69%) (P= 0.0013). Patients with serum HBV DNA load≥108 copies/ml showed no statistical difference in the number of cells expressing PD-1 on CD4+T cells compared with patients with serum HBV DNA load<108 copies/ml (61.18±12.02% VS 54.36±7.88%, respectively, P=0.0552), but the level of PD-1 expression on CD4+T cells in patients with serum HBV DNA load≥108 copies/ml was increased compared with patients with serum HBV DNA load<108 copies/ml.③the percentage of CD4+ BTLA+ T cells in CHB patients (mean±SD: 2.79±0.29 %) showed no statistical difference compared to normal controls (mean±SD: 2.386±0.4810%) (P=0.085). we also tested BTLA expression at the mRNA level. The relative BTLA mRNA levels in PBMCs from normal controls (n=10) and CHB patients (n =16) were 1.010±0.1449 and 1.097±0.0634, respectively. There was no significant difference between groups (P=0.916).④The percentage of CD4+ CD25highPD-1+ T cells in CHB patients (mean±SD: 97.78±5.657%) was no significant difference compared with normal controls (mean±SD: 99.11±1.133%) (P=0.37). the percentage of CD4+ CD25high BTLA+ T cells in CHB patients (mean±SD: 9.811±6.013 %) showed no statistical difference compared to normal controls (mean±SD: 13.97±10.84%) (P=0.11).⑤we analyzed the increase in circulating CD4+CD25high Tregs and CD4+PD-1+ T cells correlated with HBV replication level, Spearman analysis showed that the frequency of CD4+ CD25 high Tregs (r= 0.3314, P = 0.0393), and CD4+PD-1+ T cell (r = 0.3712, P=0.02) was positively correlated with serum HBV DNA load. Coclusion:Our findings suggested that the increased frequency of CD4+CD25high Tregs and PD-1 expression on CD4+ T lymphocytes may inhibit cellular immune response against HBV and affect viral clearance, leading to the persistence of chronic HBV infection.二:Circulating CD4+CD25high regulatory T cells,PD-1 and PD-L1 expre- ssion on CD4+T cells ,TLR2 and TLR4 expression during antiviral therapy of CHB patientsAdaptive immunity and innate immunity play a key role in chronic HBV infection,but we know litter about the influence of adaptive and innate immunity after antiviral treatment. In the study,The frequency of circulating Tregs ,PD-1 and PD-L1 expression of CD4+ T cells,TLR2 and TLR4 expression on mononuclear cells were analyzed by 28 chronic hepatitis B patients after telbivudine treatment.28 CHB patients were given nucleoside analogues (telbivudine) anti-viral treatment of total six months, PBMCs were obtained peripheral blood at baseline, after three and six months of treatment, The circulation CD4+CD25high Treg cells,PD-1and PD-L1 expression of CD4+ T cells, TLR2 and TLR4 expression on mononuclear cells were performed to assess by flow cytometry in different periods; IFN-γand IL-9 level wad detected in plasma by ELISA .Results:①Serum HBVDNA and ALT level were determined at the baseline, as well as after three and six months of treatment. HBVDNA load(log10) were:7.672±0.3055 copies/ml, 3.688±0.2711copies/ml and 2.628±0.3176 copies/ml, a significant difference was observed among the three groups (P<0.001, P<0.001); ALT level respectively was : 111.7±9.206u / L, 68.19±12.38u / L and 30.38±3.273u / L, there were significant difference between at baseline and after 3 months treaments(P<0.001) or after 6 months treaments (P <0.001).②Frequency of CD4+ CD25highTreg cells of CHB patients were at the baseline (10.48±0.4809%), after 3 months of treatment (7.290±0.4258%) and after 6 months of treatment (7.078±0.4274%),there were significantly reduced the baseline and after 3 months (P <0.001) or after 6 months of treatment( P <0.0001),There was no significant difference between after 3 monthsof treatment and 6 months of treatment (P =0.6521); The frequency of CD4+ CD25+ Treg cells accounted for CD4+ T cells : 27.95±1.553%, 24.03±1.497%, 25.69±1.437%, The frenquency of CD4+ CD25+ Treg cells of CHB patients after 3 months of treatment are decreased compared with the baseline (P = 0.0238).③CD4+PD-1+T and CD4+PD-L1+T cells were determined at the baseline, after 3 and 6 months of treatment. CD4+ PD-1+ Tcells in total CD4+T cells, as follows: 42.52±3.386%, 33.19±3.352% and 40.08±3.931%, The frenquency of CD4+PD-1+T was reduced after 3 months of treatment compared with the baseline(P = 0.0464),There was no difference between the baseline and after 6 months of treatment (P=0.9639);CD4+PD-L1+T cells respectively were: 0.9258±0.22 13% ,0.4827±0.0909%,0.5258±0.0913%,There was not significant difference among the three groups (P = 0.0747, P = 0.0631).but we observed that CD4+PD-L1+T cells were deceresed between after 3 , 6 months of treatment and the baseline.④The values of mean fluorescence intensity(MFI)of TLR2 and TLR4 of CHB at baseline and after 3 and 6 months of treatment respectively were: 89.07±6.089,96.12±8.803 and 102.9±6.633, TLR4 of CHB were:41.17±2.397,38.20±2.125,40.48±2.371.There was statistical diffeneceat the baseline and after 6 months of treatment (P =0.0399), In addition, TLR2 expression was up-regulation in after 6 months of treatment compared with the baseline, however ,there was no difference between the baseline and after 3 months of treatment(P>0.05);The MFI values corresponding to TLR4 was no significant difference among the three groups (P> 0.05).⑤The IFN-γlevel of plasma was tested at the baseline, as well as after 3 and 6 months of treatment about 20 chronic hepatitis B patients, the concentration was respectively : 24.49±3.862 pg / ml, 21.86±2.873 pg / ml, 27.28±6.581 pg / ml there were showed no significant difference among three groups (P> 0.05), The concentration of IFN-γwsa increased compared after 6 months of treament with the baseline; IL-9 concentration respectively was : 30.89±5.279pg/ml, 25.80±4.159 pg / ml, 12.76±2.524 pg / ml, There was significantly reduced after 3 and 6 months of treament compared with the baseline .( P = 0.010, P = 0.0086).Conclusion: HBV load reduction and the normalization of ALT levels by treatments with telbivudine results in a partial restoration of the impaired immune response,as indicated by a decrease in percentages of CD4+CD25high Tregs as well as PD-1 expression decline on CD4+T cells.In addition,TLR2 may also play a role in CHB antiviral therapy, the specific mechanism needs further research.