| Hepatitis B virus (HBV) infection is one of the major global public health problems.Asymptomatic carrier, chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma aredifferent disease stages of HBV infection. Studies have shown that the immune response isthe core mechanism of chronic HBV infection. Immune clearance can lead to recovery,while persistent immune injury can lead to chronic hepatitis, cirrhosis, liver cancer andother clinical phenotypes. HBV-specific CD4+T cell is a key factor to immune injury, thebalance of its different cell subsets polarization determines whether patients with hepatitisB have immune damage. HBV-specific CD4+T cell plays an important role in immuneinjury. However, the exact mechanism of immune injury in chronic HBV infection is notclear.Human leukocyte antigen (HLA) is a major genetic factor associated with disease.Currently, genetic variations of HLA-Ⅱ molecule determine the clinical outcomes likeinfection, clearance and hepatocellular carcinoma of HBV infection were found bynumerous genome-wide association studies which based on large samples of the patientpopulation. These studies showed an important role of HLA-Ⅱ molecules in HBVprotective immunity. HLA-Ⅱ molecules are expressed on antigen-presenting cells (APCs),can present antigens and activate of CD4+T cell(Th1、Th2、Th17、Treg、Tfh) response[1].HLA-Ⅱ molecules have an important role in the process of T cell recognition and epitopepeptide identification. HLA-DR, DQ, DP molecules showed different effects in geneticsusceptibility and associated with pathogenesis mechanisms of chronic HBV infection.However, there is no further explanation of the underline mechanism. Observation of theexpression patterns of HLA-Ⅱ molecule in patients with chronic HBV infection and theirindependent effect to CD4+T cells polarization type will enrich the pathogenesismechanism of HBV infection and provide the strategies for clinical personalized medicineand disease prevention. In this study, peripheral blood mononuclear cells (PBMCs) of10mild to moderatechronic hepatitis B (MCHB) patients and5healthy controls (HCs) were isolated, DC werematured and stimulated with GM-CSF、IL-4and TNF-α, then the expression patterns ofHLA-DR/DQ/DP molecules on peripheral blood myeloid dendritic cells (mDCs) wereobserved. We also observed the expression patterns of HLA-DR/DQ/DP molecules onmDCs and plasmacytoid dendritic cells (pDCs) simultaneously. HCs, Asymptomatic HBVcarriers (AsCs), Mild to moderate chronic hepatitis B (MCHB), severe hepatitis B (SCHB),acute on chronic liver failure patients were enrolled in this study. We detected theexpression patterns of HLA-Ⅱ molecules using flow cytometry, and analyzed thefrequencies of mDC and pDC in PBMCs isolated from fresh anticoagulant peripheral bloodof experimental subjects. Moreover, the expression patterns of HLA-Ⅱ molecules on mDCand pDC of SCHB (n=4) and MCHB (n=1) patients were observed at three differentconsecutive time points.To evaluate the independent effect of angtigen presentation by HLA-DR/DQ/DP onthe polarization of CD4+T cells,20patients of mild to moderate chronic hepatitis B wereselected. After blocking HLA-DR/DQ/DP respectively the effect of T cells polarizationwere observed, then HBcAg pool with overlapping peptides were used to stimulate PBMCs,and the polarization effects of T cells were observed. Combined with a comprehensiveanalysis of clinical parameters in CHB patients, we aimed to analyze the differentexpression patterns of HLA-Ⅱ and their effects to progression of chronic hepatitis B virusinfection.The main results of our study were listed as follow:1. Compared with healthy donors, the percentage of mDC and pDC were significantlydecreased in CHB patients, and correlated with severity of the disease. Results of threeconsecutive time points on SCHB patients showed increased number and proportion ofmDC and pDC with the improvement of the clinical indicators.2. The mainly expression patterns of HLA-II molecules on mDC were DR+DQ+DP+、DR+DQ+DP-、 DR+DQ-DP-and DR-DQ-DP-. Percentage of DR+DQ+DP+cells inHC(0.9-39.2)、AsC(0-20.1)、MCHB(1.3-80.7)、SCHB(0.1-90.6) and ACLF(5.2-98.0)groupswith median17.2、5.9、23.3、40.5and37.3, were increased with severity of the disease (p＜0.001). Compared with pDC, percentage of DR+DQ+DP+cells on mDC in CHB patients and healthy controls was higher, while DR-DQ-DP-cells was lower.3. The mainly expression patterns of HLA-II molecules on pDC were not only showedsame as mDC (DR+DQ+DP+、 DR+DQ+DP-、 DR+DQ-DP-and DR-DQ-DP-), butin MCHB, SCHB, and ACLF also showed the DR-DQ-DP+patterns. Percentage ofDR+DQ+DP+cells in HC(0.3-21.7)、AsC(0.1-15.2)、MCHB(0.9-57.6)、SCHB(1.2-52.1)andACLF(0.2-63.6) groups with median5.2、1.9、7.3、20.3and15.8, were significant increasedwith severity of the disease (p＜0.001). Percentage of DR+DQ+DP-cells in HC(4.8-76.5)、AsC(0-42.6)、MCHB(1.3-54.1)、SCHB(0.9-71.4) and ACLF(0.2-38.6) groups with median32.2、18.7、8.9、11.6and3.2，were significant decreased with severity of the disease (p＜0.05). Percentage of DR-DQ-DP+cells on pDC in HC(0.01-5.8)、AsC(0-0.64)、MCHB(0-54.1,1.94)、SCHB(0-13.4) and ACLF(0-56)groups with median0.07、0.01、1.9、2.1and3.9, were significant increased with severity of the disease (p＜0.05). Observation of threeconsecutive time points on SCHB patients showed consistent results. Percentage ofDR+DQ-DP-cells on pDC in HC(0-40.7)、AsC(2.2-32.2)、MCHB(0.2-15.6)、SCHB(0.6-32.0) and ACLF(0-6.36) groups with median23.9、12.2、1.9、4.4、0.39, were significantdecreased with severity of the disease (p＜0.05).4. Preliminary function experiments showed that mild to moderate chronic hepatitis Bpatients had a certain degree of antigen-specific CD4+T cell responses. HBcAg-specificCD4+T cell polarization responses were different after blocked with anti HLA-DR/DQ/DP.Percentage of IFN-γ+CD4T cells (HLA-DR,1.13-18.1vs0.02-1.26, p=0.004; HLA-DQ,0.37-19.6vs0.02-1.26, p=0.002)、IL-17+CD4T (HLA-DR,0.03-0.55vs0.02-0.16,p=0.03；HLA-DQ,0-0.57vs0.02-0.16, p=0.085) were significant increased when blockedwith anti HLA-DR/DQ. While Percentage of IFN-γ+CD4T (0.34±0.39vs0.08±0.05,p=0.042)、 IL-17+CD4T (0.17±0.14vs0.01±0.01, p=0.012) were significant decreasedand IL-4+CD4T (0.1-7.29vs0.01-3.78, p=0.044) was significant increased whenblocked with anti HLA-DP.In conclusion, based on7-color flow cytometry, we detected the expression patterns ofHLA-Ⅱ molecules on mDC and pDC in CHB patients and blood donors. We found thatexpression patterns of HLA-DR/DQ/DP molecules were different. There were four mainlyexpression patterns on mDC, and another DR-DQ-DP+pattern on pDC. Percentage ofDR+DQ+DP+cells on mDC was significant increased with severity of the disease. Percentage of DR+DQ+DP+and DR-DQ-DP+cells on pDC were significant increased withseverity of the disease, while percentage of DR+DQ+DP-and DR+DQ-DP-cells weresignificant decreased.Based on the varied expression patterns, our preliminary function experiments showedthat MCHB patients existing a certain degree of antigen-specific CD4+T cell responses.HBcAg-specific CD4+T cell polarization responses were different after blocked with antiHLA-DR/DQ and HLA-DP. HLA-DR/DQ presented HBcAg to IL-4+CD4T cellsactivation, while HLA-DP to IFN-γ+IL-17+CD4T cells.The underline mechanisms withthe role of differential expression patterns of HLA-Ⅱ molecule and the different T cellpolarization responses in chronic hepatitis B still need further research. |
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