Therapeutic polypeptides based on HBV core 18-27 epitope can induce CD8^＋ CTL-mediated cytotoxicity in HLA-A2^＋ human PBMCs

AIM: To explore how to improve the immunogenicity of HBcAg CTL epitope based polypeptides and to trigger an HBV-specific HLA I-restricted CD8^+ T cell response in vitro.METHODS: A new panel of mimetic therapeutic peptides based on the immunodominant B cell epitope of HBV PreS2 18-24 region, the CTL epitope of HBcAg18-27 and the universal T helper epitope of tetanus toxoid (TT) 830-843 was designed using computerized molecular design method and synthesized by Merrifield‘s solid-phase peptide synthesis.Their immunological properties of stimulating activation and proliferation of lymphocytes, of inducing TN1 polarization,CD8^+ T cell magnification and HBV-specific CD8^+ CTL mediated cytotoxicity were investigated in vitro using HLA-A2^+ human peripheral blood mononuclear cells (PBMCs) from healthy donors and chronic hepatitis B patients.RESULTS: Results demonstrated that the therapeutic polypeptides based on immunodominant HBcAg18-27 CTL,PreS2 B- and universal TN epitopes could stimulate the activation and proliferation of lymphocytes, induce specifically and effectively CD8+ T cell expansion and vigorous HBVspecific CTL-mediated cytotoxicity in human PBMCs.CONCLUSION: It indicated that the introduction of immunodominant T helper plus B-epitopes with short and flexible linkers could dramatically improve the immunogenicity of short CTL epitopes in vitro.     

Immunohistochemical investigation of hepatitis B virus associated antigens,HLA antigens and lymphocyte subsets in type B chronic hepatitis

HLA antigens, hepatitis B virus (HBV)-assodated antigens and lymphocyte subsets in liver tissue from 35 patients with HBs antigenemia were studied using an immunoperoxidase double staining method arid immunoelectron microscopy in order to clarify the immune mechanism of hepatocyte lysis in type B hepatitis. Immune light and electron microscopy using monoclonal antibodies to lymphocyte subsets revealed that infiltrating lymphocytes in the areas of piecemeal necrosis and focal necrosis were predominantly CD8-positive, showing direct contact with hepatocytes. In contrast, CD4(+) cells were infrequently observed in necrotizing inflammatory lesions. HLA-A,B,C antigens were mainly found on hepatocytes in areas of piecemeal necrosis and focal necrosis, in association with CD8(+) lymphocyte infiltration. HLA-DR antigens were demonstrated on a few hepatocytes in the same lesions. In cases of CAH with serum HBeAg positive, HLA-A,B,C, antigens and HBV antigens simultaneously demonstrated on the same hepatocytes. Especially, hepatocytes expressing both HLA-A,B,C antigen and HBsAg on the plasma membrane showed direct contact with CD8(+) lymphocytes. This finding fullfilled the morphological requirements for HBsAg as a target antigen. On the other hand, HBcAg was hardly demonstrated in the liver cell membrane but was demonstrated mainly in the cytoplasm. Compared with the nuclear localization of HBcAg in cases of NSR, cytoplasmic localization of this antigen may be associated with membranous expression of new antigens induced by HBV infection.     

Immunohistochemical investigation of hepatitis B virus associated antigens, HLA antigens and lymphocyte subsets in type B chronic hepatitis

HLA antigens, hepatitis B virus (HBV)-associated antigens and lymphocyte subsets in liver tissue from 35 patients with HBs antigenemia were studied using an immunoperoxidase double staining method and immunoelectron microscopy in order to clarify the immune mechanism of hepatocyte lysis in type B hepatitis. Immune light and electron microscopy using monoclonal antibodies to lymphocyte subsets revealed that infiltrating lymphocytes in the areas of piecemeal necrosis and focal necrosis were predominantly CD8-positive, showing direct contact with hepatocytes. In contrast, CD4(+) cells were infrequently observed in necrotizing inflammatory lesions. HLA-A,B,C antigens were mainly found on hepatocytes in areas of piecemeal necrosis and focal necrosis, in association with CD8(+) lymphocyte infiltration. HLA-DR antigens were demonstrated on a few hepatocytes in the same lesions. In cases of CAH with serum HBeAg positive, HLA-A,B,C, antigens and HBV antigens simultaneously demonstrated on the same hepatocytes. Especially, hepatocytes expressing both HLA-A,B,C antigen and HBsAg on the plasma membrane showed direct contact with CD8(+)lymphocytes. This finding fulfilled the morphological requirements for HBsAg as a target antigen. On the other hand, HBcAg was hardly demonstrated in the liver cell membrane but was demonstrated mainly in the cytoplasm. Compared with the nuclear localization of HBcAg in cases of NSR, cytoplasmic localization of this antigen may be associated with membranous expression of new antigens induced by HBV infection.     

Peripheral blood CD4-mediated enhancement and CD8-mediated suppression in the presence of recombinant hepatitis B virus core antigen

The proliferative response of peripheral blood T cells to hepatitis B core antigen (HBcAg) was studied in hepatitis B patients. CD4+ T cells from patients with chronic active hepatitis type B (CAH-B) exhibited a significant proliferative response to HBcAg, especially in hepatitis B envelope antigen (HBeAg)-positive patients. In contrast, there was no apparent T cell reaction to HBcAg in patients with CAH non-A, non-B, HBeAg-positive healthy carriers and in healthy volunteers. The proliferative response to CD4+ cells to bacterial extracts of Escherichia coli was always insignificant in all patients and healthy volunteers. The CD8+ cells did not proliferate in response to HBcAg in any subject, even in the presence of autologous irradiated CD4+ responder cells. The CD8+ cells, preactivated with HBcAg and HBcAg-reactive irradiated autologous CD4+ cells, suppressed the proliferative response to autologous CD4+ cells to HBcAg but not the response to phytohemagglutinin in HBcAg-responder CAH-B patients. CD4-mediated HBcAg-specific enhancement and CD8-mediated HBcAg-specific suppression in the peripheral blood compartments of HBcAg-responsive CAH-B patients are possible.     

Cellular immune responses in hepatitis B virus e antigen negative chronic hepatitis B

The immunopathogenesis of hepatitis B e antigen (HBeAg) negative chronic hepatitis B (CHB) virus (HBV) infection has not been adequately investigated. We studied the cellular immune responses of peripheral lymphocytes using proliferating assays, intracellular cytokine staining (ICS) and ELISPOT interferon-gamma (IFN-gamma) assays after non-specific and specific stimulation with whole HBV proteins and synthetic peptides. Thirty patients with HBeAg negative CHB, eleven HBsAg inactive carriers, nine patients with acute hepatitis B and 22 healthy controls were included in the study. Patients with HBeAg negative CHB demonstrated an increased number of peripheral CD8+ T cells while their peripheral blood mononuclear cells showed increased proliferation after in vitro stimulation with overlapping hepatitis B core derived peptides and an envelope derived epitope (HBs 182-191 aa), similar to those observed in acute hepatitis B. Using ICS, we found an expanded population of IFN-gamma producing T lymphocytes, CD4+ and CD8+, after non-specific stimulation, in HBeAg negative CHB compared to all other groups. HBeAg negative CHB and acute hepatitis B patients had a similarly increased number of core specific T cells measured by the IFN-gamma assays. Inactive HBsAg carriers showed minimal proliferative responses overall while they exhibited an increased number of envelope specific effector T cells (measured by ICS). In conclusion, we showed that overall CD4+ T cell responses from patients with HBeAg negative CHB were comparable to those of acute hepatitis B, while inactive HBsAg carriers despite their limited proliferative capacity the effector activity of their peripheral T cells was maintained.     

Serial observation of lymphocyte subpopulations and interleukin 2 production of T cells from patients with acute viral hepatitis and chronic active hepatitis

T cell subpopulations and interleukin 2 (IL-2) production of T cells in peripheral blood from patients with acute viral hepatitis (AVH) and chronic active hepatitis (CAH) were studied to elucidate the change of T cells functions during the exacerbation of viral hepatitis. The ratio of the number of CD4-positive cells to CD8-positive cells (CD:CD8 ratio) was increased in many patients with AVH. During exacerbation of CAH, the CD4:CD8 ratio was higher than that during remission (p less than 0.01), due to a decrease in the number of CD8-positive cells. IL2 production of T cells in AVH and CAH with bridging necrosis was higher than that of T cells in normal controls (p less than 0.01, p less than 0.05, respectively). T cells from patients with CAH produced more IL2 during exacerbation than during remission (p less than 0.01). IL2 production of T cells and the CD4:CD8 ratio (p less than 0.005, p less than 0.01). The change in T cell subpopulations in AVH and during exacerbation of CAH was found to induce an immunological condition, in which T cells easily produce IL2, which induces a proliferation of cytotoxic T cells.     

Dendritic cells from chronic hepatitis B patients can induce HBV antigen-specific T cell responses

AIM: To determine whether dendritic cells (DCs) from chronic hepatitis B patients could induce HBV antigen-specific T cell responses or not. METHODS: DCs were generated from peripheral blood mononuclear cells of patients with chronic hepatitis B (CHB) infection and healthy donors. We compared the phenotypes of these DCs and their ability to secrete cytokines and to participate in mixed lymphocyte reactions. In addition, autologous lymphocytes were cultured with DCs loaded with HBV core region peptide HBcAg8-27, an epitope recognized by cytotoxic T lymphocytes (CTL), and bearing human leucocyte antigen (HLA)-A2 for 10 d. Cytokine secretion and lytic activity against peptide-pulsed target cells were assessed. RESULTS: DCs with typical morphology were generated successfully by culturing peripheral blood mononuclear cells (PBMCs) from CHB patients with AIM-V containing GM-CSF and IL-4. Compared with DCs from normal donors, the level of CD80 expressed in DCs from CHB patients was lower, and DCs from patients had lower capacity of stimulate T cell proliferation. When PBMCs isolated from patients with chronic or acute hepatitis B infection and from normal donors were cocultured with HBcAg18-27 peptide, the antigen-specific memory response of PBMCs from acute hepatitis B patients was stronger than that of PBMCs from chronic hepatitis B patients or normal donors. PBMCs cocultured with DCs treated with HBcAg18-27 CTL epitope peptide induced an antigen-specific T cell reaction, in which the level of secreted cytokines and lytic activity were higher than those produced by memory T cells. CONCLUSION: DCs from patients with CHB can induce HBV antigen-specific T cell reactions, including secretion of cytokines essential for HBV clearance and for killing cells infected with HBV.     

Immunological aspects of chronic hepatitis

Chronic active hepatitis (CAH) is a clinically and histologically defined syndrome which is aetiologically heterogeneous. Immune reactions are involved in almost all types of chronic inflammatory liver diseases. The host's immune system is thought to contribute significantly to the degree of liver cell destruction in chronic hepatitis B—HBcAg is regarded as a major target antigen. Auto-immune type CAH is a syndrome of unknown aetiology, a loss of tolerance against self antigens is thought to be a principal pathogenetic mechanism. At least three different subgroups of auto-immune type CAH can be distinguished by circulating auto-antibodies: antinuclear antibodies (ANA), smooth muscle antibodies (SMA), liver membrane auto-antibodies (LMA); liver-kidney-microsomal antibodies (LKM); and antibodies to a soluble liver antigen (SLA). The identification of auto-immune type CAH has clinical relevance since only auto-immune type CAH seems to profit from immunosuppressive therapy. A suppressor T cell defect was demonstrated in chronic hepatitis B and auto-immune type CAH. However, this defect could only be reversed by the addition of prednisone in vitro in auto-immune type CAH. Present research concentrates on the evaluation of the precise cellular mechanisms leading to liver cell destruction. In vitro cytotoxicity assays including autologous target cell systems have several drawbacks due to the use of peripheral blood lymphocytes and the questionable viability of the hepatocytes isolated from liver biopsies. Immunohistological studies using monoclonal antibodies revealed that T₈ positive T lymphocytes account for the majority of the inflammatory cell infiltrate. In addition, T cell clones derived from liver biopsies are mainly T₈ positive and show cytotoxic function in vitro. At present, little is known about the antigen specificity of these liver-derived T cell clones.     

Histopathological analysis of chronic hepatitis B virus (HBV) infection in relation to HBV replication

The interrelationship among expression patterns of hepatitis B surface and core antigens (HBsAg and HBcAg) in the liver, hepatitis B virus (HBV) DNA in sera, HBeAg/anti-HBe status and histological features was examined in 189 liver specimens and 106 sera from Japanese patients with chronic HBV infection, utilizing immunoperoxidase methods and a spot hybridization technique. HBsAg and HBcAg were distributed uniformly among the lobules in 8 viral carriers with "normal" liver (NVC) and 30 patients with persistent hepatitis (PB) seropositive for HBcAg. This uniform staining pattern was quite distinct from the non-uniform and irregular patterns in 137 patients with chronic active hepatitis (CAH) associated with or not associated with cirrhosis. HBcAg was often found to be stained strongly in the cytoplasm as well as in the nucleus of hepatocytes in HBeAg-positive CAH, in contrast to NVC/PH, in which cytoplasmic HBcAg was very weak. Serum HBV DNA was detected in all 22 cases with HBeAg-positive NVC/PH, 41 of 46 (89.1%) cases with HBeAg-positive CAH, 6 of 23 (26.1%) casew with anti-HBe-positive CAH and none of 3 cases with anti-HBe-positive NVC/PH. The level of serum HBV DNA and staining of HBcAg were in decreasing order in these groups. While the presence of serum HBV DNA and HBcAg staining was always associated with HBeAg seropositivity in NVC/PH, this was not always found in CAH. Moreover, necroinflammatory activity in the liver did not always parallel viral replication in CAH. These findings seem to confirm that NVC/PH and CAH have different biologic processes; viral replication is always concordant in the former, but is sometimes discordant in the latter with HBeAg/anti-HBe status. The immunologic response of the host seems to suppress and distort replication of the virus to a varying degree among patients and areas of the same liver in CAH differently to that in NVC/PH. The different life cycle of the virus, including integration of viral DNA into the cellular genome, may subsequently result in discordant states of various virus markers in CAH.     

慢性乙型肝炎患者外周血树突状细胞诱导特异性T淋巴细胞应答

目的 探讨慢性乙型肝炎患者外周血树突状细胞(Dc)是否诱导特异性T细胞应答。方法(1)将研究对象分为慢性乙型肝炎患者组、急性乙型肝炎痊愈组、健康志愿者组,分离各组研究对象的外周血单个核细胞(PBMC),细胞内细胞因子染色方法检测其对细胞毒性T淋巴细胞(CTL)特异表位多肽乙型肝炎病毒核心抗原(HBcAg)18-27的记忆性免疫应答;(2)培养慢性乙型肝炎患者DC,将负载有乙型肝炎抗原表位多肽的DC诱导特异的T细胞应答。采用细胞内细胞因子染色方法检测诱导的T细胞分泌的细胞因子,乳酸脱氢酶释放法测定诱导的T细胞杀伤活性。结果(1)急性乙型肝炎患者PBMC对HBcAg 18-27 CTL特异表位多肽存在记忆的免疫应答,其分泌干扰素-γ的CD8+T细胞占CD8+T细胞总数的(4.3±2.5)％,分泌白细胞介素-2的占总细胞数的(4.8±2.2)％,分泌肿瘤坏死因子-α占总细胞数的(4.6±2.3)％。而慢性乙型肝炎患者和健康志愿者对其记忆应答很低,与急性乙型肝炎患者比较差异有显著性,t值为2.508-3.305,P<0.05。(2)用多肽共孵育过的慢性乙型肝炎患者DC多次诱导的T细胞慢性乙型肝炎患者组,加肽孵育的靶细胞比例为30:1、10:1、3:1时,其杀伤率分别为(57.0±20.3)％、(49.5±20.2)％、(21.8±12.9)％,均高于对照组,表明慢性乙型肝炎患者DC可以诱导特异的T     

急性乙型肝炎患者特异性核心抗原细胞毒性T淋巴细胞频率的变化及其意义

目的 动态观察急性乙型肝炎(AHB)患者外周血HBcAg18-27表位特异性细胞毒性T淋巴细胞(CTL)、血清ALT、HBV DNA、HBsAg和淋巴细胞亚群的变化,探讨HBV特异性CTL频率的消长在病毒清除以及肝脏损伤中的作用.方法 分别选取AHB、慢性乙型肝炎(CHB)患者外周血,根据人类白细胞抗原(HLA)-A0201结果分为两组:HLA-A0201阳性患者作为HBV特异性CTL检测组、HLA-A0201阴性患者作为特异性抗原表位对照组.用HLA-A0201限制性表位HBcAg18-27五聚体复合物通过流式细胞技术,动态定量检测外周血中HBV特异性CTL频率和T、B淋巴细胞与自然杀伤细胞(NK)和NKT淋巴细胞;以速率法检测血清ALT水平;荧光定量PCR检测HBV DNA水平; Abbott微粒子化学发光技术检测HBV血清学标志物.计量资料采用均数±标准差((x)±s)表示或中位数(P25-P75)描述,组间比较采用方差分析或非参数检验(KruskalWallis检验和Mann-Whitney U检验);两种计量指标的关系采用Pearson相关分析.结果 AHB患者入院第1、2、3周外周血HBcAg表位特异性CTL频率分别为2.11%(0.20%～3.64%)、3.56%(1.05%～5.91%)、2.03%(0.33%～3.58%),高于入院第4、5、6周的0.99%(0.12%～2.16%)、0.29%(0.05%～0.76%)、0.39%(0.05%～0.46%),也显著高于CHB的0.11%(0.06%～0.29%),z值分别为-3.258,-4.041,-3.259,P值均＜0.01.AHB患者HBcAg表位特异性CTL峰值延迟于血清HBV DNA、HBsAg和ALT等指标的峰值;在AHB患者中,HBcAg表位特异性CTL高频率患者的血清HBsAg消失时间早于CTL频率较低的患者[(1.75±1.04)周与(4.33±3.51)周,t=-2.018,P＜0.05].CD3+CD8+T淋巴细胞频率的峰值出现在入院后第2周,并与HBcAg表位特异性CTL峰值相重叠,两者动态变化规律呈相关性(r=0.420,P＜0.01).AHB患者早期外周血NK、NKT淋巴细胞数量显著低于正常对照组和CHB患者,但随着病情好转而逐渐恢复,AHB患者外周血NK细胞数量变化与HBcAg特异性CTL动态变化呈负相关(r=-0.435,P＜0.01).结论 急性HBV感染者高频率的HBcAg表位特异性CTL与HBsAg的更早消失有密切关系,动态监测外周血中HBcAg特异性CTL频率变化,可以作为预测HBV感染后临床转归的参考指标;AHB患者外周血CD8+T淋巴细胞数量的变化,可以间接反应AHB患者体内HBcAg特异性CTL频率的改变.

Abstract：

Objective This report aims to investigate the dynamical changes of HBcAg18-27 epitope specific cytotoxic T lymphocytes(CTL), alanine aminotransferase (ALT), HBV DNA and HBsAg in peripheral blood of acute hepatitis B patients, and to explore the roles of HBcAg18-27-specific CTLs in virus clearance and liver injury. Methods Acute hepatitis B (AHB) and chronic hepatitis B (CHB) patients were divided into two groups according to results of HLA-A0201. Patients with positive HLA-A0201 were classified into HBcAg-specific CTL group and those with negative HLA-A0201 were referred as control group.The specific CTLs were stained with HLA-A0201 limited HBcAg18-27 epitope MHC-Pentamer and the frequencies of CTLs, T, B, NK and NKT cells were detected by flow cytometry (FCM). The serum ALT, HBV DNA and HBsAg were examined using speed analysis, quantitative PCR and abbott chemiluminescent technology. Results The frequencies of HBcAg18-27-specific CTLs in AHB patients were higher in the early three weeks as compared to the late three weeks. The apex time of HBV-specific CTL frequencies lagged behind those of HBV DNA, HBsAg and ALT. The loss of HBsAg in patients with high frequencies of HBVspecific CTL was earlier than that in patients with low frequencies (t = 2.018, P ＜ 0.05). In the second week the peak frequencies of CD3+CD8+ cells overlapped with that of HBcAg18-27-specific CTLs and with a positive correlation between (r = 0.420, P ＜ 0.05). During the early stages of AHB, the frequencies of NK and NKT cells were found significantly lower than that of control group and CHB group and the levels were back to normal after recovery. Moreover, a negative correlation existed between the frequencies of NK cells and the dynamic changes of HBcAg18-27-specific CTLs (r = -0.435, P ＜ 0.01) in AHB group. The frequencies of HBcAg18-27-specific CTLs were significantly higher as compared to CHB group in the first three weeks (z = -3.258, -4.04, and -3.259, P ＜ 0.01). Conclusion The early loss of HBsAg was closely related to the high frequencies of HBcAg18-27 specific CTLs in AHB patients. HBcAg-specific CTL frequencies in peripheral blood could be used to predict clinical outcome after HBV infection. The frequencie of CD8+ T cells can reflect the changes of frequencies of HBcAg-specific CTL. during acute HBV infection.     

中国乙型肝炎患者外周血淋巴细胞亚群频率参考值范围

目的 调查中国乙型肝炎患者外周血淋巴细胞亚群频率参考值范围.方法 利用流式细胞术检测2846例乙型肝炎患者和117例健康人群外周血淋巴细胞亚群数值,调查我国健康人群和乙型肝炎人群的参考值范围.结果 调查了16～60岁健康人群和HBV感染相关的急性肝炎、慢性肝炎、重型肝炎和肝硬化人群外周血CD3+T淋巴细胞、CD3+CD...     

Chronic hepatitis B: The interplay between intrahepatic lymphocyte population and viral antigens in relation to liver damage

In Chronic hepatitis B (CHB) infection, virus and immune response interplay is thought to be responsible for pathogenesis. Yet, the impact of each immune cell population and viral protein expression in liver damage is still unknown. Our aim was to study the interplay between intrahepatic immune response and viral activity in relation to CHB liver damage. Immunostaining was performed in 29 liver biopsies from untreated CHB patients to characterize liver infiltrate [Th (CD4+), CTL (CD8+), Treg (FoxP3+), Th17 (IL‐17A+) and Th1 (T‐bet+)] and viral antigen expression (HBsAg and HBcAg). Inflammatory activity and fibrosis were assessed using the HAI and METAVIR scoring system. All studied populations were identified in the portal‐periportal (P‐P) areas with a CD4+ lymphocyte predominance, while only CD8+ and FoxP3+ cells were observed in the intralobular area. Both P‐P CD4+ and intralobular CD8+ cell frequencies were increased among severe hepatitis cases. Concerning HBsAg and HBcAg expression, a mutually exclusive pattern was observed. HBcAg was mainly detected among HBeAg‐positive patients and was associated with hepatitis severity and higher frequency of P‐P FoxP3+, intralobular CD8+ and FoxP3+ cells. HBsAg was identified among HBeAg‐negative cases with less severe hepatitis grade and lower frequency of P‐P CD4+ and intralobular FoxP3+ lymphocytes. In conclusion, the HBV antigen profile expression seen during CHB infection may be reflecting different stages of viral replication which impacts the host immune response and liver damage process. While HBcAg might be an inducer of a regulatory microenvironment, the intralobular CTL population seemed to have a key role in hepatitis severity.     

Inhibitory effects of hepatitis B virus antigen on induction of lymphokine-activated killer cell activity

We investigated the inhibitory effects of purified recombinant hepatitis B virus (HBV) surface antigen (rHBsAg) and core antigen (rHBcAg) on lymphokine-activated killer cell (LAK) activity. Either peripheral blood mononuclear cells (PBMCs) or CD16+ CD3- LAK precursors, both of which were pre-incubated with interleukin-2 (IL-2) and rHBsAg or rHBcAg for 72 h, showed a significant decrease in LAK cytotoxicity against Daudi cells, in comparison to the results recorded in the presence of IL-2 alone, or IL-2 and E. coli extracts. This inhibitory effect was dose-dependent and was observed to be time-dependent from 24 to 72-h-cultures with these HBV antigens. This influence was not mediated with either adherent cells or other accessory cells. The proliferative reaction of either PBMCs or the LAK precursors after being cultured with IL-2 and rHBsAg or rHBcAg for 72 h was significantly diminished compared with the levels of reaction of those cells after a 72-h culture with IL-2 alone or with IL-2 and E. coli extracts. The levels of IL-2-driven IL-2 receptor (p55) expression of either PBMCs or the LAK precursors in the presence of rHBsAg or rHBcAg were higher than the levels seen in the absence of these HBV antigens. These results suggest that HBsAg and HBcAg may inhibit the induction of LAK activity by interfering with the proliferative reaction of the LAK precursors to IL-2 without inhibiting the IL-2 receptor expression of the cells. Cytofluorographic analysis of PBMCs, cultured with rIL-2, showed lower percentages of CD3+ and CD16+ cells in the presence of these HBV antigens than those in the absence of antigens.     

Biologic and prognostic significance of hepatocyte hepatitis B core antigen expressions in the natural course of chronic hepatitis B virus infection

To elucidate the biologic significance of hepatocyte hepatitis B core antigen (HBcAg) expression and its relation to the natural course of hepatitis B virus (HBV) infection, the patterns of HBcAg were correlated with HBV virus replication state and the disease activity in 598 needle liver biopsies performed on 569 hepatitis B surface antigen (HBsAg) carriers aged 1-81 years. A good correlation of liver HBcAg with serum HBeAg and HBV DNA status was demonstrated. HBcAg was present in the hepatocyte nuclei (nHBcAg) or cytoplasm (cHBcAg), or in both (mixed). Pure nHBcAg was seen mainly in children and young adults; 86% of the patients had non-aggressive disease, but rare cases of chronic active hepatitis (CAH) and HBeAg seroconversion were observed. In contrast, cHBcAg was predominantly associated with CAH (52%) and accompanied by a significantly higher HBeAg seroconversion rate (27%). The HBeAg-negative group, particularly the liver HBcAg-negative subgroup, had a lower frequency of CAH, but an increased incidence of non-aggressive disease as well as cirrhosis and/or hepatocellular carcinoma, indicating that HBeAg seroconversion to anti-HBe does not necessarily mean a favorable prognosis. The results suggest that expression of HBcAg correlates with the liver pathology and the three phases of chronic HBV infection: (1) the early immune tolerance phase is characterized by nHBcAg, mild disease and low HBeAg seroconversion rate; (2) the virus replication/elimination phase by cHBcAg or negative HBcAg, frequent CAH, and high HBeAg seroconversion rate; and (3) the inactive virus replication phase by negative HBcAg and a bipolar disease spectrum.     

Immunoregulatory activities of human immunodeficiency virus (HIV) proteins: effect of HIV recombinant and synthetic peptides on immunoglobulin synthesis and proliferative responses by normal lymphocytes.

Recombinant and synthetic peptides corresponding to envelope proteins of the human immunodeficiency virus (HIV) were examined for their effects on the activities of lymphocytes from normal donors in vitro. Although lymphocytes cultured with env-gag peptides produced significant amounts of IgG, addition of env-gag peptides to a pokeweed mitogen-induced B-cell activation system resulted in suppression of immunoglobulin synthesis by normal lymphocytes. Recombinant antigens, env-gag and env-80 dihydrofolate reductase (DHFR), produced a substantial proliferative response by peripheral blood mononuclear cells (PBMC) as determined by [3H]thymidine incorporation. PBMC precultured with HIV synthetic peptide env 578-608 also manifested significant proliferative responses as compared to control cultures. CD3+ lymphocytes precultured with recombinant HIV antigens, env-gag and env-80 DHFR, and synthetic HIV peptide, env 487-511, showed moderate but significant proliferative responses. Both recombinant antigens and synthetic peptides also produced a dose-dependent stimulatory effect on proliferation by CD3- lymphocytes. Stimulation of CD3+ and CD3- lymphocyte subpopulations induced by env-gag peptides was specifically inhibited by goat anti-env-gag polyclonal antibodies, demonstrating the specificity of the reaction. These studies demonstrate that recombinant and synthetic peptides of the HIV genome express immunoregulatory T- and B-cell epitopes. Identification of unique HIV epitopes with immunogenic and immunoregulatory activities is necessary for the development of an effective vaccine against HIV infection.     

Proliferative responses of human intraepithelial lymphocytes to various T-cell stimuli

Human intraepithelial lymphocytes (IEL) are CD8+ T cells located between intestinal epithelial cells, capable of only minimal proliferation to mitogens but brisk proliferation to mitogens combined with sheep red blood cells. This study examines this differential response of IEL. Both IEL and CD8+ T lymphocytes from the peripheral blood are predominantly CD2+, CD3+, CD4-, CD5+, CD8+, and express the alpha beta subunits of the T-cell receptor. Human IEL express the same densities of the CD2, CD3, and CD8 antigens but a lower density of the CD5 antigen than do peripheral blood CD8+ T cells. The proliferation of IEL is significantly less than that of peripheral blood CD8+ T lymphocytes in response to phytohemagglutinin, to concanavalin A, or to anti-CD3 antibody bound to Sepharose (p less than 0.05). Supplementing IEL with interleukin-1, interleukin-2, or autologous peripheral blood macrophages does not completely reconstitute the proliferative response of IEL to these stimuli. Rather, the low proliferation of IEL to these stimuli is due to incomplete activation, as demonstrated by the low percentage of CD25 (Tac)+ lymphocytes with concanavalin A or the low density of the CD25 antigen with phytohemagglutinin. Both IEL and peripheral blood CD8+ T lymphocytes proliferate minimally in response to alloantigens or to interleukin-2, but briskly in response to stimuli of the CD2 receptor such as the combination of anti-T11(2) and anti-T11(3) antibodies or mitogen and sheep red blood cells. The sheep red blood cells enhance the mitogen-induced response of IEL by augmenting events of activation, both interleukin-2 production and interleukin-2 receptor expression. Thus, IEL represent an unusual compartment of CD2+, CD3+ T lymphocytes that are activated more completely by stimuli of the CD2 receptor than by stimuli of the CD3 receptor or by T-cell mitogens.     

HBV特异性抗原对HBV携带者T淋巴细胞免疫功能的影响

目的 通过分析不同类型HBV携带者外周血单个核细胞(PBMCs)的细胞免疫功能,分析HBV抗原对其的影响,探索HBV慢性感染的机制并寻求可能的免疫治疗方法.方法 用不同的抗原和(或)细胞因子刺激培养的无症状HBV携带者PBMCs,酶联免疫吸附法检测细胞培养上清液中不同细胞因子的水平;流式细胞术检测PBMCs的细胞表型.对数据进行t检验分析和相关性分析.结果 HBsAg刺激无症状HBV携带者PBMCs后产生的干扰素(IFN)γ为(48.3±19.8)Pg/ml,较健康对照人群低[(196.2±104.3)Pg/ml(t=3.023,P＜0.05)].HBsAg和HBcAg刺激HBeAg阳性患者PBMCs分泌的IFN y水平分别为(50.4±51.6)Pg/ml和(63.2±36.9)pg/ml,明显低于HBeAg阴性组[(86.2±42.3)Pg/ml和(101.4±32.5)pg/ml],t值分别为2.468和3.184,P值均＜0.05;HBeAg阳性患者组分泌白细胞介素(IL)-12 P70明显低于HBeAg阴性组(P＜0.05);补偿外源性IL-12可明显促进HBV携带者PBMCs分泌IFN γ(P＜0.01),IL-12协同HBV抗原可激活CD8+CD45RA+CCR7及CD8+CD45RA CD62L+细胞.结论 HBeAg阳性患者PBMCs分泌IL-12减少,这可能是HBV携带者持续感染的重要原因;外源性IL-12可促进HBV携带者PBMCs中的中枢记忆性T淋巴细胞的免疫功能.

Abstract：

Objective To investigate the effect of HBV antigens and pathological mechanism of chronic HBV infection by analyzing the cellular immune function of peripheral blood mononuclear cells (PBMCs) from HBsAg carriers. Methods PBMCs were prepared from individuals with chronic asymptomatic HBV infection and cultured in the presence of different antigens and/or cytokines. The levels of cytokines in culture supernatants were detected by ELISA method. The phenotype of the cells was detected by FACS.Results The levels of IFN γ secreted by PBMCs from HBsAg carriers were (48.3 ± 19.8) pg/ml, significantly lower than that from healthy controls (t = 3.023, P ＜ 0.05＝; The IFN γ produced by PBMCs from HBeAg positive patients due to HBsAg and HBcAg stimulation were (50.4±51.6) pg/ml and (63.2 ± 36.9)pg/ml, significantly lower than that of HBeAg negative patients (t = 2.468 and 3.184, P ＜ 0.05, respectively＝.The IL-12p70 secreted by PBMCs from HBeAg positive patients was also significantly lower than that of HBeAg negative patients (P ＜ 0.05＝; Exogenous IL-12 promoted significantly PBMCs to secrete IFN γ (P ＜0.01＝ and IL-12 combined with HBV antigens activated CD8+CD45RA+CCR7+ and CD8+CD45RA-CD62L+cells. Conclusion IL-12 secreted by PBMCs decreased in HBeAg positive patients, which may be the crucial reason of viral persistence in chronic HBV carriers. Exogenous IL-12 combined with specific HBV antigen could promote the central memory CD8+ T cells to produce IFN γ.     

Resolution of chronic hepatitis B and anti-HBs seroconversion in humans by adoptive transfer of immunity to hepatitis B core antigen

BACKGROUND & AIMS: Impaired T-cell reactivity is believed to be the dominant cause of chronic hepatitis B virus (HBV) infection. We characterized HBV-specific T-cell responses in chronic hepatitis B surface antigen carriers who received bone marrow from HLA-identical donors with natural immunity to HBV and seroconverted to antibody to hepatitis B surface antigen. METHODS: T-cell reactivity to HBV antigens and peptides was assessed in a proliferation assay, the frequency of HBV core- and surface-specific T cells was quantified directly by ELISPOT assays, and T-cell subsets were analyzed by flow cytometry. RESULTS: CD4+ T-cell reactivity to HBV core was common in bone marrow donors and the corresponding recipients after hepatitis B surface antigen clearance, whereas none reacted to surface, pre-S1, or pre-S2 antigens. Furthermore, CD4+ T cells from donor/recipient pairs recognized similar epitopes on hepatitis B core antigen; using polymerase chain reaction for the Y chromosome, the recipients' CD4+ T lymphocytes were confirmed to be of donor origin. The frequency of core-specific CD4+ and CD8+ T cells was several-fold higher than those specific for surface antigen. CONCLUSIONS: This study provides the first evidence in humans that transfer of hepatitis B core antigen-reactive T cells is associated with resolution of chronic HBV infection. Therapeutic immunization with HBV core gene or protein deserves further investigation in patients with chronic hepatitis B.