调节性T细胞和辅助性T细胞17的平衡变化在慢性乙型肝炎病毒感染中的作用

目的 研究调节性T细胞(Treg)和辅助性T细胞17(Th17)的平衡变化在慢性HBV感染中的作用.方法 应用ELISA法和流式细胞术分别对34例慢性乙型肝炎(CHB)患者、20例HBV相关慢加急性肝功能衰竭(ACHBLF)患者和20例健康对照者外周血中Treg和Th17分化相关细胞因子及外周血Th17和Treg细胞水平进行检测.计数资料应用Fisher's确切概率法,计量资料应用单因素方差分析和Tukey's多重比较检验.结果 ACHBLF组Th17分化相关因子IL-1β为(3.97±2.85) pg/mL,IL-6为(12.75±8.87)pg/mL,IL-21为(360.0±335.7)pg/mL,比健康对照组的IL-1β[(1.87±0.94)pg/mL,q=4.559,P＜0.01)、IL-6[(5.28±o.72)pg/mL,q=7.309,P＜0.01)和IL-21[(46.68±20.17) pg/mL,q=6.946,P＜0.01)均明显上调.ACHBLF组外周血Th17细胞比例明显高于健康对照组(q=3.972,P＜0.05).与健康对照组和ACHBLF组相比,CHB组Treg细胞分化相关因子TGF-β明显升高(q=4.536、5.323,均P＜0.01),外周血Treg比例也明显升高.ACHBLF组Th17细胞效应因子IL-17A水平最高,ACHBLF患者外周血Th17细胞比例与血清TBil水平呈正相关(γ=0.74,P＜0.01).结论 慢性HBV感染中,宿主免疫存在Th17和Treg失衡,ACHBLF组以Th17细胞活动为主,CHB组以Treg细胞活动为主.     

Memory B cells contribute to rapid Bcl6 expression by memory follicular helper T cells

In primary humoral responses, B-cell lymphoma 6 (Bcl6) is a master regulator of follicular helper T (T_FH) cell differentiation; however, its activation mechanisms and role in memory responses remain unclear. Here we demonstrate that survival of CXCR5⁺ T_FH memory cells, and thus subsequent recall antibody response, require Bcl6 expression. Furthermore, we show that, upon rechallenge with soluble antigen Bcl6 in memory T_FH cells is rapidly induced in a dendritic cell-independent manner and that peptide:class II complexes (pMHC) on cognate memory B cells significantly contribute to this induction. Given the previous evidence that antigen-specific B cells residing in the follicles acquire antigens within minutes of injection, our results suggest that memory B cells present antigens to the cognate T_FH memory cells, thereby contributing to rapid Bcl6 reexpression and differentiation of the T_FH memory cells during humoral memory responses.The development of high-affinity B-cell memory is essential in most effective vaccines that are in use today. Because most protein antigens require T-cell help to induce B-cell responses, understanding the mechanisms by which memory T and B cells are generated and maintained, as well as how their swift activation is executed, is of fundamental importance for vaccine development.In primary immune responses, it is widely accepted that among several differentiated helper T-cell subsets follicular helper CD4 T cells (T_FH cells) are the major subset to deliver help to B cells (1). T_FH cells express CXC-chemokine receptor 5 (CXCR5), the chemokine receptor for the B-cell homing chemokine CXCL13. Surface expression of CXCR5 enables T_FH cells to migrate into B-cell follicles, where they provide help to B cells to form germinal centers. In addition, T_FH cells are needed for the crucial affinity-maturation process of B cells in germinal centers, whereby Ag-specific B cells undergo repeated rounds of somatic hypermutation and positive selection by T_FH cells to rapidly evolve high-affinity somatically mutated B-cell receptors. B-cell lymphoma 6 (Bcl6) has recently been identified as a T_FH lineage regulator (2–4); it is highly expressed by T_FH cells and is required for their development. According to the current view, during a primary response Bcl6 expression by T cells is induced by priming with dendritic cells (5–7) and ICOS is a key coreceptor molecule for induction of Bcl6 (5, 8). The initial Bcl6 induction and subsequent CXCR5 expression allow CD4 T cells to migrate toward the T–B border, where T_FH cells interact with antigen-specific B cells. According to this model, cognate B cells are not required for the induction of Bcl6 but support the expansion of T_FH cells (9).Although the importance of Bcl6 and its expression kinetics in naïve T-cell differentiation have been well elucidated, its role and activation mechanisms in T_FH memory cells still remain obscure. Hence, in this paper we first focus upon the roles of Bcl6, demonstrating its importance for maintenance of T_FH memory cells. Then, we show that Bcl6 in memory T_FH cells was rapidly induced upon rechallenge with soluble antigen and that this response was mainly mediated through antigen presentation by the cognate memory B cells. Given the good association between Bcl6 with IL-21 expression in differentiated memory T_FH cells, our results suggest that memory B cells are the primary antigen-presenting cells (APCs) to induce the rapid differentiation of memory T_FH cells toward effector cells, further accelerating memory B-cell responses during recall.     

Regulatory T cells contribute to the impaired immune response in patients with chronic hepatitis B virus infection

Chronic hepatitis B virus (HBV) infection is characterized by a weak immune response to HBV. Regulatory T cells (T(reg)) can suppress the function of effector T cells and may thus be key players in this impaired immune response. Changes in the functionality or number of T(reg) could explain the decreased antiviral response in chronic HBV patients. To investigate the role of T(reg) in chronic HBV infection, we compared the proportional frequency and functionality of T(reg) in peripheral blood of 50 chronic HBV patients, 23 healthy controls, and 9 individuals with a resolved HBV infection. A higher percentage of T(reg), defined as CD4, CD25, CD45RO, and cytotoxic T-lymphocyte-associated antigen 4-positive cells, was detected within the population of CD4(+) cells in peripheral blood of chronic HBV patients compared with healthy controls and individuals with a resolved HBV infection. Accordingly, chronic HBV patients displayed a higher FoxP3 messenger RNA level than healthy controls. Depletion of CD25(+) cells from peripheral blood mononuclear cells (PBMC) of chronic HBV patients resulted in an enhanced proliferation after stimulation with HBV core antigen. Reconstitution of these depleted PBMC with CD4(+)CD25(+) T(reg) resulted in a dose-dependent reduction of both HBV-specific proliferation and interferon gamma production. In conclusion, chronic HBV patients harbor an increased percentage of T(reg) in peripheral blood compared with controls. T(reg) have an immunosuppressive effect on HBV-specific T helper cells. The presence of HBV-specific T(reg) could contribute to an inadequate immune response against the virus, leading to chronic infection.     

阿德福韦对HBeAg阳性慢性乙型肝炎患者Th1和Th2相关细胞因子的影响

目的 观察HBeAg阳性慢性乙型肝炎患者阿德福韦治疗前后不同时相点血清IFN-γ、L-4的水平、Th1/Th2比值以探讨阿德福韦治疗对机体的免疫功能的影响.方法 采集30例阿德福韦治疗前、治疗16周、52周和132周患者血清,其中完全应答组14例,非完全应答组16例及健康对照组10例,ELISA检测IFN-γ和L-4的水平.结果 完全应答组各时相点IFN-γ水平显著高于部分应答组(P均＜0.05)及正常对照组(P＜0.05),部分应答组与健康对照组相比无差异(P＞0.05);完全应答组IL-4水平在治疗后逐渐下降,部分应答组变化不大;两组Th1/Th2比值均有所上升,但完全应答组升高显著.结论 阿德福韦治疗后慢性乙型肝炎患者细胞免疫应答有一定程度的恢复.     

HBV前C区突变株对干扰素治疗的应答反应

目的评价干扰素对ＨＢＶ前Ｃ区突变株的应答反应。方法对１０４例慢性乙型肝炎患者，应用克隆测序和快速检测ＨＢＶ前Ｃ区终止密码突变的方法，共检出２１例突变株，其中１６例曾应用干扰素治疗；野生株２１／２５例曾应用干扰素治疗。结果１６例ＨＢＶ前Ｃ区突变株对干扰素治疗有应答者６例；野生株２１例，对干扰素有应答者９例，二组相比差异无显著性。结论ＨＢＶ前Ｃ区突变株及野生株对干扰素均有部分应答，应答率分别为３７．５％和４２．９％，二组相比差异无显著性     

Natural course and response to interferon of chronic hepatitis B accompanied by antibody to hepatitis B e antigen

The course of chronic hepatitis B was studied in 30 patients who had antibody to hepatitis e antigen and hepatitis B virus DNA in the serum and hepatitis B core antigen in the liver. Over a 2-year period, no patient experienced a sustained spontaneous remission of disease, and follow-up liver histology revealed worsening of the disease in four patients. After 2 years of observation, 24 patients were allocated randomly to one of two groups: 12 patients served as untreated controls and 12 received recombinant human alpha-interferon-2a in a dose of 9 million units intramuscularly three times weekly for 16 weeks. Patients who remained viremic after 16 weeks received 3 million units three times weekly for an additional 8 weeks. Abnormal amino-transferases and serum hepatitis B virus DNA persisted without appreciable changes in all untreated patients. Hepatitis B virus DNA rapidly became undetectable and serum aminotransferases fell to normal in eight treated patients. After the end of treatment, hepatitis B virus DNA became detectable once again in seven patients, in six of whom a peak of aminotransferases (range: 256 to 850 units per liter) ensued; subsequently, hepatitis B virus DNA disappeared, and serum aminotransferases again fell to normal in two of the seven. Overall, hepatitis B virus DNA was no longer detectable in serum and liver histology improved in three treated patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impact of liver steatosis on response to pegylated interferon therapy in patients with chronic hepatitis B

AIM:To evaluate the impact of liver steatosis upon response to given therapy in chronic hepatitis B (CHB) patients. METHODS: 84 consecutive CHB patients treated with 48-wk PEGylated interferon (PEG-IFN) therapy were enrolled. Baseline characteristics and sustained viral response (SVR) to PEG-IFN therapy were evaluated. RESULTS:Mean body mass index (BMI) was 27.36 ± 4.4 kg/m 2 . Six (7.1%) had hypertension and three (3.5%) had diabetes mellitus. Steatosis was present in 22.6% (19/84) of liver biopsy samples....     

HBsAg负载的慢性乙肝患者DCs诱导特异性Th1细胞分化的作用

目的:研究HBsAg负载的慢性乙肝患者外周血树突状细胞(dendritic cells,DCs)对自身Th1细胞分化的诱导作用.方法:Fico11密度梯度离心和贴壁法分离慢性乙肝患者外周血单核细胞,以含ThIL-4 rhGMCSF的完全培养基诱导DCs.流式细胞术检测各组DC表面CD80,CD86,CD40和HLA-DR分子的表达,CCK-8法检测各组DCs刺激同种异体淋巴细胞增殖的能力,ELISA法检测各组DCs培养液上清中IL-12的水平.免疫磁珠分选慢性乙肝患者外周血CD4 T细胞,分别与患者自身的DCs共培养,细胞内细胞因子染色,流式细胞仪检测共培养后CD4 T细胞内特征性细胞因子IFN-γ和IL-4,ELISA法检测共培养上清中IFN-γ/IL-4的水平.结果:与对照组相比,HBsAg、IFN-γ和HBsAg IFN-γ组DCs表面表达CD80,CD86,CD40和HLA-DR分子水平较高;刺激同种异体淋巴细胞增殖的能力较强;DCs分泌的上清液中、IL-12水平也较高.与对照组相比,HBsAg、IFN-γ和HBsAg IFN-γ组DCs与自身Th细胞共培养后,Th1细胞占CD4 T细胞的百分比升高(10.76%±3.98%,11.43%±4.32%,15.28%±4.73% vs 7.84%±3.10%,P＜0.01),Th2细胞占CD4 T细胞的百分比降低(1.43%±0.96%.1.68%±0.16%,0.92%±0.21% vs 2.61%±1.27%,P＜0.01),共培养上清中IFN-γ的水平增高(578±47 mg/L,496±92 mg/L,784±97 mg/L vs 342±34 meg/L,P＜0.05),IL-4的水平降低(187±52 mg/L,169±38 mg/L,89±37 mg/L vs 226±48 mg/L,P＜0.05),以HBsAg IFN-γ组最为明显.结论:经HBsAg负载的DCs可以改善患者体内因DCs功能低下而引起的Th1细胞分化不足的状态.     

慢性乙肝患者血清一氧化氮的表达与α干扰素抗病毒应答的相关性研究

目的探讨一氧化氮（NO）在慢性乙肝疾病进展中的作用及NO与α-干扰素（IFN-α）疗效的关系。方法对采用IFN-α治疗的慢性乙肝患者40例,分别在治疗前、治疗12周和24周时检测血清NO浓度;治疗结束时,依据治疗效果分为应答组和无应答组,比较两组NO浓度变化。20例健康人做为对照组,对其血清NO浓度与慢性乙肝患者基线时血清NO浓度进行比较。结果慢性乙肝患者基线时血清NO水平显著高于健康对照组,其差异具有统计学意义（P〈0.01）;慢性乙肝患者血清ALT水平和NO浓度呈正相关（r=0.673,P〈0.001）;应答组患者血清NO浓度随治疗时间的延长而下降,治疗12周和24周时与治疗前比较差异均有统计学意义（P〈0.01）,无应答组患者血清NO浓度在治疗过程中无明显变化。结论NO在慢性乙肝发病机制中可能起到重要作用,可作为IFN-α抗病毒疗效判断指标。     

Coinfection of TT virus and response to interferon therapy in patients with chronic hepatitis B or C

AIM: To investigate the serum positive percentage of TT virus (TTV) in patients with chronic hepatitis B or C and the response of the coinfected TTV to interferon (IFN) during IFN therapy for chronic hepatitis B and C. METHODS: We retrospectively studied the serum samples of 70 patients with chronic hepatitis who had received IFN-alpha therapy from January 1997 to June 2000, which included 40 cases of hepatitis B and 30 hepatitis C. All the patients had been followed up for at least 6 months after the end of IFN therapy. The serum TTV DNA was detected using the polymerase chain reaction (PCR) before and every month during the course of IFN treatment. RESULTS: TTV infection was detected in 15% (6/40) of the chronic hepatitis B group and 30% (9/30) of the chronic hepatitis C group. Loss of serum TTV DNA during IFN therapy occurred in 3 of 6 patients (50%) and 6 of 9 (67%) of hepatitis B and C groups, respectively. Seronegativity of TTV was found all during the first month of IFN therapy in the 9 patients. There was no correlation between the seroconversion of TTV and the biochemical changes of the patients. CONCLUSION: TTV is not infrequently coinfected in patients with chronic hepatitis B and C in Taiwan, and more than half of the TTV infections are IFN-sensitive. However, the loss of serum TTV DNA does not affect the clinical course of the patients with chronic hepatitis B or C.     

Long-term response to interferon plus ribavirin in patients with chronic hepatitis C refractory to interferon.

OBJECTIVE: A sustained response (SR) to interferon (IFN) is only observed in 15-20% of patients with chronic hepatitis C (CHC). The aim of this study was to determine the long-term effectiveness and safety of the treatment with IFN plus ribavirin (RIB) over two years in CHC patients without SR to IFN. DESIGN: A prospective and open longitudinal follow-up study was conducted over 3 years. PATIENTS AND METHODS: A total of 77 CHC patients were included: 63 non-responders (NR) and 14 relapsers (R) to IFN. Patients were treated with IFN (3 MU s.c. three times a week) and RIB (1,000-1,200 mg p.o. daily) for 12 months. Treatment tolerance and viral response (HCV-RNA in serum < 1,000 copies/ml) were assessed after 1, 3, 6 and 12 months of treatment. SR and relapsing rates were subsequently evaluated 6, 12 and 24 months after the end of the treatment, together with those variables capable of predicting SR. RESULTS: At the end of the treatment, 19/77 patients responded (24.7%), 9/63 (14.3%) were non-responders and 10/14 (71.4%) relapsers, and these same patients exhibited SR after 6 months. The SR rate two years after treatment was 22.1% [8/63 (12.7%) NR and 9/14 (64.3%) R]. The relapse rate after 6 months and two years was respectively 0 and 10.5% (2/77). Independent variables capable of predicting SR were negative viremia conversion within the first month of treatment, maintenance of such negative viremia after 6 months, and R status to IFN. Side effects were recorded in 90.9% of cases (70/77), the most frequent being pseudoinfluenza syndrome. Treatment had to be discontinued in 33.8% of patients (26/77). CONCLUSIONS: Combined IFN-RIB therapy for 12 months in CHC patients without SR to IFN obtains a long-term SR of 22.1%, this rate being higher in relapsers to prior IFN therapy (64.3% in R versus 12.7% in NR).     

Rh autoantigen presentation to helper T cells in chronic lymphocytic leukemia by malignant B cells

Chronic lymphocytic leukemia (CLL) is frequently associated with autoimmune diseases directed against constituents of the blood, including hemolytic anemia (AIHA). We hypothesized that CLL cells predispose to hematologic autoimmunity by acting as aberrant antigen-presenting cells (APCs). Initially, it was confirmed that all studied patients with AIHA secondary to CLL harbored activated helper T (T(H)) cells specific for epitopes on the dominant red blood cell (RBC) autoantigens in primary AIHA, the Rh proteins. Rh-specific T(H) cells were also detected in a number of patients with CLL who, although they did not have AIHA, had low levels of anti-RBC antibody in their sera. Fractionation of putative APC populations from the peripheral blood of patients by negative selection showed that CD5+ CLL cells are the most effective cell type in processing and presenting purified Rh protein to autoreactive T(H) cells. This ability was confirmed using positively selected CD5+ CLL cells. Thus, our study provides the first evidence for malignant cells driving an autoimmune response by acting as aberrant APCs.     

Patients with chronic hepatitis C express a high percentage of CD4+CXCR5+ T follicular helper cells

Background

T follicular helper (T_FH) cells are a subpopulation of T-helper cells which regulate humoral immune responses. The role of T_FH cells in viral infection is unclear. This study examined the possible involvement of CD4⁺CXCR5⁺ T_FH cells in chronic hepatitis C (HCV) infection.

Methods

The percentages of peripheral blood CD4⁺CXCR5⁺ T_FH cells, inducible T-cell costimulator cells, and/or programmed death 1-positive CD4⁺CXCR5⁺ T_FH cells in 39 HCV-infected patients, 12 patients with spontaneously resolved HCV infection (SR-HCV), and 12 healthy controls were characterized by flow cytometry analysis. The subjects’ serum HCV RNA loads and alanine aminotransferase and aspartate aminotransferase levels were measured. The potential association of the percentage of peripheral CD4⁺CXCR5⁺ T_FH cells with clinical data was analyzed.

Results

Higher percentages of peripheral blood CD4⁺CXCR5⁺ T_FH cells were found in SR-HCV and HCV-infected patients as compared with healthy controls. Interestingly, a statistically significant negative correlation was found between the percentage of CD4⁺CXCR5⁺ T_FH cells and the HCV RNA load.

Conclusions

These data suggest that CD4⁺CXCR5⁺ T_FH cells may participate in HCV-related immune responses. Increased T_FH cells in peripheral blood may help to control HCV infection.     

Activated circulating T follicular helper cells and skewing of T follicular helper 2 cells are down-regulated by treatment including an inhaled corticosteroid in patients with allergic asthma

BackgroundCXCR5⁺ T follicular helper (T_FH) cells primarily promote B cells to produce an antigen-specific antibody through germinal centers (GCs). T_FH cells exist in circulation, and circulating(c) T_FH2 cells, a subset of cT_FH cells, are able to help naïve B cells produce IgE in healthy individuals. Conversely, IL-10-producing regulatory B (Breg) cells inhibit an accelerated immune response.MethodsWe investigated the roles of cT_FH cells and cBreg cells based on a T_H2 response in patients with atopic asthma (AA). Thirty-two patients with AA and 35 healthy volunteers (HV) were enrolled. We examined cT_FH cells including their subsets, their expression of ICOS and PD-1, and cBreg cells by flow cytometry and their associations with clinical biomarkers. Plasma levels of CXCL13, which is a counterpart of CXCR5, were also measured using ELISA.ResultsIn patients with AA, cT_FH2 cells were increased and cT_FH1 cells were decreased compared with those in HV. The expression levels of ICOS on cT_FH and their subset cells were elevated and Breg cells were greatly decreased. The plasma levels of CXCL13 in patients with AA were significantly elevated and correlated well with the cT_FH2/cBreg ratio. These cells were examined in 10 patients AA before and after inhaled corticosteroid (ICS) treatment. Interestingly, the percentages and numbers of T_FH2 and ICOS⁺ cT_FH cells declined after ICS treatment together with improvements in symptoms and clinical biomarkers.ConclusionsThe percentages and numbers of cT_FH2 and ICOS⁺ cT_FH cells might be useful as biomarkers of T_H2 typed airway inflammation in patients with AA.     

Autoantibodies related to interferon therapy in chronic hepatitis B may affect the therapeutic response to interferon]

We have evaluated the problem of autoantibodies such as antinuclear antibodies (ANA), smooth muscle antibody (SMA) and antibodies to thyroid microsomal (TMA) and to thyroglobulin (TGA) related to interferon therapy in 27 patients with chronic hepatitis B. Anti-interferon antibody was also studied by Western blot method. Eight patients had ANA and 2 had SMA during interferon therapy. However, 6 of the 8 patients were ANA positive and one of the 2 was SMA positive prior to interferon treatment. No patients developed TMA, TGA or anti-interferon antibody. Eight (29.6%) of the 27 patients had clearance of both DNA polymerase and HBeAg and persistent normalization in alanine aminotransferase levels with interferon therapy. Seven of the 8 responders developed none of the autoantibodies related to interferon therapy. These results suggest that the presence of ANA or SMA during treatment may affect the therapeutic response to interferon.     

The role of follicular T helper cells in patients with malignant lymphoid disease

Objectives: To investigate the dynamic change of follicular T helper cells (T_FH) in patients with malignant lymphoid disease (MLD) and to explore its clinical significance.

Methods: The dynamic change of T_FH cells, ICOS⁺- and PD-1⁺ T_FH cells at pretreatment and different treatment periods was determined by flow cytometry in 85 MLD patients. Concentration of interleukin 21 (IL-21) was evaluated by ELISA, and the correlation between clinical prognosis and the ratio of T_FH cells was analyzed.

Results: Significantly increased ICOS⁺- and PD-1⁺ T_FH cells were found in MLD patients at pretreatment compared to healthy controls. Decreased or even close to normal levels of ICOS⁺- and PD-1⁺ T_FH cells were found at the end of treatment. However, in the patients with progressive disease, high levels of ICOS⁺- and PD-1⁺ T_FH cells were found. Moreover, a significantly increased plasma IL-21 level was found in MLD patients. Negative correlation was found between the level of ICOS⁺-, PD-1⁺ T_FH cells, as well as IL-21 and the prognosis of MLD.

Conclusions: Significantly increased T_FH cell ratios were found in patients with MLD, and decreased T_FH cells ratios could be expected in those treatment-effective patients, which could be used as the therapeutic efficacy index.     

Multicenter retrospective study of response to interferon in chronic hepatitis B.

AIM: The only agent known to have a lasting beneficial effect in chronic hepatitis B is interferon alpha, which achieves long-term remission in 25-40% of the patients. The goals of treatment are to induce clearance of HBV DNA from serum, to return serum aminotransferases to normal, and to improve histological findings in the liver. The most important factors predictive of response to treatment are high serum aminotranferases levels, low serum HBV DNA concentrations, and active histologic changes on liver biopsy. The aim of this study was to assess the response to interferon alpha in patients with chronic hepatitis B and to analyze the factors predictive of response. METHODS: We conducted a multicenter retrospective study to investigate the effect of interferon treatment in 132 patients with chronic hepatitis B in overall terms and based on HBeAg, and factors predictive of response. RESULTS: A overall sustained response was noted in 59 of 132 interferon-treated patients (45%) and 61 patients were nonresponders (46%). 12 patients relapsed (9%). None of the patients had negative HBsAg. No difference was observed in the response rate between the two treatment groups (HBeAg-positive and HBeAg-negative patients). Overall, high initial levels of serum AST and ALT predicted a good response. Age and liver biopsy findings were factors predictive of response in HBeAg-positive and HBeAg-negative patients respectively. Sex, epidemiological factors, treatment and type of virus did not correlate with the response to interferon. CONCLUSIONS: A sustained response to interferon alpha was observed in 45% of the patients with chronic hepatitis B. HBeAg seroconversion was found in up to 50% of HBeAg-positive patients. None of the patients had negative HBsAg. Overall, the response rate was higher in patients with high pretreatment serum aminotransferase levels. Age was the predictive factor in HBeAg-positive patients, and histological features were predictive in the HBeAg-negative group. However, further studies in a larger patient population are necessary to obtain well-substantiated conclusions.