PD-1表达对急性乙型肝炎患者HBV特异性CD8＋T细胞功能的影响

目的 观察急性乙型肝炎发病过程中患者体内病毒特异性CD8^＋细胞（CTL）上免疫抑制性分子程序性细胞死亡受体1（PD-1）的表达及临床意义。方法合成4种识别HBV不同抗原表位的五聚体，同时采集16例HLA-A2阳性急性乙型肝炎患者外周血，用五聚体染色后，通过流式细胞仪分析病毒特异性CTL细胞的频率及其表面PD-1分子的表达特点。同时进行肝功能、乙型肝炎病毒（HBV）血清标志物和血清HBVDNA检测。结果所有急性乙型肝炎患者发病早期均表现出多特异性的CTL反应，且PD-1在这些细胞上表达明显上调，显著高于来自同一患者的巨细胞病毒（CMV）和Flu特异性CTL上PD-1分子的表达水平。体外阻断PD-1／PD-L1间的相互作用能够恢复HBV特异性CTL分泌细胞因子的能力。相关性分析显示PD-1分子的上调表达与转氨酶水平正相关。结论在HBV急性感染过程中，尤其是在HBV基本被清除后，HBV特异性CTL细胞PD-1分子表达明显升高，有效抑制病毒特异性CTL分泌IFN-γ的能力，后者可能有利于避免急性乙型肝炎患者肝脏遭受更为严重的免疫损伤。     

急性乙型肝炎患者程序性细胞死亡受体1表达与记忆性T淋巴细胞形成的关系

目的 观察急性自限性乙型肝炎发病过程中患者体内病毒抗原特异性细胞毒性T淋巴细胞(CTL)上程序性细胞死亡受体1(PD-1)表达的动态变化特点及其与记忆性抗原特异性CD8+T淋巴细胞形成的关系. 方法 针对不同表位合成4种五聚体,长期随访收集11例人类白细胞抗原(HLA)-A2阳性的急性乙型肝炎患者的外周血,流式细胞仪检测病毒特异性CTL上免疫抑制性分子PD-1、记忆性分子(CCR7、CD45RA、CD127)和活化标志物CD38的表达情况,并分析其相关性.同时进行肝功能,HBsAg、抗-HBs和血清HBV DNA载量检测.结果所有急性自限性乙型肝炎患者发病早期均表现出高频度、病毒抗原多表位的特异性CTL反应,而晚期各表位CTL频率均明显下降.CTL上PD-1表达在早期明显上调;与早期比较,晚期PD-1分子的表达明显降低(t=4.314,P＜0.01).同时CTL高表达记忆性分子CCR7,CD45RA和CD127,而低表达活化标志物CD38.提示病毒清除后记忆性CD8+T淋巴细胞形成. 结论 在急性自限性乙型肝炎发病过程中,HBV特异性CTL上PD-1分子表达的动态变化与记忆性T淋巴细胞的形成密切相关.     

乙型肝炎相关性肝功能衰竭患者外周血PD-1表达的临床意义

目的 观察乙型肝炎相关性肝功能衰竭患者细胞程序性死亡因子-1 (PD-1)表达特点及临床意义.方法 针对不同表位合成3种pentamer,采集16例急性和慢加急性肝功能衰竭患者外周血,并收集15例急性乙型肝炎(AHB)患者外周血作为对照,流式细胞仪分析各组病毒特异性CTL细胞的频率及其表面PD-1分子表达水平,同时观察与肝功能和血清HBV DNA载量的相关性.组间比较采用Mann-whitneyU检验.结果 与AHB患者比较,肝功能衰竭患者发病早期病毒特异性CD8 T淋巴细胞PD-1表达水平显著降低,该阶段PD-1表达水平与转氨酶呈显著正相关,但与病毒载量无明显相关性.随访发现,AHB患者早期,PD-1高水平表达,随着疾病康复,PD-1表达逐渐降低；而肝功能衰竭患者早期PD-1表达较低,随后显著增加,而后随着疾病恢复,PD-1进一步降低.结论 肝功能衰竭患者发病早期外周血PD-1表达水平较AHB患者显著降低,而PD-1延迟高表达则与急性肝功能衰竭的发生密切相关.     

慢性乙型肝炎患者PD-1/PD-L1信号通路的免疫调节作用研究进展

程序性死亡分子-1（PD-1）是T淋巴细胞膜表面表达的负向协同刺激分子, 与其主要配体（PD-L1）形成通路后,可以减弱T淋巴细胞的免疫反应,甚至导致T淋巴细胞功能衰竭。PD-1/PD-L1信号通路在乙型肝炎病毒感染后的效应T细胞免疫耐受中具有重要作用,阻断该途径可能是抗病毒治疗的方向之一,具有良好的应用前景。     

PD-1/PD-L1信号通路与慢性乙型肝炎

目的：程序性死亡分子-1（programmed death-1,PD-1）是近年来发现的属于B7/CD28家族的重要协同刺激分子,与其配体（programmed death -1 ligand,PD-L）结合后在调节T淋巴细胞的活化、分化及增殖功能方面起着重要作用。在慢性HBV感染不同阶段,PD-1表达水平存在差异,且与肝脏炎症程度、ALT及病毒载量等密切相关。通过不同途径阻断PD-1/PD-L1通路可以使耗竭的T淋巴细胞功能得到改善,提示可能是未来抗病毒治疗的方向之一。     

PD-1/PD-L信号通路在慢性HBV感染免疫反应中的作用

慢性乙型肝炎是影响我国人群健康的主要疾病之一,HBV的慢性感染可导致疾病的进展。程序性死亡因子(PD)1是近年来发现的属于B7/CD28家族的重要协同刺激分子,与其配体(PD-L)结合后在调节T淋巴细胞的活化、分化及增殖功能方面起着重要作用。在慢性HBV感染的不同阶段,PD-1及其配体的表达存在差异。PD-1及其配体在不同的免疫细胞上的相互作用引起机体的免疫耐受,最终导致HBV感染的慢性化。通过不同途径阻断PD-1/PD-L通路可以使耗竭的T淋巴细胞功能得到改善,提示可能是未来抗病毒治疗的方向之一。     

程序性死亡受体-1在慢性病毒性肝炎中的作用

程序性死亡受体-1（PD-1）是用消减杂交技术从发生程序性死亡的T细胞株中分离得到的一种跨膜蛋白,是一种负向协同刺激分子。PD-1是活化T细胞表面的一种抑制性受体,与病毒特异性CD8^＋T细胞的功能耗竭有着密切关系。在乙型肝炎病毒和丙型肝炎病毒慢性感染过程中PD-1表达增强,阻断PD-1信号通路可以使耗竭的CD8^＋T细胞恢复免疫效应功能,有助于病毒清除,本文就PD-1与病毒性肝炎的关系及其在治疗中应用的前景作一综述。     

PD-1/PD-L1途径在慢性病毒感染中的作用

程序性死亡因子配体-1(PD-L1)分子是B7超家族的重要成员,在单核细胞、树突状细胞及大部分外周组织可以广泛诱导性表达。PD-L1可以和程序性死亡-1(PD-1)及CD80受体结合调节淋巴细胞的功能。PD-1/PD-L1信号在病毒感染后效应T细胞免疫耐受中具有重要作用,并且预示阻断该途径可能是抗病毒感染治疗的方向之一。     

慢性乙型肝炎患者抗病毒治疗过程中程序性死亡分子1及其配体表达的变化

目的 探讨慢性乙型肝炎患者在抗病毒治疗过程中程序性死亡分子1(PD-1)及其主要配体PD-L1表达的变化情况及其与HBeAg/抗-Hbe血清学转换的关系.方法 对10例人类白细胞抗原(HLA)-A2阳性的HBeAg阳性慢性乙型肝炎患者予以替比夫定抗病毒治疗24 周,分别于治疗的0、12周和24周随访,检测HBV DNA水平、外周血单个核细胞(PBMC)表面PD-1和PD-L1表达水平、HBV特异性T淋巴细胞数量与其表达PD 1水平,及PBMC体外培养上清液中干扰素(IFN)γ水平.其中HBV DNA定量采用荧光定量PCR法检测;PBMC自新鲜血分离,部分PBMC加入重组HBcAg体外培养7d,加入HBV抗原表位肽五聚体复合体;PD-1和PD-L1阳性细胞、CD8'T淋巴细胞以及CD8和PD-1双阳性细胞用流式细胞仪检测;IFN γ水平用酶联免疫吸附试验检测.比较不同时间点PBMC表面PD-1和PD L1表达水平、HBV特异性CD8'T淋巴细胞数量及其PD-1表达水平、PBMC培养上清液中IFN γ水平.对抗病毒治疗前后对应指标数值的变化采用配对t检验分析.结果 治疗前HBV DNA水平为5.16～8.77 log10拷贝/ml,治疗24周时,7例HBV DNA水平低于检测下限,3例仍可检测到,但明显低于基线水平.2例HBeAg/抗-Hbe血清学转换,2例HBeAg阴转,6例仍维持HBeAg阳性.0、12周和24周PBMC表面PD 1表达水平分别为52.1%±17.0%、39.1%±18.2%和23.4%±16.3%(24周和0周比较,P＜0.01);PD-L1分别为45.6%±15.4%、34.6%±16.2%和20.9%±9.5%(24周和0周,P＜0.01;24周和12周比较P＜0.05);HBV特异性T淋巴细胞上PD-1表达水平分别为76.2%±10.4%、66.5%±15.4%和49.5%±25.3%(24周和0周比较,P＜0.01;12周和0周比较,P＜0.05;24周和12周比较,P＜0.05);HBV特异性T淋巴细胞分别为1.3%±0.5%、1.5%±1.0%和2.2%±1.5%; IFN γ水平(pg/ml)分别为91.7±82.1、99.4+93.5和109.0+86.6.24周HBeAg/抗-Hbe血清学转换者上述指标变化明显大于无HBeAg/抗-Hbe血清学转换者.结论 直接抑制HBV复制能降低PD-1、PD-L1表达水平,并增加HBV特异性CD8'T淋巴细胞的数量和功能.早期PD-1快速下降和HBV特异性T淋巴细胞数量及功能的恢复与早期HBeAg/抗-Hbe血清学转换的关系有待于进一步研究.     

慢性乙型肝炎病毒感染者外周血单个核细胞表达程序性死亡分子-1及其配体与血清乙型肝炎病毒DNA水平的相关性

目的 研究程序性死亡分子-1(PD-1)及其配体(PD-L1)表达水平与慢性HBV感染者HBV DNA水平的相关性及抗病毒治疗对其表达的影响.方法 检测137例慢性HBV感染者的外周血单个核细胞(PBMC)表面PD-1和PD-L1,并检测其中64例人类白细胞抗原(HLA)-A2阳性者HBV特异性CTL数量.ELlSA法检测PBMC体外培养上清液中IFN-γ浓度.比较10例HBeAg阳性慢性乙型肝炎(CHB)患者予替比夫定抗病毒治疗24周前后上述指标的变化.两组间均数比较采用两独立样本的t检验,多组间的差异采用单因素方差分析,相关分析采用Pearson相关分析.结果 HBV DNA＜3 lg、3～6 lg和＞6 lg拷贝/mL组问PBMC表面PD-1和PD-L1表达均明显高于健康对照组,但差异无统计学意义;3组HBV特异性CTL表面PD-1表达分别为(69.3±11.2)%、(76.5±9.1)%和(78.0±11.7)%,HBV DNA＞6 lg拷贝/mL 组PD-1表达明显高于＜3 lg拷贝/mL组,而HBV特异性CTL数量明显低于＜3 lg拷贝/mL组;3组PBMC体外培养上清液中IFN-γ水平差异无统计学意义.HBeAg阳性组和阴性组间上述指标差异无统计学意义.替比夫定抗病毒治疗12周和24周时,PD-1、PD-L1表达较治疗前明显下降,伴有HBV特异性CTL数量逐渐增加和IFN-γ水平升高.结论 慢性HBV感染者PBMC表面PD-1的表达较健康者明显上调,且HBV特异性CTL表面表达PD-1水平与血清HBV DNA水平相关,但与HBeAg状态无关.抑制HBV复制能降低PD-1、PD-L1表达,并增加HBV特异性CTL的数量和功能.

Abstract：

Objective To study the relationship between programmed death-1 (PD-1)/programmed death-1 ligand (PD-L1) expressions and serum hepatitis B virus (HBV) DNA levels in chronic hepatitis B (CHB) patients. Methods A total of 137 CHB patients and 10 healthy controls were enrolled in the study. The peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood samples. HBV-specific cytotoxic T lymphocyte (CTL) was expanded in vitro in 64 human leucocyte antigen (HLA)-A2 positive patients. Flow cytometry was used to detect HLA-A2 type,expressions of PD-1/PD-L1 on PBMCs and PD-1 on HBV specific CTL. Interferon gamma (IFN-γ)was measured by commercial enzyme-linked immunosorbent assay (ELISA) kits. PD-1/PD-L1expressions on PBMCs, HBV-specific CTL and IFN-γ level in PBMC culture medium were compared among patients with different baseline HBV DNA levels. Ten hepatitis B e antigen (HBeAg) positive patients were treated with telbivudine for 24 weeks. The above mentioned parameters were determined and compared before and after the antiviral treatment. Independent-samples t test were used to compare means between two groups and one-way A NOVA were used to compare means among multigroups. We used the pearson corretation test to assess corretation significance. Results The PD-1 and PD-L1 expressions on PBMCs in patients with baseline HBV DNA＜3 lg copy/mL, 3-6 lg copy/mL and ＞6 lg copy/mL were all significant higher than those in healthy control group, but no statistical differences were found. PD-1 expressions on HBV-specific CTL in the three CHB patient groups were (69.3±11.2)%, (76.5±9. 1)% and (78.0±11.7)%, respectively. However, PD-1 expression on HBV-specific CTL was higher, while the frequency of HBV-specific CTL cells was lower in HBV DNA ＞6 lg copy/mL group compared to HBV DNA＜3 lg copy/mL group. The above parameters, including expressions of PD-1 and PD-L1, the frequency of HBV-specific CTL and its PD-1 expression were not significantly different between HBeAg-positive group and HBeAg-negative group. Compared with baseline, PD-1 and PD-L1 expression decreased obviously accompanying with increase of HBV-specific CTL cells frequency and IFN-γ level after 12 weeks and 24 weeks of telbivudine treatment. Conclusions PD-1 expression on HBV-specific CTL correlates with serum HBV DNA level, but not HBeAg status in CHB patients. Suppression of HBV replication can reduce PD-1/PD-L1 expressions and partially restore HBV specific CTL function.     

慢性乙型肝炎患者抗病毒治疗早期T细胞表面PD-1的表达

背景：慢性乙型肝炎病毒（HBV）感染者T细胞表面程序性死亡受体1（PD-1）呈持续性高表达。然而关于抗病毒治疗前后慢性乙型肝炎（CHB）患者T细胞表面PD-1表达变化及其与病毒载量关系的报道较少。目的：动态观察CHB患者抗病毒治疗早期外周血CD4＋和CD8＋T细胞表面PD-1表达水平,探讨其表达与血清HBV DNA载量和丙氨酸氨基转移酶（ALT）水平之间的关系。方法：以流式细胞术分别检测31例CHB患者抗病毒治疗前或基线期（T1）、治疗后4～8周（他）和12．16周（T3）的外周血CD4^＋和CD8^＋T细胞表面PD-1表达水平,以实时荧光定量聚合酶链反应（PCR）检测血清HBV DNA载量,同时检测血清ALT水平。结果：抗病毒治疗早期,CHB患者外周血CD4^＋和CD8^＋T细胞表面PD．1表达水平逐渐下调（P〈0．05）,血清HBV DNA载量和ALT水平亦逐步降低（P〈0．01）;CD4^＋和CD8^＋T细胞表面PD-1表达与HBV DNA载量（P〈0．01）和ALT水平（P〈0．05）均呈正相关。结论：有效的抗病毒治疗可通过降低CHB患者的病毒载量使T细胞表面PD-1表达下调,T细胞表面PD-1表达水平与患者疾病状态密切相关。     

In vitro priming and expansion of cytomegalovirus-specific Th1 and Tc1 T cells from naive cord blood lymphocytes

Adoptive transfer of CMV-specific cytotoxic T cells (CTLs) expanded in vitro from memory donor T cells can reduce the incidence of CMV disease in allogeneic transplant recipients. However, this approach has been unavailable in the cord blood (CB) transplantation setting because CB T cells are antigen naive and biased toward Th2/Tc2 function. We developed a protocol to in vitro prime and expand CMV-specific CTLs from CB. T cells were primed with cytokines to trigger skewing toward Th1/Tc1 lineage before encountering monocyte and CD34+ progenitor-derived dendritic cells loaded with CMV antigen and its immune complex. CMV-pulsed cultures expanded significantly more over 4 to 6 weeks than CMV cultures despite identical cytokine milieu. T cells isolated from CMV+ cultures showed a preferential expansion of CD45RA-/RO+/CD27+ T cells compared to CMV- cultures. CMV-specific IFN-gamma- and TNF-alpha-producing CD4+ (Th1) and CD8+ (Tc1) T cells were enriched after 3 to 4 weeks and CMV-specific cytotoxicity developed 1 to 2 weeks later.     

Human virus-specific effector-type T cells accumulate in blood but not in lymph nodes

It is believed that the size of the CD8(+) T-cell pool is fixed and that with every new viral challenge, the size of the pre-existing memory-cell population shrinks to make way for the new virus-specific cells. CMV-seropositive individuals have high numbers of CMV-specific resting-effector type CD8(+) T cells in their peripheral blood (PB). This prompted us to investigate whether CMV infection limits immunologic space at sites where immune reactions are initiated, such as in the lymph nodes (LNs). LN and paired PB samples were analyzed for CMV-, EBV-, and influenza-specific CD8(+) T cells. In marked contrast to blood, LNs contained significantly lower numbers of CX3CR1-expressing effector-type CD8(+) T cells, whereas the CMV-specific cells that were found in the LNs resembled polyfunctional memory-type cells. In contrast, EBV- and influenza-specific CD8(+) T cells were highly similar between PB and LNs both in number and function. Therefore, it is unlikely that CMV-specific CD8(+) T cells in the LNs restrain the immunologic space of other virus-specific cells.     

Programmed death-1 expression is associated with the disease status in hepatitis B virus infection

AIM： TO define the potential role of programmed death-i/programmed death-ligand （PD-1/PD-L） pathway in different hepatitis B virus （HBV） infection disease status; we examined the expression of PD-1 on antigen specific CD8＋T cells in peripheral blood of patients with chronic hepatitis B （CriB） and acute exacerbation of hepatitis B （AEHB） infection.
METHODS： The PD-1 level on CD8＋ T lymphocytes and the number of HBV specific CD8＋ T lymphocytes in patients and healthy controls （HCs） were analyzed by staining with pentameric peptide-human leukocyte antigen2 （HLA2） complexes combined with flow cytometry. Real-time quantitative polymerase chain reaction （PCR） was used to measure the serum HBV- DNA levels.
RESULTS： The level of PD-1 expression on total CD8＋ T cells in CHB patients （13.86% ± 3.38%） was significantly higher than that in AEHB patients （6.80%± 2.19%, P 〈 0.01） and healthy individuals （4.63% ± 1.23%, P 〈 0.01）. Compared to AEHB patients （0.81% ± 0.73%）, lower frequency of HBV-specific CD8＋ T cells was detected in chronic hepatitis B patients （0.37% ± 0.43%, P 〈 0.05）. There was an inverse correlation between the strength of HBV-specific CD8＋ T-cell response and the level of PD-1 expression. Besides, there was a significant positive correlation between HBV viral load and the percentage of PD-1 expression on CD8＋ T cells in CriB and AEHB subjects （R = 0.541, P 〈 0.01）. However, PD-1 expression was not associated with disease flare-ups as indicated by alanine aminotransferase （ALT） levels （R = 0.066, P 〉 0.05）.
CONCLUSION： Our results confirm previous reports that HBV specific CD8＋T-cell response in the peripheral blood is more intense in patients with AEHB than in chronic hepatitis B wlth persistent viral infection. Moreover, there is a negative correlation between the level of PD-1 and the intensity of virus specific CD8＋ T cell response.     

Impact of CMV and EBV seropositivity on CD8 T lymphocytes in an old population from West-Sicily

Herpes viruses (particularly CMV and to some extent EBV) might play a role in accelerating the deterioration of immune functions with age. Indeed, it has been demonstrated that chronic infection with CMV causes an expansion of specific CD8 T lymphocytes and that this is related to a shrinkage of the T cell repertoire in very elderly people, predicting mortality. We have analysed CD8 T cells in young and old healthy Sicilians who were both CMV- and EBV-seropositive. Our data confirm expansions of T cells specific for the HLA-A2-restricted pp65 (495-503) CMV epitope up to nearly 14% of total peripheral CD8 cells in certain elderly individuals (range 0-14%). However, the mean percentage of CMV-specific cells in the elderly was not greater than the young (range 0.2-3%). The CMV-specific CD8 cells in the elderly were predominantly CD45RA+, but in the young they were mostly CD45RO+. Our findings are somewhat different from published reports from Northern European populations, both in terms of mean numbers and surface phenotypes. These findings may reflect disparate hygienic and nutritional conditions 70-90 yr ago, which were very different in Northern and Southern Europe at that time, as well as a different genetic background.     

Differentiation of cytomegalovirus-specific CD8(+) T cells in healthy and immunosuppressed virus carriers

During immunosuppression, cytomegalovirus (CMV) can reactivate and cause serious clinical problems. Normally, abundant virus replication is suppressed by immune effector mechanisms. To study the interaction between CD8(+) T cells and persisting viruses, frequencies and phenotypes of CMV-specific CD8(+) T cells were determined in healthy individuals and compared to those in renal transplant recipients. In healthy donors, function of circulating virus-specific CD8(+) T cells, as measured by peptide-induced interferon gamma (IFN-gamma) production, but not the number of virus-specific T cells enumerated by binding of specific tetrameric peptide/HLA complexes, correlated with the number of CMV-specific IFN-gamma-secreting CD4(+) helper T cells. Circulating CMV- specific CD8(+) T cells did not express CCR7 and may therefore not be able to recirculate through peripheral lymph nodes. Based on coexpression of CD27 and CD45R0 most CMV-specific T cells in healthy donors appeared to be memory-type cells. Remarkably, frequencies of CMV-specific CD8(+) T cells were significantly higher in immunosuppressed individuals than in healthy donors. In these patients CMV-specific cells predominantly had an effector phenotype, that is, CD45R0(+)CD27(-)CCR7(-) or CD45RA(+)CD27(-)CCR7(-) and contained both granzyme B and perforin. Our data show that in response to immunosuppressive medication quantitative and qualitative changes occur in the CD8(+) T-cell compartment. These adaptations may be instrumental to maintain CMV latency. (Blood. 2001;98:754-761)     

Simultaneous ex vivo expansion of cytomegalovirus and Epstein-Barr virus-specific cytotoxic T lymphocytes using B-lymphoblastoid cell lines expressing cytomegalovirus pp65.

Cytomegalovirus (CMV) infection and Epstein-Barr virus (EBV)-induced lymphoproliferative disease are serious complications associated with allogeneic stem cell transplantation. Immunotherapy using ex vivo expanded, virus-specific cytotoxic T lymphocytes (CTL) has been explored and proven to be effective in therapeutic or prophylactic regimens for CMV and EBV infections. To generate CTL specific for both CMV and EBV, we engineered EBV-transformed B-lymphoblastoid cell lines (BLCL) to express CMV pp65 for use as antigen-presenting cells (APC). BLCL were transduced with a recombinant retrovirus encoding pp65, the immunodominant CMV polypeptide. Western blot analysis and immunocytochemistry confirmed the expression of pp65 in the transduced cells. Peripheral blood mononuclear cells (PBMC) from healthy CMV seropositive donors were stimulated with autologous pp65-expressing BLCL weekly for 3 weeks. Chromium release assays showed that the resulting CTL cultures possessed specific cytotoxicity against EBV and CMV. Recombinant vaccinia viruses encoding individual CMV peptides were used to demonstrate that this CMV-specific cytotoxicity was specific for pp65. Assays on CD4- and CD8-depleted CTL fractions indicated that CD8(+) CTL mediated the pp65-specific cytotoxicity. These CMV/EBV-specific CTL recognized CMV- and EBV-infected targets sharing HLA class I antigens, but not HLA mismatched targets. Our results demonstrate that BLCL can be used as APC to stimulate expansion of EBV- and CMV-specific CTL simultaneously. These findings have potential implications for posttransplant CMV and EBV immunotherapy in recipients of allogeneic stem cell transplants.