流式细胞术细胞因子分析法检测乙型肝炎患者特异性细胞免疫功能的初步研究

目的运用流式细胞术胞内细胞因子（CFC）分析法检测乙型肝炎患者外周血特异性细胞免疫功能，探讨该技术检测乙型肝炎病毒（HBV）特异性细胞免疫功能的临床应用价值。方法采用HBV核心抗原表位肽core18-27设计CFC分析法，检测急、慢性乙型肝炎患者外周血单个核细胞（PBMC）中γ-干扰素（IFN-γ）分泌细胞的数量。结果19例急性乙型肝炎、54例慢性乙型肝炎患者PBMC中IFN-γ分泌细胞的数量差异有统计学意义（P〈0．01）。结论CFC分析法能在体外直接检测乙型肝炎患者PBMC中IFN-γ分泌细胞的数量，其水平在一定程度上反映了机体特异性细胞毒性T淋巴细胞应答状况。急性乙型肝炎患者特异性细胞免疫功能明显强于慢性乙型肝炎患者，     

乙型肝炎病毒特异性CTL表位变异分析

目的初步调查乙型肝炎病毒(HBV)特异性CTL表位的序列特点,同时探讨乙型肝炎重症化和慢性化的可能机理。方法本研究采用PCR扩增法获取HBV全基因并测序,参考国外文献报道的17个热点HBV特异性CTL表位氨基酸序列,分析其与本文获得的CTL表位氨基酸序列的差别。对患者各个表位变异的差异进行卡方检验。结果获得46份HBV全基因组序列,急性乙型肝炎(AHB)15例,慢性乙型肝炎(CHB)18例,慢性重型乙型肝炎(CSHB)13例。其中40份属于B基因型,6份属于C基因型。对17个表位进行分析发现,B基因型中变异较大的表位有S183-191,S382-390,C18-27,X36-44,X52-60和X115-123;C基因型中有P816-824,C18-27,X36-44,X52-60和X115-123。P816-824表位在CHB和CSHB组的变异率为27.8%和46.2%,显著高于AHB组(0%,P0.01);C23-31表位在CHB组的变异率为22.2%,显著高于AHB和CSHB组(0%,0%,P0.05)。结论了解HBV CTL表位序列特点,分析其与文献报道的CTL表位的差异情况,对防治乙型肝炎的重症化和慢性化具有指导意义。     

慢性 HBV 感染患者特异性细胞免疫功能与病毒载量的关系研究

目的：探讨慢性乙型肝炎病毒（HBV ）感染患者特异性细胞免疫功能与病毒载量的关系。方法选择51例慢性乙型肝炎患者（A组）、43例乙型肝炎肝硬化患者（B组）和40例体检健康者（C组）外,采用流式细胞术检测人类白细胞抗原A2（HLA-A2）,采用 HLA-A2限制的 HBVcore18-27抗原表位肽五聚体（Pro5MHC）法检测Pro5MHC、CD8双阳性细胞[HBV特异性细胞毒性 T 淋巴细胞（CTL）],并比较不同 HBV DNA水平患者各指标检测结果。结果 A组、B组HLA-A2阳性率均明显高于C组（P＜0．05）。随着 HBV DNA水平的升高,HBV特异性CTL水平逐渐减低（P＜0．05）,但CD8＋细胞水平无明显变化（P＞0．05）。 HBV DNA水平大于105 copies／mL的患者,Pro5M HC／CD8＋水平明显低于 HBV DNA水平为103～105 copies／mL的患者以及HBV DNA水平小于103 copies／mL的患者（P＜0．05）。结论 HLA-A2是乙型肝炎肝硬化的易感基因之一。 HBV感染患者体内HBV特异性CTL水平随着病毒复制程度的升高而减低。Pro5M HC／CD8＋可作为判断病毒复制程度的客观指标。     

慢性乙型肝炎病毒感染患者外周血特异性CD8~+T淋巴细胞检测的临床意义

目的研究慢性乙型肝炎病毒(HBV)感染患者外周血特异性CD8+T淋巴细胞(CTL)频率的变化及临床价值。方法采用乙型肝炎病毒核心抗原(HBcAg)18-27表位肽-人类白细胞抗原(HLA)-A*0201五聚体及CD8单克隆抗体,设计流式细胞技术检测HLA-A2+6例、HLA-A2-12例非HBV感染者、HLA-A2+49例和HLAA2-42例HBV感染者外周血中针对该肽段的特异性CTL数量,占总计数CD8+细胞数百分比表示。结果 HLA-A2-HBV感染未抗病毒治疗者特异性CTL(0.30%～14.40%,中位数1.13%,n=20)与HLA-A2-非HBV感染者(0.33%～3.90%,中位数1.04%,n=12),差异无统计学意义(P>0.05),但显著低于HLA-A2-抗病毒治疗者(0.25%～20.30%,中位数2.11%,n=22,P<0.05);HLA-A2-抗病毒治疗者特异性CTL和HLA-A2+未抗病毒治疗者特异性CTL(0.20%～29.90%,中位数2.22%,n=20)均显著高于HLA-A2-非HBV感染者(P<0.01);HLA-A2+抗病毒治疗者特异性CTL(0.14%～39.22%,中位数1.33%,n=29)显著高于HLA-A2-非HBV感染者(P<0.05)。HLA-A2+未抗病毒组中血清HBV DNA<103 copy/mL、丙氨酸氨基转移酶小于40U/L者特异性CTL频率增高(P<0.05、0.01)。结论 HBV抗原肽-HLA-A*0201五聚体流式细胞技术能在体外直接检测外周血HBV特异性CTL频率的变化,其水平一定程度反映不同临床感染状态慢性HBV感染患者T淋巴细胞对特异性抗原表位免疫应答的差异。     

酶联免疫斑点法检测慢性乙型肝炎患者HBcAg特异性分泌γ-干扰素细胞及其临床意义

目的观察免疫应答不同期的慢性乙型肝炎(CHB)患者外周血中HBcAg特异性γ-干扰素(IFNγ)分泌细胞数,探讨其在评价患者体内非溶细胞机制清除乙型肝炎病毒(HBV)能力中的临床意义。方法采用酶联免疫斑点法(ELISPOT)检测83例免疫耐受期、49例免疫清除期患者和18名健康人HBcAg特异性IFNγ分泌细胞。结果健康人HBcAg特异性IFNγ分泌细胞数为11~19个/4×105个外周单个核细胞(PBMC),62.2%的免疫耐受期CHB患者低于正常值,44.9%的免疫清除期CHB患者高于正常值。HBcAg特异性IFNγ分泌细胞数与HBV DNA水平未发现有相关性,而HBcAg特异性IFNγ分泌细胞数与HBeAg S/N值在一定范围内有正相关趋势。13例免疫耐受期患者经乙型肝炎疫苗及草分枝杆菌制剂治疗3~6个月后,12例患者HBcAg特异性IFNγ分泌细胞数有不同程度提高。结论采用ELISPOT测定HBcAg特异性IFNγ分泌细胞数可从一个侧面反映大多数CHB患者免疫应答能力,并可以作为治疗效果的评价指标。     

酶联免疫斑点法检测慢性乙型肝炎患者HBcAg特异性分泌γ-干扰素细胞及其临床意义

目的观察免疫应答不同期的慢性乙型肝炎(CHB)患者外周血中HBcAg特异性γ-干扰素(IFNγ)分泌细胞数,探讨其在评价患者体内非溶细胞机制清除乙型肝炎病毒(HBV)能力中的临床意义.方法采用酶联免疫斑点法(ELISPOT)检测83例免疫耐受期、49例免疫清除期患者和18名健康人HBcAg特异性IFNγ分泌细胞.结果健康人HBcAg特异性IFNγ分泌细胞数为11～19个/4 ×105个外周单个核细胞(PBMC),62.2%的免疫耐受期CHB患者低于正常值,44.9%的免疫清除期CHB患者高于正常值.HBcAg特异性IFNγ分泌细胞数与HBV DNA水平未发现有相关性,而HBcAg特异性IFNγ分泌细胞数与HBeAg S/N值在一定范围内有正相关趋势.13例免疫耐受期患者经乙型肝炎疫苗及草分枝杆菌制剂治疗3～6个月后,12例患者HBcAg特异性IFNγ分泌细胞数有不同程度提高.结论采用ELISPOT测定HBcAg特异性IFNγ分泌细胞数可从一个侧面反映大多数CHB患者免疫应答能力,并可以作为治疗效果的评价指标.     

慢性乙型肝炎患者血清HBV DNA水平与HBV特异性CTL、非特异性CTL、Th1、Th2的关系

目的:探讨慢性乙型肝炎(CHB)患者血清HBV DNA水平与HBV特异性CTL、非特异性CTL、Th1、Th2的关系,进而探讨HBV DNA水平影响抗病毒疗效的机理.方法:123例HBV DNA阳性、HBeAg阳性、人白细胞抗原(HLA)-A2阳性的CHB患者,采用实时荧光定量PCR检测HBV DNA,并根据HBV DNA水平将123例CHB患者分为甲、乙两组,甲组59例,HBV DNA水平为104～105拷贝/mL;乙组64例,HBV DNA 水平为106～107拷贝/mL.采用流式细胞术检测两组患者的HBV特异性CTL、非特异性CTL和Th1、Th2,同步进行肝功能检测,并对检测结果进行比较分析.结果:甲组HBV DNA水平低于乙组(t=25.99,P<0.001),HBV特异性CTL高于乙组(t=12.51,P<0.001),非特异性CTL低于乙组(t=2.97,P<0.01),Th1高于乙组(t=3.945,P<0.01),ALT低于乙组(t=3.88,P<0.01),TBil低于乙组(t=2.35,P<0.05).结论:CHB患者血清HBV DNA水平与HBV特异性CTL和Th1水平有关,HBV DNA水平影响抗病毒疗效的机理可能与HBV持异性CTL和Th1水平有关.     

乙型肝炎肝内细胞间粘附分子—1（ICAM—1）和HBcAg双重表达研究

目的：探讨乙型肝炎肝内ICAM-1和HBcAg表达间的关系及意义。方法：用免疫组化双重染色技术分析70例HBV感染者肝细胞ICAM-1和HBcAg双重表达。结果：5例慢性无症状HBsAg携带者肝内只有ICAM-1^-/HBcAg^ 肝细胞;45例慢性乙型肝炎（CHB）和20例重型乙型肝炎（SHB）患者肝内均存在ICAM-1/HBcAg,ICAM-1/HBcAg及ICAM-1/HBcAg等三种类型肝细胞：ICAM-1/HBcAg肝细胞多见于炎症坏死区,是多数中、重度CHB患者肝内的主要细胞类型;ICAM-1/HBcAg肝细胞是多数轻度CHB患者肝内主要细胞类型;多数SHB患者肝内的主要细胞类型是ICAM-1/HBcAg肝细胞。结论：在乙型肝炎,ICAM-1/HBcAg肝细胞代表“细胞毒模型”,HBV感染的临床类型与肝细胞ICAM-1/HBcAg表达模式有关。     

慢性HBV感染患者治疗前后外周血HBV特异CTL、HBV DNA、ALT比较

摘要：目的：比较慢性HBV感染患者抗病毒治疗前后外周血HBV特异性细胞毒性T淋巴细胞（cytotoxic lymphocyte,CTL）、HBV DNA、丙氨酸氨基转移酶（ALT)的变化,探讨其临床意义。 方法：收集118例慢性HBV感染患者、42例非病毒性肝病患者及100例体检健康者;用流式细胞法检测HLA-A2等位型及外周血特异性CTL,套式PCR法检测血清HBV DNA及ALT水平。 结果：治疗前慢性HBV感染者HLA-A2⁺细胞检出率为61.86%（73/118）,在个体化抗病毒治疗后,检出率降为30.51%（36/118）;与肝病对照组的14.29%（6/42）和健康人对照组的5.00%（5/100）比较,差异均有统计学意义（P均<0.05）。在慢性HBV感染者治疗后HBV特异CTL显著升高,HBV DNA及ALT显著降低（P均<0.05）。 结论：外周血HBV特异CTL、HBV DNA及ALT在慢性HBV感染患者抗病毒治疗前后有明显的变化,提示这些指标可用于判断治疗效果。     

肝癌患者外周血中针对NY-ESO-1/LAGE-1表位的自发性CTL的定量检测

目的检测原发性肝细胞癌（HCC）患者外周血中针对HLA-A2限制性NY-ESO-1b表位的特异性细胞毒性T细胞（cytotoxic Tlymphocyte,CTL）的水平,评价该表位多肽诱导HCC患者自发性细胞免疫应答的能力。方法采用流式细胞技术分析HCC患者的HLA表型,并通过RT-PCR技术检测其癌组织中NY-ESO-1/LAGE-1基因的表达情况;采用HLA-A2/多肽五聚体复合物即pentramer对HCC患者外周血中NY-ESO-1b表位特异性CTL进行标记并用流式细胞仪进行定量检测。结果在HLA-A2阳性、且其癌组织中NY-ESO-1/LAGE-1mRNA为阳性29例HCC患者中,13例（41.4%）患者外周血中可检测到针对HLA-A2限制性NY-ESO-1b表位（p157-165,SLLMWITQC）的特异性CTL应答,而在19例HLA-A2阳性的健康志愿者和12例HLA-A2阳性的肝炎后肝硬化患者外周血中均未检测到相应的自发性CTL应答（P值分别为0.004和0.023）。结论NY-ESO-1b表位多肽可诱发HCC患者体内产生较高频率的自发性CTL应答,提示该多肽可作为备选疫苗用于HCC的主动免疫治疗。     

非小细胞肺癌患者癌胚抗原特异性细胞毒T细胞与临床疗效的研究

目的 通过对HLA-A0201限制性的特异性细胞毒T细胞(CTL)分析,研究晚期非小细胞肺癌患者化疗不同疗效与T淋巴细胞对特异性抗原表位免疫应答的差异.方法 鉴定HLA-A0201阳性的晚期非小细胞肺癌患者13例(Ⅲb期6例,Ⅳ期7例);给予吉西他滨+顺铂方案化疗,收集化疗前患者的外周血单个核细胞,采用酶联免疫斑点试验测定针对癌胚抗原表位肽的特异性CTL的数量和功能.结果 晚期非小细胞肺癌化疗有效患者外周血癌胚抗原(CEA)多肽特异性CTL数量和功能明显高于晚期非小细胞肺癌化疗无效患者.结论 肺癌特异性表位的CTL应答,在不同化疗疗效患者的应答特征不同.化疗有效的患者特异性CTL应答水平显著高于疗效无效者,肺癌特异性CTL应答与临床疗效有密切关系.肺癌特异性CTL在抑制肿瘤的过程中发挥着重要作用,这将有助于预测肺癌患者的临床转归和开拓肺癌的免疫治疗的新策略.     

Cytotoxic T lymphocytes recognize an HLA-A2-restricted epitope within the hepatitis B virus nucleocapsid antigen.

The absence of readily manipulable experimental systems to study the cytotoxic T lymphocyte (CTL) response against hepatitis B virus (HBV) antigens has thus far precluded a definitive demonstration of the role played by this response in the pathogenesis of liver cell injury and viral clearance during HBV infection. To circumvent the problem that HBV infection of human cells in vitro for production of stimulator/target systems for CTL analysis is not feasible, a panel of 22 overlapping synthetic peptides covering the entire amino acid sequence of the HBV core (HBcAg) and e (HBeAg) antigens were used to induce and to analyze the HBV nucleocapsid-specific CTL response in nine patients with acute hepatitis B, six patients with chronic active hepatitis B, and eight normal controls. By using this approach, we have identified an HLA-A2-restricted CTL epitope, located within the NH2-terminal region of the HBV core molecule, which is shared with the e antigen and is readily recognized by peripheral blood mononuclear cells from patients with self-limited acute hepatitis B but less efficiently in chronic HBV infection. Our study provides the first direct evidence of HLA class I-restricted T cell cytotoxicity against HBV in humans. Furthermore, the different response in HBV-infected subjects who successfully clear the virus (acute patients) in comparison with patients who do not succeed (chronic patients) suggests a pathogenetic role for this CTL activity in the clearance of HBV infection.     

Induction of cytotoxic T lymphocytes with peptides in vitro: identification of candidate T-cell epitopes in hepatitis B virus X antigen.

Cytotoxic T lymphocytes (CTL) have been suggested to contribute to viral clearance during hepatitis B virus (HBV) infection. To induce effective CTL against viral infection by peptide vaccination, it is essential to identify the epitope peptides recognized by CTL. Here, 15 peptide sequences that contain HLA-A2.1-restricted CTL binding consensus motif were identified on hepatitis B virus X (HBx) protein and synthesized for further characterization. In the binding assay, 8 of 15 synthetic peptides enhanced the expression of HLA-A2.1 molecules on the surface of T2 cells, a human transport-associated antigen processing-deficient cell line. This result implies that these eight peptides are able to bind to the HLA-A2.1 molecules. These peptides were further tested for their ability to activate CTL from peripheral blood mononuclear cells (PBMCs) isolated from HBV chronic carriers. Five of eight tested peptides activated PBMC-derived T cells, resulting in the lysis of the target T2 cells pulsed with the same peptide. Furthermore, the CTL responses to HBx antigen in HBV chronic carriers were shown to be polyclonal, multispecific, and mediated mainly by CD8+ T cells. In contrast, these responses were not detected in uninfected healthy blood donors. Although the five CTL epitope peptides identified in this study have not been proven to be the naturally processed epitopes in HBV-infected hepatocytes, they could be candidates for peptide-based immunotherapy against HBV infection.     

Human histocompatibility leukocyte antigen-binding supermotifs predict broadly cross-reactive cytotoxic T lymphocyte responses in patients with acute hepatitis.

The present study was designed to determine if highly conserved hepatitis B virus (HBV)-derived peptides that bind multiple HLA class I alleles with high affinity are recognized as cytotoxic T lymphocyte (CTL) epitopes in acutely infected patients. Peripheral blood mononuclear cells from 67 patients with acute hepatitis B, and 12 patients convalescent from acute hepatitis B, were stimulated with three panels of peptides, each of which bind with high affinity to several class I alleles from the HLA-A2-, HLA-A3-, or HLA-B7-supertypes. In these patients, 8 of the 19 peptides tested were found to represent CTL epitopes recognized by two or more alleles in each supertype. Two sets of nested peptides were recognized in the context of alleles with completely unrelated peptide binding specificities. Finally, promiscuous recognition by the same CTL of a given peptide presented by target cells expressing different A2 subtypes was also commonly observed. In conclusion, several HBV-specific CTL epitopes, recognized by acutely infected or convalescent patients in the context of a wide range of HLA alleles have been identified. These results demonstrate the functional relevance of the supertype grouping of HLA class I molecules in a human viral disease setting. Furthermore, they represent a significant advance in the development of a totally synthetic vaccine to terminate chronic HBV infection and support the feasibility of a systematic approach to development of similar vaccines for prevention and treatment of other chronic viral infections.     

拉米夫定在慢性乙型肝炎患儿外周血Th17细胞变化的研究

目的研究拉米夫定在慢性乙型肝炎(下称慢性乙肝)患儿外周血Th17细胞变化的特征及意义。方法选取该院慢性乙肝患儿45例作为慢性乙肝组,另选取23例健康儿童作为健康对照组。慢性乙肝组患儿给予拉米夫定治疗1年。抽取健康对照组、慢性乙肝组治疗前及治疗1年后外周静脉血,检测血清中乙型肝炎病毒表面标志物(HBV-M)、乙型肝炎病毒核酸定量(HBV-DNA)、丙氨酸氨基转移酶(ALT)及外周血中Th17细胞特征。结果经拉米夫定治疗,慢性乙肝患儿ALT、HBV-DNA载量明显降低,其中26例患儿出现HBV-DNA转阴,4例出现HBeAg转阴。慢性乙肝组治疗前Th17细胞频率明显高于健康对照组(P0.05),经拉米夫定治疗1年后外周血Th17细胞频率较治疗前显著降低,但仍高于健康对照组(P0.05)。治疗后HBV-DNA转阴患儿较未转阴患儿的Th17细胞频率明显降低。结论拉米夫定治疗儿童慢性乙肝能有效抑制病毒复制,且可降低患儿外周血中Th17细胞的频数。     

Hepatitis B virus (HBV) sequence variation of cytotoxic T lymphocyte epitopes is not common in patients with chronic HBV infection.

It has been suggested that immune selection pressure exerted by the cytotoxic T lymphocyte (CTL) response could be responsible for viral persistence during chronic hepatitis B virus infection. To address this question, in the current study we compared the DNA and amino acid sequences of, and the CTL responses to, multiple HLA-A2-restricted CTL epitopes in the hepatitis B virus in several HLA-A2-positive patients with acute and chronic hepatitis. Our results indicate that the CTL response to these epitopes is barely detectable in the majority of patients with chronic hepatitis. Further, we show that the weak CTL response is not secondary in infection by mutant viruses lacking these epitopes, and we show that the CTL response did not select for escape mutants in any of these patients. We conclude that an ineffective hepatitis B virus specific CTL response is the primary determinant of viral persistence in chronic hepatitis and that immune selection of viral variants is not a common event in the majority of patients.     

Efficient generation of a hepatitis B virus cytotoxic T lymphocyte epitope requires the structural features of immunoproteasomes

Interferon (IFN)-gamma-induced cells express the proteasome subunits low molecular weight protein (LMP)2, LMP7, and MECL-1 (multicatalytic endopeptidase complex-like 1), leading to the formation of immunoproteasomes. Although these subunits are thought to optimize MHC class I antigen processing, the extent of their role and the mechanistic aspects involved remain unclear. Herein, we study the proteolytic generation of an human histocompatibility leukocyte antigen (HLA)-Aw68-restricted hepatitis B virus core antigen (HBcAg) cytotoxic T lymphocyte (CTL) epitope that is recognized by peripheral blood lymphocytes from patients with acute self-limited but not chronic hepatitis B virus (HBV). Immunological data suggest that IFN-gamma-induced rather than uninduced HeLa cells process and present the HBV CTL epitope upon infection with HBcAg-expressing vaccinia viruses. Analyses of 20S proteasome digests of synthetic polypeptides covering the antigenic HBcAg peptide demonstrate that only immunoproteasomes efficiently perform the cleavages needed for the liberation of this HBV CTL epitope. Although the concerted presence of the three immunosubunits appears essential, we find that both catalytically active LMP7 and inactive LMP7 T1A support CTL epitope generation. We conclude that LMP7 influences the structural features of 20S proteasomes, thereby enhancing the activity of the LMP2 and MECL-1 catalytic sites, which provide cleavage specificity. Thus, LMP7 incorporation is of greater functional importance for the generation of an HBV CTL epitope than cleavage specificity.     

Cytotoxic T lymphocyte responsiveness after resolution of chronic hepatitis B virus infection.

Clearance of the hepatitis B virus (HBV) during acute hepatitis is associated with a strong, polyclonal, multispecific cytotoxic T lymphocyte (CTL) response to the viral envelope, nucleocapsid and polymerase proteins that persists for decades after clinical recovery. In contrast, chronically infected patients usually fail to mount a strong CTL response to this virus. In this study we demonstrate that chronically infected patients who experience a spontaneous or interferon-induced remission develop a CTL response to HBV that is similar in strength and specificity to patients who have recovered from acute hepatitis. The results suggest that specific immunotherapeutic enhancement of the CTL response to HBV should be possible in chronically infected patients, and that it could lead to viral clearance in these individuals with resolution of chronic liver disease.     

Lack of strong immune selection pressure by the immunodominant, HLA-A*0201-restricted cytotoxic T lymphocyte response in chronic human immunodeficiency virus-1 infection.

Despite detailed analysis of the HIV-1-specific cytotoxic T lymphocyte response by various groups, its relation to viral load and viral sequence variation remains controversial. We analyzed HLA-A*0201 restricted cytotoxic T lymphocyte responses in 17 HIV-1-infected individuals with viral loads ranging from < 400 to 221,000 HIV RNA molecules per milliliter of plasma. In 13 out of 17 infected subjects, CTL responses against the SLYNTVATL epitope (p17 Gag; aa 77-85) were detectable, whereas two other HLA-A*0201 restricted epitopes (ILKEPVHGV, IV9; and VIYQYMDDL, VL9) were only recognized by six and five individuals out of 17 individuals tested, respectively. Naturally occurring variants of the SL9 epitope were tested for binding to HLA-A*0201 and for recognition by specific T cell clones generated from five individuals. Although these variants were widely recognized, they differed by up to 10,000-fold in terms of variant peptide concentrations required for lysis of target cells. A comparison of viral sequences derived from 10 HLA-A*0201-positive individuals to sequences obtained from 11 HLA-A*0201-negative individuals demonstrated only weak evidence for immune selective pressure and thus question the in vivo efficacy of immunodominant CTL responses present during chronic HIV-1 infection.