烟曲霉抗原Asp f16HLA-A*0201限制性的CD8+CTL抗原表位生物信息学预测与实验室鉴定

目的 预测与鉴定烟曲霉抗原Asp f16的HLA-A *0201限制性CD8+细胞毒性T细胞(CTL)抗原表位.方法 以国人常见的HLA-A*0201位点为靶点,依据生物信息学软件扫描烟曲霉特异性抗原Asp f16的全部427个氨基酸序列.使用HLA-A *0201转基因小鼠制备骨髓来源的树突状细胞(DC)和CTL.流式细胞仪技术检测DC表面MHC Ⅱ类抗原,CD80,CD86和CD11c的表达来验证其是否成熟.ELISPOT试验检测烟曲霉抗原多肽特异性CTL产生的细胞因子IFN-γ.四聚体(Tetramer)试验证实烟曲霉特异性CTL与抗原肽,HLA-A*0201分子复合体的亲和性.结果 根据与MHC I类分子结合的半衰期评分,选择了3个HLA-A*0201限制性抗原表位.流式细胞仪分析示成熟DC高表达HLA Ⅱ类抗原,CD80,CD86和CD11c.Tetramer试验证实烟曲霉特异性T细胞受体与抗原肽,HLA-A*0201分子复合体的高亲和性.ELISPOT实验结果 表明烟曲霉抗原肽体外可以活化CD8+CTL,被负载了抗原肽的DC刺激活化后可以产生IFN-γ.结论 本研究成功鉴定烟曲霉抗原Asp f16的HLA-A*0201限制性CD8+CTL表位,可作为疫苗设计的候选表位,为进一步研发新型抗烟曲霉疫苗提供参考.     

HPV16 E7抗原CTL表位拟肽设计及活性评价

人乳头瘤病毒(human papillomavirus,HPV)早期基因E7是致癌的关键基因,其表达在宫颈癌细胞癌变进程及维持癌细胞恶性表型方面发挥重要作用,已成为宫颈癌治疗的理想靶标.目前,基于HPV16 E7抗原细胞毒性T淋巴细胞(cytotoxic lymphocyte,CTL)表位设计多肽疫苗是抗宫颈癌治疗发展的重要方向,但天然CTL表位肽普遍存在体内半衰期短、激发CTL反应效果不佳等缺点.因此,本研究基于前期HPV16 E7抗原CTL表位鉴定的基础,结合多肽酶解实验结果,进行分子动力学模拟及结合自由能计算,初步筛选了3条表位模拟肽.人工合成相关待测表位肽,并利用T2细胞株测定各肽与HLA-A2分子的结合力.研究结果表明,3条表位模拟肽体外抗酶解能力较天然HPV16 E7抗原CTL表位肽均有提高,以(d)RAHYNIVTF表位模拟肽的效果最为明显.此外,(d)RAHYNIVTF表位模拟肽与HLA-A2分子的结合力也有所提高(荧光系数为2.06).以上结果表明,基于HPV16 E7抗原CTL表位模拟肽进行结构修饰有望为宫颈癌治疗性疫苗的设计奠定基础.     

HLA-A*2402-BSP在大肠杆菌中的优化表达及其四聚体的制备和鉴定

HLA-A*2402是中国人群中最常见的等位基因之一,为研究该基因型人群的人巨细胞病毒(HCMV)特异性细胞毒T细胞(CTL)免疫应答,需要制备负载相应抗原肽的HLA-A*2402四聚体。以RT-PCR方法克隆HLA-A*2402重链基因的cDNA,并构建了羧基端融合生物素化酶BirA底物肽(BSP)的HLA-A*2402重链胞外域融合蛋白(HLA-A*2402-BSP)的表达载体,但该载体不能在大肠杆菌(E.coli)中有效表达HLA-A*2402-BSP融合蛋白;通过对氨基端(N端)区域编码区的密码子进行优化,构建了同义突变的HLA-A*2402-BSP表达载体,融合蛋白在E.coli中获得了高效表达。进而制备了负载HLA-A*2402限制性HCMVpp65341-349抗原肽(QYDPVAALF,QYD)的可溶性HLA-A*2402-QYD单体分子和四聚体,获得的四聚体具有与HLA-A24 供者抗原特异性CTL的结合活性,特异性CTL的频率为总CD8 T细胞的0·09%～0·37%。这些结果为进一步研究HLA-A*2402限制性的特异性CTL免疫应答规律奠定基础。     

自身肽结合于HLA-A2分子上能够在体外诱导抗原肽特异性的同种T细胞

在同种反应性T细胞(同种T细胞)识别的配体中, 抗原肽的作用是免疫学长期争论的问题, 即同种T细胞识别是否具有抗原肽特异性. 为了证实通过长期混合淋巴细胞培养(LTMLC)方法能够诱生抗原肽/MHC复合物(pMHC)特异性的同种T细胞, 本研究利用仅表达HLA-A2, TAP缺陷的T2细胞, 将酪氨酸激酶来源的自身抗原肽(Tyr_369-377)和EB病毒来源的病毒抗原肽(LMP2A_426-434)分别加载到T2细胞上, 使T2细胞提呈单一的T细胞抗原识别表位, 并且选择4个HLA-A2阳性(HLA-A2+ve)与4个HLA-A2阴性(HLA-A2-ve)个体的PBL样本, 与加载上述抗原肽的T2细胞混合培养. 在此实验系统中, HLA-A2+ve PBL与加载病毒抗原肽的T2细胞(T2/LMP)混合培养代表T细胞对普通抗原的反应, 而HLA-A2-ve PBL与加载自身抗原肽的T2细胞(T2/Tyr)混合培养则为T细胞对同种抗原的反应. 利用特异性pMHC四聚体染色与特异性细胞毒试验检测LTMLC诱生CTL的特异性, 其中利用HIV抗原肽(Gag_77-85)作为对照. 结果显示: (ⅰ) T2/LMP与HLA-A2+ve个体的PBL混合培养产生CTL(CTL-T2/LMP), CTL-T2/LMP对T2/LMP的杀伤显著高于对照T2/HIV的杀伤(26.52%±3.72% vs 7.01%±0.87%, P<0.001); LMP四聚体对CTL-T2/LMP染色的阳性细胞数显著高于对照HIV四聚体 (0.98%±0.33% vs 0.05%±0.01%, P=0.0014); (ⅱ) 加载自身抗原肽的T2细胞(T2/Tyr)可诱导HLA-A2-ve个体的PBL产生CTL(CTL-T2/Tyr), CTL-T2/Tyr对T2/Tyr的杀伤显著高于对T2/HIV的杀伤(28.07%±2.58% vs 6.87±1.01%, P<0.001); Tyr四聚体对CTL-T2/Tyr染色的阳性细胞数显著高于HIV四聚体(0.88%±0.3% vs 0.06±0.03%, P=0.0018). 结果说明: 结合于自身MHC分子上的病毒抗原肽与结合于同种MHC分子上的自身抗原肽都能诱导产生抗原肽特异性的CTL; 在LTMLC诱生的同种CTL中, 有相当数量的CTL具有pMHC特异性, 这些同种CTL的识别机制与普通抗原反应性CTL一样, 识别的对象也是特异性的pMHC. 支持了同种抗原的pMHC种类繁多造成同种T细胞反应强度极高的假说. 利用LTMLC诱生抗原肽特异性同种T细胞方法对于T细胞过继治疗具有潜在的应用价值.     

加载HCMV抗原肽的HLA-A*0201单体及其四聚体制备和鉴定

细胞毒T淋巴细胞(CTL)在控制病原体感染以及抗肿瘤过程中发挥重要作用,因而特异性CTL的检测相当重要;而过去检测CTL的方法都是间接的,最近发展起来的四聚体技术则是直接检测抗原特异性CTL的有效而特异的方法,成为目前研究T细胞免疫应答的关键技术。报道一种简化的四聚体制备程序,利用该程序成功制备加载人巨细胞病毒(HCMV)抗原肽的HLA-A2四聚体,具有特异性结合CTL活性。HLA-A*0201重链基因是通过RT-PCR方法从HLA-A2⁺供者白细胞中克隆,进而以PCR方法构建在羧基端融合生物素化酶BirA底物肽(BSP)的HLA-A*0201(A2)重链胞外区原核表达载体, A2重组蛋白在大肠杆菌中得到高表达,主要以包涵体形式存在。加载抗原肽的可溶性A2单体是A2胞外区在轻链β2微球蛋白和HLA-A2限制性HCMV pp65_495-503抗原肽(NLVPMVATV,NLV)存在时通过稀释法复性获得,以BirA对其进行生物素化,然后以阴离子交换树脂纯化,得到的纯化A2-NLV单体与Streptavidin_PE按4:08比例混合形成四聚体,结合程度在85％以上,流式细胞仪分析显示该四聚体具有与HLA-A2+供者的特异性CTL结合活性。总之,这种简化的四聚体制备程序,不仅有利于该技术的推广,为特异性T细胞免疫研究建立必要的技术平台,而且A2-NLV四聚体在临床监测CMV特异性CTL水平等方面也有应用价值。     

目视化纳米探针检测宫颈癌组织HPV16E6基因的研究

目的：建立目视化纳米探针检测宫颈癌组织HPV16E6基因的方法,为临床降低HPV感染者宫颈癌的发病率提供技术支持。方法：采用固定于硅烷化片基上的捕获探针,特异性结合单链人乳头瘤病毒16型新疆株E6基因(HPV16E6)扩增片断,再与互补的纳米金颗粒标记的探针结合,通过银染加强显色。结果：初步建立了金标银染法快速检测HPV16E6 DNA的技术。结论：金标银染法灵敏,可以快速特异地检测出各类宫颈组织中人乳头瘤病毒的感染,为进一步应用于临床检测降低新疆维吾尔妇女宫颈癌的发病率奠定了基础。     

MHCI类分子四聚体技术的应用研究进展

主要组织相容性复合体(major histocompatibility complex,MHC)I类分子四聚体(tetramer)技术可直接对抗原特异性细胞毒性T淋巴细胞(cytotoxic T lymphocytes,CTL)进行标记,以检测能够识别特定抗原肽-MHC I的CTL,用于临床检测、疾病诊断及相关科学研究。综述了MHC I四聚体技术应用的最新研究进展,为开展四聚体的相关研究提供新的参考。     

乙型肝炎病毒特异性细胞毒性T淋巴细胞在病毒清除过程中的作用

在乙型肝炎病毒(HBV)感染过程中,适应性免疫与病毒的致病和清除密切相关。一般认为,体液免疫产生的抗体可以清除外周循环的病毒颗粒,从而阻止病毒在宿主体内的传播,细胞免疫主要清除被感染细胞中的病毒。HBV特异性的细胞毒性T淋巴细胞(CTL)在抑制HBV复制过程中发挥着重要的作用。CTL在肝内主要通过分泌γ干扰素抑制病毒,同时,当CTL识别HBV抗原后,HBV特异性CTL募集抗原非特异性炎症细胞对肝组织浸润,造成肝细胞的损伤。对CTL抗病毒作用进行深入研究,将为乙型肝炎的治疗开辟新的途径。     

液相芯片技术在HPV血清检测中的初步应用

目的:建立一种高效的HPV病毒血清学检测方法.方法:重组质粒表达HPV16/18 E6和E7蛋白,同微球偶联后作为抗原,应用Luminex系统检测宫颈癌患者血清中的相应抗体.结果:57.4%宫颈癌患者血清至少检测到HPV16 E6、E7和HPV18 E6、E7蛋白抗体中1种,36.1%的血清检测到HPV16 E6和E7蛋白抗体中1种,31.9%的血清检测到HPV18 E6和E7蛋白抗体中1种.结论:基于GST重组HPV蛋白的液相芯片技术可进一步应用于患者血清HPV抗体的检测中.     

人乳头瘤病毒16型 E7蛋白在子宫颈癌细胞内的定位

应用重组质粒在大肠杆菌中表达人乳头瘤病毒(HPV)16 型 E7 基因.以所产生的 E7 融合蛋白为抗原免疫家兔,制得抗 E7 蛋白抗血清.在子宫颈癌组织切片中用此抗血清作免疫组化染色(胶体金标记染色法).在光学显微镜下可观察到癌细胞中存在 E7 抗原黑色颗粒,位于细胞核内.主要附着于核膜,可证明 E7 基因在 HPV16 感染的子宫颈癌细胞中有强烈表达;提示 E7 基因可能即为 HPV16的癌基因.     

Antigen processing defects in cervical carcinomas limit the presentation of a CTL epitope from human papillomavirus 16 E6.

Human papillomavirus (HPV) infection, particularly type 16, is causally associated with the development of cervical cancer. The E6 and E7 proteins of HPV are constitutively expressed in cervical carcinoma cells making them attractive targets for CTL-based immunotherapy. However, few studies have addressed whether cervical carcinomas can process and present HPV E6/E7-derived Ags for recognition by CTL. We generated HLA-A*0201-restricted CTL clones against HPV16 E6(29-38) that recognized HPV16 E6 Ags transfected into B lymphoblastoid cells. These CTL were unable to recognize HLA-A*0201(+) HPV16 E6(+) cervical carcinoma cell lines even when the level of endogenous HPV16 E6 in these cells was increased by transfection. This defect in presentation of HPV16 E6(29-38) correlated with low level expression of HLA class I, proteasome subunits low molecular mass protein 2 and 7, and the transporter proteins TAP1 and TAP2 in the cervical carcinoma cell lines. The expression of all of these proteins could be up-regulated by IFN-gamma, but this was insufficient for CTL recognition unless the level of HPV16 E6 Ag was also increased by transfection. CTL recognition of the HPV16 E6(29-38) epitope in 721.174 B cells was dependent on TAP expression but independent of immunoproteasome expression. Collectively, these findings suggest that presentation of the HPV16 E6(29-38) epitope in cervical carcinoma cell lines is limited both by the level of TAP expression and by the low level or availability of the source HPV E6 oncoprotein. These observations place constraints on the use of this, and potentially other, HPV-derived CTL epitopes for the immunotherapy of cervical cancer.     

Immunological ignorance of an E7-encoded cytolytic T-lymphocyte epitope in transgenic mice expressing the E7 and E6 oncogenes of human papillomavirus type 16.

Certain human papillomaviruses (HPV) have been implicated in the etiology of cervical malignancies, and the E7 and E6 gene products of HPV type 16 are frequently expressed in these lesions. However, cytolytic T-lymphocyte (CTL)-mediated responses to HPV are rarely detectable in patients with cervical cancer. To examine whether the T-cell response is deficient during the HPV-induced transformation, we produced lines of transgenic (Tg) mice that expressed the E6 and E7 oncogenes in keratinized epithelia. The mice developed severe hypertrophy of all keratinized epithelia, but no malignancies were observed. Although epithelial cells from Tg mice could present at least an E7-encoded CTL epitope (E7 49-57), CTLs from these mice were neither primed to nor made tolerant of this epitope. No quantitative or qualitative differences were seen in the CTL responses of the Tg mice compared to those of their littermates following immunization with the peptide E7 49-57. Immunization of Tg mice with the E7 49-57 peptide protected them against a subcutaneous challenge with tumor cells expressing a transfected E7 gene, yet the skin was unaffected, although the cultured skin epithelial cells from Tg mice expressed E7. Our results suggest that the Tg mice were immunologically ignorant of HPV oncoproteins with respect to a CTL response and that a similar type of ignorance may explain why HPV-associated cervical cancer cells can escape immunological destruction.     

Induction of human papillomavirus-specific CD4(+) and CD8(+) lymphocytes by E7-pulsed autologous dendritic cells in patients with human papillomavirus type 16- and 18-positive cervical cancer.

Human papillomavirus (HPV) type 16 (HPV 16) and HPV type 18 (HPV 18) are implicated in the induction and progression of the majority of cervical cancers. Since the E6 and E7 oncoproteins of these viruses are expressed in these lesions, such proteins might be potential tumor-specific targets for immunotherapy. In this report, we demonstrate that recombinant, full-length E7-pulsed autologous dendritic cells (DC) can elicit a specific CD8(+) cytotoxic T-lymphocyte (CTL) response against autologous tumor target cells in three patients with HPV 16- or HPV 18-positive cervical cancer. E7-specific CTL populations expressed strong cytolytic activity against autologous tumor cells, did not lyse autologous concanavalin A-treated lymphoblasts or autologous Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL), and showed low levels of cytotoxicity against natural killer cell-sensitive K562 cells. Cytotoxicity against autologous tumor cells could be significantly blocked by anti-HLA class I (W6/32) and anti-CD11a/LFA-1 antibodies. Phenotypically, all CTL populations were CD3(+)/CD8(+), with variable levels of CD56 expression. CTL induced by E7-pulsed DC were also highly cytotoxic against an allogeneic HLA-A2(+) HPV 16-positive matched cell line (CaSki). In addition, we show that specific lymphoproliferative responses by autologous CD4(+) T cells can also be induced by E7-pulsed autologus DC. E7-specific CD4(+) T cells proliferated in response to E7-pulsed LCL but not unpulsed LCL, and this response could be blocked by anti-HLA class II antibody. Finally, with two-color flow cytometric analysis of intracellular cytokine expression at the single-cell level, a marked Th1-like bias (as determined by the frequency of gamma interferon- and interleukin 4-expressing cells) was observable for both CD8(+) and CD4(+) E7-specific lymphocyte populations. Taken together, these data demonstrate that full-length E7-pulsed DC can induce both E7-specific CD4(+) T-cell proliferative responses and strong CD8(+) CTL responses capable of lysing autologous naturally HPV-infected cancer cells in patients with cervical cancer. These results may have important implications for the treatment of cervical cancer patients with active or adoptive immunotherapy.     

CD4+ tumor-infiltrating lymphocytes in cervical cancer recognize HLA-DR-restricted peptides provided by human papillomavirus-E7.

Human papillomavirus (HPV)-encoded proteins may provide targets for CD8+ or CD4+ T lymphocytes infiltrating into cervical cancer. We established an MHC class II-restricted CD4+ T cell line from a patient with cervical cancer that recognizes autologous (HPV35+, HPV59+) cervical cancer cells and the HLA-DR4-matched cervical cancer cell line Me180 (HPV68+) as determined by TNF-alpha secretion. Expression of different HPV-E7 genes in autologous B cells revealed that this T cell line defines a DR4-presented T cell epitope that is shared among the E7 genes of HPV59 and HPV68. MHC class II-presented peptides may be implemented to augment T cell responses directed against autologous tumor cells, particularly if cancer cells lack MHC class I expression, which is a frequent event in the evolution of cervical cancer.     

Immunotherapy of a human papillomavirus type 16 E7-expressing tumor by administration of fusion protein comprised of Mycobacterium bovis BCG Hsp65 and HPV16 E7

Human papillomavirus type 16 (HPV16) infection has been linked to the development of cervical and anal dysplasia and cancer. One hallmark of persistent infection is the synthesis of the viral E7 protein in cervical epithelial cells. The expression of E7 in dysplastic and transformed cells and its recognition by the immune system as a foreign antigen make it an ideal target for immunotherapy. Utilizing the E7-expressing murine tumor cell line, TC-1, as a model of cervical carcinoma, an immunotherapy based on the administration of an adjuvant-free fusion protein comprised of Mycobacterium bovis BCG Hsp65 linked to HPV16 E7 (HspE7) has been developed. Initial in vitro analyses indicate that immunization with HspE7 results in the induction of a type 1 immune response based on the pattern of secreted cytokines and the presence of cytolytic activity following antigenic recall. It has been previously shown that prophylactic immunization with HspE7 protected mice against challenge with TC-1 cells and that these tumor-free animals are also protected against rechallenge with TC-1 cells. The present report shows that a single therapeutic immunization with HspE7 induces regression of palpable tumors, confers protection against tumor rechallenge, and is associated with long-term survival (>253 days). In vivo studies using mice with targeted mutations in CD8 or MHC class II or depleted of CD8 or CD4 lymphocyte subsets demonstrate that tumor regression following therapeutic HspE7 immunization is CD8 dependent and CD4 independent. These studies extend previous observations on the induction of CTL by Hsp fusion proteins and are consistent with the clinical application of HspE7 as an immunotherapy for human cervical and anal dysplasia and cancer.     

Lethality of PAK3 and SGK2 shRNAs to Human Papillomavirus Positive Cervical Cancer Cells Is Independent of PAK3 and SGK2 Knockdown

The p21-activated kinase 3 (PAK3) and the serum and glucocorticoid-induced kinase 2 (SGK2) have been previously proposed as essential kinases for human papillomavirus positive (HPV+) cervical cancer cell survival. This was established using a shRNA knockdown approach. To validate PAK3 and SGK2 as potential targets for HPV+ cervical cancer therapy, the relationship between shRNA-induced phenotypes in HPV+ cervical cancer cells and PAK3 or SGK2 knockdown was carefully examined. We observed that the phenotypes of HPV+ cervical cancer cells induced by various PAK3 and SGK2 shRNAs could not be rescued by complement expression of respective cDNA constructs. A knockdown-deficient PAK3 shRNA with a single mismatch was sufficient to inhibit HeLa cell growth to a similar extent as wild-type PAK3 shRNA. The HPV+ cervical cancer cells were also susceptible to several non-human target shRNAs. The discrepancy between PAK3 and SGK2 shRNA-induced apoptosis and gene expression knockdown, as well as cell death stimulation, suggested that these shRNAs killed HeLa cells through different pathways that may not be target-specific. These data demonstrated that HPV+ cervical cancer cell death was not associated with RNAi-induced PAK3 and SGK2 knockdown but likely through off-target effects.     

Conservation of E6 and E7 regions of human papillomavirus types 16 and 18 present in cervical cancers

The E6 and E7 regions of human papillomavirus (HPV) type 16 were present in the DNA samples from cervical cancer cell lines, SKG-IIIa and SKG-IIIb, and those from cervical cancer tissues of three different patients. T601 cells, an NIH3T3 transformant obtained by transfection of DNA from a surgical specimen of a cervical cancer, also contained the E6 and E7 regions. The E6 region of HPV type 16 was expressed as mRNA in SKG-IIIa, SKG-IIIb and T601 cells. The E6 and E7 regions of HPV type 18 were present in the DNA samples from cervical cancer cell lines, SKG-I and SKG-II, and those from cervical cancer tissues of two different patients. SKG-I and SKG-II cells expressed the E6 region of HPV type 18 as mRNAs. These results strongly suggest that the E6 and E7 regions or the sequence surrounding these regions are important for maintaining malignant phenotype of cervical cancer cells.