肝素酶HLA-A2限制性CTL表位肽的筛选及其活性鉴定

目的 预测并鉴定新的人乙酰肝素酶(Hpa)抗原的HLA-A2限制性CTL表位,为恶性肿瘤多肽疫苗的免疫治疗提供依据. 方法 采用SYFPEITHI和BIMAS软件预测方法,对肝素酶HLA-A2限制性CTL表位进行预测,合成候选表位肽;利用T2细胞特点,对合成的候选肽与HLA-A2分子进行亲和力分析;利用乳酸脱氢酶释放试验检测待检肽特异性CTL诱导活性;利用ELISPOT检测T细胞活性. 结果 在所筛选的6条候选CTL表位肽中,Hpa(310～318)FLNPDVLDI与HLA-A2分子具有高亲和力,在体外可有效诱导肝素酶特异性CTL的产生,对肝素酶阳性且HLA-A2限制性的HCC-LM6肝癌细胞及SW-480结肠癌细胞具有明显的杀伤效应,且能有效诱导IFN-γ分泌,增强免疫活性. 结论 首次发现Hpa(310～318)FLNPDVLDI可能是肿瘤肝素酶抗原的HLA-A2限制性CTL表位.     

抗原NY-BR-1的HLA-A2限制性CTL表位的初步鉴定

目的：鉴定乳腺癌分化肿瘤抗原NY-BR-1的HLA-A2限制性CTL表位。方法：采用量化基序法初步预测NYBR-1的HLA-A2限制性CTL表位；分子动力学方法进一步筛选量化基序法中评分最高的3个表位肽；HLA-A2亲合力鉴定预测的结果。结果：分子动力学和HLA-A2亲合力实验均证明，在量化基序法预测的3个候选CTL表位中，NY-BR-11043-1051与HLA-A2亲合力最佳。结论：NY-BR-11043-1051可能为乳腺癌分化抗原NY-BR-1的HLA-A2限制性CTL表位。     

癌-睾丸抗原MAGEC2 HLA-A3限制性细胞毒性T淋巴细胞表位的鉴定

目的:预测并初步鉴定HLA-A3超型限制性MAGEC2抗原特异性细胞毒性T细胞(CTL)表位肽,为基于超型表位的MAGEC2治疗提供实验基础及新的候选靶标。方法:通过BIMAS、SYFPEITHI和IEDB软件预测打分来选取MAGEC2的HLA-A3限制性表位;结合力实验用于检测候选表位与T2A3细胞表面HLA-A3分子的结合能力,ELISPOT实验检测候选表位肽诱导的CTL分泌IFN-γ的能力,体外细胞毒实验检测侯选表位肽诱导的CTL杀伤靶细胞的能力。结果 :表位肽P147、P167、P196、P229和P251具有较好的HLA-A3结合力。ELISPOT实验结果显示表位肽P167、P196和P251诱导的CTL具有分泌IFN-γ的能力。细胞毒实验结果显示表位肽P196和P251诱导的CTL对靶细胞有一定的杀伤作用(P0.05或P0.01)。结论 :P196和P251有更高的HLA-A3分子亲和力,保留了原有的免疫原性,是优秀的MAGEC2抗原的HLA-A3限制性CTL候选表位,可以成为新的抗肿瘤多肽免疫治疗疫苗的候选表位。     

肿瘤抗原TRAG-3 HLA-A2.1限制性CTL表位的鉴定

目的寻找并鉴定TRAG-3来源的HLA-A2．1限制性CTL表位，为临床开展基于TRAG-3表位的特异性免疫治疗奠定基础。方法应用超基序和量化基序方案，预测TRAG-3 HLA-A2．1限制性CTL表位。用计算机分子模拟的方法，对预测表位进行分子模拟。用流式细胞仪分析测定各肽与HLA-A2．1分子的结合力。最后，应用HLA-A2．1阳性健康志愿者外周血单个核细胞(PBMC)对其体外诱导的CTL效应进行鉴定。结果应用超基序和量化基序方案，预测出4个候选表位肽，用计算机分子模拟发现其中只有TRAG-3（37-45)不符合HLA-A2．1限制性CTL表位的特点。在上述4个九肽中，TRAG-3(58-66)与HLA-A2．1分子的结合力最高。在进一步的CTL诱导实验中，发现TRAG-3(58-66)能够诱导健康志愿者PBMC产生特异性CTL。结论表位预测、计算机分子模拟、结合力分析和体外CTL诱导实验的一致性较好。4种方法一致认为，TRAG-3（58-66）(ILLRDA-GLV)为HLA-A2．1限制性CTL表位。该表位肽可望用于基于TRAG-3抗原多肽疫苗的临床实验。     

肝癌高表达抗原MAGEC2 CTL表位肽的鉴定

目的:观察肝癌高表达抗原MAGEC2的改造表位是否有HLA-A2限制性抗肿瘤能力。方法:通过Net CTL 1.2、SYFPEITHI和IEDB软件预测打分选取MAGEC2的HLA-A2限制性表位;替换MAGEC2抗原锚定位点氨基酸获得改造肽;结合力实验检测候选表位与T2细胞表面HLA-A2分子的结合能力,ELISPOT实验和胞内因子染色检测候选表位肽诱导细胞毒性T淋巴细胞(CTL)分泌干扰素γ(IFN-γ)的能力,体外细胞毒实验检测诱导CTL的能力。结果:P248、P248-1Y、P356、P356-1Y、P356-2L和P356-1Y2L具有较好的结合力,且P248-1Y和P356-1Y2L等改造肽与HLA-A2的结合力高于原肽。胞内因子染色和ELISPOT实验结果显示,表位肽P248、P248-1Y、P356和P356-1Y2L诱导的CTL具有分泌IFN-γ的能力,且P248-1Y和P356-1Y2L诱导特异性T细胞免疫分泌的IFN-γ略高于原肽(P0.05)。细胞毒实验结果显示表位肽P248、P248-1Y、P356和P356-1Y2L对Hep G2细胞均有一定的杀伤作用,且P248-1Y和P356-1Y2L特异性CTLs对Hep G2细胞杀伤率高于原肽特异性CTLs(P0.05)。结论:MAGEC2抗原改造表位P248-1Y和P356-1Y2L分别与天然表位P248和P356相比有更高的HLA-A2分子亲和力,保留了原有的免疫原性,并且改造肽抗肿瘤免疫效应强于天然表位。P248-1Y和P356-1Y2L是优秀的MAGEC2抗原的HLA-A2限制性CTL候选表位,可以成为新的抗肿瘤多肽免疫治疗疫苗的候选表位。     

人锌转运蛋白8的HLA-A~*0201限制性CTL表位的预测及初步验证

目的预测和初步鉴定1型糖尿病(T1DM)主要自身抗原锌转运蛋白8(ZnT8)的HLA-A*0201限制性细胞毒性T淋巴细胞(cytotoxic T lymphocytes,CTL)表位,为基于ZnT8抗原表位的特异性免疫治疗奠定基础。方法选取BIMAS预测工具预测该抗原HLA-A*0201限制性结合肽,人工合成待测表位肽,利用T2细胞株测定各肽与HLA-A*0201分子的结合力。利用酶联免疫斑点检测(enzyme-linked immunospotassay,ELISPOT)方法检测候选肽刺激T1DM患者外周血单个核细胞分泌IFN-γ和IL-2的能力,利用标准51Cr释放试验检测特异性CTL诱导活性。结果在所筛选的5个候选CTL表位中,ZnT8(107-115)、ZnT8(115-123)及ZnT8(145-153)与HLA-A*0201分子具有较高的结合荧光强度,可在体外有效诱导抗原特异性CTL的产生,刺激T1DM患者PBMC分泌IFN-γ和IL-2,并对抗原肽负载的T2细胞具有明显的杀伤效应。结论 ZnT8(107-115)、ZnT8(115-123)及ZnT8(145-153)可能是HLA-A*0201限制性CTL表位,为基于人ZnT8抗原表位的特异性免疫治疗奠定理论基础。     

胰腺癌高表达抗原MUC4 CTL表位肽的筛选与改造

目的:观察胰腺癌高表达抗原黏蛋白4(MUC4)的改造表位是否有HLA-A2限制性抗肿瘤能力。方法:首先运用RT-PCR和Western blot方法检测MUC4在胰腺癌细胞系CAPAN-2和ASPC-1的表达情况。通过Net CTL 1.2、BIMAS、SYFPEITHI和IEDB软件预测打分来选取MUC4的HLA-A2限制性表位;替换MUC4抗原锚定位点氨基酸获得改造肽;候选表位肽的合成方法为标准的Fmoc化学合成法,结合力实验用于检测候选表位与T2A2细胞表面HLA-A2分子的结合能力,ELISPOT实验检测候选表位肽诱导细胞毒性T淋巴细胞(CTL)分泌IFN-γ的能力,体外细胞毒实验活性检测候选肽诱导CTL的能力。结果:MUC4在胰腺癌细胞系CAPAN-2和ASPC-1均有表达。P1944-1Y、P1944-2L、P1944-1Y2L、P2004和P2004-1Y9V具有较好的结合力,且P2004-1Y9V、P1944-1Y2L等改造肽与HLA-A2的结合力高于原肽。ELISPOT实验结果显示表位肽P1944、P1944-1Y2L、P2004和P2004-1Y9V诱导的CTL具有分泌IFN-γ的能力。P1944-1Y2L和P2004-1Y9V诱导特异性T细胞免疫分泌的IFN-γ略高于原肽。细胞毒实验结果显示表位P1944、P1944-1Y2L、P2004和P2004-1Y9V对CAPAN-2细胞均有一定的杀伤作用。P1944-1Y2L和P2004-1Y9V特异性CTLs对CAPAN-2细胞杀伤率高于原肽特异性CTLs。结论:MUC4抗原改造表位P1944-1Y2L、P2004-1Y9V与天然表位P1944、P2004相比有更高的HLA-A2分子亲和力,保留了原有的免疫原性,并且改造肽抗肿瘤免疫效应强于天然表位。P1944-1Y2L和P2004-1Y9V是优秀的MUC4抗原的HLAA2限制性CTL候选表位,可以成为新的抗肿瘤多肽免疫治疗疫苗的候选表位。     

基于骨肉瘤高表达抗原PBF CTL表位改造及鉴定

目的:观察骨肉瘤高表达抗原乳头瘤病毒结合因子(PBF)的改造表位是否有HLA-A2限制性抗肿瘤能力,开发基于骨肉瘤的多肽免疫治疗。方法:首先运用RT-PCR和Western blot方法检测PBF在骨肉瘤细胞系U2OS和Saos-2的表达情况。然后通过Net CTL 1.2、SYFPEITHI和IEDB软件预测打分来选取PBF的HLA-A2限制性表位。替换PBF抗原锚定位点氨基酸获得改造肽。候选表位肽的合成方法是标准的Fmoc化学合成法,结合力实验用于检测候选表位与T2A2细胞表面HLA-A2分子的结合能力,ELISPOT实验检测候选表位肽诱导细胞毒性T淋巴细胞(CTL)分泌干扰素γ(IFN-γ)的能力,乳酸脱氧酶(LDH)释放实验和羧基荧光素琥珀酰亚胺脂(CFSE)细胞毒实验检测候选肽诱导CTL的能力。结果:PBF在骨肉瘤细胞系U2OS和Saos-2均有表达,候选肽P75-1Y2L、P412-1Y、P416-1Y2L9V、P107-1Y和P435-1Y2L具有较好的结合力,且改造肽与HLA-A2的结合力高于原肽。ELISPOT实验结果显示表位肽P412、P412-1Y、P416、P416-1Y2L9V和P435-1Y2L诱导的CTL具有分泌IFN-γ的能力;P412-1Y和P416-1Y2L9V诱导特异性T细胞免疫分泌的IFN-γ略高于原肽。LDH释放实验和CFSE细胞毒实验结果显示表位P412、P412-1Y、P416和P416-1Y2L9V对U2OS细胞均有一定的杀伤作用,P412-1Y和P416-1Y2L9V特异性CTLs对U2OS细胞杀伤率高于原肽特异性CTLs。结论:PBF抗原改造表位P412-1Y和P416-1Y2L9V与天然表位P412和P416相比有更高的HLA-A2分子亲和力,保留了原有的免疫原性,并且改造肽抗肿瘤免疫效应强于天然表位。P412-1Y和P416-1Y2L9V是优秀的PBF抗原的HLA-A*0201限制性CTL候选表位,可以成为新的抗肿瘤多肽免疫治疗疫苗的候选表位。     

人乳头状瘤病毒11型E7抗原HLA-A*0201限制性细胞毒性T淋巴细胞表位的功能鉴定

目的 筛选和鉴定人乳头状瘤病毒11型E7抗原(HPVllE7)HLA-A*0201限制性细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)表位.方法 预测HPVllE7抗原HLA-A*0201限制性CTL表位并合成相对应的表位多肽和四聚体(tetramer),即HPVllE7 7-15(TLKDIVLDL)、15-23(LQPPDPVGL)、47-55(PLTQHYQIL)、81-89(DLLLGTLNI)和82-90(LLLGTLNIV).从健康HLA-A*0201成人外周血单一核细胞诱导树突状细胞(DC)并负载上述表位多肽,流式细胞技术检测DC成熟分化标记及ELISA法检测DC分泌的IL-12;成熟DC负载各组多肽后观察DC激活T淋巴细胞的效应,ELISA法检测T细胞分泌的IFN-γ;四聚体检测抗原特异性CD8+ T细胞及乳酸脱氢酶(LDH)释放法评价DC诱导的CTL对靶细胞的特异性体外杀伤效应.结果 预测的5条HPVllE7表位多肽均能诱导DC的成熟分化;E7 7-15、82-90和15-23多肽负载的DC能激活T淋巴细胞分泌高水平IFN-γ;E7 7-15多肽负载的DC能刺激特异性tetramer+CD8+细胞增殖且其诱导的CTL对HPVllE7/293细胞产生高效率的特异性杀伤作用(P<0.05).结论 筛选并鉴定出1条HPVllE7HLA-A*0201限制性CTL表位E7 7-15(TLKDIVLDL),负载该表位肽的DC体外可诱导高效、特异性的CTL效应,抗原性较强,有可能作为HPV感染治疗用肽疫苗的候选表位.     

Ran HLA-A 2.1限制性CTL表位的预测及初步鉴定

目的 寻找并鉴定肿瘤相关抗原Ran来源的HLA-A2．1限制性CTL表位，为临床开展基于Ran表位的特异性免疫治疗奠定基础。方法应用超基序和量化基序相结合的方法初步预测Ran的HLA-A2．1限制性CTL表位，利用其与他细胞亲和力实验以及与T2细胞结合稳定性试验初步验证预测结果。结果应用超基序和量化基序相结合的方法得到4个候选表位IMFDVTSRV（88-96），TLGVEVHPL（42-50），YVATLGVEV（39-47）和VLCGNKVDI（118-126），亲和力试验结果显示IMFDVTSRV，TLGVEVHPL和YVATLGVEV有较高的的亲和力而VLCGNKVDI无明显亲和力，结合稳定性试验显示在这4个候选肽中IMFDVTSRV的结合稳定性最好。结论IMFDVTSRV最有可能是肿瘤抗原Ran的HLA-A2．1限制性CTL表位。     

十肽表位HPV-16E711-20氨基酸置换修饰的研究

目的 采用氨基酸置换对人乳头状瘤病毒16型口抗原的人白细胞抗原A2分子限制性细胞毒性T细胞表位HPV-16E7 11-20进行修饰和鉴定。方法 运用量化模体方案对置换后的多肽与人白细胞抗原A2分子的结合系数进行比较，以分子模拟方法确定合成序列，采用标准Fmoc方案进行合成与纯化多肽以及标准^51 Cr释放试验检测特异性细胞毒性T细胞诱导活性。结果 修饰多肽符合人白细胞抗原A2分子限制性细胞毒性T细胞的表位要求，十肽YLLDLQPEVT具有特异性细胞毒性T细胞诱导活性。结论 修饰表位YLLDLQPEVT具有更好的结合力和较强的抗原性，可以代替原有序列E7 11-20（YMLDLQPEIT）作为人乳头状瘤病毒感染治疗性肽疫苗分子设计的新表位。     

A MAGE-A4 peptide presented by HLA-A2 is recognized by cytolytic T lymphocytes.

The MAGE-encoded antigens that are recognized by cytolytic T lymphocytes (CTL) are shared by many tumors and are strictly tumor specific. Clinical trials involving therapeutic vaccination of cancer patients with MAGE antigenic peptides or proteins are in progress. To increase the range of patients eligible for therapy with peptides, it is important to identify additional MAGE epitopes. We have used a method to identify CTL epitopes, which selects naturally processed peptides. CD8(+) T cells, obtained from individuals without cancer, were stimulated with autologous dendritic cells infected with a recombinant adenovirus containing the MAGE-A4 coding sequence. Responder cell microcultures that specifically lysed autologous EBV-transformed B cells infected with vaccinia-MAGE-A4 were cloned using autologous stimulator cells infected with a Yersinia enterocolitica carrying the MAGE-A4 sequence. An anti-MAGE-A4 CTL clone was obtained and the epitope was found to be decapeptide GVYDGREHTV (amino acids 230-239) presented by HLA-A2 molecules. The CTL clone lysed HLA-A2 tumor cells expressing MAGE-A4. This is the first reported antigenic peptide encoded by MAGE-A4. It may be valuable for cancer immunotherapy because MAGE-A4 is expressed in 51% of lung carcinomas and 63% of esophageal carcinomas, whereas about 50% of Caucasians and Asians express HLA-A2.     

Generation of peptide-specific CD8+ T cells by phytohemagglutinin-stimulated antigen-mRNA-transduced CD4+ T cells

Functional analysis of antigen-specific CD8(+) T cells is important for understanding the immune response in various immunological disorders. To analyze CD8(+) T cell responses to a variety of antigens with no readily defined peptides available, we developed a system using CD4(+) phytohemagglutinin (PHA) blasts transduced with mRNA for antigen molecules. CD4(+) PHA blasts express MHC class I and II, and also CD80 and CD86 and are thus expected to serve as potent antigen presenting cells. EGFP mRNA could be transduced into and the protein expressed by more than 90% of either LCL or CD4(+) PHA blasts. Its expression stably persisted for more than 2 weeks after transduction. In experiments with HLA-A*2402 restricted CD8(+) CTL clones for either EBNA3A or a cancer-testis antigen, SAGE, mRNA-transduced lymphoid cells were appropriate target cells in ELISPOT assays or (51)Cr releasing assays. Finally, using CD4(+) PHA blasts transduced with mRNA of a cancer-testis antigen MAGE-A4, we successfully generated specific CTL clones that recognized a novel HLA-B*4002 restricted epitope, MAGE-A4(223-231). Messenger RNA-transduced CD4(+) PHA blasts are thus useful antigen presenting cells for analysis of CD8(+) T cell responses and induction of specific T cells for potential immunotherapy.     

Increased effector–target cell conjugate formation due to HLA restricted specific antigen recognition

The T cell receptor (TCR) orchestrates T cell mediated-cytotoxicity through a complex interaction that results in an antigen-specific effector–target cell conjugate formation. While it is well recognized that specific TCR/antigen interactions generate the immunological synapse, their direct contribution to the effector–target cell conjugate has not been conclusively demonstrated. Moreover, since human cytotoxic T lymphocyte (CTL) clones are also susceptible to antigen-independent adhesion to target cells, it remains unclear whether effector–target cell conjugate formation can serve as an indicator of specific antigen recognition by the TCR. To address this question, a well-characterized epitope-specific CTL clone recognizing the melanoma-associated antigen epitope gp100:209–217 in association with HLA-A*0201 was tested against melanoma cell lines lacking or expressing the HLA-A*0201 allele and/or gp100. In this model, TCR/HLA/antigen interactions cooperated with accessory/adhesion molecules to facilitate effector–target cell conjugate formation. HLA-restricted antigen recognition played a dominant role resulting in up to 2-fold increases in conjugate frequency, and a 50% increase of CTL binding to tumor cells over background. The increased number of CTL contained in conjugates correlated with the number of IFN-γ producing CTL. These results warrant further investigation to evaluate conjugate assays as a potential tool to detect and isolate viable and functionally active CTL. Since conjugate formation analysis does not require knowledge of the target antigen, this assay could potentially be used for enrichment of CTL directed against novel antigens.     

Cross-binding between Plasmodium falciparum CTL epitopes and HLA class I molecules

Plasmodium falciparum CTL epitope minigenes containing HLA-A2 and HLA-B7 subtype supermotifs were cloned into a plasmid expression vector; this expression was measured in eight human HLA class I molecule specific cell lines. Three assays for in vitro antigen presentation analysis were developed to examine the cross-binding between CTL epitopes and HLA class I molecules, including cell surface peptide-MHC class I binding assay, binding stabilization assay and MHC class I assembling assay. The results demonstrated that the HLA-B51 restricted CTL epitope of Plasmodium falciparum could be presented by other HLA class I molecules; however, no other presentation was found for HLA-A2.1 CTL epitope. This work suggests the possibility for improved vaccine-coverage rates by development of a CTL vaccine which contains epitopes capable of cross-binding among different MHC class I alleles.     

肿瘤抗原MAGE—12的表位及二级结构预测

目的 预测肿瘤抗原MAGE－12的α－螺旋二级结构、B细胞表位和MAGE－12的HLA－A2限制性CTL表位。方法 用Garnier-Robson和Chou-Fasman方案预测肿瘤抗原MAGE－12的α－螺旋二级结构;用Kyte-Doolittle亲水方案预测肿瘤抗原MAGE－12的B细胞表位;用简单基序（simple motifs)和延展基序（extended motifs）对肿瘤抗原MAGE－12的HLA－A2限制性CTL表位进行预测。结果 MAGE－12可以含有较多的α－螺旋;B细胞识别的表位可能在82－93残基（QSDEGSSNEE－QE）或附近和3－14残基（LEQRSQHCKPEE）或附近;发现1个HLA－A2限制性CTL表位为FLWGPRALV（271－279）。结论 用亲水性方案和基序法预测肿瘤抗原MAGE－12的B细胞表位和MAGE－12的HLA－A2限制性CTL表位,为实验方法探索MAGE－12的表位提供有用线索。     

Identification of an epitope from the epithelial cell adhesion molecule eliciting HLA-A*2402-restricted cytotoxic T-lymphocyte responses

Because the epithelial cell adhesion molecule (Ep-CAM) is expressed in almost all carcinomas and human leucocyte antigen (HLA)-A*2402 is the most common allele in many ethnic groups, including Japanese, the identification of peptide sequences, which elicit HLA-A*2402-restricted Ep-CAM-specific cytotoxic T-lymphocyte (CTL) responses, would facilitate specific immunotherapy for various histological types of carcinomas. An epitope was identified through the following steps: (i) computer-based epitope prediction from the amino acid sequence of Ep-CAM, (ii) major histocompatibility complex (MHC) stabilization assay to determine the affinity of the predicted peptide with HLA-A*2402 molecules, (iii) stimulation of CD8+ T cells with peptide-pulsed dendritic cells and (iv) testing the CTL specificity by means of enzyme-linked immunospot (ELISPOT) assays, CTL assays and MHC/peptide-tetramer staining. Peripheral CD8+ T cells of four of five healthy donors after three rounds of stimulation with the peptide Ep-CAM173-181 (RYQLDPKFI) secreted interferon-gamma in ELISPOT assays when exposed to the peptide. A CTL clone specific to the peptide efficiently lysed Ep-CAM-expressing cancer cell lines in an HLA-A*2402-restricted fashion. Endogenous processing and presentation of the peptide in a lung cancer cell line were confirmed by means of cold target inhibition assays. The CTL clone was also lytic to normal bronchial epithelial cells but to a lesser extent at low effector: target ratios. All these data suggest that the peptide-specific CTL responses may play some roles both in anti-cancer and autoimmune reactions. The peptide should prove useful to study anti-Ep-CAM CTL responses among population possessing HLA-A*2402.