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。     

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.     

人Ⅱ型胶原的HLA-A~*0201限制性CTL表位的预测及初步鉴定

目的预测和初步鉴定类风湿关节炎(rheumatoid arthritis,RA)主要自身抗原Ⅱ型胶原(CollagenⅡ,CⅡ)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CⅡ抗原表位的特异性免疫治疗奠定基础。方法选取BIMAS、SYFTEPITHI、IEDB、SVMHC、AntiJen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细胞株通过直接免疫荧光法测定各肽与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关节滑液单个核细胞(synovial fluid mononuclear cell,SFMC)分泌IFN-γ的能力。结果综合BIMAS、SYFTEPITHI、IEDB、SVMHC、AntiJen预测结果筛选出来可能与HLA-A*0201结合的5条肽。MHC亲和力实验表明,在候选的5条肽中,P1261、P1365及P1399具有与HLA-A*0201分子结合的能力,平均荧光强度分别为:1.35、2.53、1.78。ELISPOT试验结果表明,P1365具有刺激SFMC分泌IFN-γ的能力。结论表位预测结果与初步鉴定结果具有一致性,两者联合应用初步认为P1365是HLA-A*0201限制性CTL表位的可能性最大,为下一步表位鉴定及基于人CⅡ抗原表位的特异性免疫治疗奠定理论基础。     

HLA-A2限制性Survivin点突变肽诱导特异性抗肝癌CTL反应

目的 探讨HLA-A2限制性Survivin点突变抗原肽体外诱导CTLs的抗肝癌作用.方法 生物信息学软件BIMAS和SYFPEITHI用来鉴定点突变的HLA-A2限制性Survivin抗原九肽;流式细胞术检测突变肽与HLA-A2分子结合效率;肽体外刺激肝癌患者腹水来源的肿瘤浸润淋巴细胞(TILs)诱导反应性CTLs,用流式细胞术及ELISA检测反应性CTLs释放IFN-γ情况;肽诱导的CTLs与肝癌细胞系HepG2及BEL-7402共孵育,CytoTox 96(R)非放射性细胞毒性检测法检测对肿瘤细胞的裂解情况,倒置相差显微镜观察肝癌细胞形态学变化.结果 生物信息学分析筛选出与MHC分子结合效率评分显著提高的点突变Survivin抗原九肽Sur79M2 (KMSSGCAFL),流式细胞术检测证实负载Sur79M2的T2细胞,HLA-A2表达率显著提高,经Sur79M2体外刺激诱导的CTLs与靶细胞共孵育后能释放较高水平的IFN-γ,Sur79M2诱导的CTLs能通过HLA-A2限制性机制有效杀伤肝癌细胞系HepG2,对HLA-A2阴性的BEL-7402细胞无明显杀伤作用.结论 点突变肽Sur79M2能在体外诱导反应性CTLs产生,该CTLs能以HLA-A2限制性方式有效杀伤肝癌细胞系.     

基于HCA587的HLA-A2限制性表位肽的体内免疫原性研究

目的检测来源于HCA587的HLA-A2限制性表位肽免疫HLA-A2转基因小鼠后产生细胞应答的水平,筛选出体内具有免疫原性的HLA-A2限制性表位肽。方法将来源于HCA587的候选HLA-A2限制性肽与MHC-Ⅱ类限制性肽HBV-core128-140、不完全弗氏佐剂、Cp G ODN1826联合应用皮下免疫HLA-A2转基因小鼠,用酶联免疫斑点实验(ELISpot)检测小鼠脾细胞表位肽特异性细胞免疫应答的水平,流式细胞术检测表位肽特异性的CD8+T细胞比例。结果在检测的4个表位肽中,p248-256诱导HLA-A2转基因小鼠产生的细胞免疫应答最强,HLA-A2-H-2Kb-p248-256四聚体染色进一步表明p248-256免疫后可产生p248-256特异性的CD8+T细胞。结论来源于HCA587的HLA-A2限制性表位肽中,p248-256具有较强的体内免疫原性。     

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。     

H-2K~d四聚体的制备及其在检测特异性同种CTLs中的应用

目的制备H-2Kd限制性胰岛GAD抗原肽/H-2Kd四聚体并用于检测相应的特异性同种CTL。方法以原核表达的sH-2Kd-BSP为重链,β2m为轻链,与人工合成的GAD抗原肽在体外利用稀释法进行共折叠复性得到单体。然后在BirA酶作用下对其进行生物素化,通过凝胶过滤层析法纯化生物素化的复合物单体,再与Strep-tavidin-FITC按4∶1比例混合形成四聚体;取Balb/c小鼠(H-2Kd)巨噬细胞加载胰岛GAD抗原肽,作为刺激细胞,取C3H小鼠脾细胞(H-2KK)作为效应细胞,在体外进行混合淋巴细胞培养,以获得针对H-2Kd限制性胰岛GAD抗原肽的特异性同种CTL。结果该四聚体能够与长期混合淋巴细胞培养出的同种特异性T细胞结合。结论成功构建胰岛GAD抗原肽/H-2Kd四聚体,并可用于针对H-2Kd限制性胰岛GAD抗原肽特异性同种CTL的检测。     

肿瘤抗原PIWIL2的HLA-A2限制性CTL表位鉴定

目的:鉴定肿瘤抗原PIWIL2的人类白细胞抗原A2(HLA-A2)限制性细胞毒性T淋巴细胞(CTL)表位。方法:首先运用RT-PCR、Western blot方法检测PIWIL2在肿瘤细胞系MCF-7、SW480和HT-29中的表达情况,然后通过BIMAS、Rank Pep、Net MHC、Net CTL1.2及IEDB软件预测打分来选取PIWIL2的HLA-A2限制性的表位。候选表位肽通过标准的Fmoc化学合成法合成,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CTL分泌IFN-γ的能力,体外细胞毒性实验检测候选肽诱导CTL的能力。结果:PIWIL2在肿瘤细胞系MCF-7、SW480和HT-29中均有表达。候选肽P485、P493、P965具有中等结合力。ELISPOT实验结果显示表位肽P485和P965诱导的CTL具有分泌IFN-γ的能力。细胞毒性实验结果显示表位肽P485和P965对MCF-7细胞均有一定的杀伤作用。结论:表位肽P485和P965是优秀的PIWIL2抗原HLAA2限制性CTL候选表位,可能成为新的抗肿瘤多肽免疫治疗疫苗的候选表位。     

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。     

p210~(bcr-abl)抗原的T细胞表位鉴定及其特异性CTL细胞检测

目的:探讨p210~(bcr-abl)抗原的免疫原性,预测并鉴定该蛋白来源的HLA-A2限制性T细胞表位,并在慢性粒细胞白血病患者中检测其特异性CTL细胞分布.方法:首先利用生物信息学软件预测并选取2个p210~(bcr-abl)来源的表位:~(BCR-ABL)_(642)与~(BCR-ABL)_(926m);T2细胞亲和力实验鉴定短肽与HLA-A2分子的亲和力;然后制备分别锚合这2种表位的可溶性HLA-A2四聚体,流式检测术检测其特异性CTL细胞在CML患者外周血CD8+T细胞中的频率.结果:与健康人群相比,~(BCR-ABL)_(642)和~(BCR-ABL)_(926m)肽表位限制性CTL细胞的频率在CML患者均明显升高(P<0.01);而来自流感病毒短肽的特异性CTL细胞在两个群体中无统计学差异(P>0.05);另外,~(BCR-ABL)_(642)特异性CTL细胞的频率在CML慢性期和急变期之间有统计学差异(P<0.05).结论:所选2种肽表位均具有免疫原性,可建立基于这2种短肽的免疫治疗措施.     

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.     

Identification of a new HLA-A*0201-restricted CD8+ T cell epitope from hepatocellular carcinoma-associated antigen HCA587

For the development of peptide-based cancer immunotherapies, we aimed to identify specific HLA-A*0201-restricted CTL epitopes in hepatocellular carcinoma (HCC) associated antigen HCA587, which has been identified as a member of the cancer/testis (CT) antigens highly expressed in HCC. We first combined the use of an HLA-A*0201/peptide binding algorithm and T2 binding assays with the induction of specific CD8(+) T cell lines from normal donors by in vitro priming with high-affinity peptides, then IFN-gamma release and cytotoxicity assays were employed to identify the specific HLA-A*0201 CD8(+) T cell epitope using peptide-loaded T2 cells or the HCA587 protein(+) HCC cell line HepG2. In the six candidate synthesized peptides, two peptides showed higher binding ability in T2 binding assays. No. 2 peptide, encompassing amino acid residues FLAKLNNTV (HCA587(317-325)), was able to activate a HCA587-specific CD8(+) T-cell response in human lymphocyte cultures from two normal donors and two HCC patients, and these HCA587-specific CD8(+) T cells recognized peptide-pulsed T2 cells as well as the HCA587 protein(+) HCC cell line HepG2 in IFN-gamma release and cytotoxicity assays. The results indicate that no. 2 peptide is a new HLA-A*0201-restricted CTL epitope capable of inducing HCA587-specific CTLs. Our data suggest that identification of this new HCA587/HLA-A*0201 peptide FLAKLNNTV may facilitate the design of peptide-based immunotherapies for the treatment of HCA587-bearing HCC patients.     

Prediction of HLA class I-restricted T-cell epitopes of islet autoantigen combined with binding and dissociation assays

Identification of cognate peptides recognized by human leucocyte antigen (HLA)/T cell receptor (TCR) complex provides insight into the pathogenic process of type 1 diabetes (T1D). We hypothesize that HLA-binding assays alone are inadequate metrics for the affinity of peptides. Zinc transporter-8 (ZnT8) has emerged in recent years as a novel, major, human autoantigen. Therefore, we aim to identify the HLA-A2-restricted ZnT8 epitopes using both binding and dissociation assays. HLA class I peptide affinity algorithms were used to predict candidate ZnT8 peptides that bind to HLA-A2. We analyzed 15 reported epitopes of seven β-cell candidate autoantigens and eight predicted candidate ZnT8 peptides using binding and dissociation assays. Using IFN-γ ELISpot assay, we tested peripheral blood mononuclear cells (PBMCs) from recent-onset T1D patients and healthy controls for reactivity to seven reported epitopes and eight candidate ZnT8 peptides directly ex vivo. We found five of seven recently reported epitopes in Chinese T1D patients. Of the eight predicted ZnT8 peptides, ZnT8(153-161) had a strong binding affinity and the lowest dissociation rate to HLA-A*0201. We identified it as a novel HLA-A*0201-restricted T-cell epitope in three of eight T1D patients. We conclude that ZnT8(153-161) is a novel HLA-A*0201-restricted T-cell epitope. We did not observe a significant correlation (P = 0.3, R = - 0.5) between cytotoxic T cell (CTL) response and peptide/HLA*0201 complex stability. However, selection of peptides based on affinity and their dissociation rate may be helpful for the identification of candidate CTL epitopes. Thus, we can minimize the number of experiments for the identification of T-cell epitopes from interesting antigens.     

Recognition of HLA-DR1/DRB1*0101 molecules presenting HLA-A2 derived peptides by a human recombinant antibody,Fab-5 A1

MHC molecules present peptides in their binding groove to T-cell receptors inducing proliferation or cytotoxicity of alloreactive T cells. A previously generated human monoclonal antibody (mAb) UL-5 A1, recognizing a conformational epitope formed by HLA DR1/DRB1*0101 molecules and HLA-A2 derived peptides, demonstrates T-cell-like recognition of the peptide/MHC complex (PMC). To study the genes of the antigen binding region, the nucleotide sequences of the rearranged genes in the variable regions of UL-5 A1 were determined and the V-gene usage (VH3, Vλ2) was identified by comparison with published germlines. The genes encoding heavy (Fd) and light (L) chains of UL-5 A1 were linked and expressed in a bacterial system. Specificity of the recombinant Fab-5 A1 was determined with HLA-typed LCLs by flow cytometric analysis. As demonstrated in competitive inhibition assays, UL-5 A1 and Fab-5 A1 recognize the same PMC epitope on HLA-A2⁺, -DR1/DRB1*0101⁺ typed LCLs. Additionally, mAb UL-5 A1 and Fab-5 A1 both recognize HLA-A2^–, -DR1/DRB1*0101⁺ LCLs exogenously loaded with HLA-A2 peptides (105–117, 103–117). UL-5 A1-like antibodies against peptide/MHC complexes could prove valuable tools for research on T-cell recognition and MHC function.     

Activation of epitope-specific memory cytotoxic T lymphocyte responses by synthetic peptides

Cytotoxic T lymphocytes (CTL) recognize antigens as short peptides selected for presentation by their ability to bind to MHC class I molecules. Polyclonal Epstein–Barr virus (EBV)-specific memory CTL responses, reactivated from blood lymphocytes of HLA-A11-positive individuals by stimulation with the autologous EBV-transformed lymphoblastoid cell line (LCL), are often dominated by reactivities directed to the peptide epitope IVTDFSVIK (IVT), corresponding to amino acids 416–424 of EBV nuclear antigen-4 (EBNA4). We now report the selective activation of IVT-specific CTL by stimulation of lymphocytes with the corresponding synthetic peptide. A more than 10-fold increase in frequency of CTL clones with this specificity (from 8% to 96%) was obtained when the peptide was presented by HLA-A11-transfected T2 cells (T2/A11). Titration of synthetic peptide in cytotoxic assay demonstrated that clones activated under these conditions are as efficient as clones activated by conventional LCL stimulations. Induction of memory CTL responses required low surface density of MHC : peptide complexes, since reactivation was achieved by stimulation with T2/A11 cells pulsed with concentrations of peptide that are suboptimal for induction of target cell lysis. This protocol of activation revealed the presence of IVT-specific CTL precursors in a donor that failed to mount an IVT-specific response upon stimulation with the autologous B95·8 virus-transformed LCL. The results suggest that stimulation with synthetic peptide epitopes can be efficiently used for induction of memory CTL responses, and may be particularly helpful for the selective expansion of subdominant CTL specificities.     

Variation in cytotoxic T-lymphocyte responses to peptides derived from tyrosinase-related protein-2

In this study, we developed three optimized peptide ligands (OPL) that demonstrate increased affinities for HLA-A*0201 compared with wild-type tyrosinase-related protein-2 (TRP-2) peptide. The OPL contain amino acids from TRP-2((180-188)) and preferred primary and auxiliary HLA-A*0201 anchor residues. Cytotoxic T lymphocyte (CTL) lines were generated against wild-type TRP-2 peptide and OPL by multiple rounds of peptide stimulation of peripheral blood mononuclear cells from HLA-A2*0201(+) healthy individuals. CTL reactivity profiles to three different OPL were donor-dependent. Among donors, at least one OPL was particularly stimulatory and elicited high levels of CTL that cross-reacted with wild-type TRP-2 peptide. Cytotoxicity assays using CTL raised on wild-type TRP-2 peptide or OPL demonstrated lysis of HLA-A2-positive glioblastoma cells. Molecular models of TRP-2 and OPL peptides docked with HLA-A*0201 demonstrated that substitution of F for S at position 1 (P1) oriented the peptides favoring a pi-pi aromatic interaction with W 167 of HLA-A*0201. This in turn positions P5 and P8 aromatic rings to face solvent that may promote binding to the T-cell receptor, leading to a robust T-cell activation. The results of this study further substantiate the concept that rational design and testing of multiple peptides for the same T-cell epitope should elicit a broader response among different individuals than single peptide immunization. Our results may partially explain why some patients have better clinical responses to peptide-based immunotherapy, whereas others respond poorly.     

Cytotoxic T-lymphocyte clones, established by stimulation with the HLA-A2 binding p5365-73 wild type peptide loaded on dendritic cells In vitro, specifically recognize and lyse HLA-A2 tumour cells overexpressing the p53 protein

Mutations in the tumour suppressor gene p53 are among the most frequent genetic alterations in human malignancies, often associated with an accumulation of the p53 protein in the cytoplasm. We have generated a number of cytotoxic T lymphocyte (CTL) clones that specifically recognize the HLA-A*0201 p53 wild type peptide RMPEAAPPV [65-73], designated R9V, by the in vitro stimulation of CD8 enriched peripheral blood lymphocytes from a healthy HLA-A*0201 donor using peptide loaded autologous dendritic cells. A total of 22 CTL clones were generated from the same bulk culture and demonstrated to carry identical T-cell receptors. The CTL clone, 2D9, was shown to specifically lyse the HLA-A*0201+ squamous carcinoma cell line SCC9 and the breast cancer cell line MDA-MB-468. Our data demonstrate that human peripheral blood lymphocytes from normal healthy individuals comprise T cells capable of recognizing p53 derived wild type (self) peptides. Furthermore, the capacity of R9V specific T cell clones to exert HLA restricted cytotoxicity, argues that the R9V peptide is naturally presented on certain cancer cells. This supports the view that p53 derived wild type peptides might serve as candidate target antigens for the immunotherapeutic treatment of cancer.     

Prediction of HLA class I-restricted T-cell epitopes of islet autoantigen combined with binding and dissociation assays

Identification of cognate peptides recognized by human leucocyte antigen (HLA)/T cell receptor (TCR) complex provides insight into the pathogenic process of type 1 diabetes (T1D). We hypothesize that HLA-binding assays alone are inadequate metrics for the affinity of peptides. Zinc transporter-8 (ZnT8) has emerged in recent years as a novel, major, human autoantigen. Therefore, we aim to identify the HLA-A2-restricted ZnT8 epitopes using both binding and dissociation assays. HLA class I peptide affinity algorithms were used to predict candidate ZnT8 peptides that bind to HLA-A2. We analyzed 15 reported epitopes of seven β-cell candidate autoantigens and eight predicted candidate ZnT8 peptides using binding and dissociation assays. Using IFN-γ ELISpot assay, we tested peripheral blood mononuclear cells (PBMCs) from recent-onset T1D patients and healthy controls for reactivity to seven reported epitopes and eight candidate ZnT8 peptides directly ex vivo. We found five of seven recently reported epitopes in Chinese T1D patients. Of the eight predicted ZnT8 peptides, ZnT8_153–161 had a strong binding affinity and the lowest dissociation rate to HLA-A*0201. We identified it as a novel HLA-A*0201-restricted T-cell epitope in three of eight T1D patients. We conclude that ZnT8_153–161 is a novel HLA-A*0201-restricted T-cell epitope. We did not observe a significant correlation (P = 0.3, R = ? 0.5) between cytotoxic T cell (CTL) response and peptide/HLA*0201 complex stability. However, selection of peptides based on affinity and their dissociation rate may be helpful for the identification of candidate CTL epitopes. Thus, we can minimize the number of experiments for the identification of T-cell epitopes from interesting antigens.     

Identification of HLA-A24-restricted CTL epitope encoded by the matrix protein pp65 of human cytomegalovirus

The activation of a specific cellular immune response against human cytomegalovirus (CMV) is an important key factor to solving CMV infection after bone marrow transplantation (BMT). In the present study, our purpose was to identify the HLA-A24-restricted cytotoxic T cell (CTL) epitope from the CMV immunogenic matrix protein pp65. We selected five CMV pp65 peptides with HLA-A24 binding motif from the HLA peptide binding predictions web site. Peptide binding assay was performed using biotinylated HLA-A24-restricted MAGE-1 peptide as a reference peptide and transporter associated with antigen processing (TAP)-deficient T2-A24 cells expressing high level of HLA-A24 protein as target cells. After co-incubation of biotinylated MAGE-1 and titrated amounts of competitor peptides with T2-A24 cells, the binding of each peptide was analyzed on a flow cytometer. Peptide binding assay showed that three out of five peptides derived from CMV pp65 bound to T2-A24 cells with various affinity levels. CTL induction assay using peptide-pulsed DC derived from eight HLA-A24(+) donors revealed that the peptide (QYDPVAALF) with the highest affinity was able to elicit potent CTLs which killed peptide-pulsed TISI cells. These CTLs were found to show the killing activity against human fibroblast cells transduced with both HLA-A*2402 and CMV pp65 cDNAs, and CMV-infected HLA-A24(+) fibroblast cells. These results suggested that the peptide (QYDPVAALF) is one of HLA-A24-restricted CTL epitope derived from CMV pp65 protein and may be of therapeutic value in peptide-based vaccines against CMV infection in BMT patients.     

Limited plasticity in the recognition of peptide epitope variants by an alloreactive CTL clone correlates directly with conservation of critical residues and inversely with peptide length

Although self-restricted T cells are peptide-specific and can distinguish among closely related ligands, they have some flexibility in the recognition of sequence variants of their natural peptide epitopes. Alloreactive cytotoxic T lymphocytes (CTL) can recognize specific peptides bound to the allo-major histocompatibility complex (MHC) molecule, but their plasticity in the recognition of related peptide variants has not been properly defined. The anti-B*2705 alloreactive CTL 27S69 specifically recognizes a natural octamer ligand of HLA-B*2705. In this study, we tested the recognition of a nested set of epitope variants by this CTL clone. Although none of these peptides was recognized equally as the natural epitope, two of the peptide variants were recognized with only slightly decreased efficiency. Peptide sensitization assays showed that CTL recognition of epitope variants correlated directly with conservation of two non-anchor residues that were critical for recognition of the natural epitope, and inversely with peptide length. Molecular modeling of the peptide variants complexed with B*2705 provided a rational explanation for their differential recognition. Location of the two critical peptide residues at the right three-dimensional space favored efficient recognition by CTL 27S69. The negative effect of increasing peptide length on recognition was due to the bigger bulging surface between the two critical residues, which precluded for optimal interaction with the specific T-cell receptors (TCR). Our results demonstrate that an alloreactive CTL has a degree of plasticity in the recognition of peptide epitope variants that is comparable to that of peptide-specific self-restricted CTL, and define the structural features determining crossreaction among related peptides.