小鼠肝素酶H-2Kb限制性CTL表位的实验鉴定

目的实验鉴定H-2Kb限制性小鼠肝素酶(mHpa)CTL表位。方法5条mHpa表位分别负载C57BL/6小鼠骨髓来源树突状细胞,并进一步诱导产生表位特异性的效应细胞,采用标准4 h51Cr释放试验检测效应细胞对不同靶细胞的免疫杀伤效应;采用ELISPOT检测效应细胞分泌IFN-γ的能力。结果5条mHpa CTL表位中,仅mHpa398-405和mHpa519-526可以诱导小鼠产生特异性CTL,对同来源的B16黑色素瘤细胞、Lewis肺癌细胞和EL-4淋巴瘤细胞具有明显的免疫杀伤效应,而对H-2Kb阴性的P815肥大细胞瘤细胞不具有免疫杀伤效应,进一步研究发现,上述多肽特异性CTL对自体淋巴细胞和DC细胞不具有杀伤效应,另一方面,mHpa398-405和mHpa519-526还可促进效应细胞分泌IFN-γ的能力。结论mH-pa398-405、mHpa519-526为小鼠肝素酶来源且受H-2Kb限制性CTL表位,小鼠体内可以诱导产生表位特异性的CTL反应。     

结核杆菌抗原Rv0173的HLA-A*0201限制性CTL表位的预测及鉴定

目的 鉴定结核杆菌(Mycobacterium tuberculosis,Mtb)抗原Rv0173中的HLA-A*0201限制性CTL表位.方法 应用数据库SYFPETTHI预测结核杆菌Rv0173中可能存在的HLA-A*0201限制性CTL表位,经流式细胞术分析各抗原肽与HLA-A*0201的亲合力,经时间分辨荧光法检测外周血单个核细胞(PBMCs)对各抗原肽产生的增殖反应,经细胞毒性实验研究各抗原肽诱导的特异性T细胞的细胞毒杀伤活性,逐步鉴定Rv0173的HLA-A*0201限制性CTL表位.结果 位于Rv0173氨基酸序列肽3(21-30aa)和抗原肽7(161-170aa)与HLA-A*0201分子具有较高的亲合力,并都能刺激HLA-A*0201阳性个体PBMCs增殖,并诱导产生特异性杀伤活性.结论 肽3(RLSQSADQYL)(21-30aa)和肽7(LLRGGGLVNL)(161-170aa)是抗原Rv0173上HLA-A*0201限制性CTL的优势表位,可作为结核疫苗设计的候选表位.     

MAGE-12的HLA-A2限制性CTL表位肽预测及其三维结构构建

目的从理论上分析、预测黑色素瘤抗原(MAGE)-12的人类白细胞抗原(HLA)-A2限制性细胞毒性T淋巴细胞(CTL)表位肽,并构建其三维结构。方法以肿瘤特异性抗原MAGE-12为研究目标,采用超基序法、量化基序法、多项式法、延展基序法与三维结构构建相结合的CTL表位预测方法。结果预测出的MAGE-12的表位中有4个符合HLA-A2限制性CTL表位要求。结论预测出的4个HLA-A2限制性CTL表位为MAGE-12的表位的可能性较大,经后续实验筛选、鉴定后,可用于基于MAGE-12的肿瘤治疗性多肽疫苗的设计研究。     

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。     

隐球菌抗原MP98的HLA-A*0201限制性CD8+CTL表位的预测及鉴定

目的鉴定隐球菌抗原MP98中的HLA-A＊0201限制性CD8＋CTL表住。方法应用数据库SYFPEITHI预测隐球菌MP98中可能存在的HLA-A＊0201限制性CD8＋CTL表位，经流式细胞术分析各抗原肽与HLA-A＊0201的亲合力，经时间分辨荧光法检测外周血单个核细胞（PBMCs）对各抗原肽产生的增殖反应，经细胞毒性实验研究各抗原肽诱导的特异性T细胞的细胞毒杀伤活性，逐步鉴定MP98的HLA-A＊0201限制性CD8＋CTL表位。结果位于MP98氨基酸序列肽5（436-444aa）与HLA-A＊0201分子具有较高的亲合力，并都能刺激HLA-A＊0201阳性个体者PBMC增殖，并诱导产生具有特异性杀伤活性的CTL。结论肽5GMFDGLSGV（436-444aa）是MP98上HLA-A＊0201限制性CD8＋CTL的优势表位，可作为隐球菌疫苗设计的候选表位。     

Eps8抗原HLA-A* 0201限制性CTL表位的预测和鉴定

目的:寻找并鉴定肿瘤相关抗原Eps8来源的HLA-A*0201限制性CTL表位,为临床开展基于Eps8表位的特异性免疫治疗奠定基础。方法:通过在线生物学软件从C-末端酶切位点、MHC-I类分子结合力及TAP转运效率3个方面初步预测Eps8抗原的HLA-A*0201限制性CTL表位,通过肽/MHC分子结合力及肽/MHC分子复合物稳定性实验初步验证预测结果,利用酶联免疫斑点检测(enzyme-linked immuospot assay,ELISPOT)方法检测候选表位刺激健康志愿者外周血单个核细胞(PBMNC)分泌IFN-γ的能力,以及LDH细胞毒实验验证其体外杀伤肿瘤细胞的功能,最后在HLA-A*0201/Kb转基因小鼠中检测候选表位的体内免疫功能。结果:通过在线生物学软件筛选得到4个候选表位。结合力实验结果显示,p360-368具有高结合力,p101-109、p276-284具有中等结合力。稳定性实验结果显示,该3个表位的DC50均大于8 h。LDH细胞毒实验中p101-109、p276-284、p360-368都能诱导产生特异性CTL,对靶细胞具有显著的杀伤效应,还能提高效应细胞分泌IFN-γ的水平。在HLA-A*0201/Kb转基因小鼠体内免疫功能检测中同样证实该Eps8抗原表位能产生强烈的免疫应答。结论:天然表位p101-109、p276-284、p360-368可能是Eps8抗原HLA-A*0201限制性CTL表位。     

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.     

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。     

生物信息学预测GPⅡb／Ⅲa抗体CTL、Th的混合表位

背景:GPb/Ⅲa是特发性血小板减少性紫癜患者最常见的血小板糖蛋白,已证实该蛋白独特型抗体结合血小板后会激活补体,破坏血小板.目的:以GPⅡbⅢa抗体为靶抗原,应用生物信息学软件对其CTL,Th细胞表位进行多参数预测.设计、时间及地点:开放性实验,于2008-03/08在珠江医院血液科实验室完成.材料:GPⅡb/Ⅲa抗体的氨基酸序列由GENBANK公司提供.方法:分别应用SYFPEITHI,RANKPEP,BIMAS,SVMHC,PREDEP,MHCPRED,PROPRED软件对人和鼠源的血小板糖蛋白GPⅡb/Ⅲa抗体蛋白进行HLA-A*0201,HLA-A*1101,HLA-A*2401限制性表位预测,经去除能引起自身免疫的已发表的多肽,取前者覆盖后二者的多肽为混合CTL表位,再联合软件predTAP、TAPPred的TAP结合预测结果,以及NetChop、MAPPP、PAProC软件的蛋白酶切位点预测结果,取能包含二者的多肽为CTL修正表位.用基于肽MHC-Ⅱ结合的算法Syfpeithi,RANKPEP,Mhcpred,HLAPRED进行HLA-DR的Th表位预测,最后取Th表位覆盖CTL修正表位,得到CTL、Th细胞混合表位.结果:从1 740条多肽中筛选出5条人源抗人血小板糖蛋白GPⅡb/Ⅲa抗体的CTL、Th细胞混合表位肽,即Anti-GPⅡb/Ⅲa-Human的第1-15,24-38,50-64,65-81,109-121位:筛选出5条鼠源抗人血小板糖蛋白GPⅡb/Ⅲa抗体的CTL、Th细胞混合表位肽,即Anti-GPⅡb/Ⅲa-Mice的第1-15,26-40,46-60,68-82,93-107位.结论:根据血小板糖蛋白GPⅡb/Ⅲa抗体预测的CTL、Th细胞优势抗原表位,可用于制备特异性抗体,可成为特发性血小板减少性紫癜新疫苗研究的靶点.     

汉滩病毒核蛋白HLA-A*02限制性CTL表位的预测及实验鉴定

目的:采用人工神经网络模型及在线软件BIMAS和SYFPEITHI共同预测汉滩病毒核蛋白HLA-A)02限制的CTL表位,并对CTL表位进行实验鉴定。方法:①选取2004-11解放军第四军医大学唐都医院住院的肾综合征出血热患者1例,汉滩病毒特异性IgM抗体为阳性。无菌抽取肾综合征出血热患者外周血,分离外周血单个核细胞,液氮冻存备用。②采用JavaNNS软件模拟神经系统对信息的处理过程,建立人工神经网络模型。训练人工神经网络的9肽包括584种HLA-A)02结合肽和130种HLA-A)02非结合肽,随机数字表法分为3部分:80%为训练肽(用来训练模型,使其学习其中隐含的规律),10%为确认肽(防止训练过度,使训练在适当的时候终止),10%为试验肽(对训练好的模型进行初步的评价)。无、低、中、高亲和力9肽训练人工神经网络的输出值分别为0.1,0.4,0.6,0.8。因此,人工神经网络模型的默认阈值为0.4。此外,还应用在线软件BIMAS和SYFPEITHI,共同预测汉滩病毒核蛋白的CTL表位。③采用ELISPOT鉴定汉滩病毒核蛋白特异的HLA-A)02限制的CTL表位。结果:①人工神经网络、BIMAS和SYFPEITHI预测的准确度:3种方法预测的灵敏度分别达到0.89,0.78,0.97,特异性分别为0.90,0.87,0.20。三者共同预测到23个汉滩病毒核蛋白特异的CTL表位。②HLA-A)02限制的CTL表位的实验鉴定:在可刺激肾综合征出血热患者外周血单个核细胞产生阳性反应的3条15肽中,预测到了4个HLA-A)02限制的CTL表位。经ELISPOT实验证实,其中的2种9肽可刺激HLA-A)02阳性的肾综合征出血热患者外周血单个核细胞分泌γ干扰素。结论:肾综合征出血热患者中存在着汉滩病毒特异的CTL应答。汉滩病毒核蛋白CTL表位的有效预测,大幅度减少了合成候选9肽的数量,降低了成本。为肾综合征出血热细胞免疫应答的研究以及多肽疫苗的设计,奠定了重要基础。     

Vaccines targeting the cancer-testis antigen SSX-2 elicit HLA-A2 epitope-specific cytolytic T cells

The cancer-testis antigen synovial sarcoma X breakpoint-2 (SSX-2) is a potentially attractive target for tumor immunotherapy based upon its tissue-restricted expression to germline cells and its frequent expression in malignancies. The goal of this study was to evaluate genetic vaccine encoding SSX-2 to prioritize human leukocyte antigen (HLA)-A2-specific epitopes and determine if a DNA vaccine can elicit SSX-2-specific cytotoxic T lymphocytes (CTLs) capable of lysing prostate cancer cells. HLA-A2-restricted epitopes were identified based on their in vitro binding affinity for HLA-A2 and by the ability of a genetic vaccine to elicit peptide-specific CTL in A2/DR1 (HLA-A2.1+/HLA-DR1+/H-2 class I-/class II-knockout) transgenic mice. We found that SSX-2 peptides p41-49 (KASEKIFYV) and p103-111 (RLQGISPKI) had high affinity for HLA-A2 and were immunogenic in vivo; however, peptide p103-111 was immunodominant with robust peptide-specific immune responses elicited in mice vaccinated with a plasmid DNA vaccine encoding SSX-2. Furthermore, p103-111-specific CTLs were able to lyse an HLA-A2+ prostate cancer cell line. The immunodominance of this epitope was found not to be due to a putative HLA-DR1 epitope (p98-112) flanking p103-111. Finally, we demonstrated that SSX-2 epitope-specific CTLs could be detected and cultured from the peripheral blood of HLA-A2+ prostate cancer patients, notably patients with advanced prostate cancer. Overall, we conclude that SSX-2 peptide p103-111 is an immunodominant HLA-A2-restricted epitope, and epitope-specific CD8 T cells can be detected in patients with prostate cancer, suggesting that tolerance to SSX-2 can be circumvented in vivo. Together, these findings suggest that SSX-2 may be a relevant target antigen for prostate cancer vaccine approaches.     

Multiple antigenic peptides based on H-2K(b)-restricted CTL epitopes from murine heparanase induce a potent antitumor immune response in vivo

Accumulating research suggests that heparanase may be a universal tumor-associated antigen (TAA). Several heparanase T-cell epitopes from humans and mice have already been identified. However, because of low immunogenicity, polypeptide vaccines usually have difficulty inducing effective antitumor immune responses in vivo. In this study, to increase the immunogenicity of polypeptide vaccines, we designed and synthesized two four-branch multiple antigenic peptides (MAP) on the basis of mouse heparanase (mHpa) T-cell epitopes (mHpa398 and mHpa519). The dendritic cells (DC) from mice bone marrow loaded with above MAP vaccines from heparanase were used to evaluate immune response against various tumor cell lines, compared with immune response to their corresponding linear peptides, ex vivo and in vivo. We further assessed IFN-γ release both in CD4(+) T-cell-depleted and nondepleted mice. The results showed that effectors generated from DCs, loaded with MAP-vaccinated mice splenocytes, induced a stronger immune response against target cells expressing both heparanase and H-2K(b) than did effectors generated from mice vaccinated with their corresponding linear peptides. Heparanase-specific CD8(+) T-cell responses induced by MAP and linear peptide vaccination required synergy of CD4(+) T cells. In addition, heparanse-derived MAP vaccines significantly inhibited the growth of B16 murine melanoma in C57BL/6 mice, while also increasing the survival rate of tumor-bearing mice. Our data suggest that MAP vaccines based on T-cell epitopes from heparanase are efficient immunogens for tumor immunotherapy.     

Identification of the immunodominant peptides of the MART-1 human melanoma antigen recognized by the majority of HLA-A2-restricted tumor infiltrating lymphocytes

Four melanoma proteins, MART-1, gp100, tyrosinase, and tyrosinase- related protein-1 (gp75) were evaluated for recognition by HLA-A2- restricted melanoma-specific cytotoxic T lymphocytes (CTLs) derived from the tumor-infiltrating lymphocytes (TIL) of 10 different patients. 9 of 10 TIL recognized MART-1, 4 recognized gp100 (including 3 that also recognized MART-1), but none of the TIL recognized tyrosinase or gp75. Based on the known HLA-A2.1 peptide binding motifs, 23 peptides from MART-1 were synthesized in an attempt to identify the epitopes recognized by TIL. Three peptides were recognized by TIL when pulsed on T2 target cells. One of the 9-mer peptides, AAGIGILTV, was most effective in sensitizing the T2 cells for TIL lysis. This peptide was recognized by 9 of 10 HLA-A2-restricted melanoma-specific CTLs. Therefore, this peptide appears to be a very common immunogenic epitope for HLA-A2-restricted melanoma-specific TIL and may be useful for the development of immunotherapeutic strategies.     

Resistance of cytotoxic T lymphocytes to the lytic effects of their toxic granules

A cytotoxic T lymphocyte (CTL) characteristically kills target cells one after the other by releasing toxic granules that contain one or more cytolytic components. To determine how CTLs avoid destroying themselves when they release granules and lyse target cells, 7 murine CD8+ CTL cell lines were compared with 19 other cell lines for susceptibility to lysis by the isolated toxic granules. Murine CD8+ CTLs were clearly the most resistant cells: granules did not lyse them even after they were exposed to azide, cyanide, and 2-deoxyglucose, conditions that were found to enhance the susceptibility of all the other cells tested, including other T cells. Thus, resistance of CD8+ CTLs to cytotoxic granules appears to be independent of cellular ATP. To reconcile these findings with other observations that, under some circumstances, CTLs can be lysed by other CTLs, we suggest a model in which a CTL releases only a limited proportion of its toxic granules at each antigen-specific encounter with a target cell; the amount released is sufficient to kill most target cells but to leave the CTL undamaged and with enough granules to attack other target cells.     

Competition among peptides in melanoma vaccines for binding to MHC molecules

The effectiveness of peptide-based cancer vaccines depends on the ability of peptides to bind to MHC molecules on the surface of antigen-presenting cells, where they reconstitute epitopes for cytotoxic T lymphocytes (CTLs). Multivalent vaccines have advantages over single-peptide vaccines; however, peptides may compete for binding to the same MHC molecules. In particular, it is possible that peptides with high affinity for MHC molecules prevent the binding of lower-affinity peptides. However, only small numbers of peptide/MHC complexes per cell are required for CTL recognition. Thus, the authors hypothesized that competition of peptides for MHC binding would not significantly reduce CTL recognition of individual peptides within a multiple-peptide mixture, and this hypothesis was tested by a series of experiments performed in vitro. In multiple experiments, two peptides with different affinities for HLA-A*0201 molecules were mixed at various concentrations and pulsed onto HLA-A2 cells, which were then evaluated for susceptibility to lysis by HLA-A*0201-restricted CTLs. CTL recognition of the melanoma peptides gp100(154-162) (KTWGQYWQV), gp100(280-288) (YLEPGPVTA), and tyrosinase(369-377D) (YMDGTMSQV) was maintained even when target cells were co-pulsed with equimolar concentrations of peptides with comparable or higher affinity for HLA-A2. In some cases, CTL recognition was maintained even when the higher-affinity peptide was present at concentrations several orders of magnitude higher than the target peptide. In addition, CTLs generated by in vitro stimulation with a peptide mixture developed reactivity to three different peptides, at a level comparable to that obtained by stimulation with each individual peptide separately. These data suggest that CTLs can respond to multiple peptides presented on the same antigen-presenting cells and justify further investigation, in clinical trials, of multiple-peptide cancer vaccines.     

New peptide vaccine candidates for epithelial cancer patients with HLA-A3 supertype alleles

We previously identified 2 cancer-associated antigens, immediate early response gene X-1 (IEX) and small GTPase (Ran), and their 5 epitopes using human leukocyte antigen (HLA)-A33-restricted and tumor-infiltrating T cells from a colon cancer patient. In this study, we examined whether or not these peptides can induce cytotoxic T lymphocytes (CTLs) in HLA-A11+ or HLA-A31+ epithelial cancer patients because the HLA-A11, HLA-A31, and HLA-A33 alleles share binding motifs as an HLA-A3 supertype family, which is widely distributed in many ethnic populations. Among them, the 2 peptides, IEX 47-56 and IEX 61-69, induced peptide-specific CTLs from peripheral blood mononuclear cells of cancer patients with the HLA-A11 and HLA-A31 alleles more efficiently than the other 3 peptides. Antibody blocking and cold inhibition experiments revealed that the cytotoxicity of peptide-induced CTLs against cancer cells was attributable to peptide-specific and CD8+ T cells. Together with our previous findings, these results indicate that the 2 IEX peptides could be appropriate vaccine candidates for HLA-A11, HLA-A31, and HLA-A33 positive epithelial cancer patients. This information could expand the chance of a peptide-based cancer vaccine for epithelial cancer patients of many ethnic populations.     

Major histocompatibility complex-restricted lysis of neuroblastoma cells by autologous cytotoxic T lymphocytes

Vigorous host immune reactivity to neuroblastoma may correlate with better prognosis, but identification of human cytotoxic T-lymphocyte (CTL) responses has been relatively unsuccessful. We generated neuroblastoma-reactive CTL lines from two human leukocyte antigen (HLA) A2+ neuroblastoma patients by stimulation of peripheral blood lymphocytes (PBLs) with irradiated autologous tumor cells pretreated with interferon-gamma in the presence of low concentrations of interleukin-2 (5 U/mL). These lines lyse autologous tumor cells but do not kill HLA mismatched allogeneic tumor cells, Epstein-Barr virus-transformed autologous B cells, or standard natural killer cell targets. Cytotoxic T lymphocytes generated from one patient recognize tumor cells from several HLA-A2 matched children, although the other patient's CTLs do not kill tumor cells from other HLA-A2+ individuals. Pretreatment of CTLs or target cells with appropriate standard monoclonal antibodies demonstrates that these CTLs are major histocompatibility complex class I (HLA-A2) restricted and that the effector cell population is CD8+. Our findings suggest that these tumor cells express at least one common HLA-A2 restricted antigen and at least one unique private epitope. Autologous tumor-specific CTLs can be readily generated from patients' PBLs and maintained in long-term culture using standard techniques.     

Induction of human leukocyte antigen-A26-restricted and tumor-specific cytotoxic T lymphocytes by a single peptide of the SART1 antigen in patients with cancer with different A26 subtypes

Peptide antigens available for use in specific immunotherapy of patients with cancer have not been fully determined. Although the authors have reported the SART1 gene encoding epitopes recognized by HLA-A2601-restricted and tumor-specific cytotoxic T lymphocytes (CTLs), the HLA-A26 allele is mainly subdivided into A2601, A2602, and A2603 subtypes. In this study, the authors attempted to determine whether the SART1-derived peptide at position 736-744 (KGSGKMKTE) is suitable to induce HLA-A26-restricted and tumor-specific CTLs in patients with cancer who have these subtypes. This peptide induced the HLA-A26 subtype-restricted and tumor-specific CTLs in HLA-A2601+ or HLA-A2603+ peripheral blood mononuclear cells, respectively. It also induced the HLA-A26-restricted CTL activity in HLA-A2602+ peripheral blood mononuclear cells. Therefore, this peptide could be useful for specific immunotherapy of patients with cancer who have any of the three HLA-A26 subtypes.     

Cross-reactive lysis of trinitrophenyl (TNP)-derivatized H-2 incompatible target cells by cytolytic T lymphocytes generated against syngeneic TNP spleen cells

Normal spleen cells, when cultured with irradiated trinitrophenyl (TNP)-derivatized syngeneic spleen cells, develop cytotoxic effectors that lyse most effectiviely a TNP-derivatized target that is H-2 compatible with the effector. However, these effectors also lyse to a lesser extent TNP tumor and TNP spleen targets that are H-2 incompatible. This cross-reactive lysis correlates with the degree of cytolysis seen on the TNP-derivatized syngeneic target; it appears to be medicated by Thy 1.2-bearing cells and is inhibited by antisera to the K and/or D loci of the target cell and not by antisera to non-K or non-D surface antigens. Nonradiolabeled TNP-derivatized lymphoid cells syngeneic to either the stimulator or the target are able to competitively inhibit cross-reactive lysis, while TNP chicken red blood cells are unable to specifically inhibit lysis. These data on cross-reactive lysis of TNP-conjugated targets are most consistent with the altered-self hypothesis.