免疫球蛋白重链框架区九肽诱导抗急性B淋巴细胞白血病的细胞毒T细胞应答

目的 用免疫球蛋白(Ig)重链框架区几肽体外诱导抗急性B淋巴细胞白血病(B-ALL)的细胞毒T细胞应答。方法 合成Ig重链可变区第1家族框架区3～11位置上的九肽QLVQSGAEV(IgHV13-11)，进行T2细胞结合实验。IgHV13-11负荷抗原呈递细胞(APC)，体外刺激HLA-A*0201正常外周血单个核细胞(PBMNC)，每周1次，共3次：HLA-A*0201／IgHV13-11四聚体监测培养细胞中IgHV1-311特异性CD8^ T细胞的增殖。对B-ALL初治患儿的7个IgHV基凶家族进行PCR扩增及PCR产物的测序，选IgHV1和IgHV3家族单等位基冈功能性重排克隆，并鉴定其HLA-A*0201位点。MTT比色法分析IgHV13-11，特异性CD8^ T细胞对HLA-A*0201^ IgHV1和IgHV3家族B-ALL细胞克隆的杀伤活性。结果 IgHV13-11可上调HLA-A*0201分子在T2细胞表面的表达1．63倍、HLA-A*0201*正常PBMNC中IgHV13-11特异性CD8^ T细胞的频率由2轮刺激后1．64％增至3轮刺激后82．57％。IgHV13-11特异性CD8^ T细胞在效靶细胞比(E：T)20：1时对IgHV1家族B-ALL细胞的杀伤率为18．24％，是IgHV3家族B-ALL细胞的1．8倍(P=0．01)。结论 体外诱导的Ig重链框架区九肽特异性CD8*T细胞可杀伤表达该肽的B-ALL细胞。     

负载肝癌肿瘤抗原的B淋巴细胞诱导特异性抗肿瘤免疫的实验研究

目的:探讨体外经小鼠肝癌细胞不同抗原致敏的CD40配体活化的B淋巴细胞诱导的特异性细胞毒T淋巴细胞(CTL)抗肿瘤免疫的能力.方法:分离、纯化T、B混合淋巴细胞,并在CD40L、rmIL-4联合作用下培养.然后分离T、B淋巴细胞以备用.将凋亡的肝癌细胞及其冻融裂解物作为实验组,1640培养基作为空白对照组分别与B淋巴细胞共同培养,检测培养后各组B细胞表面抗原呈递细胞标记(CD40、CD80、CD86)及主要组织相容性抗原的表达情况.利用混合淋巴细胞增殖实验检测T细胞增殖情况.以负载肿瘤抗原的B淋巴细胞诱导的CTL作为效应细胞,肝癌细胞Hepal-6为靶细胞,LDH释放实验检测杀伤活性.结果:负载肿瘤抗原的B淋巴细胞具有刺激T细胞增殖的能力,实验组的B淋巴细胞,其组织相容性分子及其刺激分子表达明显高于空白对照组,其对靶细胞的杀伤率与空白对照组比较经统计学分析差异有显著性.结论:负载肝癌肿瘤抗原的B淋巴细胞作为抗原呈递细胞诱导的CTL可有效产生特异性抗肝癌免疫作用.     

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。     

表达新疆出血热病毒S基因片段的重组腺病毒特异性CTL功能测定

目的通过表达新疆出血热病毒(XHFV)S基因片段的重组腺病毒特异性细胞毒性T淋巴细胞(CTL)功能测定,探求由XHFVS基因片段编码的核衣壳蛋白作为抗原诱导的特异性CTL细胞对重组腺病毒致敏的靶细胞杀伤能力大小。方法PCR扩增XHFVS基因的cDNA片段,将扩增后的cDNA片段克隆入腺病毒自主载体,使其成为重组腺病毒,用重组腺病毒感染Balb/c小鼠后,利用非放射性乳酸脱氢酶(LDH)释放法测定重组腺病毒的特异性CTL功能。结果在一定比例范围内,效应细胞与靶细胞的比例越高,特异性CTL%越高。结论由XHFVS基因片段编码的核衣壳蛋白抗原诱导的特异性CTL细胞,能特异性地杀伤重组腺病毒致敏的靶细胞;证实由腺病毒自主载体本身所表达的一些蛋白质也可诱导细胞免疫,由该载体所引发的细胞免疫对疫苗接种不利。     

儿童急性B淋巴细胞白血病免疫球蛋白重链可变区基因的分子特征

目的 探讨儿童急性B淋巴细胞白血病(B—ALL)的起源及其免疫球蛋白重链可变区(IgHV)上可被细胞毒性T淋巴细胞(cTL)识别的表位(epitope)。方法 PCR扩增108例儿童ALL的7个IgHV基因家族；PCR产物直接测序并翻译成氨基酸序列；利用生物信息资源分析B—ALL IgHV基因重排类型、胚系基因片段利用及体细胞突变情况，并预测CTL细胞识别的表位。结果 66％的ALL患儿检测到IgHV基因重排，其中单等位基因重排37例(52．1％)，双等位基因重排26例(36.6％)，寡克隆基因重排8例(11．3％)。40份B-ALL IgHV基因序列中，不含终止密码子的读码框内(in frame)重排8份(20．0％)。重排利用最多的VH家族为VH3、VH4和VH1，占75%；VH4—59和VH4—34是VH4家族中利用频率最高的片段。优先利用的D家族为D3(35．9％)和D2(28.2％),D7—27的利用率(15．4％)明显高于正常外周血淋巴细胞(P=0．02)。JH6是B-ALL利用最多的JH基因片段(47．5％)，JH4(27．5％)次之。20％的B—ALL在DJH连接区缺乏N核苷酸的插入，高于正常外周血淋巴细胞(P=0．02)。17．5％B—ALL IgHV基因发生替代突变，突变碱基散布于IgHV全长，互补决定区替代突变与静寂突变的比例≤1。26例B—ALL IgHV的HIA—I类分子结合肽80％以上位于框架区，同一IgHV家族的B-ALL有相同的1～2条九肽。结论 B—ALL起源于前B祖细胞或前B细胞，恶性转化往往发生于胚胎期，它们的IgHV基因具有胚系特征；生物信息分析B-ALL IgHV的T细胞表位主要位于框架1区和框架3区。     

表达新疆出血热病毒S基因片段的重组腺病毒特异性CTL功能测定

目的 通过表达新疆出血热病毒（XHFV）S基因片段的重组腺病毒特异性细胞毒性T淋巴细胞（CTL）功能测定，探求由XHFVS基闪片段编码的核衣壳蛋白作为抗原诱导的特异性CTL细胞对蓖组腺病毒致敏的靶细胞杀伤能力大小。方法 PCR扩增XHFVS基因的cDNA片段，将扩增后的cDNA片段克隆入腺病毒自主载体，使其成为重组腺病毒，用重组腺病毒感染Balb／C小鼠后，利用非放射性乳酸脱氰酶（LDH）释放法测定重组腺病毒的特异性CTL功能。结果 在一定比例范围内，效应细胞与靶细胞的比例越高，持异性CTL％越高。结论 由XHFVS基因片段编码的核衣壳蛋白抗原诱导的特异性CTL细胞，能特异性地杀伤重组腺病毒致敏的靶细胞；证实由腺病毒自主载体本身所表达的一些蛋白质也可诱导细胞免疫，由该载体所引发的细胞免疫对疫苗接种不利。     

肿瘤抗原特异性细胞毒T细胞和肿瘤免疫治疗

肿瘤抗原特异性细胞毒T细胞(CTL)是重要的抗肿瘤细胞。本文介绍了抗原呈递及CTL识别的分子基础，并对肿瘤相关抗原(TAA)及T细胞表位的鉴定、肿瘤相关抗原肽诱导特异性CTL应答的免疫原性、肿瘤相关抗原肽疫苗治疗肿瘤的原理以及T细胞免疫应用前景做一综述。     

外周血单核细胞来源树突状细胞体外扩增及诱导抗乳腺癌免疫的研究

目的研究外周血来源树突状细胞(DC)的体外扩增及诱导特异性抗乳腺癌细胞的免疫效应。方法(1)从外周血中分离单个核细胞后,获得单核细胞(monocyte,Mo)。粒-单集落刺激因子(GM-CSF)和白介素4(IL-4)诱导分化扩增培养7d后,应用流式细胞术进行表型分析。(2)诱导单核细胞分化的第3天加入人乳腺癌细胞株3A0的冻融抗原培养4d后,获得负载肿瘤抗原的成熟DC;将致敏DC与从外周血中分离的同种异体T淋巴细胞共培养3d,获得细胞毒T淋巴细胞(CTL);四甲基偶氮唑蓝法检测CTL及上清液对人乳腺癌细胞株3A0、人胚肾细胞株293T(对照细胞)、人肝癌细胞株HCCC-9810的细胞毒作用。结果(1)外周血来源Mo在GM-GSF和IL-4作用下,第7天后可分化生成成熟的DC,高表达DC特异性抗原CDla、CD80(B7-1)、CD86(B7-2)HLA-DR、CD83。(2)DC可负载并递呈肿瘤抗原,激活同种异体T淋巴细胞,诱导肿瘤特异性CTL产生。不同浓度CTL及上清液对乳腺癌细胞3A0有特异性杀伤、抑制作用(P<0.05)。结论外周血中Mo可体外分化扩增为成熟的功能性DC,并诱导出特异性杀伤乳腺癌细胞的免疫效应。     

MHC-肽四聚体技术在造血干细胞移植中的应用

MHC-肽四聚体技术是一种直接监测抗原特异性细胞毒性T细胞(CTL)的新方法,具有较强的特异性和敏感性。本文就MHC-肽四聚体技术在造血干细胞移植后巨细胞病毒特异性CTL、次要组织相容性抗原(mHags)特异性CTL以及肿瘤抗原特异性CTL的检测及其意义进行综述。     

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。     

Generation of melanoma-specific cytotoxic T lymphocytes for allogeneic immunotherapy

To exploit alloreactive T-cell responses known as the graft-versus-tumor effect, allogeneic hematopoietic stem cell transplantation is being explored as experimental therapy in selected solid tumors, including metastatic melanoma. However, donor T-cell responses are often delayed and associated with severe graft-versus-host disease. Posttransplant adoptive immunotherapy using tumor-specific cytotoxic T lymphocytes (CTL) of donor origin might provide immediate graft-versus-tumor effects but not graft-versus-host disease. Therefore, the aim of the current study was to define in vitro conditions for the expansion of allogeneic major histocompatibility complex-matched CTLs targeting melanoma-associated antigens (MAA). The CTLs were generated from peripheral blood mononuclear cells (PBMCs) of HLA-A*0201+ healthy donors by repetitive stimulations with HLA-A*0201-restricted MAA-derived peptides. Melanoma reactivity, as determined by lysis of peptide-pulsed T2 cells and HLA-A2+/Ag+ melanoma cells, was detected using in vitro expanded CTL targeting MAA peptides AAGIGILTV(MT(27-35)), IMDQVPFSV(G(209-2M)), and YMDGTMSQV(T(368-376)). In contrast, FLWGPRALV(MAGE3(271)-(279)) and VLPDVFIRCV(GnT-V(nt38-67)) induced peptide-specific recognition of T2 target cells only, whereas ITDQVPFSV(G(209-217)), KTWGQYWQV(G9(154)), MLLAVLYCL(T(1-9)), and tumor lysate could not induce specific CTLs. Specific cytolytic activity was accompanied by interferon-gamma secretion. Peptide-pulsed dendritic cells were required only for the initial stimulation of CTLs and could be substituted by PBMCs during restimulations. The median expansion rate of CTL was five to six times, regardless of whether dendritic cells or PBMCs were used after the initial stimulation. The results delineate the conditions for effective ex vivo expansion of melanoma-specific CTLs from PBMCs of healthy donors to be used as an adjunct in allogeneic cell therapy of metastatic melanoma.     

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.     

Expansion of melanoma-specific cytolytic CD8+ T cell precursors in patients with metastatic melanoma vaccinated with CD34+ progenitor-derived dendritic cells

Cancer vaccines aim at inducing (a) tumor-specific effector T cells able to reduce/eliminate the tumor mass, and (b) long-lasting tumor-specific memory T cells able to control tumor relapse. We have shown earlier, in 18 human histocompatibility leukocyte antigen (HLA)-A*0201 patients with metastatic melanoma, that vaccination with peptide-loaded CD34-dendritic cells (DCs) leads to expansion of melanoma-specific interferon gamma-producing CD8+ T cells in the blood. Here, we show in 9 out of 12 analyzed patients the expansion of cytolytic CD8+ T cell precursors specific for melanoma differentiation antigens. These precursors yield, upon single restimulation with melanoma peptide-pulsed DCs, cytotoxic T lymphocytes (CTLs) able to kill melanoma cells. Melanoma-specific CTLs can be grown in vitro and can be detected in three assays: (a) melanoma tetramer binding, (b) killing of melanoma peptide-pulsed T2 cells, and (c) killing of HLA-A*0201 melanoma cells. The cytolytic activity of expanded CTLs correlates with the frequency of melanoma tetramer binding CD8+ T cells. Thus, CD34-DC vaccines can expand melanoma-specific CTL precursors that can kill melanoma antigen-expressing targets. These results justify the design of larger follow-up studies to assess the immunological and clinical response to peptide-pulsed CD34-DC vaccines.     

Detection of T helper responses, but not of human papillomavirus-specific cytotoxic T lymphocyte responses, after peptide vaccination of patients with cervical carcinoma

Human papillomavirus type 16 (HPV16)-encoded E7 oncoprotein is constitutively expressed in cervical carcinoma cells and is required for cellular transformation to be maintained. The E7 protein, therefore, forms an attractive target for T-cell-mediated immune intervention to prevent or treat HPV16+ tumors. The authors performed a peptide-based phase I/II vaccination trial to induce anti-tumor immune responses in patients with recurrent or residual cervical carcinoma. Fifteen HLA-A*0201+ patients with HPV16+ cervical carcinoma received vaccinations with synthetic peptides representing 2 HPV16 E7-encoded, HLA-A*0201-restricted cytotoxic T lymphocyte epitopes and a pan-HLA-DR-binding T-helper epitope, PADRE, in adjuvant. No signs of toxicity were observed. Two patients had stable disease for more than 1 year after vaccination, 3 patients died of the disease during or shortly after the vaccination period, and 10 patients maintained progressive cervical carcinoma. Specific immune responses directed against the vaccine components were analyzed in peripheral blood samples. No cytotoxic T lymphocyte responses against the HPV16 E7 peptides were detectable. After vaccination, strong PADRE helper peptide-specific proliferation was detected in 4 of 12 patients. In conclusion, peptide vaccination with 2 HPV16 E7 cytotoxic T lymphocyte epitopes and a universal T helper epitope is well tolerated by patients with advanced cervical carcinoma. Despite a reduction of in vitro cytolytic or proliferative recall responses to some, but not all, conventional antigens in this patient group, peptide-specific proliferative responses were induced in 4 patients. Based on the current study, it is now feasible to perform peptide vaccination in earlier stages of HPV16-induced cervical disease.     

可溶性HLA-A*0201-PR1复合物的构建与鉴定

为制备HLA-A＊0201-PR1四聚体,本课题构建了可溶性的HLA-A＊0201-PR1复合物。以原核表达的HLA-A＊0201-BSP融合蛋白为重链,β2-微球蛋白（β2m）为轻链,与人工合成的HLA-A2限制性抗原肽PR1（蛋白酶3的第169-177氨基酸,VLQELNVTV）进行共折叠复性,以生成可溶性HLA-A＊0201-PR1复合物。应用BirA酶对复性混和物进行生物素化,再经阴离子交换柱纯化,得到生物素化的可溶性HLA-A＊0201-PR1复合物。应用凝胶过滤高效液相色谱分析可溶性HLA-A＊0201-PR1复合物的生成率。利用HLA-A2构象特异性抗体（BB7.2）及链霉亲和素进行Western blot和ELISA分析,对生物素化的可溶性HLA-A＊0201-PR1复合物进行鉴定。结果表明：在折叠体系中,主要含有HLA-A＊0201-BSP聚合体、HLA-A＊0201-PR1复合物及β2m,其中可溶性复合物生成率约18%。获得的HLA-A＊0201-PR1复合物可在BirA酶作用下被有效生物素化。结论：成功地获得了生物素化的可溶性HLA-A＊0201-PR1复合物,为进一步制备HLA-A＊0201-PR1四聚体创立了基础。     

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.     

Dendritic cells in vaccination therapies of malignant diseases.

Dendritic cells (DC) are the most potent APC with the unique capacity to initiate primary immune responses. For clinical use DC can be generated in vitro from CD34+ peripheral blood progenitor cells or monocytes. Vaccination of patients with cancer using DC was shown to be effective for B-cell lymphoma, renal cell carcinoma (RCC), prostate cancer and malignant melanoma. We provide evidence that patients with advanced breast and ovarian cancer can be efficiently vaccinated with autologous DC pulsed with HER-2/neu- or MUC1-derived peptides. In 5 of 10 patients, peptide-specific cytotoxic T lymphocytes (CTL) could be detected in the peripheral blood using both intracellular IFN-gamma staining and Cr-release assays. In addition, in one patient vaccinated with the MUC1-derived peptides, CEA- and MAGE-3 peptide-specific T-cell responses were detected after several vaccinations. In a second patient immunized with the HER-2/neu peptides, MUC1-specific T lymphocytes were induced after seven immunizations, suggesting that antigen spreading in vivo might occur after successful immunization with a single tumor antigen. Currently we are analyzing the effect of T-helper epitopes and IL-2 on the CTL induction using peptide pulsed DC. In this ongoing trial one patient with metastatic RCC developed a partial remission of the metastatic sites was induced after the first four vaccinations with MUC1 peptides pulsed DC, that was ongoing after the next cycles containing IL-2. Vaccine-induced peptide-specific T-cell responses in vivo were detected in the PBMNC of this patient and in peptide-specific DTH reactions. This studies demonstrate that peptide pulsed DC can be effective in cancer patients and induce significant clinical and immunological responses.     

The matrix protein pp65(341-350): a peptide that induces ex vivo stimulation and in vitro expansion of CMV-specific CD8+ T cells in subjects bearing either HLA-A*2402 or A*0101 allele

BACKGROUND: The stimulation of PBMNCs with HLA Class I restricted synthetic peptides derived from CMV phosphorylated matrix protein 65 (pp65) evokes CMV-specific cytotoxic T lymphocyte (CTL) activity, a necessary condition for initiating adoptive immunotherapy against CMV-related diseases in immune-compromised patients. It was previously demonstrated that the CMV decamer (10-mer) peptide pp65(341-350), QYDPVAALFF, was able to induce CMV-specific CTLs in HLA-A*2402 CMV-seropositive individuals. STUDY DESIGN AND METHOD: We investigated the ability of the peptide pp65(341-350) to reactivate memory CD8+ T cells in CMV-seropositive subjects bearing either the HLA-A24 or A1 allele. CTL responses were measured by IFN-gamma mRNA expression and IFN-gamma protein production as well as cytotoxic activity. RESULTS: In this study it was found that peptide pp65(341-350) induced a specific reactivation of memory CD8+ T cells from CMV-seropositive donors expressing either HLA-A*2402 and/or HLA-A*0101. Moreover, a pp65(341-350)-specific selection and expansion using PBMNCs of CMV-seropositive donors bearing both HLA-A*2402 and HLA-A*0101 alleles produced cytotoxic CTLs to both HLA-A24 and A1 peptide-pulsed and autologous CMV-infected target cells. CONCLUSION: The results demonstrate that pp65(341-350) induced a specific CTL activity at both molecular and protein levels and that the peptide is specifically processed, presented, and recognized by subjects bearing HLA-A*2402 and/or A*0101. These findings suggest that it may be possible to use this single immune dominant peptide to induce and expand CMV-reactive CTLs for the treatment of individuals with both HLA-A24 and A1 types.     

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.