联合表位肽诱导HLA-A2+人PBMC产生抗原特异性CTL及杀伤活性研究

目的:探讨MAGE-3,MAGE-n抗原表位体外联合诱导的CTL,并研究其特异性杀伤活性。方法:候选抗原表位以固相多肽合成技术合成,并用HPLC进行纯化,质谱法(MS)鉴定,以流式细胞仪筛选HLA-A2 人外周血PBMC,T2细胞负载抗原肽反复刺激活化诱导抗原特异性CTL,LDH检测其杀伤活性。结果:联合表位肽体外刺激人PBMC,能够较强地诱导抗原特异性CTL并产生特异性杀伤。结论:MAGE-3与MAGE-n的HLA-A2限制性CTL表位肽的联合应用能够产生较强的体外抗肿瘤免疫反应。     

树突状细胞负载MAGE-n表位肽的体外免疫学效应研究

目的:研究树突状细胞(dendritic cell,DC)负载MAGE-n表位肽QLVFGIEVV体外诱导特异性CTL的能力及其抗肿瘤效应。方法:MAGE-n表位肽以固相多肽合成技术合成,并用HPLC进行纯化,质谱法(MS)鉴定,以流式细胞仪筛选HLA-A2+人外周血PBMC,连续贴壁法分离培养人外周血来源树突状细胞,用成熟的DC负载MAGE-n表位肽QLVFGIEVV反复刺激活化诱导抗原特异性CTL,用51Cr释放法检测CTL的杀伤活性,并用抗HLA-A2分子单抗进行杀伤抑制实验。结果:用DC负载MAGE-n表位肽QLVF-GIEVV可诱导特异性CTL反应,对MAGE-n阳性表达细胞有较强的杀伤作用。结论:DC负载抗原肽QLVF-GIEVV在体外可诱发较强的特异性免疫反应。     

树突状细胞负载MAGE-n表位肽的体外免疫学效应研究

目的：研究树突状细胞（dendritic cell,DC）负载MAGE-n表位肽QLVFGIEVV体外诱导特异性CTL的能力及其抗肿瘤效应。方法：MAGE-n表位肽以固相多肽合成技术合成,并用HPLC进行纯化,质谱法（MS）鉴定,以流式细胞仪筛选HLA-A2＋人外周血PBMC,连续贴壁法分离培养人外周血来源树突状细胞,用成熟的DC负载MAGE-n表位肽QLVFGIEVV反复刺激活化诱导抗原特异性CTL,用51Cr释放法检测CTL的杀伤活性,并用抗HLA-A2分子单抗进行杀伤抑制实验。结果：用DC负载MAGE-n表位肽QLVF-GIEVV可诱导特异性CTL反应,对MAGE-n阳性表达细胞有较强的杀伤作用。结论：DC负载抗原肽QLVF-GIEVV在体外可诱发较强的特异性免疫反应。     

人细胞毒T淋巴细胞杀伤胃癌细胞及诱发凋亡的实验研究

目的　测定特异性细胞毒 T淋巴细胞 (CTL )对胃癌细胞的体外杀伤活性及观察形态学改变。方法　用分离胃腺癌患者的癌细胞经丝裂霉素 C处理后 ,作为抗原与抗CD3单克隆抗体、重组白细胞介素 - 2刺激患者自体外周血单个核细胞 ,体外诱导 CTL,用 MTT比色法测定 CTL 对自体癌细胞、人胃癌细胞株 (BGC- 82 3和 MGC- 80 3)的体外杀伤活性。将 CTL与 BGC- 82 3共同温育 ,通过透射和扫描电镜观察杀伤靶细胞的形态改变。结果　 CTL对自体胃癌细胞有特异性杀伤作用 ,可使靶细胞凋亡或溶解坏死。结论　体外混合细胞培养诱导出的 CTL,对靶细胞有明显的杀伤作用     

修饰的肿瘤抗原肽CEA_（610D）疫苗的体外抗肿瘤作用

目的:评价修饰的CEA610D抗原肽疫苗体外抗肿瘤免疫的有效性,为其临床应用提供依据.方法:多肽固相合成法合成HLA-A2 CEA修饰肽(CEA610D)、HLA-A2天然肽CEA605-613(天然肽)和HLA-A2无关肽MAGE-3(无关肽),采用同种异体混合淋巴细胞与肽共孵育的方法,诱导肽特异性细胞毒性T淋巴细胞(CTL),利用MTT法检测CTL的增殖和杀伤活性;流式细胞术观察细胞表型;RT-PCR检测细胞穿孔素的表达;ELISA法检测CTL上清中IFN-γ的水平.结果:CEA610D疫苗产生肽特异性CTL的能力最强(P<0.05),其CD8+T细胞数也高于天然肽组和无关肽组;在效靶比为40∶1时,CEA610D和天然肽所诱导的CTL对CEA+HLA-A2限制性人结肠癌细胞T84的杀伤活性可达(56.7±3.73)%和(51.2±1.86)%,而3种肽特异性CTL细胞对CEA+HLA-A2人结肠癌lovo细胞杀伤活性维持在本底水平;CEA610D组的CTL所释放的杀伤相关介质穿孔素和上清中IFN-γ的水平也显著高于其余两组(P<0.01).结论:与天然肽相比,CEA610D疫苗可打破自身表位的免疫耐受,在体外抗肿瘤免疫中更具优势.     

自体宫颈癌-树突细胞疫苗激活的CTL杀伤效应

背景与目的:树突细胞(dendriticcells,DC)是目前已知的功能最强的抗原递呈细胞(antigen-presentingcell,APC),它可以在体内、外向T淋巴细胞递呈抗原,并诱发细胞毒T淋巴细胞(cytotoxicTlymphocyte,CTL)反应。本研究旨在探讨负载自体宫颈癌抗原的DC体外激发的CTL对自体宫颈癌细胞的杀伤效应。方法:先冻融宫颈癌细胞制备抗原,然后以GM-CSF、IL-4诱导自体外周血单个核细胞(peripheralbloodmononuclearcell,PBMC)获得DC并负载抗原,刺激自体T淋巴细胞制备宫颈癌抗原特异性CTL,观察CTL对宫颈癌细胞的杀伤活性。结果:负载自体宫颈癌抗原DC诱导的特异性CTL对自体宫颈癌细胞的体外杀伤率高达79.32%～89.27%,显著高于淋巴因子激活的杀伤细胞(lymphokine-activatedkillingcells,LAK)的杀伤率(t≥2.89,P<0.05);且对宫颈癌HeLa细胞株具有一定杀伤效应(40.35%～58.09%),但低于自体癌细胞组(t≥2.97,P<0.05);特异性CTL对HepG2、MCF7、A549、MGC803细胞无明显杀伤效应。结论:自体宫颈癌-树突细胞疫苗体外诱导的CTL具有高效而特异的抗自体宫颈癌细胞免疫活性,可望成为宫颈癌生物治疗的一个有力手段。     

MAGE-3/CEA(HLA-A2/A24+)肽疫苗冲击树突状细胞诱导消化道肿瘤患者CTL的研究

目的:树突状细胞(DC)是一种重要的抗原递呈细胞,在T细胞参与的HLA限制性免疫应答中起重要作用.已证实在动物模型中DC递呈抗原可以诱导出T细胞介导的免疫反应.我们在体外扩增获得肿瘤患者DC,观察其特点,并以MAGE-3/CEA(HLA-A2/A24+)肽疫苗冲击DC诱导特异性CTL,研究其杀伤活性.方法:选择MAGE-3-HLA-A2/A24+或CEA-HLA-A24+肿瘤患者,收集PBMNC中的贴壁细胞,在含有rhGM-CSF、rhIL-4的1640中培养诱导DC细胞,第7天加入HLA-A2-MAGE-3、HLA-A24-MAGE-3、HLA-A24-CEA肽冲击.以肽冲击后的DC与未经纯化的T细胞混合培养(T-DC-P)诱导的CTL作为效应细胞,Mel526,803,Raji,K562作为靶细胞,以LDH法检测特异性CTL的杀伤力.结果:体外扩增可获得高纯度的DC,高表达DC特异性表面标志CD40(74.18%±15.76%)、CD86(94.6%±3.80%)、HLA-DR(88.6%±10.94%).不同肿瘤患者DC得率表现出较大差异(7.07%±3.24%),反映出免疫功能的不同.T-IL-2细胞与T-DC-P细胞对Mel526和803细胞株的杀伤活性有显著性差异(P＜0.01),可以将特异性杀伤力提高25%～35%,而对Raji和K562细胞株的杀伤活性无显著性差异(P＞0.01).结论:rhGM-CSF、rhIL-4体外联合应用可扩增出高纯度DC,肽疫苗冲击后的DC可诱导出MAGE、CEA特异性的CTL应答.表明DC为基础的疫苗是肿瘤免疫治疗重要的新方法,因其具有简便、快速的特点,有良好的前景.     

基于Survivin的模拟病毒瘤苗体外激发CTL杀伤活性的实验研究

目的探讨基于Survivin的模拟病毒瘤苗体外激发细胞毒性T细胞(cytotoxicTlymphocytes,CTL)对乳腺癌细胞的杀伤作用。方法制备含有Survivin的模拟病毒瘤苗,体外刺激HLA-A2·1阳性健康者外周血单个核细胞(peripheralbloodmononu-clearcell,PBMC),用酶联免疫斑点实验(enzymelinkedimmunospotassay,ELISPOT)和标准51Cr释放实验分别检测瘤苗所诱导的特异性CTL活性。结果Survivin模拟病毒瘤苗能够在体外诱导出特异性CTL,对HLA-A2·1阳性的乳腺癌细胞系MCF-7有明显的杀伤毒性,杀伤率为56·4%;并可诱导CTL产生IFN-γ等细胞因子,ELISPOT显示斑点数为287个/105细胞,P<0·05。结论模拟病毒瘤苗能有效激发出特异性CTL应答,为新型乳腺癌治疗性疫苗的设计提供了新思路。     

MAGE—3／CEA（HLA—A2／A24＋）肽疫苗冲击树突状细胞诱导消化道肿瘤患者CTL的研究

目的树突状细胞(DC)是一种重要的抗原递呈细胞,在T细胞参与的HLA限制性免疫应答中起重要作用.已证实在动物模型中DC递呈抗原可以诱导出T细胞介导的免疫反应.我们在体外扩增获得肿瘤患者DC,观察其特点,并以MAGE-3/CEA(HLA-A2/A24+)肽疫苗冲击DC诱导特异性CTL,研究其杀伤活性.方法选择MAGE-3-HLA-A2/A24+或CEA-HLA-A24+肿瘤患者,收集PBMNC中的贴壁细胞,在含有rhGM-CSF、rhIL-4的1640中培养诱导DC细胞,第7天加入HLA-A2-MAGE-3、HLA-A24-MAGE-3、HLA-A24-CEA肽冲击.以肽冲击后的DC与未经纯化的T细胞混合培养(T-DC-P)诱导的CTL作为效应细胞,Mel526,803,Raji,K562作为靶细胞,以LDH法检测特异性CTL的杀伤力.结果体外扩增可获得高纯度的DC,高表达DC特异性表面标志CD40(74.18%±15.76%)、CD86(94.6%±3.80%)、HLA-DR(88.6%±10.94%).不同肿瘤患者DC得率表现出较大差异(7.07%±3.24%),反映出免疫功能的不同.T-IL-2细胞与T-DC-P细胞对Mel526和803细胞株的杀伤活性有显著性差异(P＜0.01),可以将特异性杀伤力提高25%～35%,而对Raji和K562细胞株的杀伤活性无显著性差异(P＞0.01).结论rhGM-CSF、rhIL-4体外联合应用可扩增出高纯度DC,肽疫苗冲击后的DC可诱导出MAGE、CEA特异性的CTL应答.表明DC为基础的疫苗是肿瘤免疫治疗重要的新方法,因其具有简便、快速的特点,有良好的前景.     

卵巢癌细胞系SKOV3冻融抗原诱导CTL特异性杀瘤效应的研究

目的探讨卵巢癌冻融抗原对T淋巴细胞的增殖作用及其诱导的细胞毒性T淋巴细胞(CTL)在体外杀伤卵巢癌细胞的抗原特异性细胞毒性效应。方法采用免疫磁珠分离法(magnetic activated cell sorting，MACS)分离纯化正常人外周血CD3^ 细胞，体外以SKOV3卵巢癌细胞中提取的可溶性肿瘤抗原加以刺激。溴标法检测肿瘤抗原对T细胞增殖的影响；MTT法测定肿瘤抗原诱导的CTL体外杀伤卵巢癌细胞的能力；利用绒毛膜癌细胞抗原对比观察肿瘤抗原诱导CTL杀伤肿瘤细胞的抗原特异性。结果卵巢癌细胞抗原有很强的促进T淋巴细胞增殖的作用，且存在时间依赖性，在24h时促进作用最强。SKOV3抗原诱导的CTL在体外对SKOV3细胞的杀伤率为68．38％，显著高于它对JAR绒毛膜癌细胞的杀伤率(18．31％)；而JAR细胞抗原诱导的CTL对JAR和SKOV3细胞的杀伤率分别为61．02％和14．79％，有显著的抗原特异性(P=0．0001)。结论卵巢癌细胞冻融抗原诱导的CTL在体外具有很强的增殖能力和抗原特异性杀伤卵巢癌细胞的作用。     

Induction of Cytotoxic T Lymphocytes from Peripheral Blood of Human Histocompatibility Antigen (HLA)-A31+ Gastric Cancer Patients by in vitro Stimulation with Antigenic Peptide of Signet Ring Cell Carcinoma

Antigenic peptides have been used as a cancer vaccine in melanoma patients and have led to a drastic regression of metastatic tumors. However, few antigens have been identified in non-melanoma tumors. We recently purified a new natural antigenic peptide, designated F4.2, by biochemical elution from a human gastric signet cell carcinoma cell line and showed that it is recognized by an autologous human histocompatibility antigen (HLA)-A31-restricted cytotoxic T lymphocyte (CTL) clone. Here we describe in vitro induction of F4.2-specific CTLs from peripheral blood T lymphocytes of HLA-A31⁺ gastric cancer patients. The T cells of seven HLA-A31⁺ patients with gastric cancers were stimulated in vitro by F4.2-pulsed autologous dendritic cells which had been induced from peripheral blood of each patient by incubation in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-4. We tested the cytotoxicity of the T cells against F4.2-loaded C1R-A *31012 by a 6-h ⁵¹Cr release assay after 3 stimulations with F4.2-pulsed dendritic cells. F4.2-specific cytotoxicity was detectable in the stimulated T cells from two of the seven HLA-A31⁺ patients. Further, both F4.2-specific CTLs also lysed the gastric cancer cell line, HST-2, from which F4.2 was derived. These results suggest that F4.2 peptide may be useful as an HLA-A31-restricted peptide vaccine in certain patients with gastric cancer.     

Histocompatibility Leukocyte Antigen-A2-restricted and Tumor-specific Cytotoxic T Lymphocytes from Tumor-infiltrating Lymphocytes of a Patient with Testicular Embryonal Cancer

T lymphocytes play an important role in tumor rejection. To understand T cell-mediated specific immunity at the tumor site of testicular embryonal cancer, we investigated whether interleukin-2 (IL-2)-activated tumor-infiltrating lymphocytes (TIL) of a patient with testicular embryonal cancer show histocompatibility leukocyte antigen (HLA)-class I-restricted and tumor-specific cytotoxicity. We established a CD3⁺CD4^-CD8⁺ cytotoxic T lymphocyte (CTL) line from the IL-2-activated TIL of a 37-year-old patient with testicular embryonal cancer. A 6 h ⁵¹Cr-release assay was performed to measure the cytotoxicity of the CTL. The CD3⁺CD4^-CD8⁺ CTL line showed cytotoxicity against HLA-A2⁺ tumor cells, including freshly isolated autologous tumor cells, adenocarcinoma cell lines from various organs (lung, breast, pancreas, colon and kidney) and squamous cell carcinomas (esophagus and oral cavity). No other cell lines examined, including an autologous tumor cell line and HLA-A2" tumor cell lines, were lysed by this CTL line. These results suggest the existence of HLA-A2-restricted and tumor-specific CTL at the tumor site of testicular embryonal cancer.     

Induction of cytotoxic T lymphocytes from peripheral blood of human histocompatibility antigen (HLA)-A31(+) gastric cancer patients by in vitro stimulation with antigenic peptide of signet ring cell carcinoma.

Antigenic peptides have been used as a cancer vaccine in melanoma patients and have led to a drastic regression of metastatic tumors. However, few antigens have been identified in non-melanoma tumors. We recently purified a new natural antigenic peptide, designated F4. 2, by biochemical elution from a human gastric signet cell carcinoma cell line and showed that it is recognized by an autologous human histocompatibility antigen (HLA)-A31-restricted cytotoxic T lymphocyte (CTL) clone. Here we describe in vitro induction of F4. 2-specific CTLs from peripheral blood T lymphocytes of HLA-A31( +) gastric cancer patients. The T cells of seven HLA-A31( +) patients with gastric cancers were stimulated in vitro by F4.2-pulsed autologous dendritic cells which had been induced from peripheral blood of each patient by incubation in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-4. We tested the cytotoxicity of the T cells against F4.2-loaded C1R-A*31012 by a 6-h (51)Cr release assay after 3 stimulations with F4.2-pulsed dendritic cells. F4.2-specific cytotoxicity was detectable in the stimulated T cells from two of the seven HLA-A31( +) patients. Further, both F4.2-specific CTLs also lysed the gastric cancer cell line, HST-2, from which F4.2 was derived. These results suggest that F4.2 peptide may be useful as an HLA-A31-restricted peptide vaccine in certain patients with gastric cancer.     

In vitro generation of cytotoxic T lymphocytes against HLA-A2.1-restricted peptides derived from human thymidylate synthase.

5-Fluorouracil (5-FU) is a pyrimidine antimetabolite active against colorectal carcinoma and other malignancies of the digestive tract. Over-expression or mutation of thymidylate synthase (TS), the target enzyme of the 5-FU metabolite, 5-fluorodeoxyuridine monophosphate, is strictly correlated with cancer cell resistance to 5-FU. On this basis we investigated whether TS is a potential target for active specific immunotherapy of human colon carcinoma, which acquires resistance to 5-FU. Three TS-derived epitope peptides which fit defined amino acid consensus motifs for HLA-A2.1 binding were synthesized and investigated for their ability to induce human TS-specific cytotoxic T cell (CTL) responses in vitro. CTL lines specific for each peptide were established by stimulating peripheral blood mononuclear cells (PBMC) from an HLA-A2.1+ healthy donor with autologous dendritic cells loaded with TS peptide. Specific CTL lines showed HLA-A2.1-restricted cytotoxicity in vitro to HLA-A2.1+ target cells pulsed with the specific TS peptide and to HLA-class I matching colon carcinoma target cells over-expressing TS enzyme after exposure to 5-FU. Recognition by CTL lines suggests that these TS peptides may be potential candidates for use in a peptide-based vaccine against 5-FU resistant colon carcinoma.     

Cytotoxic T lymphocytes that recognize decameric peptide sequences of retinoblastoma binding protein 1 (RBP-1) associated with human breast cancer.

Retinoblastoma binding protein 1 (RBP-1) is a 143-kDa nuclear phosphoprotein that promotes cell growth by inhibiting the product of retinoblastoma tumour suppressor gene (pRB). We recently found that RBP-1 contains KASIFLK, a heptameric peptide (250-256) recognized by human antibodies and overexpressed by breast cancer cells. In the present study, we demonstrate that human T-cells stimulated with RBP-1 decameric peptides containing KASIFLK can kill human breast cancer cells. These decamers, GLQKASIFLK (247-256) and KASIFLKTRV (250-259), have anchor motifs for both HLA-A2 and HLA-A3. Peripheral blood lymphocytes from 41 normal donors were stimulated by these peptides in culture media containing 15 IU ml(-1) interleukin-2, 25 IU ml(-1) interleukin-7 and 500 IU ml(-1) granulocyte-macrophage colony-stimulating factor. Cytotoxic activity of the T-cells was assessed against autologous B lymphoblastoid cells pulsed with each peptide. Stimulation by GLQKASIFLK generated specific cytotoxic T lymphocyte (CTL) lines from HLA-A2, A3 donors, HLA-A2 donors and HLA-A3 donors. Stimulation with KASIFLKTRV generated specific CTL lines from HLA-A2 donors. No HLA-A2-, A3 CTL line showed specific cytotoxicity against these target cells. These CTL lines were also cytotoxic against HLA-A2 and HLA-A3 breast cancer cells but not against normal fibroblastoid cell lines, normal epidermal cell lines, or a melanoma cell line. RBP-1 peptide antigens may be of clinical significance as a potential peptide vaccine against human breast cancer.     

基因腺病毒感染树突状细胞诱导CTL对胶质瘤细胞的杀伤作用

目的:研究EPHA2基因修饰的树突状细胞(dendritic cell,DC)疫苗诱导细胞毒性T淋巴细胞(cytotoxic T lympho-cyte,CTL)对U251胶质瘤细胞的杀伤效应,为胶质瘤的免疫治疗提供新的方法。方法:将重组EPHA2腺病毒rAd-EPHA2感染HLA-A2阳性的人外周血来源的DC,制备EPHA2基因修饰的DC疫苗,Western blotting和FACS方法检测感染后DC的EPHA表达。以DC疫苗体外刺激HLA-A2阳性的单个核细胞,酶联免疫斑点实验(enzyme-linked immunospot assay,ELISPOT)和标准51Cr释放实验分别检测DC疫苗所诱导的CTL活性和对HLA-A2阳性的U251细胞的杀伤作用(另设rAd-Lac Z感染的DC组和PBS组作为对照)。结果:成功制备了EPHA2基因修饰的DC疫苗,其可有效表达EPHA2蛋白。与感染rAd-LacZ的DC和PBS组相比,感染rAd-EPHA2的DC疫苗能有效激发CTL活性[(187±21)vs(12±4)、(18±5)个,P<0.01];所诱导的CTL对胶质瘤U251细胞有明显的杀伤效应[(45.7±6.8)%vs(7,1±4.5)%,P<0.01],对自身淋巴细胞没有杀伤效应。结论:EPHA2基因修饰的DC疫苗能有效激发CTL活性,并对胶质瘤U251细胞有明显的杀伤活性。     

Histocompatibility Leukocyte Antigen-A2402-restricted Cytotoxic T Lymphocytes Recognizing Adenocarcinoma in Tumor-infiltrating Lymphocytes of Patients with Colon Cancer

To cast light on T cell-mediated specific immunity at the tumor site of colon cancer, we investigated whether interleukin-2 (IL-2)-activated tumor-infiltrating lymphocytes (TIL) from colon cancer show histocompatibility leukocyte antigen (HLA)-class I-restricted cytotoxicity against adenocarcinoma. IL 2-activated TIL from all four HLA-A24 patients examined lysed HLA-A2402+ adenocarcinomas, but not HLA-A2402 tumors. Those of two of the four cases also lysed HLA-A2402⁺ squamous cell carcinomas. CD8⁺ cytotoxic T lymphocyte (CTL) clones recognizing HLA-A2402⁺ adenocarcinomas were established from one CTL line. This CTL line produced IFN-γ upon recognition of an HLA-A2402^- adenocarcinoma transfected with HLA-A2402 cDNA. These results suggest the presence of HLA-A2402-restricted CTL recognizing adenocarcinoma at the tumor site of colon cancer. Furthermore, HLA-A31-restricted CTL activity was found in IL-2-activated TIL from one of two HLA-A31⁺ patients, suggesting the existence of HLA-class I-restricted CTL involving an allele other than A24     

人工抗原提呈细胞体外激活肝癌CD8+T细胞

目的:探讨人工抗原提呈细胞(artificial antigen presenting cell,aAPC)K32/4-IBBL/CD86对肝癌CD8+T细胞的活化作用。方法:磁珠法分选HLA-A 2阳性肝癌患者外周血CD8+T细胞,aAPC与CD8+T细胞按不同比例(1∶1、1∶2、1∶3)混合培养,诱导CTL。锥虫蓝拒染法测定CTL的生长曲线,MTS/PMS法检测CTL的增殖,流式细胞术检测CTL分泌IFN-γ的能力,MTT法检测CTL对人肝癌细胞株BEL7402和自体肝癌细胞的杀伤作用。结果:肝癌患者外周血CD8+T细胞经aAPC作用后,增殖能力明显增强,按1∶1、1∶2、1∶3比例混合培养后第8天分别为(21.2±2.5)×105、(17.6±3.2)×105、(15.3±2.8)×105,明显高于对照组的(8.5±0.15)×105(P<0.01),混合培养后分泌IFN-γ的CTL比例分别为(26.2±1.91)%、(21.3±1.38)%、(18.6±1.20)%,明显高于对照组的(0.1±0.02)%(P<0.01)。aAPC活化的CTL对BEL7402细胞和自体肝癌细胞的杀伤率较对照组显著增强,按1∶3混合培养后得到的CTL对BEL7402细胞和自体肝癌细胞的杀伤率分别为(21.8±4.3)%和(25.6±3.6)%,明显高于对照组的(3.8±1.8)%和(3.8±2.0)%(P<0.01);效靶比为50∶1时,1∶1混合培养组CTL对BEL7402细胞的杀伤率(56.8±3.0)%和对自体肝癌细胞的杀伤率(64.8±4.2)%明显高于1∶2混合培养组的(44.3±2.6)%、(56.1±3.4)%和1∶3混合培养组的(38.9±4.7)%、(46.2±4.7)%(P<0.05)。结论:aAPC在体外可高效活化肝癌患者CD8+T细胞,诱导CTL分泌IFN-γ,且CTL对人肝癌细胞株BEL7402和自体肝癌细胞具有明显的杀伤作用。     

HER-2/neu-derived peptide epitopes are also recognized by cytotoxic CD3(+)CD56(+) (natural killer T) lymphocytes

The human HER-2/neu gene encodes a 185 kDa transmembrane glycoprotein recognized by MHC class I-restricted CTLs. Here, we report that HER-2/neu peptide CTL epitopes can also be recognized by cytotoxic NK-T lymphocytes. Unfractionated peptides derived from HLA-A2(+), HER-2/neu(+) tumor cells acid cell extract (ACE), collected from patients with metastatic ovarian cancer, were used as antigen to generate in vitro cytotoxic effectors. ACE was able to elicit from cancer patients' PBMCs both alphabetaTCR(+)CD3(+)CD56(-) and alphaTCR(+)CD3(+)CD56(+) (NK-T) CTLs that lysed ACE-sensitized T2 cells in an HLA-A2-restricted manner. The same CTL lines also recognized T2 cells pulsed with HER-2/neu-derived CTL peptide epitopes, a HER-2/neu-transfected HLA-A2(+) cell line and autologous tumor cells. alphaTCR(+)CD3(+)CD56(+) CTL lines also exhibited NK-like cytotoxicity against autologous tumor cells. CTL clones were isolated from alphaTCR(+)CD3(+)CD56(+) bulk cultures displaying both MHC- and non-MHC-restricted cytotoxicity, thus confirming the dual cytolytic function of such cells. Our data demonstrate that ACE from metastatic ovarian tumors can be used as multiepitope vaccines for generating in vitro, besides classical CTLs, NK-T cells exerting efficient MHC- and non-MHC-restricted cytotoxicity against autologous tumor targets. Such NK-T cells expressing dual cytotoxic activity may prove advantageous in cancer immunotherapy.     

Cytotoxicity of Cord Blood Derived Her2/neu-specific Cytotoxic T Lymphocytes against Human Breast Cancer in vitro and in vivo

The Her2/neu oncogene encodes a transmembrane protein with homology to the epidermal growth factor receptor. Overexpression of this gene contributes to the aggressiveness of breast cancer and poor prognosis. Therefore, Her2/neu is an ideal target molecule for generating effective cytotoxic T lymphocytes (CTLs) against breast cancers. This study reports on the generation of Her2/neu-specific CTL from umbilical cord blood mononuclear cells (UCBC) using dendritic cells primed with Her2/neu-derived peptide (KIFGSLAFL, E75) for immunostimulation. The CTLs showed specific cytotoxicity to Her2/neu high expressing MDA-453 but not toward Her2/neu low expressing MDA-231 human breast cancer cells. Similarly generated CTLs stimulated with irrelevant peptide pulsed dendritic cells did not show significant cytotoxicity towards breast cancer targets. The phenotypes of cells in culture showed high percentage of CD3+, CD4+ and CD8+T cells as determined by flow cytometry. However, the antibody mediated blocking assay demonstrated that only HLA-Class I restricted CD8+ cells are involved in the cytotoxicity. Furthermore, in vivo studies showed that treatment of SCID mice bearing MDA-453 tumor with Her2/neu-specific CTLs resulted in significant inhibition of tumor growth compared to untreated tumor bearing control mice. These results demonstrate that human umbilical cord blood mononuclear cells are a good source for generating Her2/neu-specific CTLs against human breast cancer both in vitro and in vivo.