Schreurs Marco W J, Scholten Kirsten B J, Kueter Esther W M, Ruizendaal Janneke J, Meijer Chris J L M, Hooijberg Erik
Department of Pathology, Vrije Universiteit Medical Center, 1081 HV Amsterdam, The Netherlands.
J Immunol. 2003 Sep 15;171(6):2912-21. doi: 10.4049/jimmunol.171.6.2912.
Human papillomavirus (HPV) type 16 infection is strongly associated with the development of cervical carcinoma (CxCa) in women. The HPV16-derived oncoproteins E6 and E7, responsible for both onset and maintenance of malignant transformation, are expressed constitutively in CxCa cells and represent tumor-associated Ags. As a result, E6 and E7 constitute potential targets for adoptive CTL-mediated immunotherapy of CxCa. However, the availability to date of well-characterized HPV16-specific, CxCa-reactive human CTLs is extremely limited. The current study describes the in vitro generation and isolation of HPV16 E7-specific, CxCa-reactive human CTL clones from low-frequency healthy donor-derived CD8beta-positive precursors. For this purpose, an in vitro CTL induction protocol was used involving mature monocyte-derived dendritic cells as stimulator cells loaded with an HLA-A2.1-restricted, E7(11-20)-derived high-affinity altered peptide ligand. A double tetramer-guided isolation procedure and subsequent limiting-dilution cloning resulted in Ag-specific CTL clones. Stringent CTL characterization clearly indicated Ag-specific, HLA-A2.1-restricted reactivity against different HPV16-transformed CxCa cell lines. To allow expansion of E7(11-20)-specific CTL clones to numbers required for prolonged in vitro as well as in vivo application, their life span was significantly extended by ectopic expression of human telomerase reverse transcriptase. Collectively, our results show that optimized CTL induction and stringent CTL selection procedures, followed by human telomerase reverse transcriptase-mediated life span extension will allow continued availability of low-frequency HPV16-specific, CxCa-reactive human CTL clones. This may enhance the prospects of HPV16-specific adoptive CTL immunotherapy in CxCa patients.
人乳头瘤病毒16型（HPV16）感染与女性宫颈癌（CxCa）的发生密切相关。HPV16衍生的癌蛋白E6和E7负责恶性转化的起始和维持，在CxCa细胞中持续表达，是肿瘤相关抗原。因此，E6和E7构成了CxCa过继性CTL介导免疫治疗的潜在靶点。然而，迄今为止，具有充分特征的HPV16特异性、CxCa反应性人CTL的可用性极其有限。本研究描述了从低频健康供体来源的CD8β阳性前体细胞中体外生成和分离HPV16 E7特异性、CxCa反应性人CTL克隆方法。为此，使用了一种体外CTL诱导方案，该方案涉及成熟单核细胞来源的树突状细胞作为刺激细胞，负载有HLA - A2.1限制性、E7（11 - 20）衍生的高亲和力改变肽配体。双重四聚体引导的分离程序和随后的有限稀释克隆产生了抗原特异性CTL克隆。严格的CTL鉴定清楚地表明了针对不同HPV16转化的CxCa细胞系的抗原特异性、HLA - A2.1限制性反应性。为了使E7（11 - 20）特异性CTL克隆扩增到体外长期以及体内应用所需的数量，通过人端粒酶逆转录酶的异位表达显著延长了它们的寿命。总的来说，我们的结果表明，优化的CTL诱导和严格的CTL选择程序，随后是人端粒酶逆转录酶介导的寿命延长，将使低频HPV16特异性、CxCa反应性人CTL克隆持续可用。这可能会提高CxCa患者中HPV16特异性过继性CTL免疫治疗的前景。
