Choi Joshua, Meilleur Courtney E, Haeryfar S M Mansour
Department of Microbiology and Immunology, Western University.
Department of Microbiology and Immunology, Western University; Department of Medicine, Division of Clinical Immunology and Allergy, Western University; Department of Surgery, Division of General Surgery, Western University; Lawson Health Research Institute;
J Vis Exp. 2019 May 6(147). doi: 10.3791/59531.
Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8 cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 "immunodominance" in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.
基于羧基荧光素琥珀酰亚胺酯（CFSE）的体内细胞毒性测定能够灵敏且准确地定量针对肿瘤和病原体衍生肽引发的CD8细胞毒性T淋巴细胞（CTL）反应。与传统杀伤测定相比，它们具有多个优势。首先，它们允许在结构完整的二级淋巴器官（通常是脾脏）内监测CTL介导的细胞毒性。其次，它们能够在CTL反应的启动、效应和回忆阶段进行机制研究。第三，它们为在真正的体内环境中进行疫苗/药物疗效测试提供了有用的平台。在此，我们提供了一个优化方案，用于检测针对模型肿瘤抗原（Ag）（即猿猴病毒40（SV40）编码的大T抗原（T Ag））的多个肽表位的伴随CTL反应。与大多数其他临床相关肿瘤蛋白一样，T Ag含有许多潜在的免疫原性肽。然而，在C57BL / 6小鼠中，只有四种这样的肽能诱导可检测到的CTL反应。这些反应根据其强度始终按等级顺序排列，这构成了这个强大系统中TCD8“免疫显性”的基础。因此，大部分T Ag特异性TCD8反应集中在单个免疫显性表位上，而其他三个表位仅被微弱识别和反应。免疫显性损害了抗肿瘤TCD8反应的广度，因此，许多人认为这是成功进行癌症疫苗接种的障碍。因此，了解决定或塑造TCD8免疫显性 的细胞和分子因素及机制很重要。我们在此描述的方案是针对T Ag免疫模型中这一现象的研究量身定制的，但可以很容易地修改和扩展到其他肿瘤模型的类似研究。我们提供了如何使用体内细胞毒性测定来测量实验性免疫治疗干预影响的示例。
