Ingulli Elizabeth
Center for Immunology, Department of Pediatrics, University of Minnesota Medical School, Minneapolis, MN, USA.
Methods Mol Biol. 2007;380:365-76. doi: 10.1007/978-1-59745-395-0_23.
Tracking antigen-specific cytotoxic T lymphocyte (CTL) function in vivo can be difficult due to the need to monitor the presence and subsequent destruction of antigen-bearing target cells. In this report, we describe a simple method using the fluorescent dye 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) to evaluate CD8+ T-cell effector function in vivo by flow cytometry. In this assay, peptide-pulsed and control target cells are labeled to different levels with CFSE and coadministered to animals that have been previously immunized or tolerized to the cognate antigen. Because naive antigen-specific CD8+ T cells cannot acquire effector function within the time frame of this assay, adoptively transferred nonimmunized animals are used as negative controls for in vivo CTL function. Target cells are syngeneic splenocytes pulsed with peptide antigen and control cells are unpulsed syngeneic splenocytes. The loss of antigen-specific target cells is indicative of cytotoxicity and immunity, whereas the lack of killing in the setting of antigen recognition is suggestive of tolerance.
由于需要监测携带抗原的靶细胞的存在及随后的破坏情况，在体内追踪抗原特异性细胞毒性T淋巴细胞（CTL）功能可能具有挑战性。在本报告中，我们描述了一种简单的方法，即使用荧光染料5-（及6-）羧基荧光素二乙酸琥珀酰亚胺酯（CFSE），通过流式细胞术在体内评估CD8 + T细胞效应器功能。在该实验中，用CFSE将肽脉冲处理的靶细胞和对照靶细胞标记至不同水平，并共同给予先前已针对同源抗原进行免疫或耐受的动物。由于未致敏的抗原特异性CD8 + T细胞在此实验的时间范围内无法获得效应器功能，因此将过继转移的未免疫动物用作体内CTL功能的阴性对照。靶细胞是用肽抗原脉冲处理的同基因脾细胞，对照细胞是未脉冲处理的同基因脾细胞。抗原特异性靶细胞的损失表明存在细胞毒性和免疫性，而在抗原识别情况下缺乏杀伤作用则提示存在耐受性。
