Jakic Bojana, Kimpel Janine, Olson William J, Labi Verena, Hermann-Kleiter Natascha
Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
Institute of Virology, Medical University of Innsbruck, Innsbruck, Austria.
Bio Protoc. 2021 Dec 5;11(23):e4247. doi: 10.21769/BioProtoc.4247.
Pathogens such as bacteria, viruses, fungi, or protozoa can cause acute and chronic infections in their hosts. The intracellular bacterium serves as a model pathogen to assess the molecular mechanisms regulating CD8 T cell activation, differentiation, and function. We set up an experimental workflow to investigate cell-intrinsic roles of the nuclear receptor NR2F6 in CD8 T cell memory formation upon (LmOVA) infection ( Jakic , 2021 ). The current protocol details how to cultivate ovalbumin-expressing LmOVA, infect naïve C57BL/6 mice with these bacteria and determine the bacterial load in host organs. Furthermore, we describe how to evaluate antigen-specific CD8 T cell responses and discriminate between short-lived effector and memory precursor cells following LmOVA infection (Figure 1). To assess CD8 T cell-intrinsic molecular mechanisms, we integrated an adoptive cell transfer (ACT) experiment of genetically modified naïve OT-I CD8 T cells into congenic hosts before LmOVA infection. Graphic abstract: Figure 1.Experimental workflow depicting the steps for infection of mice with Listeria and subsequent analysis of antigen-specific CD8 memory responses. Bacteria (ovalbumin expressing ) are thawed and grown on lysogeny broth (LB) plates overnight (ON). A single colony is picked and grown in LB medium ON. Bacteria from the exponential growth phase are then injected into a C57BL/6 mouse via tail vein injection. Colony forming units (CFU) of the bacteria can be detected in the spleen on day 3 post injection. Antigen-specific CD8 T cell immune response can be investigated during the acute phase (d3 after infection), during the peak of the adaptive immune response (d7), the clearance phase (d26), or the memory phase (d70) by flow cytometry. Created with BioRender.com.
细菌、病毒、真菌或原生动物等病原体可在其宿主中引发急性和慢性感染。细胞内细菌作为一种模式病原体，用于评估调节CD8 T细胞激活、分化和功能的分子机制。我们建立了一个实验流程，以研究核受体NR2F6在感染（LmOVA）后CD8 T细胞记忆形成中的细胞内在作用（Jakic，2021年）。本方案详细介绍了如何培养表达卵清蛋白的LmOVA，用这些细菌感染未感染过的C57BL/6小鼠，并确定宿主器官中的细菌载量。此外，我们还描述了如何评估抗原特异性CD8 T细胞反应，并区分LmOVA感染后的短期效应细胞和记忆前体细胞（图1）。为了评估CD8 T细胞内在分子机制，我们在LmOVA感染前将基因改造的未感染过的OT-I CD8 T细胞的过继性细胞转移（ACT）实验整合到同基因宿主中。图形摘要：图1.描述用李斯特菌感染小鼠并随后分析抗原特异性CD8记忆反应步骤的实验流程。细菌（表达卵清蛋白）解冻后在溶原肉汤（LB）平板上培养过夜。挑取单个菌落并在LB培养基中培养过夜。然后将处于指数生长期的细菌通过尾静脉注射到C57BL/6小鼠体内。注射后第3天可在脾脏中检测到细菌的菌落形成单位（CFU）。可在急性期（感染后第3天）、适应性免疫反应高峰期（第7天）、清除期（第26天）或记忆期（第70天）通过流式细胞术研究抗原特异性CD8 T细胞免疫反应。由BioRender.com创建。
