National Center for Cancer Immune Therapy, Department of Oncology, University Hospital Herlev.
National Center for Cancer Immune Therapy, Department of Oncology, University Hospital Herlev; Department of Immunology and Microbiology, Inflammation and Cancer Group, University of Copenhagen.
J Vis Exp. 2021 Oct 19(176). doi: 10.3791/62984.
During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is indispensable for T cell activation, and the survival of memory T cells is dependent on mitochondrial remodeling. Consequently, this affects the long-term clinical outcome of cancer immunotherapies. Changes in T cell quality are often studied by flow cytometry using well-known surface markers and not directly by their metabolic state. This is an optimized protocol for measuring real-time mitochondrial respiration of primary human T cells using an Extracellular Flux Analyzer and the cytokines IL-2 and IL-15, which differently affect T cell metabolism. It is shown that the metabolic state of T cells can clearly be distinguished by measuring the oxygen consumption when inhibiting key complexes in the metabolic pathway and that the accuracy of these measurements is highly dependent on optimal inhibitor concentration and inhibitor injection strategy. This standardized protocol will help implement mitochondrial respiration as a standard for T cell fitness in monitoring and studying cancer immunotherapies.
在激活过程中，T 细胞的代谢会适应影响其命运的变化。线粒体氧化磷酸化的增加对于 T 细胞的激活是必不可少的，而记忆 T 细胞的存活则依赖于线粒体的重塑。因此，这会影响癌症免疫疗法的长期临床结果。T 细胞质量的变化通常通过使用已知的表面标志物的流式细胞术进行研究，而不是直接通过其代谢状态进行研究。本优化方案使用细胞外通量分析仪和细胞因子 IL-2 和 IL-15 来测量原代人 T 细胞的实时线粒体呼吸，这两种细胞因子会对 T 细胞的代谢产生不同的影响。结果表明，通过测量抑制代谢途径中的关键复合物时的耗氧量，可以清楚地区分 T 细胞的代谢状态，并且这些测量的准确性高度依赖于最佳抑制剂浓度和抑制剂注射策略。本标准化方案将有助于将线粒体呼吸作为监测和研究癌症免疫疗法中 T 细胞适应性的标准。
