鉴定调节钙依赖性CD8 T细胞细胞毒性的分子候选物。

Identification of molecular candidates which regulate calcium-dependent CD8 T-cell cytotoxicity.

作者信息

Zöphel Sylvia, Schäfer Gertrud, Nazarieh Maryam, Konetzki Verena, Hoxha Cora, Meese Eckart, Hoth Markus, Helms Volkhard, Hamed Mohamed, Schwarz Eva C

机构信息

Biophysics, Center for Integrative Physiology and Molecular Medicine, School of Medicine, Saarland University, Building 48, 66421 Homburg, Germany.

Center for Bioinformatics, Saarland Informatics Campus, Saarland University, 66041 Saarbrücken, Germany.

出版信息

Mol Immunol. 2023 May;157:202-213. doi: 10.1016/j.molimm.2023.04.002. Epub 2023 Apr 17.

DOI:10.1016/j.molimm.2023.04.002

PMID:37075611

Abstract

Cytotoxic CD8 T lymphocytes (CTL) eliminate infected cells or transformed tumor cells by releasing perforin-containing cytotoxic granules at the immunological synapse. The secretion of such granules depends on Ca-influx through store operated Ca channels, formed by STIM (stromal interaction molecule)-activated Orai proteins. Whereas molecular mechanisms of the secretion machinery are well understood, much less is known about the molecular machinery that regulates the efficiency of Ca-dependent target cell killing. CTL killing efficiency is of high interest considering the number of studies on CD8 T lymphocytes modified for clinical use. Here, we isolated total RNA from primary human cells: natural killer (NK) cells, non-stimulated CD8 T-cells, and from Staphylococcus aureus enterotoxin A (SEA) stimulated CD8 T-cells (SEA-CTL) and conducted whole genome expression profiling by microarray experiments. Based on differential expression analysis of the transcriptome data and analysis of master regulator genes, we identified 31 candidates which potentially regulate Ca-homeostasis in CTL. To investigate a putative function of these candidates in CTL cytotoxicity, we transfected either SEA-stimulated CTL (SEA-CTL) or antigen specific CD8 T-cell clones (CTL-MART-1) with siRNAs specific against the identified candidates and analyzed the killing capacity using a real-time killing assay. In addition, we complemented the analysis by studying the effect of inhibitory substances acting on the candidate proteins if available. Finally, to unmask their involvement in Ca dependent cytotoxicity, candidates were also analyzed under Ca-limiting conditions. Overall, we identified four hits, CCR5 (C-C chemokine receptor type five), KCNN4 (potassium calcium-activated channel subfamily N), RCAN3 (regulator of calcineurin) and BCL (B-cell lymphoma) 2 which clearly affect the efficiency of Ca dependent cytotoxicity in CTL-MART-1 cells, CCR5, BCL2, and KCNN4 in a positive manner, and RCAN3 in a negative way.

摘要

细胞毒性CD8 T淋巴细胞（CTL）通过在免疫突触处释放含穿孔素的细胞毒性颗粒来清除被感染的细胞或转化的肿瘤细胞。此类颗粒的分泌依赖于通过由基质相互作用分子（STIM）激活的Orai蛋白形成的储存操纵性钙通道的钙内流。虽然分泌机制的分子机制已得到充分了解，但对于调节钙依赖性靶细胞杀伤效率的分子机制却知之甚少。考虑到对用于临床的修饰CD8 T淋巴细胞的研究数量，CTL杀伤效率备受关注。在此，我们从原代人细胞中分离出总RNA：自然杀伤（NK）细胞、未刺激的CD8 T细胞以及金黄色葡萄球菌肠毒素A（SEA）刺激的CD8 T细胞（SEA-CTL），并通过微阵列实验进行全基因组表达谱分析。基于转录组数据的差异表达分析和主调节基因分析，我们鉴定出31个可能调节CTL中钙稳态的候选基因。为了研究这些候选基因在CTL细胞毒性中的假定功能，我们用针对已鉴定候选基因的小干扰RNA（siRNA）转染SEA刺激的CTL（SEA-CTL）或抗原特异性CD8 T细胞克隆（CTL-MART-1），并使用实时杀伤试验分析杀伤能力。此外，如果有可用的作用于候选蛋白的抑制性物质，我们通过研究其作用效果来补充分析。最后，为了揭示它们在钙依赖性细胞毒性中的作用，还在钙限制条件下对候选基因进行了分析。总体而言，我们鉴定出四个关键基因，即C-C趋化因子受体5型（CCR5）、钾钙激活通道亚家族N成员4（KCNN4）、钙调神经磷酸酶调节因子3（RCAN3）和B细胞淋巴瘤2（BCL2），它们明显影响CTL-MART-1细胞中钙依赖性细胞毒性的效率，CCR5、BCL2和KCNN4起正向作用，而RCAN3起负向作用。