Effect of STm-delta-aroA on CD4+ T cell activation

The experiment was designed to test how tumour conditoned media from STm-delta-aroA infected tumour organoids would modulate T cell activation. Using splenocytes from Nr4a3-Tocky Great Smart-17A mice, T cells were activated with soluble anti-CD3 and anti-CD28 antibodies. At 4 and 16 h live CD4+ T cells were FACS sorted and RNA extracted. Analysis revealed that T cells exposed to STm-delta-aroA conditioned tumor medium failed to upregulate metabolic pathways associated with canonical T cell activation. Overall design: Tumour organoids (colonic and small intestinal) derived from APCmin mice were cultured in Matrigel and infected with either an attenuated STm mutant (aroA, containing a deletion in the shikimate pathway) or left non-infected (non-treated) for 2 hours. After 2 hours, the organoids were washed twice with PBS and then re-cultured in fresh media containing gentamicin, in order to kill any extracellular bacteria. Tumours were incubated at 37C for 48hours, after which time the tumour-conditioned media (TCM) was aspirated and pooled. Meanwhile, splenocytes from Nr4a3-Tocky-GS mice were isolated and seeded into a 96-well plate. Splenocytes (1x10^6/well) were cultured in 100ul TCM from non-treated or STm-infected tumours for 4 and 16h, with 1 ug/mL a-CD3 and 5 ug/mL a-CD28 antibodies added to each well to induce T cell activation. Live CD4+ T cells were then FACS-sorted and RNA extracted. Libraries were made using Lexogen's Quant-Seq 3' mRNA FWD UMI kits according to the manufacturer's instructions. Libraries were pooled and sequenced on a NextSeq 500 machine.

本实验旨在探究鼠伤寒沙门氏菌aroA基因缺失突变株（STm-delta-aroA）感染的肿瘤类器官所制备的条件培养基，对T细胞活化的调控作用。实验使用来自Nr4a3-Tocky Great Smart-17A小鼠的脾淋巴细胞，以可溶性抗CD3、抗CD28抗体激活T细胞。分别于培养4小时和16小时时，通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）分离活的CD4+ T细胞并提取核糖核酸（RNA）。分析结果显示，暴露于STm-delta-aroA条件化肿瘤培养基的T细胞，未能上调与经典T细胞活化相关的代谢通路。总体实验设计如下：从APCmin小鼠中分离得到的结肠及小肠肿瘤类器官，接种于基质胶（Matrigel）中培养，分别用减毒STm突变株（aroA基因缺失，涉及莽草酸途径）感染2小时，或不做感染处理作为空白对照。感染2小时后，用磷酸盐缓冲液（Phosphate Buffered Saline, PBS）洗涤类器官两次，随后转移至含庆大霉素的新鲜培养基中培养，以杀灭胞外残留细菌。将肿瘤样本置于37℃孵育48小时，之后收集肿瘤条件培养基（TCM）并混合均匀。与此同时，分离来自Nr4a3-Tocky-GS小鼠的脾淋巴细胞，接种于96孔板中。每孔接种1×10^6个脾淋巴细胞，加入100μL来自空白对照或STm感染肿瘤的TCM进行培养，同时每孔添加1 μg/mL抗CD3抗体与5 μg/mL抗CD28抗体以诱导T细胞活化，分别培养4小时和16小时。随后通过FACS分离活的CD4+ T细胞并提取RNA。参照Lexogen公司Quant-Seq 3' mRNA FWD UMI试剂盒的制造商说明书构建测序文库，将文库混合后在NextSeq 500测序仪上完成测序。