Transcriptomic profile of WT and Mthfd2 KO alloreactive CD4+ T cells [RNA-seq]

To investigate the effect of Mthfd2 KO in alloreactive CD4+ T cells. We performed in vitro mixed lymphocyte to induce alloreactive T cells. CD4+ T cells were respectively purified from the spleens of 8-week-old TEaWT and TEaMthfd2 KO mice by magnetic-activated cell sorting (MACS) positive selection. APCs were sorted from the spleens of 8-week-old F1 (BALB/c x C57BL/6) mice. CD4+ T cells were cocultured with APCs three days and then performed RNA-sequencing. Overall design: CD4+ T cells were respectively purified from the spleens of 8-week-old TEaWT or TEaMthfd2 KO mice by magnetic-activated cell sorting (MACS) positive selection. APCs were sorted from the spleens of 8-week-old F1 (BALB/c x C57BL/6) mice and stained with mitomycin for 30 min at 37 °C with 5% CO2. Then, CD4+ T cells were then cocultured with APCs for 3 days at 37 °C and 5% CO2 in RPMI 1640 medium supplemented with 10 mM HEPES, 50 mM 2-mercaptoethanol, 100 U/mL penicillin/streptomycin, and 2 mM glutamine.

为探究Mthfd2基因敲除（Mthfd2 knockout, KO）对同种反应性CD4+ T细胞的影响，我们通过体外混合淋巴细胞培养诱导同种反应性T细胞。我们采用磁激活细胞分选（magnetic-activated cell sorting, MACS）阳性分选法，分别从8周龄TEa野生型（TEaWT）与TEaMthfd2基因敲除（TEaMthfd2 KO）小鼠的脾脏中纯化CD4+ T细胞；抗原呈递细胞（antigen-presenting cells, APCs）则从8周龄F1（BALB/c×C57BL/6）小鼠的脾脏中分选获得。将CD4+ T细胞与APCs共培养3天后，进行RNA测序（RNA-sequencing）。

整体实验设计：我们通过磁激活细胞分选（MACS）阳性分选法，分别从8周龄TEaWT或TEaMthfd2 KO小鼠的脾脏中纯化CD4+ T细胞；APCs从8周龄F1（BALB/c×C57BL/6）小鼠脾脏中分选获得后，于37℃、5%CO₂条件下用丝裂霉素处理30分钟。随后将CD4+ T细胞与APCs于37℃、5%CO₂条件下共培养3天，所用培养基为添加了10mM HEPES、50mM β-巯基乙醇、100U/mL青霉素/链霉素以及2mM谷氨酰胺的RPMI 1640培养基。