RNA-seq profiling of in vitro FRC-conditioned, IL6-conditioned or control (anti-CD3/CD28)-stimulated CD8+ T cells

To broadly assess which pathways FRCs regulate in CD8+ T cells, we sorted CD8+ T cells for RNA-seq following activation in whole splenocyte mixtures via soluble anti-CD3/CD28 for 48 hours (Stim), activated with anti-CD3/CD28 in the presence of Nos2–/– FRCs (FRC) or activated with anti-CD3/CD28 plus 100 ng/ml recombinant IL-6 (IL-6). Here we demonstrate that FRC-derived signals, including IL-6, act in concert with TCR signaling and co-stimulation to promote the expression of MYC and HIF-1-dependent glycolytic genes and vital pro-survival genes (encoding BCL-2 family members, inhibitors of apoptosis [IAPs] members and Cflar) in activated CD8+ T cells. Overall design: CD8+ T cells were sorted for RNA-seq following activation in whole splenocyte mixtures via soluble anti-CD3/CD28 for 48 hours (Stim), activated with anti-CD3/CD28 in the presence of Nos2–/– FRCs (FRC) or activated with anti-CD3/CD28 plus 100 ng/ml recombinant IL-6 (IL-6). The following number of biological replicates were used for each condition: Stim (2), FRC (3), IL-6 (2)

为全面评估成纤维网状细胞（FRCs）在CD8+ T细胞中调控的信号通路，我们将经不同方式激活的CD8+ T细胞分选后进行RNA测序（RNA-seq）：将CD8+ T细胞与全脾细胞混合物共同培养，使用可溶性抗CD3/CD28抗体激活48小时（记为Stim组）；或在Nos2–/–成纤维网状细胞存在下，以抗CD3/CD28抗体激活（记为FRC组）；或使用抗CD3/CD28抗体联合100 ng/ml重组白细胞介素6（IL-6）激活（记为IL-6组）。本研究证实，成纤维网状细胞来源的信号（包括IL-6）可与T细胞受体（TCR）信号及共刺激信号协同作用，促进激活态CD8+ T细胞中MYC、缺氧诱导因子1（HIF-1）依赖的糖酵解相关基因，以及关键促存活基因（编码BCL-2家族蛋白、凋亡抑制蛋白（IAPs）家族成员及Cflar）的表达。实验整体设计：将CD8+ T细胞经以下三种方式激活后分选进行RNA测序（RNA-seq）：1. 与全脾细胞混合物共同培养，使用可溶性抗CD3/CD28抗体激活48小时（Stim组）；2. 在Nos2–/–成纤维网状细胞存在下，以抗CD3/CD28抗体激活（FRC组）；3. 使用抗CD3/CD28抗体联合100 ng/ml重组IL-6激活（IL-6组）。各实验组的生物学重复数如下：Stim组2例，FRC组3例，IL-6组2例。