Regulation of CD8 T cell differentiation by the RNA-binding protein DDX5. Regulation of CD8 T cell differentiation by the RNA-binding protein DDX5

The RNA-binding protein DDX5 is a polyfunctional regulator of gene expression, but its role in CD8+ T cell biology has not been extensively investigated. In this study, we demonstrate that deletion of DDX5 reduced differentiation of terminal effector (TE), effector memory (TEM), and terminal effector memory (t-TEM) cells while increasing the generation of central memory (TCM) cells; forced expression of DDX5 elicited the opposite phenotype. DDX5-deficient CD8+ T cells exhibited increased expression of genes that promote TCM cell differentiation, including Tcf7 and Eomes. Together, these findings reveal a role for DDX5 in regulating the differentiation of effector and memory CD8+ T cell subsets in response to microbial infection. Overall design: WT CD45.1 and Ddx5fl/flCd4-Cre+ CD8+ CD45.1.2 P14 cells were adoptively co-transferred into congenically distinct recipient mice. The recipient mice were infected with 2×105 PFU LCMV 24 hours after transfer and sacrificed on days 4, 7, and 30 post-infection. Splenic P14 T cells were isolated by FACS. About 10,000 cells per sample were loaded into Single Cell G chips (10x Genomics) and partitioned into Gel Bead In-Emulsions in a Chromium Controller (10x Genomics). Single-cell RNA libraries were prepared according to the 10x Genomics Chromium Next GEM Single Cell 3’ v3.1 (Dual Index) User Guide and sequenced on a HiSeq 4000 (Illumina).

RNA结合蛋白（RNA-binding protein）DDX5是一种多效性基因表达调控因子，但其在CD8+ T细胞（CD8+ T cell）生物学中的作用尚未得到广泛研究。本研究证实，敲除DDX5会抑制终末效应细胞（terminal effector, TE）、效应记忆细胞（effector memory, TEM）及终末效应记忆细胞（terminal effector memory, t-TEM）的分化，同时促进中央记忆细胞（central memory, TCM）的生成；而强制过表达DDX5则会诱导出相反的表型。DDX5缺陷型CD8+ T细胞中，促进TCM细胞分化的基因（包括Tcf7和Eomes）的表达水平显著上调。综上，本研究揭示了DDX5在微生物感染应答过程中调控效应性及记忆性CD8+ T细胞亚群分化中的作用。
整体实验设计：将野生型（wild type, WT）CD45.1小鼠与Ddx5fl/flCd4-Cre+ CD8+ CD45.1.2 P14细胞通过过继共转移的方式移植至同基因背景不同的受体小鼠中。受体小鼠在细胞移植24小时后，以2×10^5 蚀斑形成单位（plaque forming unit, PFU）的淋巴细胞脉络丛脑膜炎病毒（lymphocytic choriomeningitis virus, LCMV）进行感染，并分别于感染后第4、7、30天处死小鼠。通过荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）分离脾脏中的P14 T细胞。每个样本取约10000个细胞，加载至Single Cell G芯片（10x Genomics）中，在Chromium控制器（10x Genomics）中进行凝胶微珠乳液（Gel Bead In-Emulsions, GEM）的分区操作。随后按照10x Genomics Chromium Next GEM Single Cell 3’ v3.1（双索引）用户指南制备单细胞RNA测序文库，并在HiSeq 4000测序仪（Illumina）上完成测序。