Early Notch signaling programs effector CD8+ T cell differentiation [RNA-Seq]

A better understanding of the mechanisms regulating CD8+ T cell differentiation is essential to develop new strategies to fight infections and cancer. Using genetic mouse models and blocking antibodies, we uncovered cellular and molecular mechanisms by which Notch signaling favors efficient generation of effector CD8+ T cells. Using transcriptional and epigenetic studies, we identified a unique Notch-driven T cell-specific signature. Early Notch signals were associated with chromatin opening in regions occupied by bZIP transcription factors, specifically BATF, known to be important for CD8+ T cell differentiation. Overall, we show that the delivery of early Notch signals controls the molecular and functional fate of CD8+ T cells after infection. For RNA-seq of naive WT vs Notch1/2 KO CD8+ T cells, naive OT-I cells (Zombie dye− CD8+ CD45.2+ CD25− CD69− CD44−) from pooled lymph nodes and spleens were sorted, and a total of 3 individual biological samples from 3 independent experiments were collected for each genotype (6 x 10^5 to 3 x 10^6 cells per sample). For RNA-seq of day 3 WT vs Notch1/2 KO CD8+ T cells, B6-CD45.1 mice were adoptively transferred with WT vs Notch1/2 KO OT-I cells and infected with Lm-OVA. 72h post-infection, activated OT-I cells (Zombie dye− CD8+ CD45.2+ CD44+) from spleens were sorted, and a total of 8 individual biological samples from 3 independent experiments were collected for each genotype (1.9 x 10^4 to 1.1 x 10^5 cells per sample). For RNA-seq of OP9 vs OP9-DLL4-cocutured CD8+ T cells, OT-I cells from pooled lymph nodes were activated in vitro with aCD3/aCD28 for 72h before being co-cultured on OP9 or OP9-DLL4 fibroblasts for 24h. OT-I were harvested and co-cultured on fresh OP9 or OP9-DLL4 for another 24h. 5 days post-activation, OT-I cells were harvested and sorted (Zombie dye−CD8+ CD45.2+ CD44+). A total of 3 individual biological samples from one independant experiment were collected for each condition (1 x 10^5 cells per sample). For RNA-seq of day 3 WT vs Notch1/2 KO CD8+ T cells transduced with empty-RV or BATF-RV, OT-I cells were isolated from pooled lymph nodes and cultured with IL-7, IL-15 and IL-2 to induce homeostatic proliferation. After 48h in culture, cells were spin-transduced with RV supernatant and put back in culture with IL-7, IL-15 and IL-2 for 48h. Transduced OT-I cells were adoptively transferred into B6-CD45.1 mice and infected with Lm-OVA. 72h post-infection, transduced OT-I cells (Zombie dye− CD8+ CD45.2+ CD44+ GFP+) were sorted from spleens. A total of 4 individual biological samples from one independent experiment were collected for each condition (6 x 10^4 to 1 x 10^5 cells per sample).