Single-Cell RNA Sequencing of Thymic KN6 ?d T-Cells Modulated by TCR Signal Strength and Notch Signaling

?d T-cells form an integral arm of the immune system through their rapid and potent effector functions, and are critical players during both protective and destructive immunity. However, the factors that dictate ?d T-cell functional programming in vivo remain to be fully elucidated. Here, we employed RBPJ-inducible and KN6-transgenic mice to assess the roles of ontogenic timing, T-cell receptor (TCR) signal strength, and Notch signaling in the generation of ?d T-cell functional subsets in vivo. We found skewed generation of V?1+ cells toward the PLZF+ ?d T-cell lineage at the fetal stage. Similarly, generation of interleukin (IL)-17 producing ?d T-cells was favored during, although not exclusive to, the fetal stage. Strong TCR signals, in conjunction with Notch, were necessary for the generation of IL-4 producing ?d T-cells. Conversely, weak TCR signals were amenable for the generation of IL-17 producing ?d T-cells, which was Notch-independent. Additionally and surprisingly, Notch signaling was also dispensable for peripheral ?d T-cell IL-17 production. Thus, our results precisely defined the roles of ontogenic timing, TCR signal strength, and Notch signaling in ?d T-cell functional programming in vivo. Overall design: CD3+V?4+ KN6 cells were sorted from thymi using BD FACSAria Fusion. Library construction was done using 10x Genomics Chromium Controller v3. Single-cell RNA sequencing was performed using Illumina NovaSeq 6000. Raw data were aligned to GRCm38, and raw read counts were obtained using Cell Ranger version 5.0.