RNA Sequencing of Fetal and Adult Mature Thymic Vγ1+ γδ T-Cells

γδ 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 γδ 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 γδ T-cell functional subsets in vivo. We found skewed generation of Vγ1+ cells toward the PLZF+ γδ T-cell lineage at the fetal stage. Similarly, generation of interleukin (IL)-17 producing γδ 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 γδ T-cells. Conversely, weak TCR signals were amenable for the generation of IL-17 producing γδ T-cells, which was Notch-independent. Additionally and surprisingly, Notch signaling was also dispensable for peripheral γδ T-cell IL-17 production. Thus, our results precisely defined the roles of ontogenic timing, TCR signal strength, and Notch signaling in γδ T-cell functional programming in vivo. Mature CD24- Vγ1+ γδ T-cells were sorted from thymi using BD FACSAria Fusion. RNA was extracted using TRIzol. Library construction was done using Takara SMARTer Stranded Total RNA-Seq Kit v3 – Pico Input Mammalian. RNA sequencing was performed using Illumina NovaSeq 6000. Raw data were aligned to GRCm38 using HISAT2 version 2.1 and raw read counts were obtained using HTSeq version 0.1.