Fig. 2.

Developmental pathway of CD24⁻CD73⁻ γδT17 cells. a Schematic of the precursor-product experiment designed to determine the differentiation pathway of CD24⁻CD73⁻ γδT17 cells. Thymic lobes from E14.5 CD1 embryos were collected and cultured in FTOC for 3 days. Next, CD24⁺CD73⁻, CD24⁺CD73⁺, and CD24⁻CD73⁺ γδ T cells (all CD3⁺TCRγδ⁺) were sorted by FACS and incubated with E14.5 TCRδ-deficient thymic lobes overnight in hanging drop culture. The TCRδ-deficient lobes were prepared in advance by treatment with 2′-deoxyguanosine monohydrate (dGuo) for 2 days followed by resting for 1–2 days in media. The colonized lobes were transferred to FTOC and analyzed 2 and 5 days later. b Representative FACS plots of the developmental outcomes of the CD24⁺CD73⁻, CD24⁺CD73⁺, or CD24⁻CD73⁺ γδ T-cell precursors in FTOC for 2 or 5 days. Numbers in FACS plots indicate frequency within each gate. All plots are gated on CD3⁺TCRγδ⁺ cells. c Frequencies of CD24⁻CD73⁻ γδ T cells obtained from CD24⁺CD73⁻, CD24⁺CD73⁺, or CD24⁻CD73⁺ γδ T cells precursors at d2 and d5. d Schematic of the routes through development taken by γδ T-cell precursors on Pathway 1 (blue) vs. Pathway 2 (orange). Data are representative of at least three independent samples. Center values indicate mean, error bars denote s.e.m. p-values were determined by two tailed Student's t-test