Figure 3. PLZF is required for the effector maturation of Vγ6⁺ γδT-cells in vitro.

a) Gating strategy indicating the subpopulations of Vγ6⁺ γδT-cells that were sorted and placed for 3 days in OP9-DL1 co-cultures in the presence of IL-7 to evaluate their ability to generate different subpopulations. FACs analysis showing the phenotypic profile of sorted cells post-sort and after co-culture. The numbers indicate the proportion of cells within the quadrants. b) FACs analysis for intracellular IL-17 staining after PMA plus Ionomycin stimulation of sorted CD27⁺CD44⁻Vγ6⁺γδT-cells from fetal (E16) thymus after sort (Ex vivo) or after a 3 day differentiation in OP9-DL1 co-cultures as in panel (a). The percentage of IL-17⁺ cells is indicated. c) FACs analysis of Anexin V positive cells after 3 day OP9-DL1 co-culture. The percentage of anexinV positive cells is indicated. d) Histogram showing the dilution of cell trace violet on sorted wild type (+/+) and PLZF-deficient (−/−) CD27⁺CD44⁻ Vγ6⁺ γδT-cells after 3 day co-culture as in (a). The gray histogram represent non-proliferating T-cells. e) histograms showing the incorporation of BrdU in fetal E16 Vγ6⁺ γδT-cells 24 hours after a single BrdU injection to the mother. f) Histogram showing the dilution of cell trace violet on sorted wild type CD27⁺CD44⁻ Vγ6⁺ γδT-cells after co-culture in the presence of the CDK4/6 inhibitor (PD0332991). g) FACs analysis showing the phenotypic profile of sorted cells after co-culture. h) FACs analysis showing intracellular IL-17 staining in sorted cells after co-culture. The numbers indicate the proportion of cells within the quadrants. i) RT-PCR analysis of sorted immature E16 Vγ6⁺CD27⁺CD44⁻ γδT-cells for the indicated genes. Data correspond to two independent experiments. * Vγ6⁺ cells are identified as GL3⁺2C11⁺Vγ5⁻17D1⁺ cells.