Role of c-Myc in IL-15–dependent differentiation of DN TCRαβ⁺ precursors of CD8αα TCRαβ IELs. (A) IEL reconstitution on transfer of electronically sorted DN B220⁻ NK1.1⁻ TCRαβ⁺ thymocytes from WT and c-myc^ΔCD4 mice into Rag2⁻γ_c⁻ recipient mice. PBS alone was injected as negative control. Mice were analyzed 1 month after the transfer. TCRαβ IELs (identified in top panels) were stained with CD8α versus CD8β (bottom panels). Bar graph represents absolute numbers (mean ± SD; n = 5) of CD8αα TCRαβ IELs. (B) Histograms represent surface expression of IL-15R chains on DN NK1.1⁻B220⁻ TCRαβ⁺ thymocytes from WT and c-myc^ΔCD4 mice. The number in each histogram corresponds to the mean fluorescence intensity (MFI) of the IL-15R staining (empty histogram) minus the MFI of the unstained control (gray filled histogram) (mean ± SD; n = 5). (C) DN B220⁻ NK1.1⁻ TCRαβ⁺ thymocytes from WT and c-myc^ΔCD4 were electronically sorted and cultured with 100 ng/mL hIL-15 for 6 days. After culture, cells were counted (the absolute number of viable cells was similar for WT and c-myc^ΔCD4 mice) and stained with TCRαβ, CD8α, and CD8β. Dot plots show CD8α versus CD8β profile of TCRαβ⁺ cells. Numbers in the dot plots indicate percentages. Data are representative of 2 independent experiments. (D) Histograms show surface expression of IL-15R chains on DN NK1.1⁻B220⁻ TCRαβ⁺ thymocytes from control C57BL/6 and IL15^−/− mice. The number in each histogram corresponds to the MFI of the IL-15R staining (empty histogram) minus the MFI of the unstained control (gray filled histogram) (mean ± SD; n ≥ 4). Bar graph represents absolute number of DN NK1.1⁻ TCRαβ⁺ thymocytes in control C57BL/6 and IL-15^−/− mice (mean ± SD; n ≥ 4).