Figure 1: Demonstration of the DN-stage-thymocyte origin of NKT cells by a novel fate-mapping approach using E8_III-Cre⁺Rosa26-YFP mice, in which YFP expression is controlled by the DP-stage-specific expression of E8_III-Cre.

(a) Flow cytometry of cells from E8_III-Cre⁺Rosa26-YFP mice (n = 6), showing the gating of viable TCRβ⁺B220^– splenocytes by expression of CD4 and CD8 (far left), and expression of the YFP reporter by the electronically gated CD4⁺ TCRβ⁺ fraction (middle left), CD8⁺ TCRβ⁺ fraction (middle right) and DN TCRβ⁺ fraction (far right). Numbers in top corners of plots (middle and right) indicate percent YFP^– cells (left corner) or YFP⁺ cells (right corner) in gates bracketed below, as follows (values, mean ± s.e.m.): YFP^– cells, 5.3% ± 0.8% (CD4⁺), 4.0% ± 0.6% (CD8⁺) and 9.6% ± 1.0% (DN); YFP⁺ cells, 95.0% ± 0.8% (CD4⁺), 96.0% ± 0.6% (CD8⁺) and 90.0% ± 1.0% (DN). (b,c) Flow cytometry of DN thymocytes (n = 3 mice per genotype) (b) and splenocytes (n = 6 mice) (c) from C57BL/6 mice (B6) or E8_III-Cre⁺Rosa26-YFP mice, purified by magnetic depletion with anti–mouse CD4 and CD8 microbeads and assessed with unloaded CD1d dimers (far left, b) or α-GalCer (GC)-loaded CD1d dimers. Numbers adjacent to outlined areas indicate percent CD1d-dimer⁺ NKT cells among DN TCRβ⁺ splenocytes (left) or among gated YFP^– (middle) or YFP⁺ (right) DN TCRβ⁺ splenocytes (b), or percent YFP^– cells (left) or YFP⁺ cells (right) among DN TCRβ⁺ splenocytes (c, top), or CD1d-dimer⁺ NKT cells among those gated DN TCRβ⁺ splenocytes (c, below). Data are from three independent experiments.