Fig. 1: Characterization of CD8^Dual mice.

a, Top: schematic of double-positive thymocytes from CD8^Dual mice. The altered Cd4 gene locus encodes CD8α.1 co-receptors (composed of CD8α.1CD8β dimers), and the endogenous Cd8 gene locus encodes CD8α.2 co-receptors (composed of CD8α.2CD8β dimers). Bottom: schematic of the altered Cd4^CD8 and endogenous Cd8^CD8 genes in CD8^Dual mice; IRES, internal ribosome entry site; poly A, polyadenylation signals. b, Flow cytometry analysis of whole thymocytes from CD8^Dual (n = 16) and B6 (n = 15) mice (representative of 15 independent experiments). Top: Cd4-encoded co-receptors (CD8α.1 or CD4) versus Cd8-encoded co-receptor CD8α.2 profile, showing four thymocyte subsets; double-negative (DN), double-positive (DP) and two single-positive T cell subsets. Total cell number (mean ± s.e.m) is shown above profiles. Middle: TCRβ histogram, showing TCRβ^hi (TCR^hi) thymocytes. Bottom: CD24 versus TCRβ profile, showing CD24⁻TCR⁺ mature thymocytes. c, Flow cytometry analysis of TCR^hi thymocytes and TCR⁺ LN T cells from CD8^Dual (n = 16) and B6 (n = 15) mice, showing Cd4-encoded co-receptor (CD8α.1 or CD4) versus Cd8-encoded co-receptor (CD8α.2) or CD8β profiles. TCR expression of LN cells are shown in Extended Data Fig. 1b. Numbers (mean ± s.e.m.) of TCR^hi thymocytes and LN T cells are shown above profiles (representative of 15 independent experiments). d, Numbers of CD8β⁺ T cells among CD24⁻TCR⁺ mature thymocytes (T) and TCR⁺ LN T cells (L) in CD8^Dual mice with the indicated MHC deficiencies. CD8β histograms are shown in Extended Data Fig. 1d (WT: T (n = 6), L (n = 9), MHC-II^KO: T (n = 9), L (n = 9), β2m^KO: T (n = 7), L (n = 9), MHC^KO: T (n = 3), L (n = 5), 3–7 independent experiments). e, Mean fluorescence intensity (MFI) of Runx3d-YFP and ThPOK-GFP reporter expression in CD8.1, CD8.2, CD4⁺ and CD8⁺ T cells among TCR^hi thymocytes and TCR⁺ LN T cells in CD8^Dual and B6 mice based on histograms shown in Extended Data Fig. 1e (Runx3d-YFP: CD8^Dual T (n = 4), L (n = 4), B6 T (n = 5), L (n = 4), ThPOK-GFP: CD8^Dual T (n = 3), L (n = 3), B6 T (n = 4) L (n = 3), representative of 3–5 independent experiments). f, RNA-seq analysis of CD62L⁺TCR⁺ LN T cells from CD8^Dual and B6 mice. Genes differentially expressed between B6 CD4⁺ and CD8⁺ LN T cells were evaluated in the heat map for analysis of gene expression in CD8.1 and CD8.2 LN T cells from CD8^Dual mice (n = 3 per group, P < 0.05, 5-fold change). Numbers within profiles and histograms indicate frequency of cells in each box or lines (b and c). ***P < 0.001, **P < 0.01, *P < 0.05 (two-tailed unpaired t-tests); mean ± s.e.m. (d and e).