Supplementary Figure 1: Impact of Tcf1 and Lef1 deficiency on the development and gene expression of CD8⁺ T cells.

(a) Derepression of the CD4 coreceptor in Tcf7^–/–Lef1^–/– CD8⁺ T cells. Thymocytes from Tcf7^–/–Lef1^–/– and control mice were surface-stained. Shown is the gating strategy to identify TCRβ^hi CD69^–CD44^– mature thymocytes and further fractionation to identify CD4⁺ and CD8⁺ single positive cells. The CD8⁺ mature thymocytes were further analyzed for the CD44^hiIL-2Rβ⁺ memory-phenotype subset. Our previous study (Steinke et al. Nat. Immunol. 15, 646-656, 2014) demonstrated that the CD4⁺CD8⁺ population within the mature thymocyte subset was due to derepression of the CD4 coreceptor in CD8⁺ cells, but not aberrant expression of the CD8 coreceptor in CD4⁺ cells. This gating strategy was also used to sort control CD4⁺, CD8⁺, and Tcf7^–/–Lef1^–/– CD8⁺ T cells for transcript and histone mark analyses.

(b) Tcf7^–/–Lef1^–/– CD8⁺ T cells preserved the expression of CD8⁺ signature genes. CD4⁺ and CD8⁺ mature thymocytes were sorted from control mice, and Tcf7^–/–Lef1^–/– CD8⁺ mature thymocytes were also sort-purified. The sorted cells were analyzed for gene expression with quantitative RT-PCR. Nkg7 and Itgae are CD8⁺-specific genes, and the Runx3d transcript from its distal promoter is specifically utilized in CD8⁺ T cells.

(c) Aberrant upregulation of CD8⁺ effector molecules in naïve Tcf7^–/–Lef1^–/– CD8⁺ T cells. Cells were sorted and analyzed for gene expression as in (b). Prdm1 and Fasl are virtually not expressed, and Prf1 is minimally expressed in naïve CD8⁺ T cells. Data of (b) and (c) are means ± s.d. from 4 independent experiments (n ≥ 6).

(d) Tcf7^–/–Lef1^–/– splenic CD8⁺ T cells were neither aberrantly activated nor enriched in memory-phenotype cells. CD8⁺TCRβ⁺ splenocytes from Tcf7^–/–Lef1^–/– and control mice were identified (top panels) and further fractionated to CD62L⁺CD44^lo naïve, CD62L⁺CD44^hi memory-phenotype, CD62L^–CD44^hi effector-phenotype cells (bottom panels). The CD8⁺ T cells were also analyzed for the CD44^hiIL-2Rβ⁺ memory-phenotype subset. The frequency of each subset is shown in representative contour plots. Data are from ≥ 3 independent experiments with similar results.

Note that CD44^hiIL-2Rβ⁺ memory-phenotype population was not enriched but actually reduced in both Tcf7^–/–Lef1^–/– mature CD8⁺ thymocytes and splenic CD8⁺ T cells. This observation is consistent with our previous finding that longevity of memory CD8⁺ T cells depends on Tcf1 (Zhou et al. Immunity 33, 229, 2010).

(e)-(f) Survey of Vβ TCR repertoire in Tcf7^–/–Lef1^–/– CD8⁺ T cells. CD8⁺ mature thymocytes from control and Tcf7^–/–Lef1^–/– mice were further stained with mouse Vβ TCR screening panel, and the percentage of each Vβ subtype was determined. Representative data for select Vβ TCRs are shown in histograms (e), and cumulative data from 2 experiments (n = 3) are summarized in (f). *, p<0.05; **, p<0.01 by student’s t-test.