text: Isolation of CD8+ T cells from Runx3-/- mice.
  Runx3-deficient T cells fail to silence CD4 expression normally (Fig. S1) (12, 13). We therefore further fractionated the positively selected CD8+ T cells from Runx3 KO mice into CD8+CD4- SP or CD8+CD4+ DP cells by separation using anti-CD4 magnetic beads. This yielded a Runx3 KO SP "enriched" population that contained 75% CD8+CD4- cells and a KO DP enriched population that contained 85% CD8+CD4+ cells (Fig. S1). The cells were stimulated with anti-CD3+ anti-CD28 for 2 d before removing them from the TCR stimulus and culturing them in media containing 100 U/ml IL-2. As previously reported, TCR-induced proliferation of Runx3-/- CD8+ T cells was severely impaired, irrespective of CD4 expression (Fig. S1) (12, 13). However, the Runx3-/- cells showed cell-surface expression patterns indicative of activated cells, including up-regulation of CD25 and CD69 (Fig. S1). As expected from their ability to up-regulate CD25, Runx3-/- CD8+ T cells responded to IL-2 supplementation after day 2 and efficiently expanded until day 6 of the culture period, albeit at slower rates compared with WT cells (Fig. S1). Although a fraction of the KO DP cells silenced CD4 expression after activation, the ratio of SP/DP cells in each enriched population remained constant thereafter, and we did not observe any major differences between these two populations throughout the culture period, indicating that in terms of effector CTL differentiation and under our culture conditions, Runx3-/- CD8+ T cells that also coexpress CD4 are indistinguishable from those that do not. The data presented in Fig. S2 are from Runx3 KO SP cells, whereas those shown in Figs. 3 and 4 are from total Runx3 KO CD8 cells. 
  