Fig. 3.

Recruitment of Gro/TLE is required for Runx3-mediated CD4 silencing but not for Runx3-dependent CD8⁺ T cell proliferation. (A) Mature thymic and splenic CD8⁺ T cells of Runx3^VWRPY−/− mice also expressed CD4. Thymocytes (Upper) and splenocytes (Lower) from WT, Runx3^VWRPY−/−, and Runx3^−/− mice were stained with anti-TCR-β, anti-HSA, anti-CD8α, and anti-CD4 mAb and analyzed by FACS. Mature cells were gated as TCR^highHSA^−/low. The frequencies of SP CD8, SP CD4, and DP CD4⁺CD8⁺ populations are indicated. A representative experiment of five independent analyses is shown. Note the profound reduction of splenic CD8⁺ T cells in Runx3^−/− but not in Runx3^VWRPY−/− or WT mice. (B) Frequency of mature splenic CD8⁺ cells that also expressed CD4 is significantly (∗) increased in Runx3^VWRPY−/− (n = 5; P < 0.0001) and Runx3^−/− mice (n = 5; P < 0.0003) compared with WT. Frequency of splenic CD4⁺CD8⁺ in Runx3^VWRPY−/− mice was significantly higher (n = 5; P < 0.015) compared with Runx3^−/− mice. (C) Runx3^VWRPY−/− splenic CD8⁺ T cells display normal proliferation. 5,6-Carboxyfluorscein diacetate, succinimidyl ester (CFSE) cell division assay of CD8⁺ splenocytes isolated from WT (blue), Runx3^VWRPY−/− (green), and Runx3^−/− (pink) mice. The black line represents a control experiment of cells incubated without anti-CD3 and IL2 and, thus, were nondividing. The overall cell division rate of WT or Runx3^VWRPY−/− was similar, whereas that of Runx3^−/− cells was much lower. Cells from WT or Runx3^VWRPY−/− mice underwent four population divisions compared with only three divisions of Runx3^−/− cells. After 72 h in culture, >20% of Runx3^−/− cells remained undivided compared with ≈1% and 2% of WT and Runx3^VWRPY−/−, respectively.