Figure 3.

Genetic evidence for a role of Runx proteins in the maintenance of Foxp3 expression in nT reg cells. (A) CD4⁺ CD25⁺ LN T cells were sorted from control Cbfb^wt/wt and Cbfb^lox/lox mice, activated for 18 h with anti-CD3/anti-CD28 beads in the presence of IL-2, and retrovirally transduced with vector-IRES-GFP (vector) or Cre-IRES-GFP (Cre). 4 d later, Foxp3 expression had declined selectively in Cbfb^lox/lox cells transduced with Cre-IRES-GFP (red). Foxp3 expression remained unaffected in vector-transduced Cbfb^lox/lox cells (black) and in Cbfb^wt/wt cells transduced with either vector-IRES-GFP (black) or Cre-IRES-GFP (red). Data are representative of three independent experiments. (B) Time course of Foxp3 expression by GFP⁺ Cre-IRES-GFP–transduced relative to vector-IRES-GFP–transduced Cbfb^lox/lox nT reg cells (red) and Cbfb^wt/wt nT reg cells (black; mean ± SD of three independent experiments). (C) GFP-positive and -negative nT reg cells were sorted 4 d after transduction with vector-IRES-GFP (vector) or Cre-IRES-GFP (Cre), and Cbfb loci were analyzed by genomic PCR. The position of bands corresponding to the wild-type (wt), floxed (lox), and deleted (Δ) locus are indicated. One of three independent experiments is shown. (D) GFP-positive and -negative Cbfb^lox/lox cells were sorted 4 d after transduction with vector-IRES-GFP (vector) or Cre-IRES-GFP (Cre), and Cbfb protein expression was analyzed by Western blotting. Tubulin was used as a loading control. One of three independent experiments is shown.