Fig 1: T-bet and Eomes compete for binding to and repressing Pdcd1 transcription(A) Immobilized T-bet bound to a T-box consensus sequence was competed with recombinant Eomes, and binding of both T-bet and Eomes was determined using an ELISA-based binding assay (described in STAR Methods and Figure S2B). The bar graph displays the fold change in binding of T-bet or Eomes following competition with Eomes+ lysate relative to each uncompleted protein.(B) Immobilized Eomes bound to a T-box consensus sequence was competed with recombinant T-bet, and binding of both T-bet and Eomes was determined using an ELISA-based binding assay (described in STAR Methods and Figure S2B). The bar graph displays the fold change in binding of Eomes or T-bet following competition with T-bet+ lysate relative to each uncompleted protein.(C) T-bet or Eomes binding to a consensus T-box sequence was competed with oligos corresponding to a canonical T-box half site sequence or the T-box sequences found in Pdcd1 or Il2. The bar graph displays the fold change in binding of Eomes or T-bet following competition with various T-box-containing oligos relative to each uncompleted protein.(D) PCR amplification of immunoprecipitated DNA sequences in EL4 cells expressing exogenous T-bet or Eomes is shown. Ifng(positive) and Il4 (negative) regions were used as controls.(E) Dual-luciferase reporter assays were performed using a reporter plasmid containing two conserved regions within the Pdcd1 promoter region (CR-B+C). A bar graph shows the luciferase activity observed in PMA/ionomycin-stimulated EL4 cells nucleofected with wild-type T-bet, Eomes, or co-nucleofected T-bet and Eomes plasmids. Data were normalized to EL4 cells containing the firefly luciferase reporter only.(F) Endogenous PD-1 expression in unstimulated (left) or PMA/ionomycin-stimulated (right) EL4 cells is shown in T-bet+ (green), Eomes+ (blue), or T-bet+ Eomes+ (pink) cells relative to T-bet-, Eomes-, or T-bet-Eomes- cells (gray shaded). Representative histograms from one experiment are shown. Data in bar graphs are represented as mean ± SEM; p values were determined using a one-way ANOVA test (****p < 0.0001; ***p < 0.001, *p < 0.05).