FIGURE 5. Differential engagement of T-bet and NFκB transcription factors on the IFN-γ promoter in naive and memory CD4 T cells.

(A) Chromatin immunoprecipitation (ChIP) analysis of T-bet bound to the IFN-γ promoter. OVA-specific naïve (“Nai”) and memory (“Mem”) CD4 T cells were crosslinked and lysed directly ex vivo or following activation for 6–48hrs with anti-CD3/anti-CD28 antibodies. DNA was sheared and immunoprecipitated with protein A-sepharose alone (negative control, “−“), anti-histone H3 (positive control, “+”) and anti-T-bet (“T”). Sequences on the IFN-γ promoter corresponding to a 360bp region of the T-bet binding sites were PCR-amplified from immunoprecipitates or input DNA (“N”) as an additional positive control. (B) ChIP analysis of NFκB binding to the IFN-γ promoter in memory CD4 T cells. OVA-specific memory CD4 T cells were isolated and activated as in (A), and prepared for ChIP analysis. Gel shows a 388bp product corresponding to the NFκB binding site on the mouse IFN-γ promoter that was PCR-amplified from immunoprecipitates using protein A agarose alone (lanes marked “−“), anti-histone H3 (lanes marked “+”), or anti-p50(NFκB) antibody (lanes marked “κB”). (C) Quantitative Comparison of NFκB versus T-bet binding to the IFN-γ promoter in naive and memory CD4 T cells. ChIP analysis was performed on resting and activated naive (N) and memory (M) CD4 T cells as in (A) and (B), except real-time qPCR was used for amplification. Graph shows the average fold induction over negative control immunoprecipitates from 4 independent experiments.