CD4+ T-regulatory (Treg) cells maintain self-tolerance and control immune responses to nonself-antigens, and thereby limit excessive inflammation. High level and stable expression of Foxp3 transcription factor are necessary for maintaining the phenotype and suppressive function of Treg cells. Foxp3 expression is stabilized through DNA demethylation at conserved noncoding sequences (CNS2, TSDR) within the Foxp3 locus, and associated CREB/ATF binding to this CNS2 site. We now show that the histone acetyltransferase, CBP, also binds to the Foxp3 CNS2 region in murine Tregs and have identified the importance of this binding in maintaining Foxp3 stability. Previous studies showed CBP acetylates CREB and promotes CREB transcription and DNA binding. We found that CBP further increased pCREB expression and DNA binding to CNS2 region upon Treg activation in vitro using PKA. To investigate the role of CBP in regulating Foxp3 expression, we generated mice with conditional deletion of CBP in Tregs (CBPflfl/Foxp3cre mice). Tregs from these mice had only mildly impaired suppressive function as reflected by the presence of activated Teff cells in secondary lymphoid tissues, and increased Teff proliferation in a standard in vitro Treg assay. However, though levels of Foxp3 in CBPflfl/Foxp3cre Tregs were similar to that of WT Tregs, Tregs from CBPflfl/Foxp3cre mice markedly downregulated Foxp3 upon CD3/CD28 stimulation in vitro. Consistent with in vitro findings, compared to WT Tregs, CBPflfl/Foxp3cre mice Tregs showed decreased Foxp3 in vivo during inflammatory and immune responses, including colitis and cardiac transplantation. Thus, injection of CBPflfl/Foxp3cre mice Tregs could not rescue mice developing colitis following adoptive transfer of WT T cells, in contrast to control of inflammation by WT Tregs. Likewise, CBPflfl/Foxp3cre C57BL/6 mice acutely rejected BALB/c cardiac allografts when treated with CD154 mAb/DST, whereas mice with WT Tregs maintained their allografts for >100 days (p<0.01). In summary, pCREB is activated during inflammation and recruits CBP to the Foxp3 CNS2 region; the actions of CBP increase pCREB DNA binding and promote Foxp3 expression. Thus, CBP cooperates with pCREB in maintaining Foxp3 stability during inflammatory and immune responses, and is thereby important for long-term, Treg-dependent, allograft survival.

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