Based on in vitro studies and limited in vivo data from patients with mutations in FOXP3 or animals with depletion of circulating Tregs, Tregs are considered central to immune homeostasis. How the practical nuts and bolts of how these cells function in vivo remain largely unexplored, i.e. we have a reasonable cellular understanding but lack biochemical insights into their regulation and activities. We have shown that the histone/protein acetyltransferase, Tip60, interacts with the N-terminal repressor domain of Foxp3. We now present data showing how Tip60 is vital for Treg survival and function. Conditional deletion of Tip60 in Foxp3+ Tregs did not affect their intrathymic development but decreased peripheral Tregs (3.7% vs 10.7% in WT mice) and almost completely erased Treg suppressive function in vitro. Accordingly, immune activation of conventional CD4 and CD8 T cells (CD69+, CD62L^lowCD44^high) was markedly increased in mice with Treg depletion of Tip60, and they developed severe autoimmunity and died within 3 weeks of birth. Tregs from these mice showed markedly decreased mRNA levels of key Treg genes, including Foxp3, CTLA4 and GITR, and increased expression of IL-2, IFN-g, IL-4, IL-6 and IL-17 mRNA (qPCR), and Western blots showed decreased Foxp3 protein expression in KO vs. WT Treg. Biochemical studies showed that Tip60 acetylates and stabilizes Foxp3 expression, such that without Tip60, Foxp3 was ubiquitinated and subject to proteasomal degradation. Microarray data showed up-regulation of many pro-apoptotic genes, consistent with the poor survival of Tip60-/- vs. WT Tregs following their adoptive transfer into immunodeficient mice. Lastly, in translational studies, we found that pharmacologic inhibition of Tip60 (i) impaired Treg suppressive function in vitro, but had little effect on conventional T cell proliferation; and (ii) prevented induction of allograft tolerance (BALB/c->C57BL/6) induced by CD154 mAb plus donor splenocyte transfusion, i.e. abrogated Treg function in the most powerful tolerance strategy yet described in rodents. In conclusion, Tip60 is the most important HAT in Tregs, proving essential for acetylation, dimerization and function of Foxp3, and in its absence or upon pharmacologic inhibition of Tip60, Foxp3 is subject to ubiquitination and proteasomal degradation, with consequent loss of Treg function.

CITATION INFORMATION: Wang L, Beeler A, Dahiya S, Han R, Hancock W. A Single Histone/protein Acetyltransferase, Tip60, Is Absolutely Essential for Treg Survival and Function, and Its Inhibition Abrogates Induction of Treg-Dependent Allograft Tolerance. Am J Transplant. 2016;16 (suppl 3).

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