*Purpose: Most of our ideas about Treg cells are extrapolated from findings in mice, with actual clinical data typically involving flow cytometric enumeration of CD4+FOXP3+ cells, with the implication that Treg cell numbers correlate with Treg function. However, FOXP3 is subject to many post-translational modifications that critically determine the function of Treg cells, and those modifications have not yet been studied in primary human Treg cells.

*Methods: Tregs and plasma were isolated from healthy controls or patients listed for lung, liver or kidney transplantation (Tx). We studied FOXP3 protein by Taqman protein assay. In addition, in studies of lung ischemia/reperfusion injury (IRI), IL-18-treated or control Tregs from normal C57BL/6 mice were adoptively transferred into immunodeficient (Rag1-/-) mice.

*Results: Obese (but not non-obese) patients listed for lung, kidney and liver Tx had impaired pre-Tx Treg suppressive function and increased levels of IL-18 compared to healthy individuals. In healthy donor Tregs, ~50% of FOXP3 protein was present as dimers and/or oligomers, ~25% of total FOXP3 was ubiquitinated, and ~80% of total FOXP3 was acetylated. Exposure of Treg cells to IL-18 led to a significant decrease of total FOXP3 protein level (~69% of initial levels) and a 2-fold reduction of FOXP3 dimerization and/or oligomerization; such assembly of FOXP3 into higher-ordered structures is critical for Treg function. We also observed a substantial increase of FOXP3 ubiquitination, but not FOXP3 acetylation, and increased TRAF6-STUB1 complexes, decreased TRAF6-FOXP3 complexes and increased STUB1-FOXP3 complexes in Treg after exposure to IL-18. UbiTest confirmed that in the presence of IL-18, FOXP3 ubiquitination was significantly increased, with increased K48-linked ubiquitination and STUB1 involvement. IL-18 also induced expression of the pro-inflammatory cytokines, IL-1β and IL-6, in Treg cells. In conjunction with increased FOXP3 ubiquitination and disrupted di- and oligomerization of FOXP3, the stability of FOXP3 expression under stimulatory conditions was significantly altered. Lastly, we assessed direct effects of IL-18 on Treg function in vivo using a murine IRI model. We found that adoptively transferred Tregs that had been pretreated with IL-18 were unable to prevent lung IRI, whereas control Tregs significantly suppressed such injury, revealing previously unknown direct effects of Tregs on the innate immune system.

*Conclusions: We provide quantitative insights into human FOXP3 biology and how such parameters can be modulated by IL-18. The effects of IL-18 on Tregs have relevance to both the innate and adaptive immune systems, and for the pre-Tx evaluation of patients.

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