*Purpose: Estrogen signaling is thought to play a role in nearly every cell type, but the biological significance of estrogen signaling via estrogen receptors in immune cells remains unclear.

*Methods: In preliminary studies, we found that female Tregs showed weaker Treg suppressive function than male Tregs, leading us to undertake tamoxifen-induced deletion of estrogen receptor-α (ERα) and estrogen receptor-β (ERβ) in Foxp3+ T-regulatory (Treg) cells of adult female mice.

*Results: In the absence of ERβ, the ratios of T-effector (Teff) to Treg cells, and follicular Teff (Tfh) to follicular Treg (Tfreg) cells were drastically increased within the spleens (48% increase of Tfh:Tfreg; p=0.0029), superficial lymph nodes (sLNs) (29% with p=0.0049 and 68% with p<0.0001, respectively), and mesenteric lymph nodes (mLNs) (26% with p=0.04 and 42% with p=0.0002, respectively). ERβ KO mice also had decreased proportions of Tregs and Tfregs, most prominently in mLNs (37% with p<0.0001 and 60% with p=0.0014, respectively). While ERα KO mice had fewer and less pronounced differences in immune cell proportions compared to WT mice, there were again significantly fewer Tregs in mLNs (31% with p=0.0007). Importantly, and very surprisingly given our findings of reduced Treg populations upon ERα or ERβ deletion, CD4+CD25+ Tregs lacking ERα, ERβ, or both ERα/β displayed an enhanced ability to suppress the proliferation of CD4+ (p<0.05, 0.001, and 0.05, respectively) and CD8+ (p<0.05, 0.001, and 0.01, respectively) T cells in vitro. Moreover, when C57BL/6 Rag-1-/- mice bearing BALB/c cardiac allografts underwent adoptive transfer with ERα or ERβ KO Tregs at a 1:4 ratio with WT Teff cells, the allografts survived for more than 100 days (p<0.01 vs. acute rejection at <10 days using WT Tregs:Teffs at this same ratio). Histologic examination of cardiac allografts harvested at >100 days after adoptive transfer showed that the grafts from mice receiving ERα KO Tregs had undergone significant immune cell infiltration, focal necrosis and vascular injury, whereas those receiving ERβ KO Tregs showed only minor cellular infiltrates and well-preserved myocardium and vessels.

*Conclusions: These data show that in the absence of estrogen signaling via ERα or ERβ, the Foxp3+ Treg population is reduced but develops considerably more potent suppressive function, as seen in vitro and in vivo, especially in the case of deletion of ERβ. While studies are ongoing regarding the differing effects of ER deletion on Treg biochemistry and gene expression, as well as the utility of selective estrogen receptor modulators such as raloxifene in transplant models, the current data suggest potential roles for estrogen receptor targeting to promote a tolerogenic immune environment suitable for prolongation of transplant survival.

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