Abstract Allogeneic hematopoietic cell transplantation (allo-HCT) is an essential therapeutic modality for patients with hematological malignancies and benign blood disorders. Unfortunately, acute graft-versus-host disease (aGVHD) remains the major complication of allo-HCT and is associated with high mortality. Soluble STimulation-2 (sST2) is increased during aGVHD while regulatory T cells (Tregs) that express membrane-bound ST2 prevent GVHD through unknown mechanisms. Herein, we studied nonlymphoid tissue ST2+ Tregs in murine models of allo-HCT. Transplantation of Foxp3- T cells and Tregs sorted from different Foxp3 reporter mice indicated that ST2+Tregs isolated from aGVHD mice were of donor origin, thymus-derived, and predominantly intestine localized (data not shown). To further understand the mechanism, we performed a transcriptome analysis of sorted Foxp3GFP Tregs from aGVHD mice receiving wild type (WT) CD25-depleted total T cells with either WT or ST2-/- Foxp3GFP Tregs in both major (B6, H-2bàBALB/c, H-2d, Mhc) and minor (B6, H-2bàC3H.SW, H-2b, miH) mismatched aGVHD models. ST2-/- Foxp3GFP Tregs showed decreased expression of key molecules needed for Treg function such as Foxp3, Il2ra, Icos, Il27, Ctla4, and Ahr. Strikingly, transcription of RAR-related-orphan-receptor-c (Rorc), essential for proinflammatory IL-17+ T cells development, was the most upregulated transcript in ST2-/- Foxp3GFP Treg isolated from the gut in both models (Figure 1A). The transcriptome finding led us to investigate the frequency and function of ST2+Tregs in the gut of naive mice deficient for Rorc. Isolation and staining of intestinal T cells showed a significant increase of ST2+Treg frequency in the gut of Rorc-/- mice compared to WT mice: 55% vs. 12%, respectively (Figure 1B). Rorc-/- T cells transplantation enhanced the day 10 post-HCT frequency of intestinal ST2+Tregs (Figure 1C, left) that co-expressed TIGIT and Helios expression (data not shown), and decreased aGVHD score and mortality (Figure 1C, right). Previous studies have shown that IL-33 signaling in Tregs elicits their amphiregulin expression and increases their ability to suppress effector T cells (Teffs). IL-23, a key proinflammatory cytokine in the maintenance and amplification of Th17/Tc17, can restrain Treg responses by inhibiting IL-33 responsiveness. Using ex vivo polyclonal Tregs from donor B6 cultured with IL-2 (control Tregs) vs IL-2+IL-33 (TregIL-33) vs IL-2+IL-33+IL-23+IL-17, we found that adding IL-33 doubled the frequencies of ST2+ Tregs compared to control Tregs, and adding IL-23+IL-17 reversed this activity (Figure 2A). Markers of Tregs activation (KLRG1) (Figure 2B) and function (Helios, LAG3) (data not shown) were augmented in TregIL-33 compared to other Treg conditions. TregIL-33 also expressed more amphiregulin than Tregs cultured in the two other conditions (Figure 2C). Next, we evaluated their function in vitro and in vivo. TregIL-33 displayed better immunosuppression than other Tregs on CD4+ T cells at a physiological Tregs:Teffs ratio of 1:12; differences were not seen at higher ratios or on suppression of CD8+ T cells (Figure 3A). Then we examined the in vivo capacity of donor TregIL-33 to protect from aGVHD intestinal damage by co-adoptive transfer of Tregs cultured in the three aforementioned conditions with CD25-depleted WT T cells in Mhc and miH mismatch aGVHD models. We used a physiological ratio of 1 donor Treg per 20 donor Teffs (5% of total T cells). Mice receiving TregIL-33 showed significantly less severe aGVHD and better survival compared to those cultured either without IL-33 or with IL-33 and IL23/IL17 in both Mhc (Figure 3B) and miH (data not shown) mismatched aGVHD models. Analysis of intestinal T cells of these mice showed decreased total lymphocytes infiltration in the gut at day 10 after HCT, and decreased intestinal Tbet and RORγt expressing Teffs in both Mhc (Figure 3C) and miH (not shown) mismatched aGVHD models. Altogether, we conclude that within GVHD target organs, ST2+Tregs are predominantly intestine localized, and inversely correlated with RORgt in steady state and inflammatory conditions. Triggering the ST2/IL-33 pathway in Tregs prevents severe aGVHD, possibly through a dual mechanism: upregulation of amphiregulin and immunosuppression on effector T cells. Thus, TregIL-33 is a potential cellular therapy avenue for preventing or treating aGVHD. Disclosures Blazar: Kadmon Corporation, LLC: Consultancy, Research Funding. Paczesny:Viracor IBT Laboratories: Patents & Royalties.