Abstract Abstract 338 Graft-versus-host disease (GVHD) is the major limitation of allogeneic hematopoietic bone marrow transplantation (BMT). Donor T cells play pivotal roles in GVHD and graft-versus-leukemia (GVL) effects and following BMT all T cell fractions, including regulatory T cells (Treg) express the DNAX accessory molecule-1 (DNAM-1, CD226) and T cell Immunoglobulin and ITIM domain (TIGIT) molecule. DNAM-1 is a co-stimulatory and adhesion molecule, expressed mainly by NK cells and CD8+ T cells at steady state to promote adhesion to ligand (CD155, CD112)–expressing targets and enhance cytolysis. TIGIT is a regulatory ligand expressed predominantly by Treg as steady state which competes for CD155 binding, We have analyzed the role of this pathway in GVHD and GVL. Lethally irradiated C3H/Hej (H-2k) mice were injected with bone marrow cells and T cells from MHC disparate wild-type (wt) or DNAM-1–/– C57Bl6 (H-2b) mice. Recipients of DNAM-1–/– grafts were protected from GVHD (survival 67% vs. 7%, P < .0001). We also confirmed the role of DNAM-1 in GVHD in a MHC-matched BMT model (B6 → BALB/B (H-2b)) where GVHD is directed to multiple minor histocompatibility antigens. Next we examined the donor populations expressing DNAM-1 which mediate this effect. DNAM-1 had little impact on acute GVHD severity in the B6 → bm1 BMT model where GVHD is directed against an isolated MHC class I mismatch and is CD8-dependent. In contrast, recipients of wt bone marrow and DNAM-1–/– CD4 T cells survived long-term (compared to recipients of wt CD4 T cells, survival 81% vs. 25%, P = .003) in the B6 → B6C3F1 BMT model, confirming the protection from GVHD is CD4-dependent. Donor CD4 T cell expansion and effector function (Th1 and Th17), and CD8 T cell expansion and cytotoxic function were equivalent in recipients of wt and DNAM-1–/– grafts. However the percentage and number of Treg were significantly increased in recipients of DNAM-1–/– grafts compared to those of wt grafts. The depletion of Treg from donor grafts eliminated the protection from GVHD seen in the absence of DNAM-1 signalling (median survival 16 days vs. 15.5 days, P = 0.53). Adoptive transfer experiments using FACS-sorted Treg were undertaken to compare the relative ability of B6.WT and B6.DNAM-1–/– Treg to suppress GVHD. The majority of recipients of DNAM-1–/– Treg survived beyond day 50 (median survival; day 56), demonstrating a superior ability to suppress acute GVHD relative to wt Treg where the median survival was day 36 (survival 47% vs. 0%, P = .001). These data demonstrate that donor DNAM-1 expression promotes GVHD in a CD4+ T cell-dependent manner via the inhibition of donor Foxp3+ Treg. Finally, the absence of donor DNAM-1 did not influence leukemia-specific mortality in multiple GVL models, regardless of whether the tumor expressed CD155 or not. Thus we demonstrate that the DNAM-1 pathway promotes GVHD, putatively due to competition with TIGIT on Treg, thereby inhibiting regulatory function. This provides support for therapeutic DNAM-1 inhibition to promote tolerance not only after transplant but also in relevant inflammatory based diseases characterized by T cell activation. Disclosures: No relevant conflicts of interest to declare.