434 SatM Involved in Chronic Gvhd after Bone Marrow Transplantation. Keisuke Seike 1 , Kyosuke Saeki MD 1 , Hisakazu Nishimori MD, PhD 1 , Takashi Satoh MD, PhD 2,3 , Yoshinobu Maeda MD, PhD 1 . 1 Department of Hematology and Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan; 2 Laboratory of Host Defense, World Premier Institute Immunology Frontier Research Center, Osaka University, Osaka, Japan; 3 Department of Host Defense, Research Institute for Microbial Diseases (RIMD), Osaka University, Osaka, Japan Background: GVHD is a result of immune attack of host tis- sues, such as the skin, gut, liver, and lung, by various immune cells in hematopoietic stem cell transplantation (HSCT). Chronic GVHD (cGVHD) is the main cause of late death and morbidity after allogeneic HSCT. Fibrosis constitutes the end stage of the inflammatory process in cGVHD leading to major morbidity. Recently, segregated-nucleus-containing atypical monocytes (SatM) that involved in lung fibrosis has been iden- tified. We hypothesized that SatM involved in tissue fibrosis of chronic GVHD after bone marrow transplantation. Materials and Methods: Lung cGVHD model: B10.BR mice received were conditioned with Cyclophosphamide (120mg/ kg/day, intraperitoneally, day-3 and -2) and TBI (8.3Gy, day-1), followed by infusion of 10»10 6 C57BL/6j T celldepleted BM (TCD-BM) plus 60000 purified splenic T cell. Respiratory func- tion test and flowcytometry of BALF were analyzed at BMT day56. Sclerodermatous cGVHD model: BALB/c mice received a single dose of 5.8 Gy x-ray total body irradiation. Recipient mice were injected with 2»10 6 purified splenic T cells and 8»10 6 TCD-BM cells from B10.D2 donors. Donor cells were injected intravenously into the recipients on day 0. The SatM in the skin and the spleen are analyzed by flow cytometry at BMT day28. Results: Compared to syngeneic group of the lung cGVHD model, allogeneic recipient developed fibrosis around the bronchi. In the respiratory function test, allogeneic recipient showed higher respiratory resistance. No difference was observed in the number of splenic monocytes by flowcytome- try. However, Ly6c low monocyte, Ly6c + monocyte and SatM increased in BALF of allogeneic recipient. In the scleroderma- tous cGVHD model, SatM increased in ear of allogeneic recipi- ent but not in spleen (Figure 1). Discussion: In this study, SatM observed in tissues targeted by cGVHD, but not in spleen. This result may indicate that SatM involved in fibrosis of cGVHD model. To demonstrate that SatM causes the fibrosis of cGVHD, we currently establish cGVHD model with SatM deficiency mouse (Cebpb ¡/¡ mouse). 435 Sequential Cyclophosphamide and Trametinib Improve Clinical Graft Versus Host Disease and Survival in Murine Hematopoietic Stem Cell Transplant George Nahas DO 1 , Henry Barreras MPH 2 , Cameron S. Bader BS 2 , Sabrina Copsel PhD 2 , Dietlinde Wolf PhD 3 , Brandon Kale 2 , Robert B. Levy PhD 2 , Krishna V. Komanduri MD 4 . 1 University of Miami, Miami, FL; 2 Microbiology & Immunology, Miller School of Medicine, Univ of Miami, Miami, FL; 3 Sylvester Comprehensive Cancer Center, Miller School of Medicine, Univ of Miami, Miami, FL; 4 Adult Stem Cell Transplant Program, University of Miami Hospital and Clinics, Sylvester Comprehensive Cancer Center, Miami, FL Background: Ongoing acute graft versus host disease (aGVHD) after allogeneic stem cell transplantation (alloSCT) causes sig- nificant morbidity and mortality with no standard therapy except corticosteroids, which are broadly immunosuppressive and toxic. Cyclophosphamide (Cy) given day 3 and day 4 is effective for prophylaxis of aGVHD but has proven ineffective when given later as in ongoing aGVHD in both murine and human studies. We previously demonstrated that the MEK inhibitor trametinib (Tram) is effective for aGVHD prophylaxis in murine models of aGVHD through the novel mechanism of inhibition of na € ıve T cells (CD44 ¡ CD62L + ). Our hypothesis is that ongoing aGVHD could be better inhibited by sequential Cy and post-Cy Tram inhibition (to prevent na € ıve T cells from re- initiating alloreactivity) without compromising graft-versus- malignancy (GVM) responses. Methods: Mice were administered sequential Cy and Tram post SCT starting day 12. Efficacy of treatment was assessed in a murine model of MHC-mismatched alloSCT. Overall survival was assessed and clinical GVHD was scored three times weekly. Peripheral blood, spleen, and lymph node cells pheno- types were determined by flow cytometry. GVM was also assessed with A20 luciferase/YFP (A20 luc/YFP ) lymphoma cells imaged with in vivo imaging system (IVIS). Results: Overall survival following sequential Cy and Tram was identical to that seen with T cell depleted bone marrow alone and significantly superior to treatment using either single-agent Cy or Tram (p<0.05). Clinical GVHD scores were improved by sequential Cy and Tram compared to treatment with either sin- gle agent Cy (p=0.002) or single agent Tram (day 38 p=0.03). Timing of initiation of sequential Cy and Tram starting day 12 was significantly better than in the GVHD control group (p=0.002) and also significantly better than after later initiation at day 19 (p=0.005). Initiation of treatment on day 6 resulted in 100% mortality by day 15. Higher doses of Trametinib at 0.3mg/ kg were significantly better than the GVHD control (p=0.006). Analysis of peripheral CD4 + and CD8 + T cells demonstrated a more physiologic CD4 + CD8 + ratio and a more normal distribution of na € ıve and effector T cells in the sequential Cy and Tram within CD4 + and CD8 + T cell subsets. Sequential Cy and Tram did not adversely affect peripheral CD4 + FOXP3 + regulatory T cells. Using A20 luciferase/YFP (A20 luc/YFP ) lymphoma cells in a transplant model demonstrated that GVM was still effective with sequential Cy and Tram. Figure 1. Flowcytometry of dermal SatM Figure 1. Sequential Cyclophosphamide and Trametinib (Cy+Tram) improved clinical GVHD score and overall survival in a preclinical model. *p<0.05; **p<0.01 S296 Abstracts / Biol Blood Marrow Transplant 25 (2019) S290S442