ABSTRACT Graft-versus-host disease (GvHD) is one of the most significant barriers to success for allogeneic hematopoietic cell transplantation (allo-HCT). The goal of this project is to identify novel genes whose genetic/pharmacologic blockade may selectively prevent GvHD while maintaining or enhancing anti-leukemia activities (aka graft- versus-leukemia or GvL effect). Allo-HCT remains the most effective treatment for patients with hematologic malignancies, as the therapeutic benefit of allo-HCT is primarily derived from GvL. However, allo-HCT also carries the risk that the donor T cells in the transplant (graft) will become overzealous and begin to attack not only the leukemia but also the patient's skin, intestines and liver, resulting in GvHD. Because of a strong association between the two donor T cell-mediated immune responses, specifically limiting GvHD remains the goal of allo-HCT. Managing the threat of GvHD would broaden the scope and benefit of allo-HCT. Several groups, including ours, have proposed therapeutic strategies to reduce GvHD without abrogating the beneficial GvL effect in animal models and human patients. However, the mechanisms by which allogeneic donor T cells differentially modulate GvHD and GvL remain largely unknown. This gap in our mechanistic understanding hinders our ability to specifically prevent/treat GvHD. We hypothesize that the genes we identify as being differentially associated with donor T cells that infiltrate tumors versus GvHD organs will be critical targets for the prevention of GvHD without negatively affecting GvL. To this end, we performed an unbiased genome-wide CRISPR/Cas9 library screen. As a result, we have for the first time identified 487 novel GvHD-associated genes that could potentially serve as molecular targets in GvHD, as the guide RNAs for these genes were enriched only in T cells obtained from tumor nodules but not from any GvHD target organs. We found that five sets of genes that are involved in known pathways are most significantly enriched in these 487 genes. All of these five pathways are closely linked to the RELN-RAP1-FGFR3 axis that has not been actively investigated in the allo- HCT field. Thus, we will determine if genetic/pharmacologic inhibition of the RELN-RAP1-FGFR3 axis results in GvHD-specific immune modulation while maintaining or enhancing GvL in our mouse models of murine GvHD/GvL and human PBMC/T cell-mediated xeno-GvHD/GvL. If successful, our findings of novel therapeutic targets to selectively and optimally control GvHD will be potential game-changers. Our proposed studies will provide significant mechanistic insights into GvHD and its prevention, which may have transformative implications for therapeutic development not only for solid organ transplant rejection but also inflammatory diseases such as inflammatory bowel disease, psoriasis and rheumatoid arthritis. In addition, our studies may also help improve the efficacies of anti-cancer therapies and immunotherapeu...