Project Summary Acute myeloid leukemia (AML) has thus far proven resistant to T cell redirecting therapies such as bispecific antibodies or chimeric antigen receptor (CAR) T cells, which is unexpected given the proven success of these in other hematologic malignancies. Preliminary studies show that the anti-tumor activity of CD19-targeting CAR T cells (CART19) is enhanced in the presence of myeloid cells, and that targeting these myeloid cells with CD33-targeting CART cells (CART33) leads to diminished long-term anti-tumor effect. Furthermore, removing CD33 from normal myeloid cells improves CART33 expansion and disease control. Based on these findings, the central hypothesis of this proposal is that bystander myeloid cells enhance CART cell activity, and directly targeting myeloid cells compromises this effect. The objective of this proposal is to delineate how myeloid cells influence CAR T cell behavior when they are targets of therapy, as compared to when they are simply bystanders, so that we can intervene within these interactions and improve CAR T cell therapy for AML. This will be achieved through the following specific aims: 1) Determine how bystander myeloid cells modulate CAR T cell function; 2) Determine how targeting myeloid cells changes CAR T cell profile. To this end, hematopoietic stem cells (HSCs) will be engineered through either viral gene transfer or CRISPR/Cas9 gene knockout to generate myeloid cells that are positive or negative for the antigen targeted by the CAR. The effects of wild-type or genetically modified myeloid cells on CAR T cells will be interrogated by in vitro culture and in vivo mouse models, using flow cytometry, single-cell RNA-seq, and functional studies. The innovations of this project are that it draws attention to host environmental factors that influence CAR T cell activity, and advances the concept that the nature of the cell being targeted can influence CAR T cell behavior. This research is significant because it will contribute to a better understanding of how CAR T cells work with the immune environment, and illuminate methods to intervene within these interactions to improve the outcomes of therapy. The long-term goal of this proposal is to establish the applicant Dr. Miriam Kim's career as an independent researcher focused on developing novel cell therapies for AML. The proposed research and career development plan will provide Dr. Kim with training in HSC/myeloid biology, immunology, and bioinformatics. Her primary mentor, Dr. John DiPersio, and co-mentors Drs. Robert Schreiber, Todd Fehniger and Carl DeSelm, offer complementary expertise in cell engineering and immunotherapy. Additionally, Dr. Kim's collaborators Dr. Li Ding and Dr. Feng Gao will contribute to developing her skills in bioinforrnatics and biostatistics. Furthermore, Washington University provides an ideal environment for Dr. Kim to successfully establish herself as an independent investigator.