Abstract The parent R21 proposal 1R21CA245468-01A1 "Innovative Cell Therapy for Pediatric Acute Myeloid Leukemia" is designed to address the mechanism of pediatric acute myeloid leukemia (pAML) blast resistance to LV-10 cell-mediated killing. LV-10 cells are engineered type 1 regulatory T cells (Tr1) that represent a promising therapy for pAML because of their inherent anti-myeloid tumor cytotoxicity, but in pilot experiments, a subset of primary pAML blasts resisted LV-10 cell killing. RNA-seq revealed two candidate genes that could contribute to pAML resistance to killing. These genes could also be used as biomarkers of resistant pAML, which is important for downstream clinical translation of LV-10 cells. However, a larger RNA-seq dataset is required for sufficiently powered biomarker discovery. Aim 1 of the parent proposal will assess the sensitivity of 45 new pAML blasts to LV-10 mediated killing, which in addition to the pilot samples, provides sufficient power for biomarker discovery. Aim 1 of the supplemental proposal will perform bulk RNAseq analyses on these 45 pAML blasts to identify potential new biomarkers of resistance. Further, the LV-10 cells are currently expanded using a mixture of healthy human donor-derived peripheral blood mononuclear cells (PBMC) as feeder cells, which introduces donor-to- donor variability to LV-10 expansion, phenotype and function. Before the clinical translation of LV-10 cells, it is essential to identify an alternative artificial expansion system that provides a functionally uniform and GMP com- pliant cell product. These systems are already in place for other T cell-based therapies. In aim 2 of the supple- mental proposal, we will compare the effect of 2 artificial antigen-presenting cell (aAPC) systems, TransAct and ImmunoCult, to the traditional PBMC feeders, on LV-10 cell expansion, phenotype, cytokine production, degran- ulation and AML killing. Altogether, this data will reveal: 1) additional genes that play a role in pAML resistance to LV-10 killing, which can be used in the future to reverse this resistance or screen pAML patients eligible for LV-10 cell therapy; and 2) optimal aAPC system for LV-10 cell expansion. As such, this supplemental proposal will complement the aims of the parent proposal, and it's focus on pAML transcriptome analysis and a novel cell therapy for pAML. Furthermore , execution of the experiments required to achieve these goals will provide the Candidate with essential background and skills in cancer immunology and immunotherapy, paving the way to his enrollment into graduate school and a career path as a cancer immunologist.