FOXO1 enhances CAR T cell stemness, metabolic fitness and efficacy Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of haematological malignancies such as acute lymphoblastic leukaemia, B cell lymphoma and multiple myeloma, but the efficacy of CAR T cell therapy in solid tumours has been limited. This is due to factors such as the immunosuppressive tumour microenvironment that leads to poorly persisting and metabolically dysfunctional T cells. Analysis of anti-CD19 CAR T cells used clinically has shown that positive treatment outcomes are associated with a more 'stem-like' phenotype and increased mitochondrial mass. We sought to identify transcription factors that could enhance CAR T cell fitness and efficacy against solid tumours. Here we show that overexpression of FOXO1 promotes a stem-like phenotype in CAR T cells derived from either healthy human donors or patients, which correlates with improved mitochondrial fitness, persistence and therapeutic efficacy in vivo. This work reveals an engineering approach to genetically enforce a favourable metabolic phenotype that has high translational potential to improve the efficacy of CAR T cells against solid tumours. In the solid tumour microenvironment, CAR T cells are predisposed to terminally differentiate in response to chronic antigen stimulation, metabolic competition and a lack of appropriate co-stimulatory signals. Terminally differentiated CAR T cells are similar to exhausted endogenous T cells that do not eliminate tumours owing to dysfunction, attenuated effector function and poor persistence. Such cells are characterized by reduced expression of effector molecules, the expression of immune checkpoints including PD-1, LAG3 and TIM3 and transcriptional regulators associated with exhaustion, such as IRF4. Less differentiated CAR T cells, defined by a phenotype of CD45RA+, persist for longer. This is because less differentiated T cells maintain higher multipotency and greater self-renewal capacity and thus can generate increased numbers of effector-like progeny to facilitate improved tumour control. Accordingly, CAR T cells with higher initial frequencies of less differentiated cells elicit improved persistence and therapeutic potential. The processes of T cell differentiation, metabolism and epigenetic reprogramming are highly interdependent. T cell exhaustion is characterized by sub-optimal effector functions that are enforced by epigenetic regulation such as repressive DNA methylation at key gene loci, processes that are directly controlled by intracellular metabolites. In the context of CAR T cells, improved oxidative metabolism and increased mitochondrial biogenesis is associated with enhanced persistence and function. A variety of approaches have been explored to favourably modulate CAR T cell differentiation. These include the use of homeostatic cytokines, epigenetic regulation, and more recently, the overexpression of transcriptional regulators. However, none of these genetic reprogramming approaches have identified a transcriptional regulator candidate that can rewire CAR T cells to enhance their metabolic fitness and protect them from exhaustion. We therefore sought to identify key transcription factors that areFOXO1 enhances CAR T cell stemness, metabolic fitness and efficacy Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of haematological malignancies such as acute lymphoblastic leukaemia, B cell lymphoma and multiple myeloma, but the efficacy of CAR T cell therapy in solid tumours has been limited. This is due to factors such as the immunosuppressive tumour microenvironment that leads to poorly persisting and metabolically dysfunctional T cells. Analysis of anti-CD19 CAR T cells used clinically has shown that positive treatment outcomes are associated with a more 'stem-like' phenotype and increased mitochondrial mass. We sought to identify transcription factors that could enhance CAR T cell fitness and efficacy against solid tumours. Here we show that overexpression of FOXO1 promotes a stem-like phenotype in CAR T cells derived from either healthy human donors or patients, which correlates with improved mitochondrial fitness, persistence and therapeutic efficacy in vivo. This work reveals an engineering approach to genetically enforce a favourable metabolic phenotype that has high translational potential to improve the efficacy of CAR T cells against solid tumours. In the solid tumour microenvironment, CAR T cells are predisposed to terminally differentiate in response to chronic antigen stimulation, metabolic competition and a lack of appropriate co-stimulatory signals. Terminally differentiated CAR T cells are similar to exhausted endogenous T cells that do not eliminate tumours owing to dysfunction, attenuated effector function and poor persistence. Such cells are characterized by reduced expression of effector molecules, the expression of immune checkpoints including PD-1, LAG3 and TIM3 and transcriptional regulators associated with exhaustion, such as IRF4. Less differentiated CAR T cells, defined by a phenotype of CD45RA+, persist for longer. This is because less differentiated T cells maintain higher multipotency and greater self-renewal capacity and thus can generate increased numbers of effector-like progeny to facilitate improved tumour control. Accordingly, CAR T cells with higher initial frequencies of less differentiated cells elicit improved persistence and therapeutic potential. The processes of T cell differentiation, metabolism and epigenetic reprogramming are highly interdependent. T cell exhaustion is characterized by sub-optimal effector functions that are enforced by epigenetic regulation such as repressive DNA methylation at key gene loci, processes that are directly controlled by intracellular metabolites. In the context of CAR T cells, improved oxidative metabolism and increased mitochondrial biogenesis is associated with enhanced persistence and function. A variety of approaches have been explored to favourably modulate CAR T cell differentiation. These include the use of homeostatic cytokines, epigenetic regulation, and more recently, the overexpression of transcriptional regulators. However, none of these genetic reprogramming approaches have identified a transcriptional regulator candidate that can rewire CAR T cells to enhance their metabolic fitness and protect them from exhaustion. We therefore sought to identify key transcription factors that are