SUMMARY: Exercise as an Immune Adjuvant for gd T-cell Therapies in Hematologic Malignanciesgd T-cells are being considered as an alternative to standard CAR ab T-cells for treating leukemic relapse afterhematopoietic stem cell transplantation (HSCT) largely due to their ability to function across MHC barrierswithout causing graft-versus-host disease (GvHD)1. gd T-cells can be readily expanded in vitro and in vivo usingzoledronate (ZOL) and have demonstrated anti-tumor activity in preclinical and early phase clinical trials buttheir efficacy against CD19-expressing tumors including acute lymphoblastic leukemia (ALL) and non-Hodgkinslymphoma (NHL) has been modest3. Recently CD19 CAR gd T-cells were found to have profound effects againstCD19+ tumors in vitro and in xenogeneic mice albeit inferior to CD19 CAR ab T-cells although CD19 CAR gdT-cells were more effective at eliminating CD19 negative escape variants5 6. As such if the natural cytotoxicityof gd T-cells could be enhanced they would become a highly attractive off the shelf therapeutic option for ALLand NHL. Our goal is to improve gd T-cell therapeutics by collecting superior gd T-cells that have been mobilizedto peripheral blood by exercise or a synthetic b2-adrenergic receptor (AR) agonist and arming them with a CAR.We will build on several novel and important observations we have made: (i) a single exercise boutinstantaneously mobilizes gd T-cells bearing a cytotoxic co-stimulatory and tissue migration phenotype allowingtheir ex vivo manufacture with ZOL+IL-2 to increase by 100-300%4; (ii) exercise expanded gd T-cells have higherin vitro cytotoxicity against several hematologic tumors4 and are more capable of inhibiting K562 leukemic growthin xenogeneic mice particularly when combined with ZOL sensitization; (iii) exercise skews expanded gd T-cellstoward an activated phenotype with heightened NKG2D TRAIL DNAM-1 and lowered NKG2A expression andblocking these activating receptors or their ligands on K562 cells abrogates the exercise effects on gd T-cellcytotoxicity; and (iv) the mobilization of these superior gd T-cells with exercise is driven by b2-AR activation4. Wehypothesize that exercise will also enhance the quality of CAR gd T-cells by mobilizing gd T-cells with sustainedactivation of cytotoxicity co-stimulation oxidative phosphorylation homing and proliferation related genes andthat this mobilization will be precipitated by increased cAMP signaling. Our aims are: 1) Determine if a singleexercise bout can improve the quality of CAR gd T-cells expanded from healthy donors. 2) Explore thetranscriptomic basis for the enhanced expansion and cytotoxicity of exercise mobilized gd T-cells and expandedproducts. 3) Identify the b2-AR signaling pathways responsible for mobilizing gd T-cells with enhanced expansionand cytotoxicity potential. Our approach involves the use flow cytometry xenogeneic mouse models single cellRNA sequencing and comparisons with CD19 CAR ab T-cells in human trials involving exercise with b-blockersand b-agonist infusion models. We expect these aims to identify underpinning mechanisms and pave the wayfor a clinical trial whereby exercise/b-agonist mobilized gd T-cells can be collected from donors and cancerpatients to increase the potency of CAR T-cell therapies to treat refractory disease and relapse after HSCT.