Exercise as an Immune Adjuvant for Gamma Delta T-cell Therapies in Hematologic Malignancies

SUMMARY: Exercise as an Immune Adjuvant for gd T-cell Therapies in Hematologic Malignancies gd T-cells are being considered as an alternative to standard CAR ab T-cells for treating leukemic relapse after hematopoietic stem cell transplantation (HSCT), largely due to their ability to function across MHC barriers without causing graft-versus-host disease (GvHD)1. gd T-cells can be readily expanded in vitro and in vivo using zoledronate (ZOL) and have demonstrated anti-tumor activity in preclinical and early phase clinical trials, but their efficacy against CD19-expressing tumors including acute lymphoblastic leukemia (ALL) and non-Hodgkin’s lymphoma (NHL) has been modest3. Recently, CD19 CAR gd T-cells were found to have profound effects against CD19+ tumors in vitro and in xenogeneic mice, albeit inferior to CD19 CAR ab T-cells, although CD19 CAR gd T-cells were more effective at eliminating CD19 negative escape variants5, 6. As such, if the natural cytotoxicity of gd T-cells could be enhanced they would become a highly attractive “off the shelf” therapeutic option for ALL and NHL. Our goal is to improve gd T-cell therapeutics by collecting “superior” gd T-cells that have been mobilized to 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 bout instantaneously mobilizes gd T-cells bearing a cytotoxic, co-stimulatory and tissue migration phenotype, allowing their ex vivo manufacture with ZOL+IL-2 to increase by 100-300%4; (ii) exercise expanded gd T-cells have higher in vitro cytotoxicity against several hematologic tumors4 and are more capable of inhibiting K562 leukemic growth in xenogeneic mice, particularly when combined with ZOL sensitization; (iii) exercise skews expanded gd T-cells toward an activated phenotype with heightened NKG2D, TRAIL, DNAM-1 and lowered NKG2A expression, and blocking these activating receptors, or their ligands on K562 cells, abrogates the exercise effects on gd T-cell cytotoxicity; and (iv) the mobilization of these superior gd T-cells with exercise is driven by b2-AR activation4. We hypothesize that exercise will also enhance the quality of CAR gd T-cells by mobilizing gd T-cells with sustained activation of cytotoxicity, co-stimulation, oxidative phosphorylation, homing and proliferation related genes, and that this mobilization will be precipitated by increased cAMP signaling. Our aims are: 1) Determine if a single exercise bout can improve the quality of CAR gd T-cells expanded from healthy donors. 2) Explore the transcriptomic basis for the enhanced expansion and cytotoxicity of exercise mobilized gd T-cells and expanded products. 3) Identify the b2-AR signaling pathways responsible for mobilizing gd T-cells with enhanced expansion and cytotoxicity potential. Our approach involves the use flow cytometry, xenogeneic mouse models, single cell RNA sequencing, and comparisons with CD19 CAR ab T-cells in human trials involving exercise with b-blockers and b-agonist infusion models. We expect these aims to identify underpinning mechanisms and pave the way for a clinical trial whereby exercise/b-agonist mobilized gd T-cells can be collected from donors and cancer patients to increase the potency of CAR T-cell therapies to treat refractory disease and relapse after HSCT.