106 Increasing AMPK activity in human T cells enhances memory subset formation without sacrificing in vitro expansion

Background The ideal adoptive cell therapy consists of memory-like T cells with enhanced oxidative potential. However, current expansion protocols drive T cells towards terminal differentiation, decreasing the number of T cells fit for the in vivo environment. AMP-activated protein kinase (AMPK), whose activity increases in memory cells, is a key regulator of mitochondrial biogenesis and oxidative metabolism, making AMPK activation an attractive candidate to improve adoptive T cell function. Methods To increase AMPK activity, AMPKγ, which controls the phosphorylation status of AMPKa and therefore activity of the AMPK complex, was cloned into a lentiviral plasmid downstream of the elongation factor 1a (EF1a) promoter and upstream of green fluorescent protein (GFP). An empty vector, containing GFP only, served as a negative control. Human T cells were transduced and expanded in vitro in the presence of IL-2. AMPK activity was assessed via immunoblot for phosphorylation of AMPKa on Thr172 and S555 on downstream target Unc-51-like kinase 1 (ULK1). Memory-marker expression and mitochondrial density (using Mitotracker Red) were analyzed by flow cytometry. Oxidative metabolism and spare respiratory capacity (SRC) were determined using the Seahorse Metabolic Analyzer. Fold changes of in vitro expansion were calculated by adjusting manual cell counts for GFP positivity and CD4+/CD8+ staining. Results AMPKγ was efficiently transduced and expressed by human T cells, which significantly increased AMPK activity (AMPKa phosphorylation 1.93 ± 0.05 vs 0.6 ± 0.09, p Conclusions Increasing AMPK activity endows T cells with a variety of characteristics ideal for adoptive cell therapy, including increased memory-marker expression, enhanced SRC and oxidative metabolism, equivalent to augmented in vitro expansion, and improved CD4+ T cell yields. Further studies are ongoing to assess the activity and function of AMPK-transduced CAR-T cells both in vitro and in vivo.