BackgroundThe 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.MethodsTo 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.ResultsAMPKγ 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<0.001, ULK1 phosphorylation 1.28 ± 0.11 vs 0.67 ± 0.08, p<0.01). AMPKγ-overexpressing T cells augmented expression of memory markers CD62L, CD27, and CCR7, with an increased yield of stem cell memory-like T cells marked by co-expression of CD45RA and CD62L (figure 1). Mitochondrial density, SRC, and maximal oxygen consumption rates were similarly increased in AMPKγ-transduced cells (figure 2A,B). Further, while enhanced memory cell production is often linked with reduced proliferation, T cells with increased AMPK activity maintained and even trended towards increased rates of expansion compared to empty-transduced controls (figure 3A), with a measurable increase in CD4+ T cell percentages by flow cytometry (figure 3B).Abstract 106 Figure 1AMPK-transduced T cells increase expression of memory surface markers. Human T cells were transduced with AMPK-GFP or GFP-only control (Empty). Memory markers were assessed by flow cytometry on Days 7–14 of in vitro culture following expansion with IL-2. Plots are representative of 3 separate donorsAbstract 106 Figure 2AMPK-transduced T cells show enhanced mitochondrial density and SRC. (A) Human T cells transduced with AMPK-GFP or GFP-only (Empty) were stained with Mitotracker Red and fluorescence intensity compared between transduced cells and GFP- controls within the same culture to account for variability in Mitotracker dye staining. (B) AMPK and Empty transduced T cells were assessed via Seahorse Metabolic Analyzer using the Mito Stress Test. Results are representative of 3 separate donors. OCR = O2 consumption rateAbstract 106 Figure 3Proliferation is maintained in AMPK-transduced T cells, with enhanced recovery of CD4+ T cells. (A) Primary human T cells transduced with AMPK-GFP or GFP-only (Empty) were expanded in vitro in the presence of IL-2. Cells were manually counted and the ratio of day 7 to day 5 cell counts calculated to assess fold expansion over time. (B) At the same, CD4+ and CD8+ percentages were measured in GFP+ cells by flow cytometryConclusionsIncreasing 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.
Simultaneous assessment of eight phosphorylated STAT residues in T-cells by flow cytometry.
