Bulk RNA-Seq analysis of TCF1 Reporter eGFP+ and TCF1 Reporter eGFP- CD8+ T cells generated with different metabolic manipulations and isolated from in vitro co-culture with HKP1-ova-GFP mouse lung adenocarcinoma cells

To investigate how metabolic manipulation alters differentiation and the transcriptional landscape in tumor-specific CD8+ T cells, we isolated OT-I+ CD8+ T cells from spleens of transgenic OT-I+ Tcf7GFP+ mice expressing a TCF1 eGFP Reporter, and activated them for one day on plates coated with 2μg/ml of both anti-CD3 and anti-CD28 in the presence of 50U/mL mouse IL-2. We then treated them with equal volumes of DMSO vehicle control, 3μM glucose 6-phosphate dehydrogenase agonist AG1, 25μM glucose 6-phosphate dehydrogenase inhibitor G6PDi-1, or 2mM hexokinase inhibitor 2-DG, and expanded them for 1 more day on anti-CD3/anti-CD28 coated plates in the presence of 50U/mL mouse IL-2. We subsequently co-cultured these cells with HKP1-ova-GFP tumor cells at a 5:1 T cell:tumor cell ratio for an additional 4 days with continuing drug treatment in the presence of 50U/mL mouse IL-2, passing cultures every other day. At day 6 post-initial-stimulation, CD8+ T cells were harvested, filtered by passing through a 70uM cell strainer, washed, and stained for CD8β and Thy1.2. Samples were dyed with DAPI at 0.2μg/mL, and sorted on a Becton-Dickinson FACS Aria II sorter for DAPI- CD8β + Thy1.2+ eGFP+ or DAPI- CD8β + Thy1.2+ eGFP- cells. We then performed gene expression profiling analysis using data obtained from RNA-seq of sorted cells, comparing across treatment groups and TCF1 eGFP Reporter expression. Comparative gene expression profiling of biological replicates of OT-I+ TCF1 Reporter eGFP+ and OT-I+ TCF1 Reporter eGFP- CD8+ T cells isolated from in vitro co-culture with HKP1-ova-GFP mouse lung adenocarcinoma cells in the presence of different metabolism-altering drugs (DMSO, AG1, G6PDi-1, 2-DG) at 6 days post-initial-stimulation. Grouped by treatment and TCF1 eGFP Reporter expression. DMSO eGFP+ [n=2]; DMSO eGFP- [n=2]; AG1 eGFP+ [n=2]; AG1 eGFP- [n=2]; G6PDi-1 eGFP+ [n=2]; G6PDi-1 eGFP- [n=2]; 2-DG eGFP+ [n=2].