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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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE238203 | GEO | 2023/10/19

REPOSITORIES: GEO

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Project description:Mapttm1(EGFP)Klt/J mice (Mapt-EGFP; The Jackson Laboratory, Bar Harbor, ME, USA; stock 004779) carry a knock-in of the EGFP coding sequence in the first exon of the microtubule-associated protein tau (Mapt) gene producing a cytoplasmic EGFP fused to the first 31 amino acids of MAPT. EGFP expression marks neurons including enteric neurons regardless of their lineage, closely patterning the expression of neuron-specific beta-tubulin III (TUBB3). Mapt-EGFP ice were backcrossed to C57BL/6J (Jackson Laboratory strain #:000664) for three to five generations at Mayo Clinic. Six male and six female Mapt-EGFP mice (54-98 days of age) underwent surgical laparotomy in 3 groups (surgery #1: 1 male and 1 female, surgery #2: 3 males and 1 female, surgery #3: 2 males and 4 females) under pentobarbital (50mg/kg) anesthesia. The celiac ganglion of each mouse was injected with 3-5 μL of 25 mg/mL Alexa Fluor 647-labeled cholera toxin subunit B (CTB-AF647; Thermo Fisher Scientific, Waltham, MA, USA) with the intention of labeling the cell soma of intestinofugal neurons in the myenteric plexus of the colon. The animals were killed 3-4 days after surgery. The muscularis externa of the colon from each Mapt-EGFP mouse was pooled together between all mice of the same surgery date (2, 4, and 6 mice) and mechanically and enzymatically dissociated into single cells with a two-step process that first enriches for cells within myenteric ganglia (PMCID: PMC8114175). The pooled cells from each group of mice formed one biological replicate and subjected to FACS immediately after dissociation to generate populations of Mapt-EGFP+ neurons with or without the CTB-AF647 tracer and Mapt-EGFP− non-neuronal cells. The frequency of Mapt-EGFP+CTB-AF647+ neurons was approximately 125-fold lower than that of Mapt-EGFP+CTB-AF647− neurons and RNA from these preparations did not pass quality control. Therefore, only data from Mapt-EGFP+CTB-AF647− neurons were analyzed and referred to as Mapt-EGFP+ cells. Total RNA was isolated from Mapt-EGFP+ colonic neurons and Mapt-EGFP− myenteric cells using RNA-Bee (AMSBIO, Cambridge, MA, USA) and purified with RNeasy Mini Kit (Qiagen, Germantown, MD, USA). RNA quality was tested using Agilent Electropherogram (Agilent Technologies, Santa Clara, CA, USA) and hybridized to Affymetrix Mouse Genome 430.2 gene expression microarrays (Thermo Fisher Scientific, Waltham, MA, USA). This study utilized Affymetrix Mouse Genome 430.2 oligonucleotide microarray analysis to charaterize the transcriptome of Mapt-EGFP+ neurons and Mapt-EGFP- non-neuronal myenteric cells isolated from the colon of Mapttm1(EGFP)Klt/J mice.

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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

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