Next Generation Sequencing Facilitates Quantitative Analysis of The Transcriptome of Microbiota-Specific Memory CD4 Cells With vs Without CAMCI Treatment

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare the transcriptomes of microbiota-specific memory CD4 T cells that were treated with vs without the combination of cell activation and metabolic checkpoint inhibition (CAMCI) strategy in vivo. Methods: mRNA profiles of donor-derived CBir1 TCR Tg CD4+ cells were generated by deep sequencing, with cells from 3-4 mice polling together per group, using Illumina HiSeq. The sequence reads that passed quality filters were analyzed with DESeq2 for gene expression comparisons between groups of samples. The Wald test was used to generate p-values and log2 fold changes. Results: Using an optimized data analysis workflow, we mapped about 50 million sequence reads per sample to the mouse genome (GRCm38). Depending on the groups that were compared, a range from 17-2868 transcripts showed differential expression, with an absolute log2 fold change >1 and an adjusted p value <0.01. Differentially expressed genes (DEGs) were used to generate a heatmap, a principle component analysis plot, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Overall design: Comparison of donor-derived CBir1 TCR Tg CD4+ cells isolated from No treatment, MEP1+CMC treatment, MEP1+Metformin treatment, MEP1+Rapamycin treatment, and MEP1+Metformin+Rapamycin treatment groups.