Transcriptome analysis reveals changes between total Teff and HLA-DR^- Teff cell subsets from PTB subjects.

Total and HLA-DR- Teff cells from PTB subjects were isolated by FACS and activated with anti-CD3/anti-CD28 activator beads (Teff:bead ratio of 1:1) for 2, 24 and 96 hrs. Unstimulated cells were used as 0 hr control. RNA was isolated at each time and subjected to RNA sequencing. A list of up-regulated DEGs (cut-off of log2 FC > 2.5, P - Teff cells. (A) Venn diagrams were drawn to identify unique and common genes up-regulated upon activation in the two cellular subsets of total and HLA-DR- Teff cells. (B) Pathway analysis of unique genes over-expressed at 2 hrs post activation was done using DAVID Functional Annotation Bioinformatics Microarray Analysis tool. A heatmap was constructed using FPKM values of genes in enriched pathways using R software. As FPKM values are unevenly distributed, z score was calculated per row i.e. per gene for proper visualization. The colour key was adjusted accordingly. Mean fold change in expression of selected genes from enriched pathways at 2, 24 and 96 hrs post activation in total (C) and HLA-DR- (D) Teff cells was plotted. A total of N = 5 at each time point for total and HLA-DR- Teff cells was used for obtaining all data.