Daan Pieren

132 Chapter 5 distinguishes these cells from NKG2A + RA + T cells that had previously been regarded to be part of the same CD8 + T-cell subset. -6 -4 -2 0 2 4 6 0 2 4 6 8 10 12 18 20 log2FC -Log10 p-value TIGIT KIR IKZF2 KLRC1 TOX KLRG1 FCRL3 HAVCR2 CTLA4 GZMH CD28 KLRA1P KIR + RA + T cells NKG2A + RA + T cells Regulatory markers KIRs Cytotoxic markers Exhaustion markers NCR1 FCRL6 versus A B C E D PCA: 34,745 genes -200 -100 0 100 200 -100 -50 0 50 100 KIR + RA + T NAIVE T EMRA NKG2A + RA + PCA1 (25.5%) PCA2 (13.4%) 0 5 10 15 Normalized gene expression (Log2-scale, counts per million) Co-inhibitory / Exhaustion CTLA-4 TOX KLRG1 TIGIT EOMES TIM-3 TCF7 Senescence Sestrin 3 FAIM CD57 ATM p65 Rel B Sestrin 2 Cytotoxicity Granzyme B Granzyme H Perforin Granzyme A Fas Ligand Regulatory FCRL3 KLRA1P RORa Helios KIR + RA + NKG2A + RA + * * * * * * * * * * * FCRL6 0 5 10 15 Normalized gene expression (Log2-scale, counts per million) CD27 CD28 CCR7 TCR/co-stimulatory CD3 * * NKG2A + RA + KIR + RA + Figure 2. The gene expression profile of KIR + RA + T cells is distinct from that of NKG2A + RA + T cells and shows features of aging and regulation. KIR + RA + , NKG2A + RA + , T NAIVE , and T EMRA cell subsets were sorted from healthy donor PBMCs (n=6) and their gene expression profiles were analyzed by RNA sequencing. ( A ) Unsupervised principal component analysis (PCA) on the total transcriptome (34,745 genes) showing clustering of the four sorted cell subsets along components 1 and 2. ( B ) Venn diagram showing the unique and shared molecular and cellular functions for each of the three cell subsets compared to T NAIVE cells as identified by ingenuity pathway analysis (IPA). ( C ) Comparison of detected transcripts associated with TCR- and co-stimulation (shown by the names of the proteins they encode) between KIR + RA + T cells and NKG2A + RA + T cells. ( D ) Volcano plot indicating differences in gene expression between NKG2A + RA + and KIR + RA + T cells. The different colors indicate genes related to regulation, exhaustion, cytotoxicity,