Daan Pieren

131 Regulatory KIR+RA+ CD8+ T cells accumulate with age The gene expression profile of KIR + RA + T cells is distinct from that of NK2A + RA + T cells and shows features of aging and regulation To further understand differences between KIR + RA + and NKG2A + T cells, we sorted these cell subsets ( Supplementary Figure 2A ) and analyzed their gene expression profiles by RNA sequencing (RNA-seq). As a reference, we included T­ NAÏVE and T EMRA cells in this analysis ( Supplementary Figure 2B ). Unsupervised principal component analysis (PCA) on the total transcriptome (34,745 genes) reveals that T NAIVE cells are distinct from the other three cell subsets ( Figure 2A ), but also reveals differences between KIR + RA + , NKG2A + RA + , and T EMRA cells. In addition, the PCA indicates that the variation among these memory subsets is highly donor-dependent ( Supplementary Figure 2C ). To asses if KIR + RA + and NKG2A + RA + T cells may have acquired distinct molecular and cellular functions, we first identified differentially expressed genes (DEGs) between the four T-cell subsets using a paired analysis (matched donor pairs). These DEGs were used to identify enrichment of unique pathways of molecular and cellular functions in KIR + RA + , NKG2A + RA + , and T EMRA memory cells, as compared to T NAIVE­ cells ( Supplementary Figure 3 ). Besides 83 pathways shared by these three memory subsets, sixteen pathways are shared between KIR + RA + and NKG2A + RA + cells but absent in T EMRA cells, which are mainly linked to chemotaxis and recruitment of cells ( Figure 2B ; Supplementary Figure 3 ). KIR + RA + T cells are uniquely enriched for seven pathways ( Figure 2B ), including pathways related to killing of lymphocytes and mononuclear leukocytes ( Supplementary Figure 3 ), whereas NKG2A + RA + T cells are enriched for six unique pathways, associated with innate antigen presenting cell features ( Supplementary Figure 3 ). These findings indicate that both KIR + RA + and NKG2A + RA + T cells have acquired distinct molecular and cellular functions. As numbers of KIR + RA + T cells increase with age, we focused our analyses on the characterization of this cell subset. Compared to NKG2A + RA + T cells, genes involved in co-stimulation ( CD28 , CCR7 ) are downregulated in KIR + RA + T cells ( Figure 2C ). In addition, KIR + RA + T cells showed enrichment for transcripts related to a regulatory cell phenotype, such as IKZF2 (encoding Helios) [35], FCRL3 [36], and KLRA1P (Ly46 in mice) [37] ( Figure 2D,E ). Moreover, the transcriptome of KIR + RA + T cells was enriched for several exhaustion-related transcripts ( TIGIT , TOX , KLRG1 , and decreased CD28 ) and transcripts of the senescence-related anti-apoptotic gene FAIM ( Figure 2D,E ), which may indicate cellular senescence [38], although other senescence-associated genes (e.g. SESN2 , SESN3 , B3GAT1, ATM , RELA , RELB, BCL2 ) were not differentially expressed. Thus, our transcriptome analysis shows that the transcriptome of KIR + RA + T cells is enriched for aging- and regulatory-associated genes, which 5