Daan Pieren

137 Regulatory KIR+RA+ CD8+ T cells accumulate with age ( H ) Proliferation of CD8 + T cells that are KIR - NKG2A - , KIR + NKG2A - , or KIR - NKG2A + and the representative flow cytometry plot (n=15). ( I ) Proportion and representative flow cytometry histograms of KIR - NKG2A - , KIR + NKG2A - , and KIR - NKG2A + cells within the CD8 + T-cell population cultured with (+) or without (-) stimulatory anti-CD3. Correlations ( r values) were assessed by Spearman test. Statistical significance of data presented in bar graphs (means ± s.d.) was determined using row-matched one-way ANOVA (with Geisser-Greenhouse correction and Dunnett’s post-test) ( A - D ), Friedman test (with Dunn’s post-test) ( E, H ), or Wilcoxon matched- pairs test ( I ). (* p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001, ns=not significant). KIR + RA + T cells show a distinct regulatory CD8 + T-cell phenotype and sup- press proliferation of KIR - NKG2A - CD8 + T cells The negative correlation between the proportion of KIR + RA + T cells and CD8 + T-cell proliferation may also imply that KIR + RA + T cells regulate proliferation of other CD8 + T cells. TIGIT and Helios are markers for CD4 + regulatory T cells (Tregs) [35,40,45]. Since KIR + RA + T cells highly express TIGIT at the cell surface and our RNA-seq data indicate enhanced TIGIT and expression of Helios transcripts, we explored the possibility that KIR + RA + T cells might be regulatory CD8 + T cells. We first confirmed the regulatory phenotype of KIR + RA + T cells by high intracellular expression of Helios protein by flowcytometry ( Figure 5A ) as well as by high cell-surface expression of the CD8 + Treg-associated marker CD122 [46] ( Figure 5B ). KIR + RA + T cells showed the highest frequency of Helios + and CD122 + cells compared to all other CD8 + T-cell subsets ( Figure 5A,B ), as well as expression of these markers per cell ( Supplementary Figure 6A,B ). Combined expression of TIGIT and Helios even further delineated KIR + RA + T cells from all other subsets ( Figure 5C ), indicating a distinct regulatory T-cell phenotype of KIR + RA + T cells. We next investigated whether KIR + RA + T cells could functionally regulate T-cell proliferation in a suppression assay. Addition of KIR + RA + T cells reduced proliferation of responder T cells (KIR - NKG2A - CD8 + T cells) ( Figure 5D ) in a dose-dependent manner ( Figure 5E ). Adding responder cells instead of KIR + RA + T cells to the responder cells did not reduce their proliferation, indicating suppression by KIR + RA + T cells ( Figure 5D left and right panels). Together, we show that KIR + RA + T cells are a CD8 + T-cell subset with regulatory phenotype and suppressive capacity. Therefore, accumulation of KIR + RA + T cells with age may suggest that these cells contribute to suppressing immunity in older adults. KIR + RA + T cells are highly activated during acute respiratory infection in older adults as bystander cells To investigate whether KIR + RA + T cells play a role in dampening protection in the elderly, we analyzed the presence and activation status of KIR + RA + T cells 5