Daan Pieren

105 Co-expression of TIGIT and Helios by CD8+ T cells (n=50). ( E ) Relationship between the frequency of proliferated CD8 + T cells and the frequency of TIGIT + Helios + cells within the CD8 + T-cell population after three days of stimulation. All frequencies of proliferating cells were relative to their unstimulated control sample. Correlations ( r and p values) were assessed by Spearman test. Statistical significance of data presented in the bar graphs (means ± s.d.) was determined using Friedman test (with Dunn’s post-test). Wilcoxon test was used in panels B and C for the comparison between the time points within each subset. (* p <0.05, ** p <0.01, *** p <0.001, ns=not significant). CD226 (BV785) TIGIT + Helios + TIGIT + Helios - TIGIT - Helios + TIGIT - Helios - 0 500 1000 1500 2000 CD226 expression (MFI) in subset *** *** *** ns ns ns TIGIT Helios + + + - - + - - 0 20 40 60 80 100 %CD226 + cells of subset *** *** *** ns ns ns TIGIT Helios + + + - - + - - A B D C ns 0 20 40 60 80 100 % CD57 + cells of subset ns ns ** * ns TIGIT Helios + + + - - + - - 0 20 40 60 80 100 % KLRG1 + cells of subset ns ns ns ns * * TIGIT Helios + + + - - + - - E Figure 2. Low expression of the co-stimulatory receptor CD226 by immunosenescent CD8 + T cells. ( A ) Representative histogram of CD226 expression within each of the TIGIT/Helios cell subsets. Bar graphs show ( B ) the frequency of CD226 + cells and ( C ) expression (median fluorescent intensity, MFI) of CD226 per cell within each of the four TIGIT/Helios cell subsets (n=50). Bar graphs show the frequency of ( D ) CD57 + and ( E ) KLRG1 + cells within each of the four TIGIT/ Helios cell subsets (n=6). Statistical significance of data presented in the bar graphs (means ± s.d.) was determined using Friedman test (with Dunn’s post-test). (* p <0.05, ** p <0.01, *** p <0.001, ns=not significant). 4