Daan Pieren

107 Co-expression of TIGIT and Helios by CD8+ T cells 0 20 40 60 80 100 0 20 40 60 80 100 Age (years) %TIGIT + cells in CD8 + T cells 0 20 40 60 80 100 0 20 40 60 80 100 Age (years) % Helios + cells in CD8 + T cells 0 20 40 60 80 100 0 20 40 60 80 100 Age (years) % TIGIT + Helios + cells of CD8 + T cells A B TIGIT + TIGIT + Helios + Helios + r 0.492 p <0.001 r 0.157 p 0.28 r 0.389 p 0.005 Early Intermediate Late 0 20 40 60 80 100 % TIGIT + cells in subset *** *** * Early Intermediate Late 0 20 40 60 80 100 % Helios + cells in subset *** *** ns Early Intermediate Late 0 20 40 60 80 100 % TIGIT + Helios + cells in subset ns *** *** Figure 4. TIGIT + Helios + cells are enriched in the intermediate- and late-differentiated CD8 + T-cell populations. ( A ) Relationship between the frequency of TIGIT + , Helios + , or TIGIT + Helios + cells within the CD8 + T-cell population and age. ( B ) Bar graphs show the frequency of TIGIT + , Helios + , and TIGIT + Helios + cells within the early-, intermediate-, and late-differentiated cell subsets. Correlations ( r and p values) were assessed by Spearman test. Statistical significance of data presented in the bar graph (means ± s.d.) was determined using Friedman test (with Dunn’s post-test). (* p <0.05, *** p <0.001, ns=not significant). Progression of CD8 + T-cell senescence (TIGIT + Helios + ) during aging occurs in the intermediate phase of differentiation We next assessed whether expression of TIGIT and Helios within early-, intermediate-, and late-differentiated CD8 + T-cell subsets changes at older age. The frequency of TIGIT + cells amongst CD8 + T cells significantly increases with age in both the early- and intermediate-differentiated cell subsets ( Figure 5A ). The proportion of Helios + cells (irrespective of their TIGIT expression) amongst CD8 + T cells showed an increase with age specifically within the intermediate-differentiated cell subset ( Figure 5B ). Interestingly, the frequency of TIGIT + Helios + in the CD8 + T-cell population increased within the early- and intermediate-differentiated cells subsets with age, but not within the late- differentiated subset ( Figure 5C ). These findings show that a significant fraction 4