Daan Pieren

106 Chapter 4 A B CD27 (BV510) CD28 (BV711) 51.2 4.34 20.0 24.5 C Early Intermediate Late 0 20 40 60 80 100 % Subset in CD8 + T cells ****** ns 0 20 40 60 80 100 0 20 40 60 80 100 Age (Years) % Subset in CD8 + T cells Early Intermediate Late r p 0.432 ** 0.0415 -0.395 ** ns Figure 3. Late-differentiated T cells accumulate with age at the expense of early-differen- tiated T cells. Expression of CD27 and CD28 by CD8 + T cells was analyzed in healthy individuals ranging from 21-82 years of age (n=50) using flow cytometry. ( A ) Representative flow-cytometry plot illustrating expression of CD27 and CD28 by CD8 + T cells. ( B ) Bar graphs show the frequency of early (CD27 + CD28 + ), intermediate (CD27 + CD28 - ), and late (CD27 - CD28 - ) differentiated cells within the total CD8 + T-cell population of each individual. ( C ) Frequency of each of the three differentiation subsets among CD8 + T cells and their relationship with age. 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 <0.01, *** p <0.001, ns=not significant). TIGIT + Helios + cells are enriched in the intermediate- and late-differentiated CD8 + T-cell populations We next explored how expression of TIGIT and Helios relates to age and to the differentiation status of CD8 + T cells. Clustering of CD8 + T cells based on their expression of CD27, CD28, TIGIT, and Helios by viSNE analysis indicated that only part of the CD8 + T-cell pool co-expresses TIGIT and Helios and that expression of these molecules may relate to distinctive expression of the differentiation markers CD27 and CD28 ( Supplementary Figure 2A ). The proportion of TIGIT + cells amongst CD8 + T cells increased at older age, whereas the proportion of Helios + cells did not considerably change with age ( Figure 4A ). Interestingly, the subset of CD8 + T cells that co-expressed TIGIT and Helios increased with age ( Figure 4A ). Notably, these TIGIT + Helios + cells appeared to be present mostly in the intermediate- and late-differentiated cell subsets ( Figure 4B ) . These findings indicate that co-expression of TIGIT and Helios defines an aging-related population of cells that accumulate in the intermediate- and late-differentiated CD8 + T-cell subsets.