Daan Pieren

102 Chapter 4 based on measurements in one donor to illustrate the overlap of markers within CD8 + T cells. A number of 6,325 CD8 + T cells was included in this analysis. Statistics Statistical analysis was performed using GraphPad Prism version 8.4.1. The appropriate parametric or non-parametric tests were used based on the tested normality of distribution of the data. Paired analyses were performed with non-parametric Wilcoxon test or Friedman Test with Dunn’s post-test. Correlations between variables were analyzed using Spearman’s rank correlation coefficient ( r ). Linear regression analysis was performed to generate lines of best fit. Statistical significance was considered when p < 0.05, with * p <0.05, ** p <0.01, *** p <0.001, and **** p <0.0001. All data presented in bar graphs are depicted as mean ± s.d. RESULTS Co-expression of TIGIT and Helios defines immunosenescent T cells We first questioned if co-expression of TIGIT and Helios links to dysfunctional immunosenescent CD8 + T cells. We therefore explored if co-expression of TIGIT and Helios ( Figure 1A ) would relate to reduced capacity of CD8 + T cells to get activated and proliferate in response to in vitro stimulation of PBMCs with anti-CD3. The proportion of TIGIT + Helios + T cells within the CD8 + T-cell population remained stable after one day of culturing with anti-CD3, although this proportion slightly declined after three days ( Figure 1A ). TIGIT + Helios + cells were activated within the first day of culture (shown by induction of classic activation markers CD69 and CD25), but to a lesser extent than the TIGIT + Helios - and TIGIT - Helios - cell subsets ( Figure 1B, C, and Supplementary Figure 1 ). Two days later, expression of the early activation marker CD69 had significantly dropped in the cells that were single positive or double negative for TIGIT and Helios. Such rapid return towards lower CD69 expression during a response in vitro is typically observed after activation of T cells [5]. However, TIGIT + Helios + cells did not show a decline of CD69 expression, but the induced CD69 expression appeared to persist on these cells instead ( Figure 1B ) . From day 1 to day 3, expression of the activation marker CD25 increased further in all four subsets, but this upregulation was lower in TIGIT + Helios + cells ( Figure 1C ). Together, these data indicate slower and reduced activation potential of CD8 + T cells co-expressing TIGIT and Helios. Although all TIGIT + subsets showed reduced proliferation, TIGIT + cells co- expressing Helios proliferated less compared to all other subsets ( Figure 1D ) .