Daan Pieren

108 Chapter 4 of immunosenescent cells as defined by TIGIT + Helios + already accumulates in the intermediate-differentiated CD8 + T-cell population at older age and that these cells are not only present in the late-differentiated stage. Moreover, despite age-related decline of the fraction of early-differentiated cells ( Figure 3C ), the minor fraction of TIGIT + Helios + cells among the early-differentiated population increases with age ( Figure 5C ). These findings suggest that CD8 + T cells develop into immunosenescent cells during the course of aging already before reaching the late state of differentiation. TIGIT + Helios + A B C Helios + TIGIT + 0 20 40 60 80 100 0 20 40 60 80 100 Age (Years) % Helios + cells in subset Early Intermediate Late r p 0.0508 ns 0.306 * -0.0457 ns 0 20 40 60 80 100 0 20 40 60 80 100 Age (Years) % TIGIT + Helios + cells in subset Early Intermediate Late r p 0.0919 ns 0.377 ** 0.428 ** 0 20 40 60 80 100 0 20 40 60 80 100 Age (Years) % TIGIT + cells in subset Early Intermediate Late r p 0.278 0.508 *** 0.360 * 0.05 Figure 5. Progression of CD8 + T-cell senescence (TIGIT + Helios + ) during aging occurs in the intermediate phase of differentiation. Frequency of ( A ) TIGIT + , ( B ) Helios + , and ( C ) TIGIT + Helios + cells within the early-, intermediate-, and late-differentiated cell subsets and their relationship with age (n=50). Correlations ( r and p values) were assessed by Spearman test with p <0.05 considered as statistically significant. (* p <0.05, ** p <0.01, *** p <0.001, ns=not significant). TIGIT + Helios + intermediate- and TIGIT + Helios + late-differentiated cells accumulate by aging within the CD8 + T-cell population Lastly, we addressed whether early-, intermediate-, and late-differentiated cells expressing TIGIT and/or Helios increase by aging within the CD8 + T-cell population. The frequency of TIGIT + late-differentiated cells amongst CD8 + T cells increased with age, and also the TIGIT + intermediate-differentiated cells amongst the CD8 + T-cell population showed a trend towards higher levels with age ( Figure 6A ). The frequency of Helios + early- and Helios + late-differentiated cells amongst total CD8 +  T cells significantly declined and increased, respectively ( Figure 6B ). Interestingly, the frequency TIGIT + Helios + intermediate- and late- differentiated cells amongst total CD8 + T cells increased with age ( Figure 5C )