Daan Pieren

110 Chapter 4 showed features of immunosenescence identified by low capacity to proliferate and become activated, low expression of the co-stimulatory receptor CD226, and a trend towards higher frequencies of senescence-associated markers CD57 and KLRG1. These findings indicate that Helios more accurately defines immunosenescent cells among the TIGIT + CD8 + T-cell population reported on previously [13]. Including analysis of Helios expression may therefore advance future studies on TIGIT + CD8 + T cells in immunosenescence. Our finding that predominantly the Helios + subset of TIGIT + cells comprises the immunosenescent cells also implies that interventions to overcome immunosenescence by targeting the co-inhibitory receptor TIGIT [14,30] may be refined by particularly targeting this most immunosenescent subset rather than all TIGIT + CD8 + T cells. Moreover, our findings warrant exploration of TIGIT + Helios + CD8 + T cells with respect to other known markers of aging and immunosenescence in CD8 + T cells, such as NK cell-related receptors, low capacity to produce IL-2, and low telomerase expression [8,31]. We found that the proportion of late-differentiated T cells rises with age and is highly enriched with immunosenescent cells as defined by TIGIT + Helios + . This indicates that the CD8 + T-cell pool becomes enriched with these immunosenescent cells over the years by accumulation of cells with a late- differentiated phenotype that express immunosenescent features. Previous work has reported that CD8 + T cells follow a linear differentiation pathway from early- to late-differentiated state through the intermediate- differentiated phase [9]. Interestingly, although the proportion of cells in the intermediate- differentiation state did not alter with age, we found that this intermediate CD8 + T-cell population becomes enriched with immunosenescent TIGIT + Helios + cells at older age. Moreover, the proportion of TIGIT + Helios + intermediate-differentiated cells among the CD8 + T-cell population increased by aging. These findings indicate that CD8 + T cells may become immunosenescent before entering the late-differentiation state and that aging-related increase of immunosenescence cannot merely be classified by accumulation of highly differentiated CD27 - CD28 - cells. Interestingly, it has been shown that elevated numbers of intermediate- differentiated CD8 +  T cells is a predictor of frailty [32], emphasizing the significance of this subset during aging. The applicability of differentiation state based on CD27 and CD28 expression as proxy for immunosenescence may thus depend on age. Our findings suggest that especially at older age, part of the intermediate-differentiated population may be regarded immunosenescent in addition to late-differentiated T cells. Hence, this indicates that differentiation and development of immunosenescence with age do not fully progress in parallel and warrants further research on factors that drive these two processes.