Daan Pieren

111 Co-expression of TIGIT and Helios by CD8+ T cells Loss of CD27 and CD28 expression and concomitant gain of senescence features by CD8 + T cells has often been linked to CMV seropositivity [9]. Our results show that CD8 + T cells significantly lose expression of CD27 and CD28 and gain features of immunosenescence over the years in CMV seronegative individuals, indicating that expansion of highly differentiated T cells is not solely due to CMV. However, since latent CMV infection can promote differentiation of T cells it would be interesting to explore to what extent such induction of differentiation contributes also to the induction of immunosenescent TIGIT + Helios + at old age. A recent study suggested that Helios may be part of immunosenescence in T cells [18] and our data now show that Helios expression clearly marks immunosenescence in CD8 + T cells along with co-expression of TIGIT. How Helios may contribute to altered function of CD8 + T cells at old age and how it becomes induced in the process of aging is unclear. Helios is known as a transcription factor that is expressed by regulatory T cells and needed for their suppressive function [16,17]. Since immunosenescent T cells have gained several features known from regulatory T cells such as Helios and TIGIT, it is tempting to hypothesize that altered functions of a T cell gained at old age may be represented by gain of immunosuppressive functions. Targeting Helios may be a way to investigate the importance of Helios expression in functionality of these TIGIT + Helios + CD8 + T cells. As Helios is an intracellular transcription factor which impedes sorting of TIGIT + Helios + T cells, targeting Helios in future studies with small molecule inhibitors or protein mimetics would be an alternative way to study the role of Helios expression. Additionally, it would be helpful to find markers on the cell surface that may either specifically result from Helios activity or be a specific inducer of Helios. Together, our study indicates that co-expression of TIGIT and Helios provides a novel functional marker for immunosenescent CD8 + T cells. Moreover, we show that these cells already accumulate in the intermediate-differentiation stage in older individuals. These findings challenge the current dogma of late- differentiation stage as proxy for T-cell immunosenescence by showing that immunosenescent T cells accumulate also within earlier differentiation stages. These insights may refine future interpretation of studies on impaired CD8 + T-cell mediated immunity to viral infections at old age. ACKNOWLEDGEMENTS We thank the participants of the ILI-3 and NVI-255 cohort studies, and the blood donors at Sanquin Blood Supply Foundation. We thank Elske Bijvank, 4