Sara van den Berg

139 6 T-cell dynamics in CMV of two older CMV-seropositive (CMV+) individuals, it has previously been proposed that CMV-specific CD8 + T-cells are relatively long-lived and thereby accumulate over time [21]. In contrast, the one aged individual that was CMV+ in a deuterated water labelling study [22] had much higher CD8 + memory T-cell production rates than the four CMV- individuals, which could possibly be explained by chronic, age-related immune activation [23] as was suggested in mice [24]. As CMV has such a broad impact on the appearance of the T-cell pool, understanding the functional implications and mechanisms underlying CMV-induced changes is important. In this study, we aimed to investigate the effect of CMV infection on CD4 + and CD8 + T-cell dynamics in healthy older adults, and to unravel the mechanisms of maintenance of large numbers of CMV-specific CD8 + T-cells. Hereto, we extensively characterized the lifespan- associated phenotype of different T-cell subsets, based on flow cytometric markers for senescence, proliferation, and apoptosis, in a cohort of 32 CMV+ and 22 CMV- healthy older adults. In five CMV- and five CMV+ healthy older adults, we additionally performed a long-term, in vivo deuterated water ( 2 H 2 O) labelling study to assess the in vivo production rate of cells. We show that the changes in the T-cell pool phenotype of CMV+ individuals cannot solely be explained by the presence of large numbers of CMV-specific CD8 + T-cells. CD4 + T EM , CD4 + T EMRA , and CD8 + T EM cells differ most between CMV+ and CMV- individuals in terms of lifespan-associated phenotype, e.g. by increased expression of senescence markers and decreased Ki-67 expression in CMV+ individuals. A trend towards lower CD4 + T EM/EMRA production rates in CMV+ individuals compared to CMV- individuals was found, but we found no significant difference between the CD8 + T-cell dynamics of CMV+ versus CMV- individuals, nor for CMV-specific CD8 + T-cells versus bulk memory CD8 + T-cells. We found a significant correlation between in vivo production rates and the expression of senescence markers. MATERIALS AND METHODS Study design Fifty-four healthy older adults were included based on very strict health criteria to ensure that we were investigating a steady-state situation. Individuals were excluded from participation if they had a condition that influenced the immune system (e.g. infection with human immunodeficiency virus, hepatitis B or C virus, Lyme disease, malaria, or chronic diseases such as asthma or diabetes mellitus, or (a history of) cancer) or drug use (with the exception of occasional use of paracetamol or ibuprofen)). For the heavy water labelling study, we sub-selected five CMV+ individuals based on high T-cell responses against CMV, as well as five age-matched CMV- individuals. All participants gave written informed consent. This study was approved by the local ethical committee of the University Medical Center Utrecht (UMCU) (METC 15/745), The Netherlands, and was conducted in accordance with the Helsinki Declaration, last amended in 2013. We obtained heparinised blood by venepuncture from all fifty-four participants at one time point. The ten participants that were included in the deuterium labelling study donated blood an additional eight times.