Sara van den Berg

178 Chapter 7 round 5 and round 6 (missing n=1 for round 5 and n=12 for round 6). Increase in frailty index was defined as the difference between the frailty indices assessed in rounds 5 and 6. Cytomegalovirus (CMV)-specific antibodies CMV-specific IgG antibody levels were measured in plasma by a multiplex immunoassay developed in-house [38]. A cutoff of 5 arbitrary units mL -1 was shown to discriminate best between CMV- and CMV+ study groups [39]. To decrease the chance of false-positive or false-negative results, samples with antibody levels <= 4 mL -1 were defined as CMV- seronegative (CMV-) and those with antibody levels >7.5) mL -1 CMV-seropositive (CMV+), while all levels in between were considered inconclusive. To reduce intra-assay variation, all samples from the same individual were measured on the same plate Cell numbers by flow cytometry Fresh whole blood samples collected in 2016-2017 were used to quantify cell numbers of T-cell subpopulations by flow cytometry, as previously described in more detail [39]. Briefly, absolute cell numbers were determined using TruCOUNT tubes (BD Biosciences, San Jose, CA, USA), in which whole blood was stained with CD3(UCHT1)-BV711 (BD Biosciences). After erythrocyte lysis with FACS Lysing Solution (BD Biosciences), tubes were measured directly on a flow cytometer (FortessaX20). Another tube was used with the following antibodies: CD3(UCHT1)-BV711, CD8(SK1)-APC-H7, CCR7(150503)-PECF594, CD27(M-T271)-BV421, CD28(CD28.2)-PerCPCy5.5 (all BD Biosciences), CD4(RPA-T4)-BV510, CD45RA(HI100)- BV650 (all Biolegend). Using the bead count of the TruCOUNT tube and the CD3 cell count of both tubes absolute cell numbers were calculated. T-cell subsets of naive (T N ), central memory (T CM ), effector memory (T EM ) and effector memory re-expressing CD45RA (T EMRA ) were defined based on the expression of CD45RA and CCR7, after which T EM and T EMRA cells were categorized as early, intermediate and late differentiated based on the expression of CD27 and CD28 [40]. PBMC isolation Peripheral blood mononuclear cells from the DCS subcohort were obtained by Lymphoprep (Progen) density gradient centrifugation from heparinized blood, according to the manufacturer’s instructions. After isolation of the PBMCs, the cells were washed with PBS medium containing 0.2% FCS (Sigma), and then frozen in a solution with 90% FCS and 10% dimethyl sulfoxide at -135°C until further use. CMV-specific functional T-cell analysis CMV-specific T-cell responses were analyzed for a selected subpopulation (n=30): first, ten CMV+ individuals were selected with the shortest duration of CMV infection for which PBMCs were available. Ten CMV- individuals were subsequently selected based on matched age and sex only, and ten CMV+ individuals with long term CMV infection also based on CMV-specific antibody levels. Cryopreserved PBMCs were rapidly thawed at 37 °C and washed once in AIM-V (Gibco) 2% human AB serum medium (AIM-V 2% hAB). The cells