Sara van den Berg

140 Chapter 6 Cytomegalovirus (CMV)-specific antibodies CMV-specific antibody (Ab) levels were measured in serum by a sensitive multiplex immunoassay (Tcheriaeva 2018, Journal Immunological Methods). A cut-off value of 5 (RU) mL -1 was used to define CMV-seropositivity. To decrease the chance of false-positive or false- negative results, we only considered samples to be CMV-seronegative if the Ab level was ≤ 4 (RU) mL -1 and CMV-seropositive if the Ab level was >7.5 (RU) mL -1 . All of our samples were clearly CMV-seropositive or CMV-seronegative according to these definitions. Cell numbers and HLA typing by flow cytometry Whole blood was used to calculate absolute leukocyte counts by Trucount™ analysis (BD Biosciences) according to manufacturer’s protocol. The following antibodies were used: CD45-PerCP (BioLegend), and CD3-APC-R700, CD4-BV711, CD8-BV786, all purchased from BD Biosciences. Using the bead count of the Trucount™ tube and the CD3 + cell count, absolute cell numbers of the proliferation/apoptosis and senescence panel were also calculated. In addition, for all individuals, HLA typing on whole blood by HLA-antibodies was performed for the most relevant HLA-types describing dominant CMV epitopes by flow cytometry at the first visit. Antibodies used were HLA-A1/36-biotin/strep-PE-CF594, HLA-A2-V450, HLA-A3-APC, HLA-A24-PE, HLA-B7-FITC, and HLA-B8-PE-Cy7. Cells were measured on a Fortessa™ flow cytometer (BD Biosciences) and analysed with FlowJo V10 software. PBMC, neutrophilic granulocytes, and serum isolation and storage Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque PLUS (GE) density centrifugation according to manufacturer’s protocol. After isolation of the PBMCs, cells were washed with phosphate buffered saline (PBS) supplemented with 0.2% FCS, and then frozen in a solution with 90% fetal calf serum and 10% dimethyl sulfoxide in liquid nitrogen or -135°C until further use. Neutrophilic granulocytes were obtained from the Ficoll- Paque pellet after erythrolysis with shock buffer (8.3 g NH 4 Cl, 1 g KHCO 3 , and 37 mg EDTA in 1 L Aqua Millipore). Serum was collected separately and stored at -80⁰C. Proliferation/apoptosis and senescence markers by flow cytometry Thawed PBMCs of all 54 individuals were stained with a proliferation/apoptosis and a senescence panel for flow cytometry. The apoptosis panel consisted of an extracellular antibody mix (CD3-PerCP and CD45RO-PE-Cy7 (both from Biolegend), CD27-APC-R700, Live/dead-APC-Cy7, CD28-BV510, CD56-BV711, CD8-BV786, , CCR7-BUV395, CD4-BUV737 (all from BD Biosciences)), and, after fixing and permeabilising (Cytofix/Cytoperm; BD Biosciences), an additional intracellular antibody mix (Ki-67-FITC and Bcl-2-PE/Dazzle594 (both from BD Biosciences)). Washing steps were performed using Perm/Wash buffer (BD Biosciences). The senescence panel contained CD57-FITC (eBioscience), CD3-AF700, Live/ dead-APC-Cy7, CD95-BV421, CD8-BV510, CD27-BV786, CCR7-BUV395, and CD4-BUV737 (all from BD Biosciences), CD127-BV650, CD45RO-BV711, and KLRG1-PE-Cy7 (all from Biolegend). In all flow cytometric panels, CMV+ donors were first stained with the specific