Sara van den Berg

179 7 Duration of CMV infection were then resuspended in AIM-V 2% hAB and rested at 37 °C at 5% CO2 for 30 min before they were dispended in 10 6 PBMCs/150ul per well in a 96 wells plate with stimulation, anti- CD107a PerCP Cy5.5 and Monensin (1:1500) (BD bioscience, GolgiStop) and Brefaldin A (1:1000) (BD bioscience, GolgPlug). Stimulation for CMV-specific responses was done using one of the CMV overlapping peptide pools (15-mers with 11 overlap) of UL55 (1 μ g/ml) (JPT peptides), IE-1 (1 μ g/ml) (JPT peptides), or pp65 (1 μ g/ml) (JPT peptides). Medium was used as negative control. Following 6-hour incubation, cells were washed once and stained with the extracellular markers Fixable Viability Staining-780 (Thermofisher), CD3(UCHT1)- FITC, CD8+(RPA-T8)-BV510, CD4+(SK3)-BUV737 (BD bioscience), CD27(O323)-BV785 (Biolegend), CD45RO(UCHL1)-BUV395 (BD bioscience) and CD107a(H4A3)-PerCP Cy5.5. Next, cells were washed once in FACS buffer and twice in perm/wash buffer (fix/perm kit BD, diluted 10x in MilliQ H 2 O). Intracellular staining was performed with IFN- γ (4S.B3)- PE-Cy7 (Thermofisher), IL-2(MQ1-17H12)-BV650 (BD bioscience), TNF-a(MAb11)-BV711 (Biolegend), Perforin(B-D48)-BV421, MIP-1 β (D21-1351)-AlexaFluor700 (BD bioscience)and Granzyme B(GB11)-PE-CF594 (BD bioscience). Cells were subsequently washed three times and analyzed by flow cytometry (on an LSRII Fortessa). Sum of the three CMV peptides pools minus the negative control medium is calculated. Quantification of CMV-specific T-cells CMV-specific T-cells were stained using HLA-class A2 tetramers specific for the NLV epitope of the CMV-pp65 protein in the HLA-A2+, CMV+ individuals of the selected sub- population for CMV-specific functional T-cell analysis (n=30). First, HLA-A2 staining by HLA- A2(BB7.2)-V450 (BD Bioscience) was performed on the CMV+ individuals (n=20) to select all HLA-A2 + individuals. For the HLA-A2+ individuals (12/20) , tetramer staining was performed for 15 minutes at room temperature with CMV-(A*0201/NLVPMVATV)-APC (Immudex) and next extracellular staining was performed for 20 minutes at 4 °C with Fixable Viability Staining-780 (APC-Cy7) (Thermofisher), CD3 APC-R700(SK7)-AF700(BD bioscience), CD8+(RPA-T8)-BrilliantViolet510 (Biolegend), CD45RO+(UCHL1)-BrilliantViolet711 (Biolegend), CD27(O323)-BrilliantViolet786 (Biolegend), CCR7(150503)-BrilliantUV395(BD bioscience), KLRG-1(13F12F2)-PE-Cy7 (eBioscience), PD-1(EH12.2H7)-PerCP Cy5.5 (Biolegend). Cells were measured by flow cytometry (on an LSRII Fortessa). Statistical analysis Statistics on CMV-specific antibody levels were performed by parametric testing on log- transformed data. Comparison within individuals between two time points was done by paired testing and comparison among different groups by non-paired testing (paired t-test and independent Student’s t-test or Mann Whitney test for non-normally distributed data). Associations between continuous variables were tested by Pearson’s or Spearman’s correlation depending on the distribution of the data. Seroconversion rate was calculated by dividing the number of seroconversion cases by the sum of the total timespan until seroconversion or until end of follow up of all people who were seronegative at baseline. Sex differences in seroconversion rate were tested using a chi-square test. To estimate the