Sara van den Berg

184 Chapter 7 conversion) compared with those of long-term CMV+ individuals (> 25 years CMV+). E. Age at seroconversion: Antibody levels of individuals that seroconverted at younger age ( ≤38yr of age) or older age (≥45yr of age, mean age 58.5±8.1, shortly after CMV-seroconversion (<max 5.5 years)). F. Antibody levels associated with age at CMV seroconversion. For the selection of long-term CMV+ individuals, CMV+ individuals of round 1 were included that were ≤38yr of age and age of seroconversion was set at 38 years. G. Antibody levels of all CMV+ individuals at end point. Interval represents geometric mean level ±geometric standard deviation. H. Variable importance when predicting CMV antibody levels at endpoint with a random forest algorithm. % increase in MSE: proportion increase in mean squared error when the variable is removed from the model. Slope of Ab level: log-linear variation in CMV-specific antibody levels after first CMV+ measurement, until timepoint 6. *=P<0.05 **=P<0.01 Dotted line in A) and C) is the upper boundary of the cutoff for CMV seropositivity. Changes in the CD8+ T-cell pool are established early after CMV infection In addition to effects on antibody levels, we investigated the effect of CMV-seropositivity and duration of CMV infection on the CD8+ T-cell pool at endpoint. Both factors did not affect the total number of naive (T N ) or central memory (T CM ) CD8+ T-cells ( Figure 3A ). As expected, numbers of effector memory (T EM ) and terminally differentiated effector (T EMRA ) T-cells were significantly higher in CMV+ compared to CMV- individuals ( Figure 3A ). This was mainly explained by an increase in the number of late-stage differentiated (CCR7-CD45RA+CD27- CD28-) T-cells (data not shown) in CMV+ individuals. No significant association was observed between duration of CMV infection and numbers of T EMRA cells, even within ST CMV+ individuals ( Figure 3B ). Also no significant difference was found in CD8+ T EMRA cell numbers at end point between individuals who seroconverted at a young age (≤38yrs of age, exact date unknown) and those who seroconverted at older age (≥45yrs of age, range 47-88 years) ( Figure 3C ). Next, we investigated whether CMV-specific CD8+ T-cell responses differed between ST CMV+ and LT CMV+ individuals (n=27 in total, matched for age and sex). Based on tetramer staining (using HLA-class I pp65 NLV-epitope) in all HLA-A2 positive individuals (n=11) , we found no significant differences in CMV-specific CD8+ T-cell numbers ( Figure 3D ) , percentages of CMV-specific CD8+ T-cells in the total CD8+ T-cell pool ( Figure 3E ) , percentages of T EMRA cells in the CMV-specific T-cell pool ( Figure 3F ) and expression of the senescence marker KLRG-1 of the CMV-specific CD8 T-cells between ST CMV+ and LT CMV+ individuals ( Figure 3G ). Also, after CMV-specific stimulation using overlapping CMV peptide pools, no significant differences were observed in IFN γ production between ST CMV+ and LT CMV+ participants ( Figure 3H ). We also studied the polyfunctionality of the CMV-specific T-cell response and identified that CMV-specific CD8+ T-cell responses were mainly IFN γ +TNFa+MIP-1 Β 1 β +CD107a+ but lacked IL-2 ( Supplementary Figure 4A,B ) , suggestive of an end-stage highly functional T-cell phenotype. The height of these functional CMV-specific CD8+T-cells did not differ between ST CMV+ and LT CMV+ individuals ( Figure 3I ). This suggests that high CD8+ T EM and T EMRA cell numbers are induced shortly after primary CMV infection and that CMV-specific T-cell numbers, phenotype and polyfunctionality are not much dependent on duration of CMV infection.