Sara van den Berg

152 Chapter 6 were obtained by fitting a single-exponential model to the data sets per individual (see Materials and methods and Supplementary Figure 5 . Note that steady state is assumed in these cell populations and loss rates are expected to equal the production rate. Horizontal lines represent median values. P- values <0.1 are presented in the figure. Data from each individual are represented by unique symbols. Kinetics of CMV-specific CD8+ memory T-cells are similar to those of bulk memory T-cells We investigated if the high numbers of CMV-specific CD8 + T-cells can be explained by accumulation of long-lived cells. Therefore, we compared the dynamics of CMV-specific CD8 + T-cells to those of T EM/EMRA CD8 + T-cells. The frequency of tetramer-positive cells in the total CD8 + T-cell population remained fairly constant during the follow-up time of ~1,5 years ( Figure 4A upper panel ). Although there were relatively large fluctuations in absolute numbers of CMV-specific CD8 + T-cells ( Figure 4A lower panel ) this could be explained by fluctuations in total T-cell counts (data not shown). For each individual, the incorporation of deuterium by CMV-specific CD8 + T-cells was largely overlapping with that of total CD8 + T EM/ EMRA cells ( Figure 4B upper panel ). There is a risk that potential kinetic differences between CMV-specific CD8 + T-cells and non-CMV-specific CD8 + T-cells may go unnoticed in this comparison as bulk T EM/EMRA CD8 + T-cells from CMV+ individuals in fact include lots of CMV- specific CD8 + T-cells. We therefore also compared the dynamics of CMV-specific CD8 + T-cells to those of bulk T EM/EMRA CD8 + T-cells of CMV- individuals, that contain simply no CMV-specific CD8 + T-cells. Again, no obvious differences in deuterium incorporation between CMV-specific CD8 + T-cells and non-CMV-specific T EM/EMRA CD8 + T-cells were observed ( Figure 4B lower panel ). Indeed, the estimated average production rates of CMV-specific CD8 + T-cells were not significantly different from those of total CD8 + T EM/EMRA cells in CMV+ nor CMV- individuals ( Figure 4C, Table 2 ). Because CMV-specific CD8 + T-cells are thought to increase over time [14], in the analyses of CMV-specific CD8 + T-cells we did not assume thaT-cell numbers were in steady state. Thus, we deduced the average loss rates of CMV- specific CD8 + T-cells from the changes in cell numbers over time and using the average production rates as estimated by deuterium labelling (see Materials and methods ). We found that the average loss rates of CMV-specific CD8 + T-cells were nearly identical to their average production rates ( Figure 4D ), suggesting that CMV-specific CD8 + T-cells are not substantially longer-lived than other memory T-cells. Although the sizes of the CMV- specific CD8 + T-cell expansions differed more than threefold between individuals, we found no significant correlation between absolute numbers (or frequencies) of CMV-specific CD8 + T-cells and their average production or loss rates ( Figure 4E ). This suggests that the large size of clonally expanded CMV-specific CD8 + T-cell subsets is neither due to increased T-cell production nor to a clearly increased lifespan of these cells. Expression of senescence markers and Ki-67 associate with production, while Bcl-2 does not Finally, we wondered if the estimated production rates of memory T-cell populations that we quantified using deuterium labelling was in part reflected by the expression of senescence, proliferation, and apoptosis markers.