Cytomegalovirus Infection Leads to Development of High Frequencies of Cytotoxic Virus-Specific CD4+ T Cells Targeted to Vascular Endothelium
Fig 5
CMV-specific CD4+ T cells are highly cytotoxic directly ex vivo.
PBMCs were stained with LIVE/DEAD fixable dye, before staining with HLA class II tetramer followed by anti-CD3, CD4 and CX3CR1. After fixation and permeabilisation Granzyme B and Perforin were stained intracellularly. (A) Pie charts represent the proportion of CD4+ EM T cells (in CMV seronegative individuals) or the TM+ population expressing the indicated markers and combinations thereof. To compare pie charts permutation analysis was performed in SPICE. (B) Expression of Granzyme B, perforin and CX3CR1 on pp65-specific (on the left) and gB-specific (on the right) T cells in relation to donor age. Spearman‘s rank correlation was used to analyse the strength of associations between variables. (C) Percentage killing of peptide-loaded target cells (HLA-matched LCLs) mediated by ex vivo separated CD4+TM+ or CD4+EM T cells following over night co-culture at effector:target (E:T) ratios indicated. All conditions were performed in triplicate; bars represent means with SEM. (D) Proportion of cells expressing NKG2D within the CD4+ EM or the CMV-specific T cell population in young, middle aged or older adults. Error bars represent medians and IQR. p-values: ** p = 0.001–0.01, *** p = 0.001–0.0001. Kruskal-Wallis test was performed with Dunn‘s multiple comparison in GraphPad Prism5 to derive p-values.