PD-1 on EBV-Specific T Cells Figure 4. PD-1 expression on CD8+ T cells during AIM: correlations with viral load and contraction at convalescence. A. Left panel: Viral load (log transformed) and the corresponding PD-1 MFI of CD8+ T cells are plotted (Spearman r = 0.698; p = 0.008). Right panel: Viral load (log transformed) and the corresponding PD-1 MFI of A2-BMLF-1 (solid squares, dotted line) and A2-BRLF-1 cells (open circles, solid line) are plotted (Spearman r = 0.170; p = 0.578 and r = 0.560; p = 0.046, respectively). B. Left panel: PD-1 MFI of CD8+ T cells during AIM and the corresponding fold- change in CD8+ T cells (log-transformed) are plotted (Spearman r = 0.955; p,0.0001). The fold-change in CD8+ T cells is the ratio of the cell #/ml during AIM over the cell #/ml during convalescence. Right panel: PD-1 MFI of A2-BMLF-1 (solid squares, dotted line) and A2-BRLF-1 cells (open circles, solid line) during AIM and the corresponding fold-change in each of these subsets (log-transformed) are plotted (Spearman r = 0.646; p = 0.037, and r = 0.764; p = 0.009, respectively). Lines of best fit are shown for each combination of variables. doi:10.1371/journal.pone.0012926.g004 BRLF-1 CD8+ T cells expressed PD-1, and PD-1 MFI were 1,585 and 985, respectively. Overall, the percentages of A2-BMLF-1 CD8+ T cells that expressed PD-1 were significantly greater than the percentages of A2-BRLF-1 CD8+ T cells, both during AIM and convalescence (Figure 2A; Wilcoxon signed rank test, p,0.01 for both comparisons). In addition, we observed higher intensity (MFI) of PD-1 staining in A2-BMLF-1 CD8+ T cells compared with A2-BRLF-1 CD8+ T cells (Figure 2B; Wilcoxon signed rank test, p,0.01 for both comparisons). Finally, significantly higher frequencies of A2-BMLF-1 CD8+ T cells expressed PD-1 at high levels (PD-1high) during AIM compared with A2-BRLF-1 CD8+ T cells (Figure 3C; Wilcoxon signed rank test, p,0.001). During both AIM and convalescence, we observed a lower frequency of PD-1negative A2-BMLF-1 CD8+ T cells compared with A2-BRLF- 1 CD8+ T cells (Figure 3C; Wilcoxon signed rank test, p,0.001 for both comparisons). Again, this analysis included measures of the intensity of PD-1 expression (MFI and PD-1high percentage) on virus-specific CD8+ T cells because they are considered to be more biologically meaningful. Given the higher degree of contraction of A2-BRLF-1 CD8+ T cells (48.7-fold decrease in mean values) following AIM, and prior reports that PD-1 expression is significantly correlated with contraction, it was surprising to find that the intensity of PD-1 expression was lower than that of A2-BMLF-1 CD8+ T cells both in AIM and convalescence. Data presented in Figure 4B (right panel) underscores the observation of consistently higher levels of PD-1 expression on A2-BMLF-1 CD8+ T cells compared with A2- BRLF-1 CD8+ T cells. It is also evident that there may be a significant correlation between PD-1 expression and degree of contraction for a particular subset of virus-specific CD8+ T cells, however this relationship may differ substantially for another subset. We (Catalina, et al 2001), and others have previously shown that EBV latent epitope-specific CD8+ T cells undergo lower levels of expansion and contraction than lytic epitope-specific CD8+ T cells [24,29]. A robust comparison of PD-1 expression on PLoS ONE | www.plosone.org 6 September 2010 | Volume 5 | Issue 9 | e12926