PD-1 on EBV-Specific T Cells Figure 3. High level PD-1 expression on CD8+ T cells during AIM and convalescence. A. Representative CD8+ T cell plots of a sample obtained during AIM demonstrating the gating strategy to define PD-1 high, dim and negative populations. B. Percentages of PD-1 high, dim, or negative CD8+ T cells in AIM and convalescence. C. Percentages of PD-1 high, dim, or negative A2-BMLF-1 and A2-BRLF-1 CD8+ T cells in AIM and convalescence. Lines define paired results for each individual. Brackets indicate that differences between selected comparisons are significant (Wilcoxon signed rank test; *p,0.05, ** p,0.01, *** p,0.001). doi:10.1371/journal.pone.0012926.g003 Expression of PD-1 on CD8+ T Cells During AIM Correlates with Contraction in Convalescence It has been reported that EBV-specific responses that are differentially ‘‘culled’’ may have different levels of PD-1 expres- sion; higher PD-1 expression was detected on certain epitope- specific CD8+ T cells at time points just prior to the disappearance of these CD8+ T cells from the peripheral circulation [31]. In animal models of uncontrolled infection, PD-1 expression decreased on epitope-specific CD8+ T cells as they decreased in frequency in the context of emerging viral escape mutations [16,34,36]. We, therefore, examined the relationship between PD- 1 expression on EBV epitope-specific CD8+ T cells during AIM and the degree to which these cell populations contracted between AIM and convalescence. We observed a significant positive correlation between the intensity of PD-1 expression and the fold-change (decrease) in the absolute number of circulating CD8+ T cells between AIM and convalescence (Figure 4B, left panel; MFI vs. fold-change; Spearman r = 0.955; p,0.0001). This relationship was also observed for the percentage of PD-1 positive CD8+ T cells; Spearman r = 0.891; p = 0.0005; data not shown). The intensity of PD-1 expression on both A2-BMLF-1 and A2-BRLF-1 CD8+ T cells also showed a significant positive correlation with the degree of contraction between AIM and convalescence (Figure 4B, right panel; Spearman correlations and p-values, respectively: r = 0.646, p = 0.037 and r = 0.764, p = 0.009). This relationship was also observed for the percentage of PD-1 positive cells in each of these subsets, but reached significance only for the A2-BRLF-1 CD8+ T cells (data not shown; Spearman correlation: r = 0.665; p = 0.031). These data are consistent with antigen driven expansion and upregulation of PD-1 on virus-specific CD8+ T cells during AIM. However, PD-1 expression on A2-BRLF-1 CD8+ T cells was more strongly associated with viral load and the degree of contraction than was PD-1 expression on A2-BMLF-1 CD8+ T cells. Consistent Differences in PD-1 Expression are Observed on EBV Epitope-specific CD8+ T Cells In our analyses, we consistently observed lower frequencies and intensities of PD-1 expression on A2-BRLF-1 CD8+ T cells compared with A2-BMLF-1 CD8+ T cells. A representative example of this is shown in Figure 5. In this example, 94% of A2- BMLF-1 and 87% of A2-BRLF-1CD8+ T cells during AIM expressed PD-1. At this time point, the MFI of PD-1 expression for A2-BMLF-1 and A2-BRLF-1 were 3,236 and 2,491, respectively. At week 52 (convalescence), 80% of A2-BMLF-1 and 65% of A2- PLoS ONE | www.plosone.org 5 September 2010 | Volume 5 | Issue 9 | e12926