Statistics GraphPad Prism version 5.0 for Mac OSX (GraphPad Software, La Jolla, CA) was used for all statistical analyses. The Wilcoxon Signed Rank test was used for paired comparisons. The Spearman rank test was used for analyses of correlations. To determine if V-beta subsets had significantly different expression of PD-1 than the overall Tetramer positive subpopulation we used the range defined by the mean +/2 3*SD of PD-1 expression on Tetramer positive cells. Results PD-1 Expression is Upregulated on EBV-specific CD8+ T Cells in AIM CD8+ T cells undergo marked expansion during AIM, primarily due to the expansion of EBV epitope-specific CD8+ T cells (reviewed in [33]). Lytic epitope-specific CD8+ T cell responses are most highly expanded during acute EBV infection; in some individuals, CD8+ T cells specific for a single lytic epitope may represent more than 25% of the total circulating CD8+ T cells [24,30,33]. Latent epitope-specific CD8+ T cells are less highly expanded during AIM and convalescence. We measured HLA A*0201-restricted, EBV-specific epitope- specific CD8+ T cells in peripheral blood samples obtained from 13 individuals during AIM (within the first two weeks after presentation with symptoms) and convalescence (6 to 12 months after presentation with symptoms; Figure 1A). CD8+ T cells were expanded in AIM and contracted (3.7-fold decrease in mean value) in convalescence. CD8+ T cells specific for an HLA-A*0201 restricted epitope in the immediate early lytic protein, BRLF-1, (YVLDHLIVV; hereafter abbreviated A2-BRLF-1 CD8+ T cells) PD-1 on EBV-Specific T Cells were most highly expanded during AIM, and underwent the greatest degree of contraction (48.7-fold decrease in mean value) in convalescence. CD8+ T cells specific for an HLA A2-restricted epitope in the early lytic protein, BMLF-1, (GLCTLVAML; hereafter abbreviated A2-BMLF-1 CD8+ T cells) were also highly expanded during AIM, but underwent a smaller contraction (12.2- fold decrease in mean value) in convalescence. CD8+ T cells specific for an HLA A2-restricted epitope in the latent antigen, EBNA-3c, (LLDFVRFMGV; hereafter abbreviated A2-EBNA-3c CD8+T cells) were even less highly expanded (detected in only 5 of 13 individuals at each time point) and underwent a 17.6-fold decrease in mean value from AIM to convalescence. These changes in circulating EBV epitope-specific CD8+ T cell numbers coincided with a 17.5-fold decrease in mean peripheral blood EBV DNA copy number over the same time period (Figure 1B). They are consistent with previous reports showing a larger decline in A2-BRLF-1 CD8+ T cell frequencies than either A2-BMLF-1 or A2-EBNA-3c CD8+ T cells [21,29]. We next examined PD-1 expression on EBV-specific CD8+ T cells in these individuals from AIM through convalescence. During AIM, 55.2 to 96.3% (median 76.3%) of A2-BMLF1-specific CD8+ T cells expressed PD-1 (Figure 2A). The frequencies of A2-BMLF1- specific CD8+ T cells expressing PD-1 remained high in convalescence (36.6 to 85.9%; median 71.5%), but were signifi- cantly lower than during AIM (Wilcoxon signed rank test, p,0.05). Similarly, high frequencies of PD-1+ A2-BRLF1-specific CD8+ T cells were detected during AIM (28.4 to 88.6%, median 61.0%), and decreased significantly in convalescence (10.3 to 80.0%, median 35.1%; Wilcoxon signed rank test, p,0.05). Similar trends were observed for two additional EBV epitope-specific responses (A2- EBNA-3c and B7-EBNA-3a CD8+ T cells, Figure 2A). Figure 1. EBV-specific CD8+ T cells and viral load during AIM and convalescence. A. Absolute values (cells per ml) of CD8+ T cells, and three different EBV-specific subsets (lytic and latent antigen specific) measured in samples from individuals during AIM (within the first two weeks after presentation with symptoms) and convalescence (CONV; 6–12 months after presentation with symptoms). A2-BMLF-1 CD8+ T cells: specific for the HLA-A*0201 restricted epitope of BMLF-1, GLCTLVAML; A2-BRLF-1 CD8+ T cells: specific for the HLA-A*0201 restricted epitope of BRLF-1, YVLDHLIVV; A2-EBNA-3c CD8+ T cells: specific for the HLA-A*0201 restricted epitope of EBNA-3c, LLDFVRFMGV. When no EBV-specific CD8+ T cells were detected with these reagents, a value of 100 cells per milliliter was assigned (less than half the lowest detected value). B. Viral load (EBV copies per million B cells) during AIM and convalescence. Lines define paired results for each individual. All paired comparisons indicated by brackets differ significantly by the Wilcoxon signed rank test; * p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pone.0012926.g001 PLoS ONE | www.plosone.org 3 September 2010 | Volume 5 | Issue 9 | e12926