Figure 2. PD-1 expression on EBV-specific CD8+ T cells at AIM and convalescence. A. The percentages of EBV-specific CD8+ T cells that expressed PD-1 are shown. Two lytic (A2-BMLF-1 and A2-BRLF-1) and two latent (A2-EBNA-3c and B7-EBNA-3a CD8+ T cells) antigen specific CD8+ T cell subsets are shown. Lines define paired results for each individual. B. The median fluorescence index (MFI) of PD-1 expression for these EBV-specific CD8+ T cells is shown. All assays were performed with the PD-1 antibody conjugated to PE. The MFI was normalized as described in methods. 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.g002 Higher intensity of PD-1 expression was also observed on EBV epitope-specific CD8+ T cells during AIM compared with convalescence (Figure 2B). The PD-1 median fluorescence index (MFI) values on A2-BMLF-1 (mean = 3.9) and A2-BRLF-1 (mean = 2.0) CD8+ T cells during AIM were significantly higher than during convalescence (mean = 2.1 for A2-BMLF-1 and 0.9 for A2-BRLF-1 CD8+ T cells); a similar trend was observed for A2-EBNA-3c CD8+ T cells. Because high levels of expression of PD-1 are associated with loss of function or ‘‘exhaustion’’ [2], we also evaluated cell populations that were PD-1high, PD-1dim, or PD-1negative. Using a gating strategy based on a consistent contour ‘‘shoulder’’ observed in CD8+ T cell population plots of PD-1 against FSC or SSC (Figure 3A), we were able to analyze our data in a manner consistent with previous reports [14]. In total, CD8+ T cells PD-1 on EBV-Specific T Cells expressed PD-1 at higher intensities during AIM (median 12.5; range 1.8 to 33.2) compared with convalescence (median 5.3; range 1.9 to 8.6; Figure 3B). These convalescent values are similar to those reported for HIV-1 infected individuals on effective antiretroviral therapy [14]. Among EBV-specific CD8+ T cells, only A2-BMLF-1 and A2- BRLF-1 CD8+ T cells were present in frequencies sufficient for analysis at this level of detail. High-level expression of PD-1 (PD- 1high) was detected on a significantly greater percentage of EBV- specific CD8+ T cells during AIM than during convalescence (Figure 3C). For example, the frequencies of A2-BMLF-1 CD8+ T cells that were PD-1high during AIM ranged from 11.3 to 46.5% (median 26.2%), and during convalescence ranged from 4.4 to 20.6% (median 8.4%). Thus the percentages of EBV epitope- specific CD8+ T cells with high-level PD-1 expression changed significantly between AIM and convalescence (Wilcoxon signed rank test, p,0.001 and 0.027 for A2-BMLF-1 and A2-BRLF-1 CD8+ T cells, respectively). In summary, we have demonstrated higher frequencies of PD- 1+ EBV-specific CD8+ T cells, as well as higher intensity of PD-1 expression during AIM compared with convalescence. These findings are consistent with the hypothesis that PD-1 expression is largely driven by antigenic stimulation. The differences in intensity of PD-1 expression (MFI and high level expression) are likely to be most biologically meaningful given prior studies demonstrating increased sensitivity to apoptosis and reduced proliferative capacity of CD8+ T cells with high intensity PD-1 staining [14,34]. Expression of PD-1 on CD8+ T Cells During AIM Correlates with Peripheral Blood EBV Load We noted substantial variability in the frequency and intensity of PD-1 staining on CD8+ T cells between individual patients. In chronic lentiviral infections, the frequency and intensity of PD-1 expression on virus-specific CD8+ T cells directly correlates with plasma viral RNA copy number (a direct measure of viral replication) [16]. EBV replication is thought to occur primarily in oropharyngeal B cell blasts and epithelial cells; direct measurement of EBV replication at these sites is difficult. Peripheral blood EBV load (EBV DNA copy numbers in latently-infected memory B-cells) is easily measurable. Hadinoto et al [35] have demonstrated that high levels of EBV lytic replication during AIM results in very high levels of circulating latently-infected memory B cells; a marked reduction in peripheral blood EBV load is observed during convalescence as EBV-specific CD8+ T cells limit rounds of lytic viral replication and resultant seeding of the peripheral blood memory B cell pool. In samples obtained during AIM (a period of active EBV replication and seeding of the peripheral blood memory B cell pool), there was a significant positive correlation between the viral load (EBV DNA copies per million B cells) and the MFI of CD8+ T cells expressing PD-1 (Figure 4A, left panel; Spearman correlation: r = 0. 698; p = 0.008). The CD8+ T cell PD-1 percentage was also significantly correlated with EBV load (data not shown; Spearman correlation: r = 0.747; p = 0.003). PD-1 expression on both A2-BMLF-1 and A2-BRLF-1 CD8+ T cells showed a weaker positive correlation with viral load during AIM, reaching statistical significance only for A2-BRLF-1 CD8+ T cells (Figure 4A, right panel; A2-BRLF-1 PD-1 MFI and viral load; Spearman correlation: r = 0.560; p = 0.046). PD-1 expression on CD8+ T cells did not correlate with viral load in samples obtained during convalescence. PLoS ONE | www.plosone.org 4 September 2010 | Volume 5 | Issue 9 | e12926