frequency of V-beta usage (i.e. immunodominance), it remains possible that avidity relates to PD-1 expression. Previous studies have emphasized a relationship between high levels of antigenic stimulation and high PD-1 expression levels on virus-specific CD8+ T cells in chronic viral infections. In a murine model of LCMV infection, graded levels of PD-1 expression on virus-specific CD8+ T cells correlated with viral clearance rates at day 30 after infection [38]. The highest levels of PD-1 expression were found on exhausted virus-specific CD8+ T cells in animals with chronically elevated LCMV titers. Whereas PD-1 expression on virus-specific CD8+ T cells in untreated HIV-1 (or SIV in animal models of AIDS) infection is high and is directly related to viral replication [8,13,34], PD-1 expression on CMV-specific CD8+ T cells is reportedly lower; presumably due to intermittent or lower levels of viral antigen expression. Relatively high levels of PD-1 expression on EBV-specific CD8+ T cells from chronically infected individuals have been reported by a number of groups [8,13]. Most studies that have compared PD-1 expression on EBV vs. HIV or CMV specific CD8+ T cells have focused on A2- BMLF-1 CD8+ T cells [8,13]. Our results are in agreement with these previous reports, however, we have also demonstrated consistent and significant differences in PD-1 expression among different epitope specific CD8+ T cell subsets. The concept that circulating virus-specific CD8+ T cells with different epitope specificities may display consistent differences in PD-1 expression has precedent in the literature. Using a broad panel of pMHCI tetramers to characterize HIV-specific CD8+ T cell responses in infected individuals, Day and colleagues observed lower levels of PD-1 expression on certain epitope-specific CD8+ T cells among a majority that expressed PD-1 at high levels [8]. A dramatic difference in PD-1 expression on virus-specific CD8+ T cells with different epitope specificities has also been reported in an individual with resolved HCV infection [10]. Our data demon- strated consistent differences in PD-1 expression on A2-BMLF-1 and A2-BRLF-1 CD8+ T cells over the course of infection from AIM to convalescence, and between different individuals. This suggests a mechanism that relates to differences in TCR signaling that are shared by HLA-A*0201 positive individuals responding to these epitopes. Possible mechanisms include: 1) differences in the stability of the peptide/MHC-1 association, or 2) a common limitation on TCR signaling (perhaps defined by the avidity of the TCR for the pMHCI complex). Our data show that exposure of epitope-specific CD8+ T cells to high (presumably saturating) peptide concentrations result in different degrees of PD-1 upregulation, consistent with PD-1 expression ex vivo. This is unlikely to be a function of limiting concentrations of pMHCI complexes on the antigen presenting cells. MHC-peptide binding algorithms actually predict that the A2-BRLF-1 epitope has a longer half-life of dissociation from HLA-A*0201 than the A2- BMLF-1 epitope. If antigen presentation were the most critical factor, the less stable pMHCI complex might be expected to be associated with lower PD-1 expression. Therefore, it is more likely that there is a determinant of PD-1 upregulation specific to the cells responding to these particular epitopes. The observation that PD-1 expression may vary significantly in different V-beta subsets that recognize the same epitope suggests that V-beta usage itself may influence PD-1 expression. Further studies are necessary to better define determinants of PD-1 expression on virus-specific CD8+ T cells and their functional consequences. Given our current understanding of EBV replication, it is likely that different EBV lytic antigens are presented to CD8+ T cells in a similar microenvironment, and in a similar time frame. The differences in PD-1 expression on A2-BMLF-1 and A2-BRLF-1 CD8+ T cells, despite highly similar differentiation phenotypes, PD-1 on EBV-Specific T Cells suggests a dissociation between signaling pathways that determine differentiation states and PD-1 expression. We have examined the relationship between PD-1 expression and CD127 in detail because a recent report indicates that CD127 expression is tightly linked with proliferative capacity, a function that is lost early as PD-1 is upregulated [7]. Furthermore, upregulation of both PD-1 and CD127 has been reported to precede disappearance of an EBV-specific CD8+ T cell from the circulation [31]. We found that few CD8+ T cells expressing high levels of PD-1 also expressed CD127 in AIM, but this relationship was not exclusive; higher frequencies of CD127+, PD-1+ cells were seen in convalescence. As reported by Salisch et al, PD-1 expression on virus-specific CD8+ T cells is a sensitive indicator of lentiviral replication [16]. Kasprowicz et al have also suggested that PD-1 staining is compatible with it being an activation marker [10]. Our analyses are consistent with these interpretations. While PD-1 levels correlated with the contraction of CD8+ T cells, we did not find a simple association between higher PD-1 levels on EBV- specific CD8+ T cells and the degree of contraction from AIM to convalescence. Within a particular epitope specific subset (e.g. A2- BRLF-1 CD8+ T cells), there can be a significant correlation, however between subsets, similar levels of contraction occur with significantly different PD-1 expression (Figure 4B, right panel). This suggests that PD-1 may be necessary, but not sufficient in this process. Sauce et al [31] have described transient increases in PD-1 expression on epitope-specific CD8+ T cells that are associated with an ‘‘accelerated’’ expression of CD127 and subsequent disappearance of that epitope-specific response from the circula- tion. Consistent differences in PD-1 staining intensity were not reported in their analysis of A2-BMLF-1 (labeled ‘‘GLC’’) than A2-BRLF-1 (labeled ‘‘YVL’’) CD8+ T cells in acutely infected individuals at all time points to convalescence. However, careful inspection of the data presented actually demonstrates higher PD- 1 expression on A2-BMLF-1 than A2-BRLF-1 CD8+ T cells in three of four individuals at multiple time points, and in the fourth subject during acute infection. Differences in PD-1 expression may have been less evident due to the binding characteristics of the anti-PD-1 antibody used. Indeed, we had collected additional earlier data using a different anti-PD-1 antibody that failed to clearly distinguish positive and negative populations, yet still demonstrated these differences in PD-1 expression at all time points (data not shown). Using another antibody (Medarex), we observed PD-1 expression profiles with clearly defined bimodal distributions (data reported here in Figures 3A, 5, 7A, 8A). The ability to distinguish differences in intensity of PD-1 expression, particularly PD-1high expression, has been previously shown to be critical for describing meaningful biological differences. The functional impact of PD-1 upregulation on EBV-specific CD8+ T cells during AIM is unclear. We found virtually no proliferation to peptide stimulation during AIM, and no enhancement of proliferation in the presence of antibody blocking the PD-1/PD-L1 interaction. While PD-1 upregulation may contribute to loss of responsiveness, it certainly cannot explain it in entirety. Our findings are consistent with other reports that PD- 1 expression alone does not explain loss of proliferative capacity or sensitivity to apoptosis [12,39]. In convalescence, the inhibitory activity associated with PD-1 is more evident; enhanced proliferative responses are observed in the presence of anti-PD- L1. Our observations thus highlight the complexities of PD-1 expression and its functional correlates; in some settings (e.g. chronic LCMV infection in mice), its expression is consistent with cellular exhaustion [6], while in others (e.g. acute hepatitis C infection), it appears primarily to be a marker of activation [10]. PLoS ONE | www.plosone.org 11 September 2010 | Volume 5 | Issue 9 | e12926