PD-1 on EBV-Specific T Cells Figure 5. Differential PD-1 expression on A2-BMLF-1 and A2-BRLF-1 CD8+ T cells at AIM and convalescence. PD-1 expression on A2- BMLF-1 and A2-BRLF-1 CD8+ T cells during AIM (upper panels) and convalescence (lower panels). Representative dot plots demonstrate differences in PD-1 percent positivity, and histograms showing differences in distribution and PD-1 MFI of these EBV-specific CD8+ T cells. Histograms overlay the two subsets on a background of the total CD8+ T cell population. doi:10.1371/journal.pone.0012926.g005 lytic and latent antigen-specific CD8+ T cells was not feasible due to the markedly lower frequencies of detectable latent antigen- specific CD8+ T cells. However, in samples where latent epitope specific CD8+ T cells were also detected in sufficient numbers (e.g., A2-EBNA-3c and B7-EBNA-3a CD8+ T cells; Figure 2), PD- 1 expression levels generally exceeded those measured on A2- BRLF1 CD8+ T cells. These data suggest that PD-1 expression may not be the sole factor influencing contraction of a CD8+ T cell response in convalescence. Differences in PD-1 Expression on EBV-specific CD8+ T Cells are not Related to Measurable Differences in Differentiation State or Activation PD-1 expression on virus-specific CD8+ T cells has been regarded as a sensitive marker of antigenic stimulation [16]. The differences in PD-1 expression on A2-BMLF-1 and A2-BRLF-1 CD8+ T cells could be consistent with quantitative or qualitative differences in antigen stimulation. If this were the case, these cells would be expected to display differences in other markers of differentiation or activation. We studied a subset of our population (n = 5) to determine whether there were any identifiable differences in differentiation between A2-BMLF-1 and A2- BRLF-1 CD8+ T cells. We found that the major differentiation states defined by CD27, CD28 and CD127 were similarly represented in these two EBV-specific CD8+ T cell subsets (data not shown). The highest PD-1 levels were observed on effector memory cells in early/intermediate stages of differentiation (CD27+28+1272) both in AIM and convalescence. High frequencies (92 to 100%) of EBV epitope-specific CD8+ T cells expressed HLA-DR during AIM. Fewer A2-BMLF-1 (16 to 48%, median 28%) and A2-BRLF-1 (6 to 41%; median 22%) CD8+ T cells expressed HLA-DR during convalescence. HLA- DR expression did not correlate with PD-1 expression during AIM or convalescence (data not shown). PD-1 Expression on EBV-specific CD8+ T Cells Does Not Correlate with CD127 Expression Previous reports have related CD127 (IL-7R) expression with the expansion and contraction of EBV-specific CD8+ T cells in AIM and convalescence [26]. Sauce et al [31] have recently reported a gradual increase in CD127, and a transient high intensity upregulation of PD-1 expression on EBV epitope-specific CD8+ T cells prior to their disappearance from the circulation. Our analysis did not detect evidence of higher PD-1 expression on PLoS ONE | www.plosone.org 7 September 2010 | Volume 5 | Issue 9 | e12926