PD-1 on EBV-Specific T Cells Figure 7. Proliferation and PD-1 expression on CD8+ T cells responding to peptide stimulation. A. Three representative examples of proliferation upon stimulation with HLA-A*0201-restricted BMLF-1 and BRLF-1 peptides. Upper and middle rows: PBMC from an individual at presentation with AIM and in convalescence; lower row: PBMC from an individual with chronic infection. Left panels (baseline prior to peptide stimulation) show PD-1 expression of the indicated tetramer positive cells (A2 BMLF-1 black line; A2 BRLF-1 grey line) superimposed on that of CD8+ T cells (grey solid). Middle panels show CD8+ T cells that proliferate (CFSE dilution) in response to the indicated peptides (A2 BMLF-1 black line; A2 BRLF-1 grey line) superimposed on unstimulated controls (grey solid). Right panels show the upregulation of PD-1 on CD8+ T cells responding to the indicated peptides superimposed on unstimulated CD8+ T cells. Marked regions on histograms depicting PD-1 expression indicate high levels of expression with corresponding percentages of A2 BMLF-1 and A2-BRLF-1 CD8+ T cells. Numbers in text boxes are the percentages of CD8+ T cells that are positive for each tetramer at baseline (left panels), or responding to peptide post-stimulation (CFSE low; middle panels). B. Upregulation of PD-1 on cells responding to stimulation with A2-BMLF-1 and A2-BRLF-1 peptides. Results shown are a mixture of chronic and convalescent samples in three separate assays. To demonstrate the extent of PD-1 upregulation observed, PD-1 MFI is expressed as a ratio of the MFI of cells that responded to peptide, relative to the MFI in control assays without peptide. Median PD-1 MFI ratio values for A2-BMLF-1 and A2-BRLF-1 were 8.1 and 2.8, respectively (Wilcoxon signed rank test; p = 0.06). Solid and open symbols demonstrate paired values for A2-BMLF-1 and A2-BRLF-1 for each sample. doi:10.1371/journal.pone.0012926.g007 peptide stimulated CD8+ T cells ranged from approximately 2 to 10 times that of medium control. Interestingly, the pattern of PD-1 expression following stimulation was similar to that observed ex vivo; CD8+ T cells responding to A2-BRLF-1 peptide had lower intensity PD-1 expression than those responding to the A2-BMLF- 1 peptide (Figure 7B). PD-1 Expression on V-beta Subsets of EBV Lytic Antigen Epitope Specific CD8+ T Cells Varies Significantly The observed differences in PD-1 expression on A2-BMLF-1 and A2-BRLF-1 CD8+ T cells raised the possibility that PD-1 expression may be influenced significantly by factors other than antigen presentation. We hypothesized that differences in the effectiveness of TCR engagement and signaling would also influence PD-1 expression. If the main determinant of PD-1 expression level were antigenic stimulation, then different clonotypes within A2-BMLF-1 or A2-BRLF-1 populations would be expected to have similar levels of PD-1 expression (assuming that all subsets of cells with a given epitope specificity are presented with a similar level of antigen in a similar context and time frame). If, however, significant variability in PD-1 expression were to exist among clonotypes with identical epitope specificity, then factors specific to particular clonotypes of responding cells would be implicated as determinants in addition to the level of antigen presentation. To address this question, we examined PD-1 expression on V- beta subsets of A2-BMLF-1 and A2-BRLF-1 CD8+ T cells from three individuals with AIM. Subsets with sufficient numbers of analyzable events (mean 129; range 24 to 702) were selected. These V-beta subsets were compared with the total tetramer positive population (representative gating and PD-1 expression shown in Figure 8A). Multiple measurements of PD-1 expression on Tetramer positive cells in a given assay enabled calculation of PLoS ONE | www.plosone.org 9 September 2010 | Volume 5 | Issue 9 | e12926