PD-1 on EBV-Specific T Cells Figure 6. Analysis of CD127 and PD-1 expression on CD8+ T cells. A. Percentages of A2-BMLF-1, A2-BRLF-1, and total CD8+ T cells that are CD127+ in AIM and convalescence (mean and SEM). B. Percentages of A2-BMLF-1, A2-BRLF-1, and total CD8+ T cells subsets that are PD-1 high or PD-1 negative that are also CD127+ (in AIM and convalescence). Brackets indicate paired comparisons that differ significantly, Wilcoxon signed rank test, *p,0.05. doi:10.1371/journal.pone.0012926.g006 a more rapidly contracting population of EBV-specific CD8+ T cells (the A2-BRLF-1 CD8+ T cells), so we examined CD127 expression in greater detail. CD127 expression was examined from AIM through convales- cence in seven individuals. The percentages of A2-BMLF-1 and A2-BRLF-1 CD8+ T cells that expressed CD127 ranged from 0.7 to 18.9% (median 5.8%) and 0.4 to 31.6% (median 6.1%) during AIM, respectively. The frequencies of CD127+ virus-specific CD8+ T cells trended upward in convalescence but reached significance only for A2-BRLF-1 CD8+ T cells (Figure 6A; Wilcoxon signed rank test, p = 0.047). Within PD-1high subpopulations, EBV-specific CD8+ T cells expressed CD127 at low frequencies that did not differ from the total CD8+ T cell population either during AIM or convalescence (Figure 6B). Within PD-1negative subpopulations, EBV-specific CD8+ T cells expressed CD127 at low frequencies that did not differ from PD-1high subpopulations at either time point. Reduced Proliferative Capacity of EBV Epitope-specific CD8+ T Cells in AIM Regardless of PD-1 Expression Levels High intensity expression of PD-1 on CD8+ T cells has correlated most consistently with poor proliferation and sensitivity to apoptosis [34]. In samples from individuals with AIM, we consistently observed dramatic EBV epitope-specific CD8+ cell loss in tissue culture when compared with samples obtained in convalescence. This cell loss occurred spontaneously (even without stimulation) and was associated with a profound lack of response to EBV peptide stimulation (Figure 7A, top panels). In AIM, neither A2-BMLF-1 nor A2 BRLF-1 CD8+ cells proliferated in response to peptide stimulation. The degree to which these EBV epitope- specific cells were lost over the course of a 6-day assay was greater than would be predicted from the percentage of cells expressing high PD-1 levels. In the representative AIM example shown in Figure 7A, the frequencies of A2-BMLF-1 and A2-BRLF-1 CD8+ T cells expressing high levels of PD-1 at baseline prior to stimulation were 37.8% and 19.8%, respectively. For each assay condition it was estimated that 217,500 A2-BMLF-1 CD8+ T cells and 322,500 A2-BRLF-1 CD8+ T cells were present initially. After 6 days with peptide stimulation, there were fewer than 200 cells that responded specifically to the BMLF-1 or the BRLF-1 peptide (by CFSE dilution). These decreases were also document- ed by similar reductions in tetramer positive cells (data not shown). From these data, it was apparent that both PD-1 positive and negative virus-specific cells failed to proliferate. The lack of responsiveness to stimulation and cell loss in AIM were virus specific; proliferative responses to Candida antigen were preserved in AIM and convalescence (data not shown). In samples obtained during convalescence, A2-BMLF-1 and A2- BRLF-1 CD8+ T cells proliferated to a similar extent with peptide stimulation. The median fold-change in frequency of peptide responsive cells relative to baseline frequency of tetramer positive cells for A2-BMLF-1 CD8+ T cells was 2.1 (range 0.4 to 23.3), and for A2-BRLF-1 CD8+ T cells was 4.3 (range 1.2 to 23.8). There was no evidence of impaired proliferation with the A2-BMLF-1 peptide compared to the A2-BRLF-1 peptide; indeed in some assays, A2- BMLF-1 proliferation was more pronounced despite higher expression of PD-1 on these cells (Figure 7A, chronic infection). The addition of antibody that blocked PD-1/PD-L1 interac- tions to assays on samples from AIM patients did not reverse the observed defects in proliferation. In contrast, samples from convalescent individuals demonstrated enhanced proliferative responses to BMLF-1 and BRLF-1 peptides (1.3–2.1-fold, mean 1.8-fold) in the presence of antibody to block PD-1/PD-L1 interactions (data not shown). PD-1 expression was upregulated on cells responding to peptide stimulation (Figure 7A). In a series of assays, the PD-1 MFI of PLoS ONE | www.plosone.org 8 September 2010 | Volume 5 | Issue 9 | e12926