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1.
Figure 6

Figure 6. Analysis of CD127 and PD-1 expression on CD8+ T cells.. From: Programmed Death-1 Expression on Epstein Barr Virus Specific CD8+ T Cells Varies by Stage of Infection, Epitope Specificity, and T-Cell Receptor Usage.

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.

Thomas C. Greenough, et al. PLoS One. 2010;5(9):e12926.
2.
Figure 5

Figure 5. Differential PD-1 expression on A2-BMLF-1 and A2-BRLF-1 CD8+ T cells at AIM and convalescence.. From: Programmed Death-1 Expression on Epstein Barr Virus Specific CD8+ T Cells Varies by Stage of Infection, Epitope Specificity, and T-Cell Receptor Usage.

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.

Thomas C. Greenough, et al. PLoS One. 2010;5(9):e12926.
3.
Figure 3

Figure 3. High level PD-1 expression on CD8+ T cells during AIM and convalescence.. From: Programmed Death-1 Expression on Epstein Barr Virus Specific CD8+ T Cells Varies by Stage of Infection, Epitope Specificity, and T-Cell Receptor Usage.

A. Representative CD8+ T cell plots of a sample obtained during AIM demonstrating the gating strategy to define PD-1 high, dim and negative populations. B. Percentages of PD-1 high, dim, or negative CD8+ T cells in AIM and convalescence. C. Percentages of PD-1 high, dim, or negative A2-BMLF-1 and A2-BRLF-1 CD8+ T cells in AIM and convalescence. Lines define paired results for each individual. Brackets indicate that differences between selected comparisons are significant (Wilcoxon signed rank test; *p<0.05, ** p<0.01, *** p<0.001).

Thomas C. Greenough, et al. PLoS One. 2010;5(9):e12926.
4.
Figure 4

Figure 4. PD-1 expression on CD8+ T cells during AIM: correlations with viral load and contraction at convalescence.. From: Programmed Death-1 Expression on Epstein Barr Virus Specific CD8+ T Cells Varies by Stage of Infection, Epitope Specificity, and T-Cell Receptor Usage.

A. Left panel: Viral load (log transformed) and the corresponding PD-1 MFI of CD8+ T cells are plotted (Spearman r = 0.698; p = 0.008). Right panel: Viral load (log transformed) and the corresponding PD-1 MFI of A2-BMLF-1 (solid squares, dotted line) and A2-BRLF-1 cells (open circles, solid line) are plotted (Spearman r = 0.170; p = 0.578 and r = 0.560; p = 0.046, respectively). B. Left panel: PD-1 MFI of CD8+ T cells during AIM and the corresponding fold-change in CD8+ T cells (log-transformed) are plotted (Spearman r = 0.955; p<0.0001). The fold-change in CD8+ T cells is the ratio of the cell #/ml during AIM over the cell #/ml during convalescence. Right panel: PD-1 MFI of A2-BMLF-1 (solid squares, dotted line) and A2-BRLF-1 cells (open circles, solid line) during AIM and the corresponding fold-change in each of these subsets (log-transformed) are plotted (Spearman r = 0.646; p = 0.037, and r = 0.764; p = 0.009, respectively). Lines of best fit are shown for each combination of variables.

Thomas C. Greenough, et al. PLoS One. 2010;5(9):e12926.
5.
Figure 2

Figure 2. PD-1 expression on EBV-specific CD8+ T cells at AIM and convalescence.. From: Programmed Death-1 Expression on Epstein Barr Virus Specific CD8+ T Cells Varies by Stage of Infection, Epitope Specificity, and T-Cell Receptor Usage.

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.

Thomas C. Greenough, et al. PLoS One. 2010;5(9):e12926.
6.
Figure 1

Figure 1. EBV-specific CD8+ T cells and viral load during AIM and convalescence.. From: Programmed Death-1 Expression on Epstein Barr Virus Specific CD8+ T Cells Varies by Stage of Infection, Epitope Specificity, and T-Cell Receptor Usage.

A. Absolute values (cells per ml) of CD8+ T cells, and three different EBV-specific subsets (lytic and latent antigen specific) measured in samples from individuals during AIM (within the first two weeks after presentation with symptoms) and convalescence (CONV; 6–12 months after presentation with symptoms). A2-BMLF-1 CD8+ T cells: specific for the HLA-A*0201 restricted epitope of BMLF-1, GLCTLVAML; A2-BRLF-1 CD8+ T cells: specific for the HLA-A*0201 restricted epitope of BRLF-1, YVLDHLIVV; A2-EBNA-3c CD8+ T cells: specific for the HLA-A*0201 restricted epitope of EBNA-3c, LLDFVRFMGV. When no EBV-specific CD8+ T cells were detected with these reagents, a value of 100 cells per milliliter was assigned (less than half the lowest detected value). B. Viral load (EBV copies per million B cells) during AIM and convalescence. 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.

Thomas C. Greenough, et al. PLoS One. 2010;5(9):e12926.
7.
Figure 8

Figure 8. PD-1 expression on V-beta subsets of A2-BRLF-1 CD8+ T cells in AIM.. From: Programmed Death-1 Expression on Epstein Barr Virus Specific CD8+ T Cells Varies by Stage of Infection, Epitope Specificity, and T-Cell Receptor Usage.

A. Representative data from one individual. Left panels show dot plots of regions defining A2 BRLF-1 CD8+ T cells, and Vb 1 and Vb 17 subsets. Right panel is a histogram showing the PD-1 expression of all A2 BRLF-1 CD8+ T cells (grey line), and the A2 BRLF-1 Vb 1 (red line), and A2 BRLF-1 Vb 17 (blue line) subsets superimposed on the PD-1 expression of the total CD8+ T cell population (grey solid). B. PD-1 expression on all V-beta subsets of A2 BRLF-1 CD8+ T cells studied in this individual. Percentage and MFI are shown in left and right panels, respectively. The corresponding measurements of the total tetramer positive population were used to calculate mean and 95% confidence intervals shown (solid horizontal lines). Also shown is the range of values defined by the mean +/− 3*SD (dotted horizontal lines). PD-1 percentage and MFI values outside the more stringent ranges are considered significantly different from the total tetramer positive population. The table inset shows the percentages of the V-beta subsets of A2-BRLF-1 CD8+ T cells in AIM and convalescence for this individual.

Thomas C. Greenough, et al. PLoS One. 2010;5(9):e12926.
8.
Figure 7

Figure 7. Proliferation and PD-1 expression on CD8+ T cells responding to peptide stimulation.. From: Programmed Death-1 Expression on Epstein Barr Virus Specific CD8+ T Cells Varies by Stage of Infection, Epitope Specificity, and T-Cell Receptor Usage.

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.

Thomas C. Greenough, et al. PLoS One. 2010;5(9):e12926.

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