PD-1 on EBV-Specific T Cells Figure 8. PD-1 expression on V-beta subsets of A2-BRLF-1 CD8+ T cells in AIM. 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 +/2 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. doi:10.1371/journal.pone.0012926.g008 mean and standard deviation (SD). For all three individuals, we observed clear examples of V-beta subpopulations that had PD-1 expression significantly higher or lower than the range defined by mean +/2 3*SD (representative data shown in Figure 8B). At early visits, these differences did not appear to predict loss or enrichment of particular V-beta subsets at subsequent visits (Figure 8B). For example, Vb 21.3 had high PD-1 expression, yet persisted at a relatively high percentage (21.1%) in convales- cence. Nor did PD-1 expression correlate with the frequency of a particular V-beta subset; high PD-1 expression was found on subsets that were present at persistently low or high frequencies (e.g. Vb 22 frequencies ranged between 2.7 and 3.1% and Vb 21.3 frequencies ranged between 33.1 and 22.1% despite both expressing high frequencies and intensities of PD-1). Discussion Consistent with prior reports, our analyses suggest that PD-1 expression is driven in large part, but not solely, by viral antigen expression. Moreover, PD-1 expression alone does not reflect the functional capacity of CD8+ T cells. Factors other than PD-1 appear to participate in: 1) modulating EBV-specific CD8+ T cell proliferative capacity during both AIM and convalescence, 2) determining states of differentiation and activation, 3) and the degree of contraction of EBV-specific CD8+ T cells in convalescence. These observations serve to refine and extend our understanding of the complex network of factors that govern immune responses and tolerance. To our knowledge, this is the first demonstration that PD-1 expression on virus-specific CD8+ T cells is influenced significantly by factors intrinsic to the responding cell that segregate with epitope specificity and V-beta usage. While these factors have yet to be defined, these observations would be consistent with variable TCR binding and signaling characteristics that may play a significant role in shaping effective immune responses. Price et al have shown that immunodominant clonotypes of EBV and CMV specific CD8+ T cells tend to have greater avidity for their cognate antigen [37]. While we have not shown a relationship between PD-1 and the PLoS ONE | www.plosone.org 10 September 2010 | Volume 5 | Issue 9 | e12926