Blackburn et al have shown that there may be multiple inhibitory molecules that contribute to an ‘‘exhausted’’ phenotype [38], and that PD-1/PD-L1 blockade primarily rescues proliferation in less terminally differentiated cells [39]. We have now shown that these complexities must be viewed through a lens that accounts not only for antigen expression, but also epitope specificity and V-beta usage. In aggregate, studies indicate that PD-1 expression and signaling play an important role in limiting pathogenic conse- quences of chronic virus infection. Efforts are underway to determine if disrupting the interaction of PD-1 with its ligands will have a beneficial effect in chronic viral infections in which cellular immunity fails to provide control. We have shown that PD-1 expression is induced by antigenic stimulation, but is dependent in part on intrinsic properties of the responding cell. Consistent differences, and significant variability in PD-1 expression observed in two lytic epitope specific CD8+ T cell subsets, and subpopulations defined by V-beta usage point to properties that are likely linked to TCR signaling, TCR avidity and recruitment References 1. Dong H, Zhu G, Tamada K, Chen L (1999) B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat Med 5: 1365–1369. 2. Sharpe AH, Wherry EJ, Ahmed R, Freeman GJ (2007) The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection. Nat Immunol 8: 239–245. 3. Ishida Y, Agata Y, Shibahara K, Honjo T (1992) Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death. Embo J 11: 3887–3895. 4. Latchman Y, Wood CR, Chernova T, Chaudhary D, Borde M, et al. (2001) PD- L2 is a second ligand for PD-1 and inhibits T cell activation. Nat Immunol 2: 261–268. 5. Brown KE, Freeman GJ, Wherry EJ, Sharpe AH (2010) Role of PD-1 in regulating acute infections. Curr Opin Immunol 22: 397–401. 6. Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, et al. (2006) Restoring function in exhausted CD8 T cells during chronic viral infection. Nature 439: 682–687. 7. Cellerai C, Perreau M, Rozot V, Enders FB, Pantaleo G, et al. (2010) Proliferation capacity and cytotoxic activity are mediated by functionally and phenotypically distinct virus-specific CD8 T cells defined by interleukin- 7R{alpha} (CD127) and perforin expression. J Virol 84: 3868–3878. 8. Day CL, Kaufmann DE, Kiepiela P, Brown JA, Moodley ES, et al. (2006) PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature 443: 350–354. 9. Freeman GJ, Wherry EJ, Ahmed R, Sharpe AH (2006) Reinvigorating exhausted HIV-specific T cells via PD-1-PD-1 ligand blockade. J Exp Med 203: 2223–2227. 10. Kasprowicz V, Schulze Zur Wiesch J, Kuntzen T, Nolan BE, Longworth S, et al. (2008) High level of PD-1 expression on hepatitis C virus (HCV)-specific CD8+ and CD4+ T cells during acute HCV infection, irrespective of clinical outcome. J Virol 82: 3154–3160. 11. Kaufmann DE, Walker BD (2009) PD-1 and CTLA-4 inhibitory cosignaling pathways in HIV infection and the potential for therapeutic intervention. J Immunol 182: 5891–5897. 12. Nakamoto N, Cho H, Shaked A, Olthoff K, Valiga ME, et al. (2009) Synergistic reversal of intrahepatic HCV-specific CD8 T cell exhaustion by combined PD- 1/CTLA-4 blockade. PLoS Pathog 5: e1000313. 13. Petrovas C, Casazza JP, Brenchley JM, Price DA, Gostick E, et al. (2006) PD-1 is a regulator of virus-specific CD8+ T cell survival in HIV infection. J Exp Med 203: 2281–2292. 14. Petrovas C, Chaon B, Ambrozak DR, Price DA, Melenhorst JJ, et al. (2009) Differential association of programmed death-1 and CD57 with ex vivo survival of CD8+ T cells in HIV infection. J Immunol 183: 1120–1132. 15. Radziewicz H, Ibegbu CC, Fernandez ML, Workowski KA, Obideen K, et al. (2007) Liver-infiltrating lymphocytes in chronic human hepatitis C virus infection display an exhausted phenotype with high levels of PD-1 and low levels of CD127 expression. J Virol 81: 2545–2553. 16. Salisch NC, Kaufmann DE, Awad AS, Reeves RK, Tighe DP, et al. (2010) Inhibitory TCR coreceptor PD-1 is a sensitive indicator of low-level replication of SIV and HIV-1. J Immunol 184: 476–487. 17. Trautmann L, Janbazian L, Chomont N, Said EA, Gimmig S, et al. (2006) Upregulation of PD-1 expression on HIV-specific CD8+ T cells leads to reversible immune dysfunction. Nat Med 12: 1198–1202. 18. Velu V, Kannanganat S, Ibegbu C, Chennareddi L, Villinger F, et al. (2007) Elevated expression levels of inhibitory receptor programmed death 1 on simian PD-1 on EBV-Specific T Cells of molecules involved in signal transduction. Properties of TCR- pMHCI interactions that influence expression of PD-1 are likely to inform the future designs of vaccines and immunotherapeutics. Acknowledgments We thank the Study Participants. For clinical coordination, we thank Rose Ciccarelli and Gail Gnatek. For excellent laboratory support, we thank James Coderre, Joyce Pepe, Linda Lambrecht, Kristina McNeal, Eric Larson, and Richard Hudson. For data management and analysis, we thank Margaret McManus. We are grateful to Wanda DePasquale for preparation of the manuscript. Author Contributions Conceived and designed the experiments: TG SCC RB SJ VSM MS KL. Performed the experiments: SCC. Analyzed the data: TG SCC RB SJ VSM. Wrote the paper: TG. Figure preparation: TG. Editing: SCC KL. Design and conduct: RB SJ VSM. Methods: RB. immunodeficiency virus-specific CD8 T cells during chronic infection but not after vaccination. J Virol 81: 5819–5828. 19. Vollbrecht T, Brackmann H, Henrich N, Roeling J, Seybold U, et al. (2010) Impact of changes in antigen level on CD38/PD-1 co-expression on HIV- specific CD8 T cells in chronic, untreated HIV-1 infection. J Med Virol 82: 358–370. 20. Zhang Z, Zhang JY, Wherry EJ, Jin B, Xu B, et al. (2008) Dynamic programmed death 1 expression by virus-specific CD8 T cells correlates with the outcome of acute hepatitis B. Gastroenterology 134: 1938–1949, 1949 e1931– 1933. 21. Hadinoto V, Shapiro M, Sun CC, Thorley-Lawson DA (2009) The dynamics of EBV shedding implicate a central role for epithelial cells in amplifying viral output. PLoS Pathog 5: e1000496. 22. Vetsika EK, Callan M (2004) Infectious mononucleosis and Epstein-Barr virus. Expert Rev Mol Med 6: 1–16. 23. Callan MF, Tan L, Annels N, Ogg GS, Wilson JD, et al. (1998) Direct visualization of antigen-specific CD8+ T cells during the primary immune response to Epstein-Barr virus In vivo. J Exp Med 187: 1395–1402. 24. Catalina MD, Sullivan JL, Bak KR, Luzuriaga K (2001) Differential evolution and stability of epitope-specific CD8(+) T cell responses in EBV infection. J Immunol 167: 4450–4457. 25. Precopio ML, Sullivan JL, Willard C, Somasundaran M, Luzuriaga K (2003) Differential kinetics and specificity of EBV-specific CD4+ and CD8+ T cells during primary infection. J Immunol 170: 2590–2598. 26. Sauce D, Larsen M, Leese AM, Millar D, Khan N, et al. (2007) IL-7R alpha versus CCR7 and CD45 as markers of virus-specific CD8+ T cell differentiation: contrasting pictures in blood and tonsillar lymphoid tissue. J Infect Dis 195: 268–278. 27. Pudney VA, Leese AM, Rickinson AB, Hislop AD (2005) CD8+ immunodo- minance among Epstein-Barr virus lytic cycle antigens directly reflects the efficiency of antigen presentation in lytically infected cells. J Exp Med 201: 349–360. 28. Hislop AD, Kuo M, Drake-Lee AB, Akbar AN, Bergler W, et al. (2005) Tonsillar homing of Epstein-Barr virus-specific CD8+ T cells and the virus-host balance. J Clin Invest 115: 2546–2555. 29. Hislop AD, Annels NE, Gudgeon NH, Leese AM, Rickinson AB (2002) Epitope- specific evolution of human CD8(+) T cell responses from primary to persistent phases of Epstein-Barr virus infection. J Exp Med 195: 893–905. 30. Catalina MD, Sullivan JL, Brody RM, Luzuriaga K (2002) Phenotypic and functional heterogeneity of EBV epitope-specific CD8+ T cells. J Immunol 168: 4184–4191. 31. Sauce D, Larsen M, Abbott RJ, Hislop AD, Leese AM, et al. (2009) Upregulation of interleukin 7 receptor alpha and programmed death 1 marks an epitope-specific CD8+ T-cell response that disappears following primary Epstein-Barr virus infection. J Virol 83: 9068–9078. 32. Luzuriaga K, McManus M, Catalina M, Mayack S, Sharkey M, et al. (2000) Early therapy of vertical human immunodeficiency virus type 1 (HIV-1) infection: control of viral replication and absence of persistent HIV-1-specific immune responses. J Virol 74: 6984–6991. 33. Callan MF (2003) The evolution of antigen-specific CD8+ T cell responses after natural primary infection of humans with Epstein-Barr virus. Viral Immunol 16: 3–16. 34. Petrovas C, Price DA, Mattapallil J, Ambrozak DR, Geldmacher C, et al. (2007) SIV-specific CD8+ T cells express high levels of PD1 and cytokines but have impaired proliferative capacity in acute and chronic SIVmac251 infection. Blood 110: 928–936. PLoS ONE | www.plosone.org 12 September 2010 | Volume 5 | Issue 9 | e12926