Programmed Death-1 Expression on Epstein Barr Virus Specific CD8+ T Cells Varies by Stage of Infection, Epitope Specificity, and T-Cell Receptor Usage Thomas C. Greenough1*, Shalyn C. Campellone3, Robin Brody1, Surbhi Jain1, Victor Sanchez-Merino1¤, Mohan Somasundaran 1 , Katherine Luzuriaga1,2 1 Pediatrics, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America, 2 Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America, 3 Medarex, Inc., Bloomsbury, New Jersey, United States of America Abstract Background: Programmed Death-1 (PD-1) is an inhibitory member of the CD28 family of molecules expressed on CD8+ T cells in response to antigenic stimulation. To better understand the role of PD-1 in antiviral immunity we examined the expression of PD-1 on Epstein-Barr virus (EBV) epitope-specific CD8+ T cells during acute infectious mononucleosis (AIM) and convalescence. Methodology/Principal Findings: Using flow cytometry, we observed higher frequencies of EBV-specific CD8+ T cells and higher intensity of PD-1 expression on EBV-specific CD8+ T cells during AIM than during convalescence. PD-1 expression during AIM directly correlated with viral load and with the subsequent degree of CD8+ T cell contraction in convalescence. Consistent differences in PD-1 expression were observed between CD8+ T cells with specificity for two different EBV lytic antigen epitopes. Similar differences were observed in the degree to which PD-1 was upregulated on these epitope-specific CD8+ T cells following peptide stimulation in vitro. EBV epitope-specific CD8+ T cell proliferative responses to peptide stimulation were diminished during AIM regardless of PD-1 expression and were unaffected by blocking PD-1 interactions with PD-L1. Significant variability in PD-1 expression was observed on EBV epitope-specific CD8+ T cell subsets defined by V- beta usage. Conclusions/Significance: These observations suggest that PD-1 expression is not only dependent on the degree of antigen presentation, but also on undefined characteristics of the responding cell that segregate with epitope specificity and V-beta usage. Citation: Greenough TC, Campellone SC, Brody R, Jain S, Sanchez-Merino V, et al. (2010) Programmed Death-1 Expression on Epstein Barr Virus Specific CD8+ T Cells Varies by Stage of Infection, Epitope Specificity, and T-Cell Receptor Usage. PLoS ONE 5(9): e12926. doi:10.1371/journal.pone.0012926 Editor: Derya Unutmaz, New York University, United States of America Received July 6, 2010; Accepted August 19, 2010; Published September 23, 2010 Copyright: 2010 Greenough et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Institutes of Health (NIH)/National Institute of Allergy and Infectious Diseases (NIAID) grant P01 AI049320-08 with additional support from NIH/CFAR (Centers for AIDS Research) P30-AI042845-10 and Medarex, Inc. Medarex, Inc. provided reagents and salary support for one author (S.C.). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of this manuscript. Competing Interests: One author (S.C.) is affiliated with Medarex, Inc. This does not alter the authors’ adherence to PLoS One policies on sharing data and materials. * E-mail: thomas.greenough@umassmed.edu ¤ Current address: Laboratorio de Retrovirologia e Inmunopatogenia Viral, HIVACAT, Barcelona, Spain Introduction Programmed death 1 (PD-1) and its ligands, PD-L1 and PD-L2, are members of the CD28 and B7 families of proteins that participate in regulating the balance between T cell activation and tolerance [1,2,3,4]. The interaction of PD-1 with its ligands delivers an inhibitory secondary signal to T cells that results in the downregulation of T cell receptor signaling and the suppression of T cell effector functions, including T cell proliferation and cytokine secretion. This downregulation of T cell responses is thought to be important in the generation of central and peripheral tolerance and in limiting immune pathology in chronic infections. In the context of acute viral infections, PD-1 may also play a role in the contraction of the immune response following acute infection [5]. High levels of PD-1 expression on virus-specific CD8+ T cells during chronic human (human immunodeficiency virus [HIV], hepatitis B virus [HBV]), monkey (simian immunodeficiency virus [SIV]), and murine (lymphocytic choriomeningitis virus [LCMV]) infections have been associated with T cell dysfunction and failure to control viral replication [6,7,8,9,10,11,12,13,14,15,16,17,18, 19,20]. However, Kasprowicz and colleagues have recently reported high levels of PD-1 expression on peripheral blood hepatitis C virus (HCV)-specific CD8+ T cells of infected individuals who clear infection, raising the question of whether PD-1 expression alone is sufficient to confer T cell dysfunction [10]. In non-persistent viral infections, PD-1 expression is downregulated with the resolution of acute infection and viral clearance. Salisch and colleagues have recently reported that the upregulation and maintenance of high PD-1 expression levels on PLoS ONE | www.plosone.org 1 September 2010 | Volume 5 | Issue 9 | e12926