antigen-specific CD8+ T cells is dependent on continued activation through the T cell receptor in lentiviral infections [16]. Epstein Barr virus (EBV) infects over 95% of the world’s population but is only rarely associated with disease despite persistent viral replication in chronically infected individuals [21]. Primary EBV infection in adolescence and young adulthood is often symptomatic, with a syndrome characterized by fever, malaise, pharyngitis and lymphadenopathy (acute infectious mononucleosis, AIM; reviewed in [22]). Vigorous EBV-specific CD4+ and CD8+ T cell responses are expanded early in AIM [23,24,25]; several lines of evidence suggest that these cellular immune responses are important for limiting primary EBV infection and controlling chronic infection such that diseases associated with EBV are relatively rare. Human EBV infection has served as a useful model in efforts to characterize the lineage relationship between effector and memory responses and the factors that influence evolution of antigen- specific CD8+ T cell responses into the memory CD8+ T cell repertoire. Current models of EBV viral immunology link the phenotypes of EBV-specific CD8+ T cells detected in the circulation with the levels of viral antigen expression and/or the qualitative differences in their presentation [24,26,27,28,29,30]. Several investigators have reported high levels of PD-1 expression on EBV-specific CD8+ T cells during chronic infection [8,13]. Sauce and colleagues have reported that EBV-specific CD8+ T cells culled from the circulation during convalescence transiently express PD-1 at high levels concurrent with gradually increasing IL-7R (CD127) expression [31]. We studied PD-1 expression levels on EBV-specific CD8+ T cells during AIM, convalescence and chronic infection to better define its relationship with viral replication, the contraction of the immune response, and CD8+ T cell functional properties. Methods Ethics Statement The Institutional Review Board of the University of Massachu- setts Medical School approved these studies, and all participants provided written informed consent. Study Population Students at the University of Massachusetts, Amherst campus were invited to participate in this study when they presented to the infirmary with symptoms consistent with acute infectious mono- nucleosis (AIM) and a positive monospot (heterophile antibody) assay. All were confirmed to have acute EBV infection by the detection of IgM antibodies to the EBV Viral Capsid Antigen. Blood samples were collected at baseline, weekly for six weeks, followed by long-term follow-up at six months and one year. Individuals with EBV-specific CD8+ T cells detected using HLA- A*0201 based, peptide-MHC Class I (pMHCI) tetramers (described below) were selected for more detailed studies. Flow Cytometry Whole blood samples were screened for EBV-specific CD8+ T cells that reacted with APC-conjugated pMHCI tetramers in- cluding HLA-A*0201 based reagents: A2-BMLF-1 (epitope: GLC- TLVAML), A2-BRLF-1 (epitope: YVLDHLIVV), A2-EBNA-3c (epitope: LLDFVRFMGV), and the HLA-B*0702-based reagent: B7-EBNA-3a (epitope: RPPIFIRRL). All pMHCI tetramer com- plexes were made in the Tetramer Core facility at UMMS as previously described [32]. PBMC samples were studied using a PE-conjugated antibody specific for PD-1 (Medarex). Antibodies from BD-Biosciences (San PD-1 on EBV-Specific T Cells Jose, CA) were used to detect: CD27 (Alexa700), CD28 (PE-Cy7), CD127 (FITC or PE), HLA-DR (FITC or PerCP). A panel of FITC-conjugated antibodies specific for different V-beta subsets (Immunotech/Beckman Coulter) was used in a four-color assay that also included CD8 (PerCP), PD-1 (Medarex; PE), and Tetramer (APC). For multi-parameter flow assays, a ‘‘dump channel’’ of cells positive for CD14 and CD19 (APC-Cy7) was used to improve specificity, and live/dead blue reagent (Invitro- gen) was used to exclude dead cells. Unstained and ‘‘fluorescence minus one’’ samples were included as controls in all experiments to optimize specificity during analyses. Assays involving#four fluorochromes utilized FACScan or FACSCalibur instruments (BD Biosciences), with CellQuest software (BD Biosciences). For experiments involving.four fluorochromes, we used a LSRII instrument (BD Biosciences). Multiparameter flow cytometry data were analyzed using FlowJo software (Treestar, Ashland OR) and programs generously provided by Mario Roederer (PESTLE and SPICE version 4.2). A complete blood count with differential was obtained at each study visit. The absolute lymphocyte count was calculated by multiplying the white blood cell count (#/ml) by the lymphocyte percentage. The absolute CD8+ T cell count was calculated by multiplying the absolute lymphocyte count by the CD8+ percentage. The absolute counts of virus-specific CD8+ T cells were calculated by multiplying the absolute CD8+ T cell count by the percentages of epitope-specific CD8+ T cells. The PD-1 median fluorescence index (MFI) values on virus- specific CD8+ T cells were expressed as normalized values: the ratio between the PD-1 median fluorescence intensity and the cutoff between PD-1 positive and negative CD8+ T cells. Peptide Stimulation Cryopreserved PBMC were thawed into RPMI 1640, contain- ing 10% heat inactivated human AB sera, 2mM L-glutamine, 100 U/ml gentamicin (R10 medium; all materials from Gibco), with 2 mg/ml DNase (Sigma) and pelleted. Cells were re- suspended at 10 million/ml, incubated with CFSE (Invitrogen) at a final concentration of 0.5mM for 10 minutes at 37uC, 5% CO2 and 95% humidity. Cells were cultured at 1.5 to 3 million cells/ml in R10 medium containing co-stimulatory antibodies, CD49d and CD28 (final concentration of 1ug/ml; both from BD Pharmingen), the EBV peptide of interest (final concentration 10 mg/ml), no peptide, or Candida antigen. Cells in each assay condition were incubated at 150,000 to 300,000 cells per well in 96-well microtiter plates, in replicates of 10. After 6 days, they were harvested, pooled, treated with 20 mM EDTA for 15 min- utes, and washed with 1% BSA/PBS in preparation for antibody staining. Cells were then stained with relevant APC-conjugated pMHCI tetramers, PD-1 (PE) (Medarex), and CD8 (PerCP; BD Biosciences). Anti-PD-L1 antibody (EBioscience, # 16-5983), when included in blocking assays, was used at a concentration of 25 mg/ml and was added simultaneously with peptide. EBV DNA Quantitation in B Cells B-cells were purified from whole blood using the RosetteSep human B-cell enrichment cocktail and manufacturer’s protocol (StemCell Technologies, Vancouver BC, Canada). Cellular DNA was extracted using QIAGEN DNeasy mini spin columns (Valencia, CA). The Roche LightCycler EBV Quantitation Kit was used to quantify EBV DNA copy number. Cell counts in each sample were determined using a previously described PCR assay to quantify the copy number of the gene encoding CCR5 (two copies per diploid cell) [25]. PLoS ONE | www.plosone.org 2 September 2010 | Volume 5 | Issue 9 | e12926