PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression Cheryl L. Day 1,2,3 *, Daniel E. Kaufmann 2 *, Photini Kiepiela 1 , Julia A. Brown 4 , Eshia S. Moodley 1 , Sharon Reddy 1 , Elizabeth W. Mackey 2 , Joseph D. Miller 5 , Alasdair J. Leslie 3 , Chantal DePierres 1 , Zenele Mncube 1 , Jaikumar Duraiswamy 5 , Baogong Zhu 4 , Quentin Eichbaum 2 , Marcus Altfeld 2 , E. John Wherry 6 , Hoosen M. Coovadia 1 , Philip J. R. Goulder 1,2,3 , Paul Klenerman 3 , Rafi Ahmed 5 , Gordon J. Freeman 4 & Bruce D. Walker 1,2,7 Functional impairment of T cells is characteristic of many chronic mouse and human viral infections. The inhibitory receptor pro- grammed death 1 (PD-1; also known as PDCD1),a negative regulator of activated T cells 1–4 , is markedly upregulated on the surface of exhausted virus-specific CD8 T cells in mice 5 . Blockade of this pathway using antibodies against the PD ligand 1 (PD-L1, also known as CD274) restores CD8 T-cell function and reduces viral load 5 . To investigate the role of PD-1 in a chronic human viral infection,we examined PD-1 expression on human immuno- deficiency virus (HIV)-specific CD8 T cells in 71 clade-C-infected people who were naive to anti-HIV treatments, using ten major histocompatibility complex (MHC) class I tetramers specific for frequently targeted epitopes. Here we report that PD-1 is signifi- cantly upregulated on these cells, and expression correlates with impaired HIV-specific CD8 T-cell function as well as predictors of disease progression: positively with plasma viral load and inversely with CD4 T-cell count. PD-1 expression on CD4 T cells likewise showed a positive correlation with viral load and an inverse correlation with CD4 T-cell count, and blockade of the pathway augmented HIV-specific CD4 and CD8 T-cell function. These data indicate thatthe immunoregulatory PD-1/PD-L1 pathway is operative during a persistent viral infection in humans, and define a reversible defect in HIV-specific T-cell function. Moreover, this pathway ofreversible T-cell impairmentprovides a potential target for enhancing the function of exhausted T cells in chronic HIV infection. Recent evidence from experimental lymphocytic choriomeningitis virus (LCMV) infection indicates a crucial role of the PD-1/PD-L1 pathway in inhibiting the function of virus-specific CD8 T cells in chronic viral infections in mice 5 . To address the potential role of this pathway in a chronic human viral infection associated with persistent viraemia and impaired T-cell function 6–13 , we examined peripheral blood from a cohort 14 of over 500 people with chronic untreated HIV infection in KwaZulu Natal Province in South Africa, where sero- prevalence rates are in excess of 30% in certain age groups. A panel of ten MHC class I tetramers —based on prevalent human leukocyte antigen (HLA) alleles and frequently targeted HIV-1 clade C epitopes 14 —was synthesized (Supplementary Table 1), allowing the direct visualization of surface PD-1 expression on tetramer-positive (tetramer þ ) cells (Fig.1a).A subsetof71 people naive for anti- retroviral therapy was studied on the basis of expression of relevan HLA alleles, and a total of 126 individual tetramer responses were examined (Fig. 1b). PD-1 expression was readily apparent on these tetramer þ cells, and was significantly higher than in the total CD8 T- cellpopulation (P , 0.0001). In turn, PD-1 expression on both tetramer þ CD8 T cells and the total CD8 T-cellpopulation was significantly higher than in HIV-seronegative controls. Significantly less PD-1 was expressed on cytomegalovirus (CMV)-specific cells compared with HIV-specific CD8 T cells (P ¼ 0.0002), whereas PD-1 expression on cells specific for a single lytic Epstein–Barr virus (EBV epitope tested was also at high levels. PD-1 expression was also analysed on CMV-specific, EBV-specific and vaccinia-virus-specific CD8 T cells from HIV-seronegative con- trols, and found to be intermediate on CMV-specific cells, high for a lytic EBV epitope, and low on vaccinia-virus-specific CD8 T cells in 18 healthy individuals (Fig. 1c),indicating a relationship between ongoing antigen exposure and PD-1 expression. High PD-1 expression on HIV-specific CD8 T cells was unique as we did not see upregulation of the inhibitory receptor CTLA-4 (cytotoxic T-lymphocyte-associated protein 4; data notshown),consistent with previous reports 15 . Next,we determined whether there was evidence for epitope- specific differences in PD-1 expression. Each HIV tetramer was used to stain peripheral blood mononuclear cells (PBMCs) from 3 to 29 individuals of the appropriate HLA type, revealing a range of PD-1 expression levels on tetramer þ cells (Fig. 1d); for a given epitope, the median percentage of PD-1-expressing tetramer þ cells ranged from 68% to 94% (Fig. 1e).Of the 71 people examined, 16 individuals had multiple tetramer responses —13 of whom showed different patterns of PD-1 expression depending on the epitope (Fig. 1f ). These data indicate that PD-1 may be differentially expressed on contemporaneous epitope-specific CD8 T cells from a single person perhaps consistent with data indicating epitope-specific differences in antiviral efficacy 16–18 . Because HIV-specific CD8 T cells also show impaired proliferative capacity 6,7 , we stimulated carboxyfluorescein diacetate succinimidyl LETTERS 1 HIV Pathogenesis Programme, Doris Duke Medical Research Institute, University of KwaZulu Natal, Durban 4013, South Africa. 2 Partners AIDS Research Center, Massachusetts General Hospital and Division of AIDS, Harvard Medical School, Boston, Massachusetts 02115, USA. 3 Nuffield Department of Medicine, The Peter Medawar Building for Pathogen Research, Oxford University, Oxford OX1 3SY, UK. 4 Department of Medical Oncology, Dana-Farber Cancer Institute, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA. 5 Emory Vaccine Center and Department of Microbiology & Immunology, Emory University School of Medicine, Atlanta, Georgia 30322, USA. 6 Immunology Program, The Wistar Institute, Philadelphia, Pennsylvania 19104, USA. 7 Howard Hughes Medical Institute, Chevy Chase, Maryland 20185, USA. *These authors contributed equally to this work. Vol 443|21 September 2006|doi:10.1038/nature05115 350 © 2006 Nature Publishing Group