© 2006 Nature Publishing Group Restoring function in exhausted CD8T cells during chronic viral infectionDaniel L. Barber1, E. John Wherry2, David Masopust1, Baogong Zhu3, James P. Allison4, Arlene H. Sharpe5,Gordon J. Freeman3& Rafi Ahmed1Functional impairment of antigen-specific T cells is a defining characteristic of many chronic infe ctions, but theunderlying mechanisms of T-cell dysfunction are not well understood. To address this question, we analysed genesexpressed in functionally impaired virus-specific CD8 T cells present in mice chronically infected with lymphocyticchoriomeningitis virus (LCMV), and compared these with the gene profile of functional memory CD8 T cells. Here wereport that PD-1 (programmed death 1; also known as Pdcd1) was selectively upregulated by the exhausted T cells, andthat in vivo administration of antibodies that blocked the interaction of this inhibitory receptor with its ligand, PD-L1(also known as B7-H1), enhanced T-cell responses. Notably, we found that even in persistently infected mice that werelacking CD4 T-cell help, blockade of the PD-1/PD-L1 inhibitory pathway had a beneficial effect on the ‘helpless’ CD8T cells, restoring their ability to undergo proliferation, secrete cytokines, ki ll infected cells and decrease viral load.Blockade of the CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) inhibitory pathway had no effect on either T-cellfunction or viral control. These studies identify a specifi c mechanism of T-cell exhaustion and define a potentiallyeffective immunological strategy for the treatment of chronic viral infections.Memory CD8 T-cell differentiation proceeds along distinct pathwaysafter an acute versus a chronic viral infection1–3. Memory CD8 T cellsgenerated after an acute viral infection are highly functional andconstitute an important component of protective immunity. Incontrast, chronic infections are often characterized by varyingdegrees of functional impairment of virus-specific T-cell responses,and this defect is a principal reason for the inability of the host toeliminate the persisting pathogen. Although functional effectorT cells are initially generated during the early stages of infection,they gradually lose function during the course of the chronicinfection. This exhaustion of virus-specific T cells was first shownduring persistent LCMV infection of mice4,5. However, these findingswere quickly extended to other model systems, as well as to chronicinfections in humans, in particular human immunodeficiency virus(HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV)infections—three chronic infections afflicting .500 million peopleworldwide6–8.PD-1 is upregulated during chronic viral infectionTo examine the mechanism of T-cell dysfunction during chronicinfection, we used the mouse model of infection with LCMV. Anadvantage of this model is the availability of LCMV strains that cancause either acute or chronic infections in adult mice; the Armstrongstrain is cleared within a week, whereas the clone 13 strain establishesa persistent infection3. As these two strains differ in only two aminoacids in the entire genome9, and neither of these mutations affectsany of the known T-cell epitopes, it is possible to track the same CD8T cell responses after an acute or chronic viral infection. In contrastto the highly robust memory CD8 T cells generated after an acuteArmstrong infection, LCMV-specific CD8 T cells become exhaustedduring a persistent clone 13 infection3–5. An example of impairedcytokine production and proliferation by the exhausted CD8 T cellsis shown in Fig. 1a. As a first step towards understanding themechanisms of T-cell dysfunction, we performed a comparativegenome-wide microarray analysis (Affymetrix) of genes expressedby exhausted versus functional LCMV-specific CD8 T cells ofthe same antigenic specificity. The most notable finding was thepronounced upregulation of messenger RNA encoding PD-1, aninhibitory receptor of the CD28 family10–13, by the exhausted LCMV-specific CD8 T cells (Fig. 1b). This was confirmed at the protein levelusing antibodies specific for PD-1 (Fig. 1b). We found that highexpression of this inhibitory receptor was a signature of the func-tionally exhausted CD8 T cells; all LCMV-specific CD8 T cells(comprising multiple epitopes) that were present in chronicallyinfected mice expressed high levels of PD-1, whereas functionalLCMV-specific memory CD8 T cells of the same antigenic specifi-cities present in mice that had cleared the acute LCMV infection didnot express any detectable levels of PD-1 (Fig. 1b and data notshown).We next analysed the kinetics of PD-1 expression during bothacute and chronic LCMV infection, and found that PD-1 wastransiently expressed on early effector CD8 T cells after LCMVArmstrong infection but then was rapidly downregulated (Fig. 1c).In contrast, PD-1 expression continued to increase on virus-specificCD8 T cells in chronically infected mice and the high level ofexpression was sustained. PD-1 has two ligands—PD-L1(B7-H1)14,15, which is widely expressed on both haematopoetic andparenchymal cells, and PD-L2 (B7-DC)12,13, which is predominantlyexpressed on macrophages and dendritic cells. We found that PD-L1was expressed at very high levels in splenocytes from persistentlyinfected mice, especially on virally infected cells (Fig. 1c). Thus, notonly did the exhausted CD8 T cells express high levels of PD-1, but itsARTICLES1Emory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.2Immunology Program, TheWistar Institute, Philadelphia, Pennsylvania 19104, USA.3Department of Medical Oncology, Dana-Farber Cancer Institute, Department of Medicine, Harvard Medical School,Boston, Massachusetts 02115, USA.4Department of Medicine, Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.5Department of Pathology, Harvard Medical School and Brigham and Women’s Hospital, Boston, Massachusetts 02115, USA.Vol 439|9 February 2006|doi:10.1038/nature04444682© 2006 Nature Publishing Group ligand was also upregulated on infected cells, suggesting a role for thisinhibitory pathway in regulating T-cell function during chronicLCMV infection.PD-L1 blockade enhances viral control and T-cell responsesTo test the above hypothesis, we treated persistently infected micewith anti-PD-L1 blocking antibody and monitored T-cell responsesand viral control