Turning on the off switch: Regulation of anti-viral T cell responses in the liver by the PD-1/PD-L1 pathway

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1 Journal of Hepatology 45 (2006) Editorial Turning on the off switch: Regulation of anti-viral T cell responses in the liver by the PD-1/PD-L1 pathway Arash Grakoui 1,2, *,#, E. John Wherry 3, Holly L. Hanson 1, Christopher Walker 4, Rafi Ahmed 1, * 1 Emory Vaccine Center and Department of Microbiology and Immunology, Atlanta, GA 30322, USA 2 Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA 3 Immunology Program, The Wistar Institute, Philadelphia, PA, USA 4 Division of Molecular Medicine, Department of Pediatrics, Children s Research Institute and The Ohio State University, USA See Article, pages * Corresponding authors. addresses: arash.grakoui@emory.edu (A. Grakoui), ra@microbio.emory.edu (R. Ahmed). # Fax: Many viruses cause acute infections and are rapidly cleared whereas others defy control and progress to chronic or persistent infections. Anti-viral T cell responses can play a dramatic role in determining whether viral infections are cleared rapidly or become chronic. For example, chronic infections are often characterized by T cell responses that are unable to fully eliminate the pathogen [1]. The principal mechanisms that account for T cell failure, and thus influence the outcome of infection are beginning to be understood. The regulation of T cell responses to infections reflects a delicate balance between effector functions required to eliminate the pathogen and their potential to cause immunopathology. In an effort to eradicate a virus, an aggressive immune response can cause damage to healthy tissue. Regulating the immune response to avoid unwanted tissue damage may be especially important when pathogen clearance is not achieved in a relatively short period of time. Indeed, the liver damage that occurs during chronic hepatitis C virus (HCV) and hepatitis B virus (HBV) infections, and that may lead to hepatocellular carcinoma (HCC), is thought to be mediated substantially by T cells [2,3]. The apparent lack of an immune response against pathogens in chronic infection may reflect mechanisms that have evolved to downregulate potentially detrimental effector functions like cell-mediated cytotoxicity and/or production of pro-inflammatory cytokines. Achieving the proper balance between positive and negative co-stimulatory signals received by antigen-specific T cells will prevent unnecessary activation and avoid tissue damage by activated T cells [4]. Recent studies have provided us with a better understanding of the pathogen host interaction through both positive and negative regulation and may help unravel the immunologic components necessary for successful clearance of infection. The PD-1/PD-L1/PD-L2 pathway is one such negative regulatory pathway that has been implicated in autoimmunity and responses to pathogens [5]. The regulation of this pathway in tissues that are the targets of human infections, however, remains poorly understood. This is an important topic since the effectiveness of anti-viral responses and also the severity of immunopathology may be strongly influenced by this pathway. In this issue, Mühlbauer et al. [6] have studied the expression of ligands for the inhibitory receptor PD-1 (programmed death-1) on hepatocytes, stellate cells and Kupffer cells. These studies demonstrate that PD-L1 is upregulated on these liver resident cell populations by viral infection, activated T cells and type 1 interferons (IFNs). Further, the authors provide evidence that elevated PD-L1 expression may enhance apoptosis of activated T cells. Thus, the PD-1/ PD-L1 pathway may represent an important facet regulating the outcome of interactions between cells in the liver, viral pathogens, and the immune system (Fig. 1). The precise balance between negative regulators, such as the /$32.00 Ó 2006 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi: /j.jhep

2 A. Grakoui et al. / Journal of Hepatology 45 (2006) Hepatocyte PD-L1 PD-1 CD8 + T cell TCR MHC I Kupffer cell Hepatotropic virus Fig. 1. Achieving T cell balance. Pathogen-specific CD8 + T cells are turned on through interaction of their TCR with MHC I presenting antigen in the presence of appropriate costimulation. These same T cells may be turned off through the interaction of PD-1 on their cell surface and PD-L1 expressed on hepatocytes and liver-resident parenchymal cells. PD-1/PD-L1/PD-L2 pathway, and positive signals may have important implications for the outcome of hepatotropic infections. Positive co-stimulatory pathways, such as CD28: CD80/CD86 interactions between antigen-specific T cells and antigen presenting cells (APCs), have been extensively studied and are known to be crucial for generating optimal T cell responses (Table 1). However, CD80 and CD86, expressed on the APCs, can also interact with the inhibitory CTLA-4 receptor on activated T cells [5]. A kinetic and affinity model has been proposed to explain how these opposing pathways result in the effective control of T cell responses [5,7]. CD28 is available to provide positive costimulation early in the response since this molecule is expressed on naïve T cells. CTLA-4, in contrast, is upregulated several days Table 1 Summary of CD28 family co-stimulatory and coinhibitory pathways (see also (5)) Receptors Receptor expression Ligands Ligand expression Hematopoetic cells CD28 T cells (naïve and some memory) Ligand expression Non-hematopoetic cells Regulation of T cell response B7-1 (CD80) B cells, T cells, Mac., DC; Rare (podocyte) Positive B7-2 (CD86) B cells, T cells, Mac., DC; Rare CTLA-4 Activated T cells, Treg B7-1 (CD80) B cells, T cells, Mac., DC; Rare (podocyte) Negative B7-2 (CD86) B cells, T cells, Mac., DC; Rare ICOS T cells (especially effector and memory), NK cells ICOSL (B7-H2, B7RP-1) B cells, T cells Mac., DC; Fibroblast, endothelial, epithelial Positive (especially CD4) PD-1 Activated T cells, B cells, Mac. PD-L1 (B7-H1) B cells, T cells Mac., DC; Endothelial, heart, lung, pancreas, placenta, some tumors (e.g. lymphoma, carcinoma, menaloma, glioblastoma) Negative PD-L2 (B7-DC) Mac., DC Not found BTLA T and B cells HVEM T cells, monocytes, immature DC? Negative? B7-H3 B cells, T cells, Mac., DC, NK??? B7-H4 (B7x, B7S1) B cells, T cells, Mac., DC Lung, tumors (ovarian)?

3 470 A. Grakoui et al. / Journal of Hepatology 45 (2006) after activation and has a much higher affinity for the B7 molecules than does CD28. This delayed expression of CTLA-4 and stronger binding to CD80 and CD86 serves to terminate the activated status of T cells via the negative signaling domains in the CTLA-4 cytoplasmic tail [8]. The importance of this negative regulatory pathway is highlighted by the fact that CTLA-4 knockout mice die early due to uncontrolled lymphoproliferative disease [9,10]. The CD28-CTLA-4/CD80-CD86 pathway has served as a paradigm by which to begin to understand related receptor/ligand pairs in this family including, ICOS/ICOSL, BTLA, and PD-1/PD-L1-PD- L2. PD-1 [11] is an Ig superfamily transmembrane protein whose cytoplasmic domain contains both an immunoreceptor tyrosine-based inhibiting motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). Although PD-1 shares 23% homology with CTLA-4, it lacks both the MYPPPY motif for binding to the B7 molecules [12] and the cysteine residue necessary for homodimerization [13]. It has been shown that PD-1 mediated inhibition of T cell proliferation is a direct result of cell cycle arrest in G0/G1 and inhibition of IL-2 production [14,15]. Unlike the expression of CD28 and CTLA-4, which is restricted to T cells, PD- 1 is expressed on T cells, B cells and myeloid cells (Table 1) [5]. In addition, expression of PD-1 peaks by 72 h post engagement of the T cell receptor in vitro. Although activation of PD-1 through crosslinking results in phosphorylation of both cytoplasmic tyrosines, the interaction between the tyrosine phosphatase src homology 2 molecule (SHP-2) with the ITSM is responsible for the negative regulatory effect of PD-1 rather than the ITIM motif usually associated with inhibition of signaling (Fig. 2) [14,16]. The ligands for PD-1, PD-L1 (B7-H1), and PD-L2 (B7-DC), are members of the B7 receptor family and have distinct expression patterns. While PD-L1 is APC T cell MHC TCR ITAM CD3 SHP-2 T cell activation PD-L1 PD-1 ITIM ITSM Fig. 2. T cell activation and proliferation require positive signaling through the TCR receptor complex. The interaction of PD-1 with PD-L1 leads to phosphorylation of the ITSM domain of PD-1, recruitment of SHP-2, and cell cycle arrest. expressed more broadly on both resting and activated T, B, myeloid, and non-hematopoietic cells such as lung, heart, pancreas, and placenta, PD-L2 is induced on DCs and macrophages [17]. Contradictory results have been published showing that PD-L1 or PD-L2 have both an inhibitory [15,18] and positive co-stimulatory [19 22] effect on activation, proliferation, and cytokine production of antigen-stimulated T cells. The reasons for these conflicting results are not known at this time, but given the similarities between the CD28-CTLA-4/B7 pathways, it has been hypothesized that a second receptor may exist, other than PD-1, that mediates a positive signal similar to the CD28 molecule. In support of the existence of a second receptor, studies have revealed mutations in PD-L1 and PD-L2 that abolish interaction with PD-1 still retain their positive co-stimulatory effect on T cells [23]. Further studies are necessary to elucidate the fine specificity and downstream signaling of the molecules in the PD-1 pathway. Interestingly, Th1 and Th2 cytokines differentially stimulate PD-L1 and PD-L2 ligands, respectively, pointing to distinct roles played by these molecules in regulating the immune responses [24]. Recent studies in mice indicate that the PD-1 pathway is an important regulator of T cell responses to chronic viral infections. PD-1 is upregulated on virus-specific CD8 + T cells during chronic LCMV infection [25]. During chronic LCMV infection the PD-1 pathway plays a prominent role controlling CD8 + T cell exhaustion and preventing optimal antiviral function. The blockade of this pathway in persistently infected mice results in improved T cell function and more efficient viral control [25]. An interesting observation from the LCMV studies was that if the PD-1 pathway was disrupted early during infection, the mice succumbed to extensive immunopathology [25]. Thus, the PD-1 pathway may play a critical role in regulating immunopathology early, but once activated also prevents immunological control of chronic infection later. In some ways, PD-1-mediated downregulation of T cell responses during chronic LCMV infection appears to have been exploited to allow pathogen persistence. An important role has also been suggested for the PD-1/PD-L1 pathway in inflammation of the liver. Using a gene targeting strategy, naïve mice lacking PD-L1 were found to have intrahepatic cell populations enriched specifically for the CD8 + but not CD4 + T cells. The absence of PD-L1 also allowed susceptibility to experimental autoimmune hepatitis when mice lacking PD-L1 were injected with Con A. Further, antigen-activated CD8 + T cells accumulated preferentially in the liver as opposed to kidney or the lung [26]. It is interesting to note that some evidence indicates that the liver may act as a site where activated T cells die [27]. Together, these observations suggest that PD-L1 may be capable

4 A. Grakoui et al. / Journal of Hepatology 45 (2006) of regulating the deletion of activated CD8 + T cells. Additional studies have shown that infection of PD-1 deficient mice with adenovirus resulted in marked expansion of antigen-specific T cells in the liver compared to wild-type mice and led to a rapid abrogation of viral infection [28]. Finally, PD-1 expression by T cells was associated with viral replication in a model of chronic HBV infection [29]. Thus, the PD-1 pathway may be preferentially important in the liver. While IFN-c has been shown to up-regulate PD-L1 on lymphocytes in vitro as well as PD-L2 on macrophages and DCs [30], the signals that control the expression of these inhibitory ligands in primary human cells and especially in the liver have not been examined. Moreover, it has been unclear whether PD-L1 and PD-L2 can be expressed on the relevant types of cells infected by hepatotropic viruses. The report by Mühlbauer et al. in this issue sheds new light on the potential importance of the PD-1 pathway in human liver. PD-L1 expression was determined at the mrna level on primary human hepatocytes and parenchymal cells such as Kupffer cell (KC), sinusoidal endothelial cells (SEC) and hepatic stellate cells (HSC). While a constitutively low level of PD-L1 cell surface expression on primary human hepatocytes was evident, upon addition of exogenous IFNc and IFNa-2a, PD-L1 expression was upregulated. PD-L1 mrna was also up-regulated by activated lymphocytes and by direct viral infection (perhaps also through IFN pathways). To address whether elevated PD-L1 levels may influence T cell survival, Mulbauer et al. examined the level of apoptosis induced in Jurkat cells in vitro by resting or IFN-activated hepatocytes. An elevated level of apoptosis was induced by the IFN-treated liver cells and this apoptosis could be blocked by antibodies to PD-L1. These studies may be relevant to hepatotropic infections such as HCV and HBV. Both infections are often characterized by priming of anti-viral CD8 + T cells that can survive in the liver for years despite persistent virus replication. At least some studies indicate that they are anergic, lacking cytotoxic function and/or the ability to produce anti-viral cytokines (reviewed in [2,3]). Both infections are a major cause of morbidity and mortality. For example, more than 70% of patients exposed to HCV will develop chronic HCV infection that may progress to cirrhosis and hepatocellular carcinoma. HCV-associated liver disease is now the leading cause of liver transplantation in the United States and the only licensed therapy for chronic HCV-associated hepatitis is alpha interferon (IFNa) in combination with ribavirin. This therapy is expensive, effective in only a minority of HCV patients, and has significant side effects [31]. The paucity of efficacious anti-hcv therapeutic options highlights the need for effective interventions aimed at augmenting or supplementing the natural immune response and that alone or in concert with therapeutic therapy can prevent HCV-associated cirrhosis and hepatocellular carcinoma. One may envision possible therapeutic interventions for hepatotropic infections that include anti-virals (peg-ifn and ribavirin for HCV, lamivudine for HBV) as well as targeted blockade of the PD-1/PD- L1 pathway. It is unclear if PD-L1 levels are elevated in chronically infected livers, but the data presented by Mulbauer et al. suggest that the PD-1 co-inhibitory pathway could be enhanced during hepatotropic infections. Such an elevation of PD-L1 may provide an explanation for the failure of T cell responses to terminate HCV or HBV infections in some individuals. This could occur via either PD-L1 mediated functional inactivation (e.g., exhaustion) or by enhanced apoptosis of activated T cells in the liver. In addition, the upregulation of PD-L1 by IFNa-2a may have important implications for antiviral therapy that includes treatment with pegylated IFN. It will be interesting in the future to determine whether there is any relationship between PD-L1 levels in the liver, antiviral therapy and outcome of HCV or HBV infection. In summary, effective T cell responses to infection represent a delicate balance of positive and negative signals. In some cases, it appears that pathogens have exploited these negative pathways to attenuate immune responses and persist in the host. Understanding the regulation of activated anti-viral T cells may be critical to elucidate targets for immune modulation including interference in the PD-1/PD-L1 pathway. As new information emerges about these pathways in the context of infectious diseases it will be important to determine how effectively they can be therapeutically manipulated to enhance pathogen control while maintaining effective regulation of immunopathology. Acknowledgements The authors would like to acknowledge the Cancer Research Institute Investigator Award, Woodruff Health Sciences Fund and Yerkes Research Center Base Grant RR (A.G.), the Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health (E.J.W.), and the Grand Challenges in Global Health Gates Foundation (R.A.). References [1] Klenerman P, Hill A. T cells and viral persistence: lessons from diverse infections. Nat Immunol 2005;6: [2] Rehermann B, Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection. Nat Rev Immunol 2005;5: [3] Bowen DG, Walker CM. Adaptive immune responses in acute and chronic hepatitis C virus infection. 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