Enhancing Virus-Specific Immunity In Vivo by Combining Therapeutic Vaccination and PD-L1 Blockade in Chronic Hepadnaviral Infection

Hepatitis B virus (HBV) persistence is facilitated by exhaustion of CD8 T cells that express the inhibitory receptor programmed cell death-1 (PD-1). Improvement of the HBV-specific T cell function has been obtained in vitro by inhibiting the PD-1/PD-ligand 1 (PD-L1) interaction. In this study, we examined whether in vivo blockade of the PD-1 pathway enhances virus-specific T cell immunity and leads to the resolution of chronic hepadnaviral infection in the woodchuck model. The woodchuck PD-1 was first cloned, characterized, and its expression patterns on T cells from woodchucks with acute or chronic woodchuck hepatitis virus (WHV) infection were investigated. Woodchucks chronically infected with WHV received a combination therapy with nucleoside analogue entecavir (ETV), therapeutic DNA vaccination and woodchuck PD-L1 antibody treatment. The gain of T cell function and the suppression of WHV replication by this therapy were evaluated. We could show that PD-1 expression on CD8 T cells was correlated with WHV viral loads during WHV infection. ETV treatment significantly decreased PD-1 expression on CD8 T cells in chronic carriers. In vivo blockade of PD-1/PD-L1 pathway on CD8 T cells, in combination with ETV treatment and DNA vaccination, potently enhanced the function of virus-specific T cells. Moreover, the combination therapy potently suppressed WHV replication, leading to sustained immunological control of viral infection, anti-WHs antibody development and complete viral clearance in some woodchucks. Our results provide a new approach to improve T cell function in chronic hepatitis B infection, which may be used to design new immunotherapeutic strategies in patients.     

PD-1 blockage reverses immune dysfunction and hepatitis B viral persistence in a mouse animal model

Persistent hepatitis B viral (HBV) infection results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Recent studies in animal models of viral infection indicate that the interaction between the inhibitory receptor, programmed death (PD)-1, on lymphocytes and its ligand (PD-L1) play a critical role in T-cell exhaustion by inducing T-cell inactivation. High PD-1 expression levels by peripheral T-lymphocytes and the possibility of improving T-cell function by blocking PD-1-mediated signaling confirm the importance of this inhibitory pathway in inducing T-cell exhaustion. We studied T-cell exhaustion and the effects of PD-1 and PD-L1 blockade on intrahepatic infiltrating T-cells in our recently developed mouse model of HBV persistence. In this mouse animal model, we demonstrated that there were increased intrahepatic PD-1-expressing CD8+ and CD4+ T cells in mice with HBV persistence, but PD-1 upregulation was resolved in mice which had cleared HBV. The Intrahepatic CD8+ T-cells expressed higher levels of PD-1 and lower levels of CD127 in mice with HBV persistence. Blockade of PD-1/PD-L1 interactions increased HBcAg-specific interferon (IFN)-γ production in intrahepatic T lymphocytes. Furthermore, blocking the interaction of PD-1 with PD-L1 by an anti-PD-1 monoclonal antibody (mAb) reversed the exhausted phenotype in intrahepatic T lymphocytes and viral persistence to clearance of HBV in vivo. Our results indicated that PD-1 blockage reverses immune dysfunction and viral persistence of HBV infection in a mouse animal model, suggesting that the anti-PD-1 mAb might be a good therapeutic candidate for chronic HBV infection.     

Longitudinal analysis of CD8+ T cells specific for structural and nonstructural hepatitis B virus proteins in patients with chronic hepatitis B: implications for immunotherapy

The cytotoxic T-cell response in chronic hepatitis B virus (HBV) infection has been described as weak and mono- or oligospecific in comparison to the more robust virus-specific T-cell response present in resolved infection. However, chronic hepatitis B is a heterogeneous disease with markedly variable levels of virus replication and liver disease activity. Here we analyzed (both directly ex vivo and after in vitro stimulation) the HBV-specific CD8 T-cell responses against structural and nonstructural HBV proteins longitudinally in patients with different patterns of chronic infections. We found that the profiles of virus-specific CD8(+)-T-cell responses during chronic infections are highly heterogeneous and influenced more by the level of HBV replication than by the activity of liver disease. An HBV DNA load of <10(7) copies/ml appears to be the threshold below which circulating multispecific HBV-specific CD8(+) T cells are consistently detected. Furthermore, CD8(+) T cells with different specificities are differentially regulated during chronic infections. HBV core-specific CD8(+) T cells are associated with viral control, while CD8(+) T cells specific for envelope and polymerase epitopes can occasionally be found in the setting of high levels (>10(7) copies) of HBV replication. These findings have implications for the design of immunotherapy for chronic HBV infections.     

Reduced hepatitis B virus (HBV)-specific CD4+ T-cell responses in human immunodeficiency virus type 1-HBV-coinfected individuals receiving HBV-active antiretroviral therapy

Functional hepatitis B virus (HBV)-specific T cells are significantly diminished in individuals chronically infected with HBV compared to individuals with self-limiting HBV infection or those on anti-HBV therapy. In individuals infected with human immunodeficiency virus type 1 (HIV-1), coinfection with HBV is associated with an increased risk of worsening liver function following antiviral therapy and of more rapid HBV disease progression. Total HBV-specific T-cell responses in subjects with diverse genetic backgrounds were characterized by using a library of 15-mer peptides overlapping by 11 amino acids and spanning all HBV proteins. The magnitude and breadth of CD4(+) and CD8(+) T-cell responses to HBV in peripheral blood were examined by flow cytometry to detect gamma interferon production following stimulation with HBV peptide pools. Chronic HBV carriers (n = 34) were studied, including individuals never treated for HBV infection (n = 7), HBV-infected individuals receiving anti-HBV therapy (n = 13), and HIV-1-HBV-coinfected individuals receiving anti-HBV therapy (n = 14). CD4(+) and CD8(+) HBV-specific T-cell responses were more frequently detected and the CD8(+) T-cell responses were of greater magnitude and breadth in subjects on anti-HBV treatment than in untreated chronic HBV carriers. There was a significant inverse correlation between detection of a HBV-specific T-cell response and HBV viral load. HBV-specific CD4(+) and CD8(+) T-cell responses were significantly (fivefold) reduced compared with HIV-specific responses. Although, the frequency and breadth of HBV-specific CD8(+) T-cell responses were comparable in the monoinfected and HIV-1-HBV-coinfected groups, HBV-specific CD4(+) T-cell responses were significantly reduced in HIV-1-HBV-coinfected individuals. Therefore, HIV-1 infection has a significant and specific effect on HBV-specific T-cell immunity.     

PD-1:PD-L1 interactions contribute to the functional suppression of virus-specific CD8+ T lymphocytes in the liver

Mechanisms contributing to the development of chronic viral infections, including chronic hepatitis B virus (HBV) infections, are not well understood. We have shown recently that production of IFN-gamma, an important antiviral cytokine, by HBV-specific CTLs is rapidly induced when they enter the liver of HBV transgenic mice, and then rapidly suppressed, despite the continued presence of Ag. Suppression of IFN-gamma production by the CTLs coincides with the up-regulation of programmed cell death (PD)-1, a cell surface signaling molecule known to inhibit T cell function. To determine whether PD-1 plays a role in the functional suppression of IFN-gamma secretion by CTLs, we treated HBV transgenic mice with blocking Abs specific for PD ligand (PD-L)1, the most widely expressed PD-1 ligand, and adoptively transferred HBV-specific CTLs. Treatment with anti-PD-L1 Abs resulted in a delay in the suppression of IFN-gamma-producing CTLs and a concomitant increase in the absolute number of IFN-gamma-producing CTLs in the liver. These results indicate that PD-1:PD-L1 interactions contribute to the suppression of IFN-gamma secretion observed following Ag recognition in the liver. Blockade of inhibitory pathways such as PD-1:PD-L1 may reverse viral persistence and chronic infection in cases in which the CTL response is suppressed by this mechanism.     

PD-L1 Expression on Retrovirus-Infected Cells Mediates Immune Escape from CD8+ T Cell Killing

Cytotoxic CD8+ T Lymphocytes (CTL) efficiently control acute virus infections but can become exhausted when a chronic infection develops. Signaling of the inhibitory receptor PD-1 is an important mechanism for the development of virus-specific CD8+ T cell dysfunction. However, it has recently been shown that during the initial phase of infection virus-specific CD8+ T cells express high levels of PD-1, but are fully competent in producing cytokines and killing virus-infected target cells. To better understand the role of the PD-1 signaling pathway in CD8+ T cell cytotoxicity during acute viral infections we analyzed the expression of the ligand on retrovirus-infected cells targeted by CTLs. We observed increased levels of PD-L1 expression after infection of cells with the murine Friend retrovirus (FV) or with HIV. In FV infected mice, virus-specific CTLs efficiently eliminated infected target cells that expressed low levels of PD-L1 or that were deficient for PD-L1 but the population of PD-L1high cells escaped elimination and formed a reservoir for chronic FV replication. Infected cells with high PD-L1 expression mediated a negative feedback on CD8+ T cells and inhibited their expansion and cytotoxic functions. These findings provide evidence for a novel immune escape mechanism during acute retroviral infection based on PD-L1 expression levels on virus infected target cells.     

Expression of the interleukin-7 receptor alpha chain (CD127) on virus-specific CD8+ T cells identifies functionally and phenotypically defined memory T cells during acute resolving hepatitis B virus infection

Virus-specific CD8+ T cells play a central role in the outcome of several viral infections, including hepatitis B virus (HBV) infection. A key feature of virus-specific CD8+ T cells is the development of memory. The mechanisms resulting in the establishment of T-cell memory are still only poorly understood. It has been suggested that T-cell memory may depend on the survival of virus-specific CD8+ T cells in the contraction phase. Indeed, a population of effector cells that express high levels of the interleukin-7 receptor alpha chain (CD127) as the precursors of memory CD8+ T cells has recently been identified in mice. However, very little information is currently available about the kinetics of CD127 expression in an acute resolving viral infection in humans and its association with disease pathogenesis, viral load, and functional and phenotypical T-cell characteristics. To address these important issues, we analyzed the HBV-specific CD8+ T-cell response longitudinally in a cohort of six patients with acute HBV infection who spontaneously cleared the virus. We observed the emergence of CD127 expression on antigen-specific CD8+ memory T cells during the course of infection. Importantly, the up-regulation of CD127 correlated phenotypically with a loss of CD38 and PD-1 expression and acquisition of CCR7 expression: functionally with an enhanced proliferative capacity and clinically with the decline in serum alanine aminotransferase levels and viral clearance. These results suggest that the expression of CD127 is a marker for the development of functionally and phenotypically defined antigen-specific CD8+ memory T cells in cleared human viral infections.     

Inhibitory Phenotype of HBV-Specific CD4+ T-Cells Is Characterized by High PD-1 Expression but Absent Coregulation of Multiple Inhibitory Molecules

Background

T-cell exhaustion seems to play a critical role in CD8⁺ T-cell dysfunction during chronic viral infections. However, up to now little is known about the mechanisms underlying CD4⁺ T-cell dysfunction during chronic hepatitis B virus (CHB) infection and the role of inhibitory molecules such as programmed death 1 (PD-1) for CD4⁺ T-cell failure.

Methods

The expression of multiple inhibitory molecules such as PD-1, CTLA-4, TIM-3, CD244, KLRG1 and markers defining the grade of T-cell differentiation as CCR7, CD45RA, CD57 and CD127 were analyzed on virus-specific CD4⁺ T-cells from peripheral blood using a newly established DRB1*01-restricted MHC class II Tetramer. Effects of in vitro PD-L1/2 blockade were defined by investigating changes in CD4⁺ T-cell proliferation and cytokine production.

Results

CD4⁺ T-cell responses during chronic HBV infection was characterized by reduced Tetramer⁺CD4⁺ T-cell frequencies, effector memory phenotype, sustained PD-1 but low levels of CTLA-4, TIM-3, KLRG1 and CD244 expression. PD-1 blockade revealed individualized patterns of in vitro responsiveness with partly increased IFN-γ, IL-2 and TNF-α secretion as well as enhanced CD4⁺ T-cell expansion almost in treated patients with viral control.

Conclusion

HBV-specific CD4⁺ T-cells are reliably detectable during different courses of HBV infection by MHC class II Tetramer technology. CD4⁺ T-cell dysfunction during chronic HBV is basically linked to strong PD-1 upregulation but absent coregulation of multiple inhibitory receptors. PD-L1/2 neutralization partly leads to enhanced CD4⁺ T-cell functionality with heterogeneous patterns of CD4⁺ T-cell rejunivation.     

Tissue-Specific Differences in PD-1 and PD-L1 Expression during Chronic Viral Infection: Implications for CD8 T-Cell Exhaustion

The PD-1/PD-L pathway plays a major role in regulating T-cell exhaustion during chronic viral infections in animal models, as well as in humans, and blockade of this pathway can revive exhausted CD8⁺ T cells. We examined the expression of PD-1 and its ligands, PD-L1 and PD-L2, in multiple tissues during the course of chronic viral infection and determined how the amount of PD-1 expressed, as well as the anatomical location, influenced the function of exhausted CD8 T cells. The amount of PD-1 on exhausted CD8 T cells from different anatomical locations did not always correlate with infectious virus but did reflect viral antigen in some tissues. Moreover, lower expression of PD-L1 in some locations, such as the bone marrow, favored the survival of PD-1^Hi exhausted CD8 T cells, suggesting that some anatomical sites might provide a survival niche for subpopulations of exhausted CD8 T cells. Tissue-specific differences in the function of exhausted CD8 T cells were also observed. However, while cytokine production did not strictly correlate with the amount of PD-1 expressed by exhausted CD8 T cells from different tissues, the ability to degranulate and kill were tightly linked to PD-1 expression regardless of the anatomical location. These observations have implications for human chronic infections and for therapeutic interventions based on blockade of the PD-1 pathway.Chronic viral infections are often associated with CD8⁺ T-cell dysfunction (30). This dysfunction, termed exhaustion, includes defects in the ability to produce antiviral cytokines, poor cytotoxicity, a loss of antigen-independent self-renewal, and the inability to vigorously re-expand following antigen exposure (30). These functional deficiencies contrast with the highly functional memory CD8⁺ T cells that are generated after acute infection and maintained via interleukin-7 (IL-7)- and IL-15-mediated homeostatic proliferation (30). During chronic viral infections, T-cell exhaustion often correlates with poor control of viral replication (3, 8, 38, 39). Thus, there is considerable interest in developing strategies to reverse exhaustion and restore function in virus-specific CD8⁺ T cells during chronic infections.Recent studies have revealed an important role for the negative regulatory molecule PD-1 in CD8 T-cell exhaustion during chronic viral infections (29). PD-1, a member of the CD28/CTLA-4 family of costimulatory/coinhibitory receptors, contains both ITIM and ITSM motifs in the intracellular tail and can deliver negative signals, at least partly via recruitment of the phosphatase Shp-2 (29). A role for PD-1 in regulating T-cell responses to chronic viral infections was first observed using lymphocytic choriomeningitis virus (LCMV) infection of mice, where PD-1 was found to be highly expressed on exhausted CD8⁺ T cells from chronically infected animals but not on functional memory CD8⁺ T cells from mice that had cleared an acute strain of the virus (3). In vivo blockade of the PD-1 pathway led to a dramatic increase in the number of virus-specific CD8⁺ T cells, improved functionality of these cells, and enhanced control of viral replication (3). These observations were extended to human chronic viral infections, and a series of studies have demonstrated that human immunodeficiency virus (HIV)-, hepatitis C virus (HCV)-, and HBV-specific CD8⁺ T cells upregulate PD-1 in humans compared to CD8⁺ T cells specific for nonpersisting viruses such as influenza virus or vaccinia virus (6-8, 24, 26, 32, 33, 42). Increasing PD-1 expression also correlates with disease status during HIV infection (8, 42). In vitro blockade of PD-1-PD-L interactions can reinvigorate exhausted virus-specific T-cell responses in humans and appears to have a prominent impact on proliferative expansion and/or prevention of apoptosis in these cases (9, 24, 32). Finally, recent results from in vivo blockade in the macaque simian immunodeficiency virus (SIV) infection model demonstrated the effectiveness of blocking PD-1 in primates during chronic viral infection (36). In these studies, PD-1 blockade enhanced virus-specific T and B-cell responses, lowered viral load, and improved the survival of chronically infected animals. Thus, PD-1 has emerged as not only a major regulator of T-cell exhaustion and viral control during chronic infection but also as an important potential therapeutic target.Despite these important studies and the clear impact of PD-1 blockade on the reversal of T-cell exhaustion, important questions remain. For example, previous work has demonstrated that PD-1 expression is not uniform on subsets of exhausted CD8 T cells (4). However, the expression of PD-1 on exhausted CD8 T cells in multiple tissues, and the relationship between PD-1 expression in these tissues to viral load, the PD-1 ligands and function has not been examined. Given the nonlymphoid accumulation of virus-specific CD8 T cells during chronic viral infections (11, 39) and the predilection of many important chronic infections for replicating in anatomically restricted locations (e.g., HCV and the liver, HIV and mucosal tissues, etc.), the dynamics of PD-1 expression by exhausted CD8 T cells outside the blood and spleen could have important therapeutic implications.In the present study we examined these issues using the mouse model of LCMV infection. Our results demonstrate that exhausted CD8 T cells have a wide range of PD-1 expression in different tissues of chronically infected mice. Virus-specific CD8 T cells in some anatomical locations such as the liver, brain, and bone marrow (BM) expressed high PD-1 for substantially longer than virus-specific CD8⁺ T cells from the spleens or blood of the same mice. Although PD-1 expression in the spleen correlated well with reduced gamma interferon (IFN-γ) and tumor necrosis factor (TNF) production, the PD-1^Hi virus-specific CD8⁺ T cells from the BM remained capable of producing antiviral cytokines ex vivo. In contrast, a strong negative correlation between PD-1 expression and cytotoxicity existed for exhausted CD8 T cells from all tissues tested. PD-L1 expression was high in the spleen, whereas in the BM antigen-presenting cell (APC) populations expressed lower amounts of PD-L1. Survival of PD-1^Hi CD8⁺ T cells from the BM was decreased in the presence of splenic APCs, suggesting that different tissue microenvironments in vivo could selectively support the persistence of PD-1^Hi exhausted CD8 T cells. Since PD-1 expression differs by anatomical location, these observations suggest that PD-1 blockade in vivo will have varying impacts on exhausted CD8 T cells from different tissues or anatomical locations. These observations have implications for human chronic infections such as HBV, HCV, and HIV.     

Liver-infiltrating lymphocytes in chronic human hepatitis C virus infection display an exhausted phenotype with high levels of PD-1 and low levels of CD127 expression

The majority of people infected with hepatitis C virus (HCV) fail to generate or maintain a T-cell response effective for viral clearance. Evidence from murine chronic viral infections shows that expression of the coinhibitory molecule PD-1 predicts CD8+ antiviral T-cell exhaustion and may contribute to inadequate pathogen control. To investigate whether human CD8+ T cells express PD-1 and demonstrate a dysfunctional phenotype during chronic HCV infection, peripheral and intrahepatic HCV-specific CD8+ T cells were examined. We found that in chronic HCV infection, peripheral HCV-specific T cells express high levels of PD-1 and that blockade of the PD-1/PD-L1 interaction led to an enhanced proliferative capacity. Importantly, intrahepatic HCV-specific T cells, in contrast to those in the periphery, express not only high levels of PD-1 but also decreased interleukin-7 receptor alpha (CD127), an exhausted phenotype that was HCV antigen specific and compartmentalized to the liver, the site of viral replication.     

Rapid Perturbation in Viremia Levels Drives Increases in Functional Avidity of HIV-specific CD8 T Cells

The factors determining the functional avidity and its relationship with the broad heterogeneity of antiviral T cell responses remain partially understood. We investigated HIV-specific CD8 T cell responses in 85 patients with primary HIV infection (PHI) or chronic (progressive and non-progressive) infection. The functional avidity of HIV-specific CD8 T cells was not different between patients with progressive and non-progressive chronic infection. However, it was significantly lower in PHI patients at the time of diagnosis of acute infection and after control of virus replication following one year of successful antiretroviral therapy. High-avidity HIV-specific CD8 T cells expressed lower levels of CD27 and CD28 and were enriched in cells with an exhausted phenotype, i.e. co-expressing PD-1/2B4/CD160. Of note, a significant increase in the functional avidity of HIV-specific CD8 T cells occurred in early-treated PHI patients experiencing a virus rebound after spontaneous treatment interruption. This increase in functional avidity was associated with the accumulation of PD-1/2B4/CD160 positive cells, loss of polyfunctionality and increased TCR renewal. The increased TCR renewal may provide the mechanistic basis for the generation of high-avidity HIV-specific CD8 T cells. These results provide insights on the relationships between functional avidity, viremia, T-cell exhaustion and TCR renewal of antiviral CD8 T cell responses.     

Upregulation of the Tim-3/Galectin-9 Pathway of T Cell Exhaustion in Chronic Hepatitis B Virus Infection

The S-type lectin galectin-9 binds to the negative regulatory molecule Tim-3 on T cells and induces their apoptotic deletion or functional inactivation. We investigated whether galectin-9/Tim-3 interactions contribute to the deletion and exhaustion of the antiviral T cell response in chronic hepatitis B virus infection (CHB). We found Tim-3 to be expressed on a higher percentage of CD4 and CD8 T cells from patients with CHB than healthy controls (p<0.0001) and to be enriched on activated T cells and those infiltrating the HBV-infected liver. Direct ex vivo examination of virus-specific CD8 T cells binding HLA-A2/peptide multimers revealed that Tim-3 was more highly upregulated on HBV-specific CD8 T cells than CMV-specific CD8 T cells or the global CD8 T cell population in patients with CHB (p<0.001) or than on HBV-specific CD8 after resolution of infection. T cells expressing Tim-3 had an impaired ability to produce IFN-γ and TNF-α upon recognition of HBV-peptides and were susceptible to galectin-9-triggered cell death in vitro. Galectin-9 was detectable at increased concentrations in the sera of patients with active CHB-related liver inflammation (p = 0.02) and was strongly expressed by Kupffer cells within the liver sinusoidal network. Tim-3 blockade resulted in enhanced expansion of HBV-specific CD8 T cells able to produce cytokines and mediate cytotoxicity in vitro. Blocking PD-1 in combination with Tim-3 enhanced the number of patients from whom functional antiviral responses could be recovered and/or the strength of responses, indicating that these co-inhibitory molecules play a non-redundant role in driving T cell exhaustion in CHB. Patients taking antivirals able to potently suppress HBV viraemia continued to express Tim-3 on their T cells and respond to Tim-3 blockade. In summary, both Tim-3 and galectin-9 are increased in CHB and may contribute to the inhibition and deletion of T cells as they infiltrate the HBV-infected liver.     

Licensing virus-specific T cells to secrete the neutrophil attracting chemokine CXCL-8 during hepatitis B virus infection

T cell functional plasticity helps tailor antiviral immunity during different phases of infections. We tested whether, during different phases of HBV infection, virus-specific T cells can acquire specific proinflammatory functions that could drive granulocyte/mononuclear cell liver infiltration. Multifunctional analysis of HBV-specific T cells during acute and chronic HBV infection revealed that HBV-specific T cells had the capacity to produce the neutrophil chemokine CXCL-8 but not IL-17. CXCL-8 producing T cells were detectable in the liver of chronic HBV patients with active hepatitis; while in acute HBV patients CXCL-8 production by T cells was temporally limited to the acute phase of disease, concomitant with the peak of liver inflammation. Characterization of the conditions necessary for the development of CXCL-8 producing T cells showed a requirement for IL-7 and IL-15 during T cell expansion. These data show that functional plasticity of virus-specific T cells spontaneously occurs during HBV infection and that an environment rich IL-7 and IL-15 can license T cells with the ability to produce CXCL-8 and potentially influence liver pathology.     

Upregulation of PD-1 expression on circulating and intrahepatic hepatitis C virus-specific CD8+ T cells associated with reversible immune dysfunction

Infection with hepatitis C virus (HCV) is associated with persistence in the majority of individuals. We demonstrate here that the inhibitory molecule programmed death-1 (PD-1) is significantly upregulated on total and HCV-specific CD8(+) cytotoxic T lymphocytes (CTLs) in the peripheral blood and livers of patients with chronic infection compared to subjects with spontaneous HCV resolution, patients with nonviral liver disease, and normal controls. PD-1 expression on cytomegalovirus-specific CTLs also varies according to HCV status and is highest in patients with chronic infection. HCV-specific CTLs that are PD-1(high) express higher levels of the senescence marker CD57 than PD-1(low) CTLs, and CD57 expression is greater in chronic than in resolved infection. In vitro blockade of PD-1 by monoclonal antibodies specific to its ligands (PD-L1 and PD-L2) results in restoration of functional competence (proliferation and gamma interferon and interleukin-2 secretion) of HCV-specific CTLs, including those residing in the liver. This reversal of CTL exhaustion is evident even in individuals who lack HCV-specific CD4(+) T-cell help. Our data indicate that the PD-1/PD-L pathway is critical in persistent HCV infection in humans and represents a potential novel target for restoring function of exhausted HCV-specific CTLs.     

HIV-mediated phosphatidylinositol 3-kinase/serine-threonine kinase activation in APCs leads to programmed death-1 ligand upregulation and suppression of HIV-specific CD8 T cells

Recent evidence demonstrates that HIV-1 infection leads to the attenuation of cellular immune responses, which has been correlated with the increased expression of programmed death (PD)-1 on virus-specific CD8(+) T cells. PD-1 is induced upon T cell activation, and its prolonged expression facilitates CD8(+) T cell inhibitory signals when bound to its B7 family ligands, PD-ligand (L)1/2, which are expressed on APCs. Importantly, early reports demonstrated that blockade of the PD-1/PD-L interaction by Abs may help to counter the development of immune exhaustion driven by HIV viral persistence. To better understand the regulation of the PD-1 pathway during HIV infection, we examined the ability of the virus to induce PD-L expression on macrophages and dendritic cells. We found a direct relationship between the infection of APCs and the expression of PD-L1 in which virus-mediated upregulation induced a state of nonresponsiveness in uninfected HIV-specific T cells. Furthermore, this exhaustion phenotype was revitalized by the blockade of PD-L1, after which T cells regained their capacity for proliferation and the secretion of proinflammatory cytokines IFN-γ, IL-2, and IL-12 upon restimulation. In addition, we identify a critical role for the PI3K/serine-threonine kinase signaling pathway in PD-L1 upregulation of APCs by HIV, because inhibition of these intracellular signal transducer enzymes significantly reduced PD-L1 induction by infection. These data identify a novel mechanism by which HIV exploits the immunosuppressive PD-1 pathway and suggest a new role for virus-infected cells in the local corruption of immune responses required for viral suppression.     

Upregulation of PD-1 expression on HIV-specific CD8+ T cells leads to reversible immune dysfunction

The engagement of programmed death 1 (PD-1) to its ligands, PD-L1 and PD-L2, inhibits proliferation and cytokine production mediated by antibodies to CD3 (refs. 5,6,7). Blocking the PD-1-PD-L1 pathway in mice chronically infected with lymphocytic choriomeningitis virus restores the capacity of exhausted CD8(+) T cells to undergo proliferation, cytokine production and cytotoxic activity and, consequently, results in reduced viral load. During chronic HIV infection, HIV-specific CD8(+) T cells are functionally impaired, showing a reduced capacity to produce cytokines and effector molecules as well as an impaired capacity to proliferate. Here, we found that PD-1 was upregulated on HIV-specific CD8(+) T cells; PD-1 expression levels were significantly correlated both with viral load and with the reduced capacity for cytokine production and proliferation of HIV-specific CD8(+) T cells. Notably, cytomegalovirus (CMV)-specific CD8(+) T cells from the same donors did not upregulate PD-1 and maintained the production of high levels of cytokines. Blocking PD-1 engagement to its ligand (PD-L1) enhanced the capacity of HIV-specific CD8(+) T cells to survive and proliferate and led to an increased production of cytokines and cytotoxic molecules in response to cognate antigen. The accumulation of HIV-specific dysfunctional CD8(+) T cells in the infected host could prevent the renewal of a functionally competent HIV-specific CD8(+) repertoire.     

PD-L1 Blockade Differentially Impacts Regulatory T Cells from HIV-Infected Individuals Depending on Plasma Viremia

Blocking the PD-1/PD-L1 pathway has emerged as a potential therapy to restore impaired immune responses in human immunodeficiency virus (HIV)-infected individuals. Most reports have studied the impact of the PD-L1 blockade on effector cells and neglected possible effects on regulatory T cells (Treg cells), which play an essential role in balancing immunopathology and antiviral effector responses. The aim of this study was to define the consequences of ex vivo PD-L1 blockade on Treg cells from HIV-infected individuals. We observed that HIV infection led to an increase in PD-1+ and PD-L1+ Treg cells. This upregulation correlated with disease progression and decreased under antiretroviral treatment. Treg cells from viremic individuals had a particularly high PD-1 expression and impaired proliferative capacity in comparison with Treg cells from individuals under antiretroviral treatment. PD-L1 blockade restored the proliferative capacity of Treg cells from viremic individuals but had no effect on its suppressive capacity. Moreover, it increased the viral production in cell cultures from viremic individuals. This increase in viral production correlated with an increase in Treg cell percentage and a reduction in the CD4/Treg and CD8/Treg cell ratios. In contrast to the effect of the PD-L1 blockade on Treg cells from viremic individuals, we did not observe a significant effect on the proliferative capacity of Treg cells from individuals in whom viremia was controlled (either spontaneously or by antiretroviral treatment). However, PD-L1 blockade resulted in an increased proliferative capacity of HIV-specific-CD8 T cells in all subjects. Taken together, our findings suggest that manipulating PD-L1 in vivo can be expected to influence the net gain of effector function depending on the subject’s plasma viremia.     

Cellular immune responses to the hepatitis B virus polymerase

CD4 T cells play an important role in hepatitis B virus (HBV) infection by secretion of Th1 cytokines that down-regulate HBV replication, and by promoting CD8 T cell and B cell responses. We have identified and characterized 10 CD4 T cell epitopes within polymerase and used them to analyze the immunological effects of long-term antiviral therapy as compared with spontaneous recovery from HBV infection. Candidate epitopes were tested for binding to 14 HLA-DR molecules and in IFN-gamma ELISPOT and cytotoxicity assays using peripheral blood lymphocytes from 66 HBV-infected patients and 16 uninfected controls. All 10 epitopes bound with high affinity to the most prevalent HLA-DR Ags, were conserved among HBV genomes, and induced IFN-gamma responses from HBV-specific CD4+ T cells. Several epitopes contained nested MHC class I motifs and stimulated HBV-specific IFN-gamma production and cytotoxicity of CD8+ T cells. HBV polymerase-specific responses were more frequent during acute, self-limited hepatitis and after recovery (12 of 18; 67%) than during chronic hepatitis (16 of 48 (33%); p=0.02). Antiviral therapy of chronic patients restored HBV polymerase and core-specific T cell responses during the first year of treatment, but thereafter, responses decreased and, after 3 years, were no more frequent than in untreated patients. Decreased T cell responsiveness during prolonged therapy was associated with increased prevalence of lamivudine-resistant HBV mutants and increased HBV titers. The data provide a rationale for the combination of antiviral and immunostimulatory therapy. These newly described HBV polymerase epitopes could be a valuable component of a therapeutic vaccine for a large and ethnically diverse patient population.