- Email: [email protected]
Hepatitis B virus-specific T cell receptors with high functional avidity redirect T cells to eliminate HBV
ORAL PRESENTATIONS developed a protocol for the direct ex vivo analysis of HBsAg-specific and total B cells. We used this novel assay to characterize B cell frequency and phenotype in healthy HBV vaccinated subjects as well as patients with resolved or chronic HBV infection. Methods: Phenotypic analysis of total and HBsAg-binding B cells was performed by flow cytometry using conjugated antibodies identifying different markers of B cell function and maturation. Recombinant HBsAg was fluorescently conjugated with either DyLight 550 or DyLight 650 and used to doubly stain peripheral blood cells of HBV vaccinated subjects (n = 17), patients with resolved (n = 13) and chronic infection. Patients were categorized as follows: HBV-DNA 107–1010 IU/ml, ALT < 40 IU/L (n = 16); eAg+/−, HBV-DNA 102–108 IU/ ml, ALT 11–367 IU/L (n = 43); HBV-DNA 101–103 IU/ml, ALT < 40 IU/L (n = 17). HBsAg was also quantified in all patients. Results: CD19 + CD27+ cells from vaccinated individuals that bound fluorochrome-conjugated HBsAg were sorted, cultured in vitro and analyzed for anti-HBs production. Anti-HBs antibodies were only detected in the supernatant of CD19 + CD27+, HBsAg-DyLight 550+ and HBsAg-DyLight 650+ cells, demonstrating that the staining protocol identifies bona-fide anti-HBs specific B cells. The frequency and phenotype of anti-HBs specific B cells were then analyzed in patients. Surprisingly, the frequency of anti-HBs memory B cells (∼0.3% of total memory B cells) were comparable in patients with resolved or chronic infection, as well as in vaccinated individuals. In contrast, B cell phenotype was different in patients with high HBsAg/HBV-DNA and no inflammation (normal ALT) relative to patients in other phases of disease and healthy vaccinated subjects. Specifically, total and anti-HBs specific B cells in patients with high HBsAg/HBV-DNA and normal ALT had higher expression of CD39 (a regulation marker) and lower expression of CD38 (an activation marker). Moreover, there was an increased frequency of atypical memory B cells (CD21-CD27-) with exhaustion properties in these patients. Conclusions: We developed a protocol for the direct ex vivo visualization of anti-HBs producing B cells. Using this new method we demonstrate that HBsAg/HBV-DNA quantity affects the phenotype of total and HBsAg-specific B cells, which may improve our understanding of the natural history of CHB. PS-050 Combined GS-4774 and tenofovir therapy can improve HBVspecific T cell responses in patients with chronic active hepatitis B C. Boni1, M. Rossi1, A. Vecchi1, D. Laccabue1, T. Giuberti1, A. Alfieri1, P. Andreone2, C. Cursaro2, M. Margotti2, A. Mangia3, R. Santoro3, V. Piazzolla3, M.R. Brunetto4, B. Coco4, D. Cavallone4, A. Lau5, A. Gaggar5, C. Ferrari1. 1Unit of Infectious Diseases and Hepatology, Azienda-Ospedaliero-Universitaria of Parma, Parma; 2Dipartimento di Scienze Mediche e Chirurgiche, University of Bologna, Bologna; 3Liver Unit, IRCCS, ‘Casa Sollievo della Sofferenza’, San Giovanni Rotondo, Foggia; 4Hepatology Unit and Liver Physiopathology Laboratory, University Hospital of Pisa, Pisa, Italy; 5Gilead Sciences, Foster City, CA, United States E-mail: [email protected] Background and Aims: HBV-specific T cells are essential for HBV control but they are functionally defective in chronic HBV infection. Thus, correction of their dysfunction may represent a rational strategy to treat chronic hepatitis B (CHB). GS-4774 is a yeast-based T-cell vaccine containing HBV core, surface and X proteins which has been shown to be immunogenic in mouse models and healthy volunteers. Aim of the study was to characterize the modulatory effect of GS-4774 on HBV-specific T cell responses in naïve HBeAg negative CHB patients. Methods: 12 HBeAg negative, naïve, viremic, genotype D infected CHB patients received 6 consecutive monthly doses of vaccine in combination with Tenofovir Disoproxil Fumarate (TDF), as part of the larger GS-US-330-1401 study. 26 chronic HBeAg negative genotype D infected patients treated with NUC alone served as controls. HBV-
specific T cell responses were studied before, during (weeks 12 and 24) and after vaccine therapy (week 48) both ex vivo (IFN-γ Elispot) and after 10 days in vitro expansion (intracellular cytokine staining for IFN-γ, TNF-a, IL-2 and CD107 degranulation) in the presence of peptides covering the overall HBV proteome or control HBV unrelated peptides. Immunological data were assessed in relation to HBsAg/HBV-DNA/ALT declines. Results: No patients had a significant HBsAg decline while all normalized ALT and suppressed HBV-DNA. Ex vivo IFN-g Elispot responses were significantly improved upon HBV core peptide stimulation at week 48 compared to baseline. Following in vitro expansion, a significant increase in the percentage of HBV-specific IFN-g and IL2 producing CD3+ T cells was detected at week 24 and 48. This functional improvement was predominantly sustained by CD8+ T cells which showed also an increased production of TNF-a. IFN-g was more improved than IL2 and TNF-a. Maximal stimulatory activity was expressed by polymerase at all time points, while a progressive increase of IFN-g production was induced by core and envelope during combined GS-4774 and NUC administration. A simultaneous improvement of more than one T cell function was detected in 11 of 12 patients; this was associated with an increased percentage of double and triple positive HBV-specific T cells. Conclusions: GS-4774 combined with TDF can improve the T cell function with a prevalent effect on CD8 cells. This seems to be however insufficient to induce a substantial decline of HBsAg which was similar in patients treated with NUC alone or combined with GS-4774. PS-051 Hepatitis B virus-specific T cell receptors with high functional avidity redirect T cells to eliminate HBV K. Wisskirchen1,2, J. Kah2,3, K. Metzger1, L. Weigand4, W. Uckert5, M. Schiemann6, T. Volz3, A. Krackhardt4, M. Dandri3, U. Protzer1. 1 Institute of Virology, Helmholtz Zentrum München / Technische Universität München, Munich; 2both authors contributed equally 3 1. Medical Clinic, University Medical Center Hamburg-Eppendorf, Hamburg; 4III. Medical Clinic, Klinikum rechts der Isar, Munich; 5 Institute of Biology, Molecular Cell Biology and Gene Therapy, MaxDelbrück-Center for Molecular Medicine, Berlin; 6Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany E-mail: [email protected] Background and Aims: Adoptive T-cell therapy of chronic hepatitis B or hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC) intends to restore antiviral T-cell immunity to clear the infection or control tumour growth. We aimed at identifying T-cell receptors (TCR) with high functional avidity to redirect T cells that have the potential to cure HBV infection. Methods: We isolated eleven TCRs that were specific for the HBV S-derived peptides S20 and S172, or for the C18 core-peptide from patients with acute and resolved HBV infection. T cells genetically modified by retroviral transduction to express HBV-specific TCRs were compared in comprehensive functional analyses in vitro and in vivo. Results: Upon TCR grafting, CD8+ as well as CD4+ T cells became polyfunctional HBV-specific effector T cells recognizing even picomolar concentrations of cognate peptide. TCR-grafted T cells secreted IFN gamma, TNF alpha and IL2, controlled HBV replication and effectively killed hepatoma cells with an integrated HBV genome as well as HBV-infected cells. Notably, these HBV-specific TCRs recognized peptide presented on different HLA-A*02 subtypes common in areas with high HBV prevalence. When co-cultured with HBV-infected cells, TCR-transduced T cells eliminated viral antigens as well as the HBV persistence form, the cccDNA. TCRs with the highest functional avidity also mediated elimination of HBV when expressed in CD4+ T cells or in T cells from patients with chronic
Journal of Hepatology 2017 vol. 66 | S1–S32
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ORAL PRESENTATIONS hepatitis B. In vivo, TCR-redirected T cells targeted the liver when transferred into HBV-infected uPA-SCID/beige/IL2rg−/− mice repopulated with HLA-A*02-matched primary human hepatocytes. After 5 days, alanine amino transferase levels were moderately increased and T-cell-mediated PHH loss after 15–18 days, when mice were sacrificed, was limited (mean 37% human serum albumin decrease). Intrahepatic analyses revealed a strong reduction of cccDNA loads (>95%) and replication activity, which resulted in a profound reduction of antigenemia, a more than 4 log decrease of virema, and negative HBcAg staining in the liver. Conclusions: Our unique set of HBV-specific TCRs with different affinities represents an interesting tool for elucidating mechanisms of TCR-MHC interaction and dissecting specific anti-HBV mechanisms exerted by T cells. Most importantly, TCR-transduced T cells with high functional avidity are very interesting for adoptive T-cell therapy of chronic hepatitis B and HBV-induced HCC, because they have the potential to mediate clearance of the virus. PS-052 Responsiveness of HBV-specific CD8+ T cells to PD1 pathway blockade is linked to TCF1 expression A. Schuch1,2, D. Wieland1,2,3, M. Hofmann1, R. Thimme1. 1University Hospital Freiburg; 2Albert-Ludwigs-University Freiburg; 3Spemann Graduate School of Biology and Medicine (SGBM), Freiburg, Germany E-mail: [email protected] Background and Aims: Chronic viral infection results in progressive loss of virus-specific CD8+ T-cell effector functions, a phenomenon called T-cell exhaustion that is characterized by upregulation of inhibitory receptors (e.g. PD1). Studies in the LCMV mouse model revealed that exhausted virus-specific CD8+ T cells are heterogeneous and consist of terminally exhausted (PD1hi) and progenitor (PD1int) subsets. As recently published, the latter was defined by expression of the memory marker TCF1 and provided the proliferative burst upon PD1 therapy. In this study we aimed to define subpopulations of exhausted virus-specific CD8+ T cells in human chronic viral hepatitis including their key transcriptional regulators and functional properties, especially in regard to PD1 pathway blockade. Methods: We analyzed HLA-A*02-restricted epitope-specific CD8+ Tcell populations in inactive chronic HBsAg carriers (n = 12) and patients chronically infected with HCV (n = 8). We applied tetramerbased magnetic bead enrichment with subsequent multi-colour flow cytometry. Additionally, we performed peptide-specific in vitro expansion assays for 14 days with and without anti-PDL1 monoclonal antibody to assess proliferative potential and cytokine production. Results: Our data revealed that exhausted epitope-specific CD8+ T cells in chronic HBV and HCV infection are heterogeneous. While the majority of HCV-specific CD8+ T cells display a terminally exhausted phenotype (CD127−PD1hi), virus-specific CD8+ T-cell populations in inactive chronic HBsAg carriers show a phenotype characteristic for progenitor subsets with high expression of the memory markers CD127 and TCF1. TCF1 expression correlated with CD8+ T-cell expansion after HBV and HCV peptide-specific in vitro culture. Interestingly, HBV-specific CD8+ T cells showed an increased polyfunctionality compared to HCV-specific CD8+ T cells, assessed by IFNγ and TNF production upon restimulation after peptide-specific in vitro expansion. Furthermore, HBV-specific CD8+ T cells were highly responsive to PD1 pathway blockade. Importantly, our preliminary data suggests a direct association of TCF1 expression and proliferative burst after PD1 pathway blockade. Conclusions: In sum, HBV-specific CD8+ T-cell populations in chronic HBsAg carriers lack terminally exhausted subsets and are defined by high expression of TCF1. This correlated with enhanced expansion upon PD1 pathway blockade, rendering TCF1 a promising prediction marker for responsiveness to PD1 pathway blockade in immunotherapy.
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PS-053 Proteasome dysfunction as a reversible defect underlying virus-specific CD8 cell exhaustion in chronic hepatitis B P. Fisicaro1, V. Barili1, G. Acerbi1, B. Montanini2, D. Laccabue1, F. Guerrieri3, M. Massari4, C. Vandelli5, D. Salerno3, G. Grossi6, A. Alfieri1, P. Lampertico6, T. Giuberti1, G. Missale1, M. Levrero7,8, S. Ottonello2,9, C. Ferrari1. 1Unit of Infectious Diseases and Hepatology, Azienda Ospedaliero-Universitaria of Parma; 2Department of Life Sciences, University of Parma, Parma; 3Center for Life Nanoscience Laboratory IIT – CNLS, Sapienza University Rome, Rome; 4Infectious Diseases Department, Azienda Ospedaliera S. Maria Nuova-IRCCS, Reggio Emilia; 5Dipartimento Integrato di Medicina e di Specialità Mediche, University of Modena and Reggio Emilia, Modena; 61st Division of Gastroenterology, Fondazione IRCCS Ca” Granda, University of Milan, Milan; 7Department of Internal Medicine (DMISM), Sapienza University Rome, Rome, Italy; 8Cancer Research Center of Lyon (CRCL), INSERM U1052, Lyon, France; 9Biopharmanet-Tec Laboratory, University of Parma, Parma, Italy E-mail: [email protected] Background and Aims: In chronic hepatitis B the HBV-specific T cell response is exhausted and its functional reconstitution may represent a rational way to control infection. Correction of immune check-point dysregulation and other pathological regulatory pathways identified in experimental models of chronic infection only leads to partial restoration of T cell function. Aim of this study was to use transcriptome analysis in HBV-specific T cells to gain a genome-wide view of misregulated genes and pathways associated with T cell exhaustion and to validate them as novel molecular targets for functional rescuing of exhausted T cells. Methods: Gene expression profiling was performed by microarray analysis (Agilent) in HBV-specific CD8 cells from 4 chronic patients, using virus-specific CD8 cells from patients in the acute (#5) and in the resolution (#4) phase of self-limited HBV infections as references. Data-mining was done by hierarchical clustering, ANOVA, GSEA and pathway/gene ontology analyses, followed by qPCR/Nanostring validation of specific gene sets. Functional validation and antiviral activity rescue assays were performed in HBVspecific CD8 cells from 16 chronic HBV patients by flow-cytometry using fluorescently labelled probes/antibodies and target-specific bioactive compounds. Results: 504 genes appeared to be differentially expressed in acute, resolved and chronic patients by ANOVA (corrected p ≤ 0.05); several misregulated biological functions were identified by GSEA in exhausted CD8 cells from chronic patients. In this study we focused on characterization and functional reconstitution of the proteasome function as a means to restore the antiviral T cell activity in exhausted CD8 cells. Enhanced aggresome accumulation confirmed the occurrence of an ubiquitin-proteasome expression defect in exhausted CD8 cells. The proteasome-active polyphenols oleuropein, quercetin and resveratrol improved proteasome function and reduced mitochondrial depolarization as well as ROS production in exhausted CD8 cells, with a concomitant enhancement (1.5- to 7.8-fold) of antiviral cytokine production in response to HBV antigens. Conclusions: Transcriptional and functional profiling of HBV-specific CD8 cells pinpointed the proteasome as a major dysregulated system in exhausted CD8 cells. Proteasome-active polyphenols significantly improved T cell function and thus represent potential candidates for novel immune reconstitution strategies in chronic HBV infection.
Journal of Hepatology 2017 vol. 66 | S1–S32
specific T cell responses were studied before, during (weeks 12 and 24) and after vaccine therapy (week 48) both ex vivo (IFN-γ Elispot) and after 10 days in vitro expansion (intracellular cytokine staining for IFN-γ, TNF-a, IL-2 and CD107 degranulation) in the presence of peptides covering the overall HBV proteome or control HBV unrelated peptides. Immunological data were assessed in relation to HBsAg/HBV-DNA/ALT declines. Results: No patients had a significant HBsAg decline while all normalized ALT and suppressed HBV-DNA. Ex vivo IFN-g Elispot responses were significantly improved upon HBV core peptide stimulation at week 48 compared to baseline. Following in vitro expansion, a significant increase in the percentage of HBV-specific IFN-g and IL2 producing CD3+ T cells was detected at week 24 and 48. This functional improvement was predominantly sustained by CD8+ T cells which showed also an increased production of TNF-a. IFN-g was more improved than IL2 and TNF-a. Maximal stimulatory activity was expressed by polymerase at all time points, while a progressive increase of IFN-g production was induced by core and envelope during combined GS-4774 and NUC administration. A simultaneous improvement of more than one T cell function was detected in 11 of 12 patients; this was associated with an increased percentage of double and triple positive HBV-specific T cells. Conclusions: GS-4774 combined with TDF can improve the T cell function with a prevalent effect on CD8 cells. This seems to be however insufficient to induce a substantial decline of HBsAg which was similar in patients treated with NUC alone or combined with GS-4774. PS-051 Hepatitis B virus-specific T cell receptors with high functional avidity redirect T cells to eliminate HBV K. Wisskirchen1,2, J. Kah2,3, K. Metzger1, L. Weigand4, W. Uckert5, M. Schiemann6, T. Volz3, A. Krackhardt4, M. Dandri3, U. Protzer1. 1 Institute of Virology, Helmholtz Zentrum München / Technische Universität München, Munich; 2both authors contributed equally 3 1. Medical Clinic, University Medical Center Hamburg-Eppendorf, Hamburg; 4III. Medical Clinic, Klinikum rechts der Isar, Munich; 5 Institute of Biology, Molecular Cell Biology and Gene Therapy, MaxDelbrück-Center for Molecular Medicine, Berlin; 6Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany E-mail: [email protected] Background and Aims: Adoptive T-cell therapy of chronic hepatitis B or hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC) intends to restore antiviral T-cell immunity to clear the infection or control tumour growth. We aimed at identifying T-cell receptors (TCR) with high functional avidity to redirect T cells that have the potential to cure HBV infection. Methods: We isolated eleven TCRs that were specific for the HBV S-derived peptides S20 and S172, or for the C18 core-peptide from patients with acute and resolved HBV infection. T cells genetically modified by retroviral transduction to express HBV-specific TCRs were compared in comprehensive functional analyses in vitro and in vivo. Results: Upon TCR grafting, CD8+ as well as CD4+ T cells became polyfunctional HBV-specific effector T cells recognizing even picomolar concentrations of cognate peptide. TCR-grafted T cells secreted IFN gamma, TNF alpha and IL2, controlled HBV replication and effectively killed hepatoma cells with an integrated HBV genome as well as HBV-infected cells. Notably, these HBV-specific TCRs recognized peptide presented on different HLA-A*02 subtypes common in areas with high HBV prevalence. When co-cultured with HBV-infected cells, TCR-transduced T cells eliminated viral antigens as well as the HBV persistence form, the cccDNA. TCRs with the highest functional avidity also mediated elimination of HBV when expressed in CD4+ T cells or in T cells from patients with chronic
Journal of Hepatology 2017 vol. 66 | S1–S32
S29
ORAL PRESENTATIONS hepatitis B. In vivo, TCR-redirected T cells targeted the liver when transferred into HBV-infected uPA-SCID/beige/IL2rg−/− mice repopulated with HLA-A*02-matched primary human hepatocytes. After 5 days, alanine amino transferase levels were moderately increased and T-cell-mediated PHH loss after 15–18 days, when mice were sacrificed, was limited (mean 37% human serum albumin decrease). Intrahepatic analyses revealed a strong reduction of cccDNA loads (>95%) and replication activity, which resulted in a profound reduction of antigenemia, a more than 4 log decrease of virema, and negative HBcAg staining in the liver. Conclusions: Our unique set of HBV-specific TCRs with different affinities represents an interesting tool for elucidating mechanisms of TCR-MHC interaction and dissecting specific anti-HBV mechanisms exerted by T cells. Most importantly, TCR-transduced T cells with high functional avidity are very interesting for adoptive T-cell therapy of chronic hepatitis B and HBV-induced HCC, because they have the potential to mediate clearance of the virus. PS-052 Responsiveness of HBV-specific CD8+ T cells to PD1 pathway blockade is linked to TCF1 expression A. Schuch1,2, D. Wieland1,2,3, M. Hofmann1, R. Thimme1. 1University Hospital Freiburg; 2Albert-Ludwigs-University Freiburg; 3Spemann Graduate School of Biology and Medicine (SGBM), Freiburg, Germany E-mail: [email protected] Background and Aims: Chronic viral infection results in progressive loss of virus-specific CD8+ T-cell effector functions, a phenomenon called T-cell exhaustion that is characterized by upregulation of inhibitory receptors (e.g. PD1). Studies in the LCMV mouse model revealed that exhausted virus-specific CD8+ T cells are heterogeneous and consist of terminally exhausted (PD1hi) and progenitor (PD1int) subsets. As recently published, the latter was defined by expression of the memory marker TCF1 and provided the proliferative burst upon PD1 therapy. In this study we aimed to define subpopulations of exhausted virus-specific CD8+ T cells in human chronic viral hepatitis including their key transcriptional regulators and functional properties, especially in regard to PD1 pathway blockade. Methods: We analyzed HLA-A*02-restricted epitope-specific CD8+ Tcell populations in inactive chronic HBsAg carriers (n = 12) and patients chronically infected with HCV (n = 8). We applied tetramerbased magnetic bead enrichment with subsequent multi-colour flow cytometry. Additionally, we performed peptide-specific in vitro expansion assays for 14 days with and without anti-PDL1 monoclonal antibody to assess proliferative potential and cytokine production. Results: Our data revealed that exhausted epitope-specific CD8+ T cells in chronic HBV and HCV infection are heterogeneous. While the majority of HCV-specific CD8+ T cells display a terminally exhausted phenotype (CD127−PD1hi), virus-specific CD8+ T-cell populations in inactive chronic HBsAg carriers show a phenotype characteristic for progenitor subsets with high expression of the memory markers CD127 and TCF1. TCF1 expression correlated with CD8+ T-cell expansion after HBV and HCV peptide-specific in vitro culture. Interestingly, HBV-specific CD8+ T cells showed an increased polyfunctionality compared to HCV-specific CD8+ T cells, assessed by IFNγ and TNF production upon restimulation after peptide-specific in vitro expansion. Furthermore, HBV-specific CD8+ T cells were highly responsive to PD1 pathway blockade. Importantly, our preliminary data suggests a direct association of TCF1 expression and proliferative burst after PD1 pathway blockade. Conclusions: In sum, HBV-specific CD8+ T-cell populations in chronic HBsAg carriers lack terminally exhausted subsets and are defined by high expression of TCF1. This correlated with enhanced expansion upon PD1 pathway blockade, rendering TCF1 a promising prediction marker for responsiveness to PD1 pathway blockade in immunotherapy.
S30
PS-053 Proteasome dysfunction as a reversible defect underlying virus-specific CD8 cell exhaustion in chronic hepatitis B P. Fisicaro1, V. Barili1, G. Acerbi1, B. Montanini2, D. Laccabue1, F. Guerrieri3, M. Massari4, C. Vandelli5, D. Salerno3, G. Grossi6, A. Alfieri1, P. Lampertico6, T. Giuberti1, G. Missale1, M. Levrero7,8, S. Ottonello2,9, C. Ferrari1. 1Unit of Infectious Diseases and Hepatology, Azienda Ospedaliero-Universitaria of Parma; 2Department of Life Sciences, University of Parma, Parma; 3Center for Life Nanoscience Laboratory IIT – CNLS, Sapienza University Rome, Rome; 4Infectious Diseases Department, Azienda Ospedaliera S. Maria Nuova-IRCCS, Reggio Emilia; 5Dipartimento Integrato di Medicina e di Specialità Mediche, University of Modena and Reggio Emilia, Modena; 61st Division of Gastroenterology, Fondazione IRCCS Ca” Granda, University of Milan, Milan; 7Department of Internal Medicine (DMISM), Sapienza University Rome, Rome, Italy; 8Cancer Research Center of Lyon (CRCL), INSERM U1052, Lyon, France; 9Biopharmanet-Tec Laboratory, University of Parma, Parma, Italy E-mail: [email protected] Background and Aims: In chronic hepatitis B the HBV-specific T cell response is exhausted and its functional reconstitution may represent a rational way to control infection. Correction of immune check-point dysregulation and other pathological regulatory pathways identified in experimental models of chronic infection only leads to partial restoration of T cell function. Aim of this study was to use transcriptome analysis in HBV-specific T cells to gain a genome-wide view of misregulated genes and pathways associated with T cell exhaustion and to validate them as novel molecular targets for functional rescuing of exhausted T cells. Methods: Gene expression profiling was performed by microarray analysis (Agilent) in HBV-specific CD8 cells from 4 chronic patients, using virus-specific CD8 cells from patients in the acute (#5) and in the resolution (#4) phase of self-limited HBV infections as references. Data-mining was done by hierarchical clustering, ANOVA, GSEA and pathway/gene ontology analyses, followed by qPCR/Nanostring validation of specific gene sets. Functional validation and antiviral activity rescue assays were performed in HBVspecific CD8 cells from 16 chronic HBV patients by flow-cytometry using fluorescently labelled probes/antibodies and target-specific bioactive compounds. Results: 504 genes appeared to be differentially expressed in acute, resolved and chronic patients by ANOVA (corrected p ≤ 0.05); several misregulated biological functions were identified by GSEA in exhausted CD8 cells from chronic patients. In this study we focused on characterization and functional reconstitution of the proteasome function as a means to restore the antiviral T cell activity in exhausted CD8 cells. Enhanced aggresome accumulation confirmed the occurrence of an ubiquitin-proteasome expression defect in exhausted CD8 cells. The proteasome-active polyphenols oleuropein, quercetin and resveratrol improved proteasome function and reduced mitochondrial depolarization as well as ROS production in exhausted CD8 cells, with a concomitant enhancement (1.5- to 7.8-fold) of antiviral cytokine production in response to HBV antigens. Conclusions: Transcriptional and functional profiling of HBV-specific CD8 cells pinpointed the proteasome as a major dysregulated system in exhausted CD8 cells. Proteasome-active polyphenols significantly improved T cell function and thus represent potential candidates for novel immune reconstitution strategies in chronic HBV infection.
Journal of Hepatology 2017 vol. 66 | S1–S32