- Email: [email protected]
c Mice Inhibited 25-Hydroxy-vitamin-D3 Alleviation of Liver NK Cytotoxicity
POSTER PRESENTATIONS CD11b/F4/80(+) CCR2 (hi) macrophages. In vitro, OPN knockdown reduced secretion and mRNA of chemokines RANTES, MCP-1 and CXCL1 (by ∼75%, 73% and 41%, respectively). Additionally, RAW264.7 macrophages cultured with conditioned media from 603B treated with OPN-neutralising aptamer exhibited a 35% decrease in migration. Conclusions: OPN is overexpressed in progressive liver disease, and enhances cholangiocyte production of key macrophage chemokines MCP-1, RANTES, and CXCL1. OPN promotes accumulation of macrophages, including a proinflammatory monocyte subset. OPN neutralization attenuates injury and fibrogenic outcomes and is therefore a promising anti-fibrotic strategy. FRI-231 HBV STRENGTHENS INTRAHEPATIC MYELOID-DERIVED CELLS MEDIATED T CELL TOLERANCE THROUGH TLR1/2 INDUCED KUPFFER CELL EXPANSION AND IL-10 PRODUCTION J. Liu1, D. Yang1, M. Lu2. 1Department of Infectious Disease, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; 2Institute for Virology, University Hospital of Essen, University of Duisburg-Essen, Essen, Germany E-mail: [email protected] Background and Aims: Hepatic antigen presenting cells (APCs) play a critical role in promoting immune tolerance in the liver. Recently, we have demonstrated that TLR1/2 stimulation on LSECs reverted their suppressive properties to induce T cell immunity. However, there is a paucity of information about how TLR1/2 stimulation modulates the immunological function of other hepatic APCs. Methods: In current study, we investigated whether TLR1/2 stimulation influences the function of intrahepatic myeloid-derived cells (iMDCs), and elucidated the mechanisms involved in iMDCsinduced T cell immunity. Results: We could show that iMDCs can potently suppress T cell activation in a cell-contact independent manner. Antigen presenting by iMDCs leads to naive CD8 T cell tolerance. To our surprise, instead of inducing cell functional maturation, TLR1/2 ligand P3C stimulation further strengthens the suppressive and tolerogenic properties of iMDCs. After P3C administration, the population of Kupffer cells (KCs) of iMDCs dramatically increased. Mechanism analysis shows that KCs are the essential for the enhanced inhibition of T cell activation by P3C stimulated iMDCs. The iMDCs-mediated CD8 T cell inhibition was mediated by soluble mediators, one of which was interleukin 10 (IL10) secreted by KCs after P3C stimulation. IL-10 blockade could partially abolish iMDCs-mediated T cell inhibition. Moreover, HBV particles stimulation on iMDCs could also induce IL-10 production by the cells. TLR2 knock out dramatically abolished the HBV-induced iMDCs IL-10 production, indicating the possibility that HBV may enroll this mechanism to facilitate its infection. Conclusions: Our results have implications for our understanding of liver-specific tolerance and for the development of strategies to overcome T cell tolerance in situations such as chronic viral liver infections. FRI-232 KIR3DL1 IN COMBINATION WITH HLA-BW4-80T IS ASSOCIATED WITH SUPERIOR FUNCTIONALITY OF NK CELLS AND SPONTANEOUS IMMUNE CONTROL OF HCV INFECTION IN PEOPLE WHO INJECT DRUGS C. Thoens1, T. Senff1, T.J. Hydes2, F.M. Heinemann3, A. Heinold3, M. Heilmann4, M. Uhrberg5, N. Scherbaum4, S. Khakoo2, J. Timm1. 1 Institute for Virology, University Hospital Düsseldorf, Düsseldorf, Germany; 2Clinical and Experimental Sciences, University of Southampton, Southampton, United Kingdom; 3Institute for Transfusion Medicine, University Hospital Essen; 4Addiction Research Group, Rheinische Kliniken Essen, Essen; 5Institute for Transplantation Diagnostics and Cell Therapeutics, University Hospital Düsseldorf, Düsseldorf, Germany E-mail: [email protected]
Background and Aims: NK cell function is regulated by inhibitory and activating receptors including killer-cell immunoglobulin-like receptors (KIRs). HLA class I-molecules act as ligands for KIRs and can be grouped into HLA-C1-alleles, HLA-C2-alleles and HLA class Ialleles carrying the HLA-Bw4 motif. It was previously reported, that homozygosity for HLA-C1 group alleles in combination with KIR2DL3 is associated with a protective effect against HCV infection. In turn, HLA-Bw4 in combination with KIR3DL1 was associated with sustained treatment response of chronic HCV infection. Here, we addressed the impact of different KIR/KIR-ligand genotypes on the outcome of HCV-infection and on NK cell function in people who inject drugs (PWID). Methods: The KIR/KIR-ligand genotype was determined in 151 chronically HCV infected PWID and 64 PWID with resolved HCV infection. NK cells of 30 PWID with chronic HCV infection and 30 PWID with spontaneously resolved HCV infection were characterized by flow cytometry. Results: Multivariate logistic regression analysis revealed that the IL28B CC-genotype ( p = 0.012; OR = 2.63) as well as KIR3DL1/HLABw4-80T ( p = 0.007; OR = 2.90) but not KIR3DL1/HLA-Bw4-80I were associated with spontaneous resolution of HCV infection in PWID. Phenotypic analysis of NK cells from 60 PWID exposed to HCV showed superior functionality (IFN-γ, TNF-α, CD107a) of KIR3DL1+ NK cells from HLA-Bw4-80T-positive donors compared to HLA-Bw6positive and HLA-Bw4-80I-positive donors ( p < 0.05) irrespective of infection outcome. Interestingly, superior NK cell function was determined by KIR3DL1-alleles associated with high expression levels on NK cells whereas KIR3DL1-alleles associated with low expression-levels did not have an influence on NK cell function. Conclusions: KIR3DL1 in combination with HLA-Bw4-80T is associated with spontaneous immune control of HCV infection in PWIDs. Superior functionality of NK cells from KIR3DL1-positive donors carrying the HLA-Bw4-80T-motif irrespective of the HCV infection status suggests that this KIR/KIR-ligand genotype associates with distinct NK cell licensing that may be beneficial for early immune control of HCV infection. FRI-233 MODULATIONS IN VITAMIN D RECEPTOR (VDR) EXPRESSIONS IN LATE FIBROSIS MODEL OF BALB/C MICE INHIBITED 25-HYDROXYVITAMIN-D3 ALLEVIATION OF LIVER NK CYTOTOXICITY A. Salhab1, J. Amer1, R. Safadi1. 1Liver & Gastroenterology Units, Hadassah-Hebrew University Hospital, Jerusalem, Israel E-mail: [email protected] Background and Aims: CD8-subsets mediate hepatic-fibrosis and NK-cells exert anti-fibrotic effects via direct interactions with hepatic-stellate-cells (HSC); mediated by phagocytosis. Low 25Hydroxy-vitamin-D; “25(OH) D3” serum and vitamin D receptor (VDR) levels were recently correlated to advanced fibrosis in Chronic Hepatitis C (CHC). However, vitamin D receptor (VDR) mechanism in liver fibrosis modulations is not well understood. We aimed to evaluate associations of VDR with NK cell modulations during fibrogenesis. Methods: Carbon-tetrachloride (CCl4) hepatic-fibrosis was induced in Balb/c -mice for 4 weeks. Along 1th to 4th weeks; 25 (OH) D3 or sham solutions were i.p injected/2x week. Liver NK cells were isolated and were identified as resident (CD49a+/CD49b−) NK1.1. NK cells were further stained for CD107a (LAMP-1; Lysosomal-associated membrane protein 1) as a marker for activation and analyzed by flowcytometry. Liver proteins were quantified for VDR and aSMA expressions by western blot and RT PCR, respectively. Livers were also assessed histologically and serum ALT levels were estimated. Results: Hepatic fibrosis were gradually increased along 4 weeks injections of CCl4 as indicated by serum ALT levels and aSMA expressions ( p < 0.02) as well as H&E staining of liver necroinflammatory lesions. These results were associated with increased activations of NK1.1 CD107a. While 25(OH) D3 administrations did
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POSTER PRESENTATIONS not modulate fibrosis of early stages (weeks 2 & 3); 25(OH) D3 significantly worsen hepatic-fibrosis of late stage (week 4) as hepatic aSMA expressions and serum ALT levels increased. In week 4, no further activations of NK cells were seen following 25(OH)D3 injections and were associated with down expressions of VDR (Fold-1.7, p < 0.01) indicating the inability of 25(OH)D3 to ameliorate hepatic fibrosis were lost due to modulations in VDR. Conclusions: 25(OH) D3 alleviate liver NK cytotoxicity in early but not in late fibrosis model of Balb/c mice due to modulations in vitamin D receptor. More studies are needed to clarify the role of VDR in attenuation of liver fibrosis. FRI-234 INTERFERON SPARING DIRECTLY ACTING ANTIVIRAL (DAA) THERAPY FOR HCV IN DECOMPENSATED CIRRHOSIS: NATURAL KILLER CELL (NK) PHENOTYPE IS ASSOCIATED WITH NONRESPONSE K. Childs1, E. Merritt2, W. Swanson1, A. Sanchez-Fueyo2, M. MartinezLlordella1,2, K. Agarwal1, I. Carey1. 1Institute of Liver Studies, King’s College Hospital; 2Institute of Liver Studies, King’s College London, London, United Kingdom E-mail: [email protected] Background and Aims: HCV activates type 1 interferon (IFN), upregulating IFN stimulated genes (ISGs) and activating natural killer (NK) cells. Elevated ISG expression and higher expression of the NK activation marker NKp30 predict treatment (Rx) failure to pegIFN. Recent data indicate that nonSVR to DAAs have lower expression of hepatic ISGs on Rx than SVR, reflecting inability of the innate IFN system to control residual HCV. We hypothesised that NK cell phenotype and changes in IP-10, a marker of hepatic ISG, may be associated with SVR to IFN free DAAs.
Methods: The UK Early Access Program gave 12 weeks of DAAs for pts with Childs-Pugh B7 cirrhosis. We recruited those treated Aug-Nov 2014. Pts who were post OLT, HIV+ or transplanted, died or had infective complications were excluded. Of 44 pts, we included 32. EDTA plasma and PBMC were sampled at baseline, treatment week 4 (TW4) and end-of-therapy (TW12). IP-10 was quantified by ELISA, Cytokine profile by cytokine chip assay. NK profile was determined by FACS Results: 24/32 achieved SVR and 8/32 relapsed post Rx (nonSVR). HCV RNA was undetectable in all by week 10. Results are shown in Table 1. IP-10 levels were higher at baseline in nonSVR. In SVR pts, most showed a rise in IP-10 from baseline to TW4 whereas in nonSVR IP-10 decreased in 100%.(p = 0.002 IP-10 fold change). There was no difference in serum IFNγ, L1, 2, 3 or other cytokines except for TGFB which was higher at TW4 in nonSVR. NK phenotype differed between SVR and nonSVR. SVR had a higher proportion of NK and lower NKp30+ NK expression at all timepoints. The pre-Rx CD56-CD16+ subset was significantly higher in SVR compared to non-SVR. By TW12 the median CD56-CD16+ had decreased in SVR but increased in non-SVR ( p = 0.001 for fold change) Conclusions: In nonSVR we saw higher baseline IP-10 and NKp30+ NK. IP-10 fell at TW4 but increased in the majority of SVR. TGF B mediates HCV induced inhibition of NK function; we found lower TGFB and higher CD56br (IFNγ producing) and CD56dim (cytotoxic) in SVR pts on Rx. The CD56-/CD16+ subset was greater in SVR at baseline than nonSVR. This is a dysfunctional NK subset with a loss of polyfunctionality likened to exhaustion seen in T-cells. This population decreased in SVR by the end of Rx but increased in nonSVR. We show that response to IFN free therapy is associated with higher levels of NK cells, increased IFN signalling and a decrease in CD56- NK on Rx. These parameters could help to identify pts at risk of non-SVR, permitting an extended duration of therapy. FRI-235 VACCINE-INDUCED HCV-SPECIFIC CD8+ T CELL RESPONSE RESTRICTED BY THE PROTECTIVE HLA ALLELE B*27: BROAD CROSSRECOGNITION OF EVOLVING VIRAL VARIANTS K. Frohnmüller1, L. Swadling2, K. Nitschke1, P. Klenerman2, R. Thimme1, E. Barnes2, C. Neumann-Haefelin1. 1Department of Medicine II, University of Freiburg, Freiburg, Germany; 2Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom E-mail: [email protected] Background and Aims: The HLA allele B*27 provides protection from the development of persistent infection after hepatitis C virus (HCV) exposure. This protective effect has been linked to a broad crossrecognition of evolving viral variants by the HLA-B*27 restricted CD8+ T cell response targeting an immunodominant and conserved viral epitope. Vaccine-induced CD8+ T cell responses with the capability to cross-recognize evolving viral variants may mediate effective protective immunity against persistent HCV infection upon challenge. We thus aimed to characterize in detail the HLA-B*27 restricted immune response induced in a human phase-I prophylactic HCV T cell vaccine trial. Methods: HLA-B*27+ healthy volunteers (n = 5) were vaccinated with a simian adenovirus prime/modified vaccinia Ankara boost regimen expressing the HCV proteins NS3 to NS5B in a prime/boost strategy given 8 weeks apart (AdCh3-NSmut at 2.5 × 1010 viral particles and MVA-NSmut at 2 × 107 (n = 2) – 2 × 108 (n = 3) plaque forming units). Peptide/HLA multimer staining and multiparametric functional and phenotypic functional analysis staining including intracellular interferon-gamma staining was performed, using 14 peptides corresponding to viral epitope variants that had been observed in persistently HCV infected patients positive for HLA-B*27 previously. Results: 4 of the 5 HLA-B*27+ vaccinees displayed strong ex vivo multimer responses targeting the immunodominant HLA-B*27
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Background and Aims: NK cell function is regulated by inhibitory and activating receptors including killer-cell immunoglobulin-like receptors (KIRs). HLA class I-molecules act as ligands for KIRs and can be grouped into HLA-C1-alleles, HLA-C2-alleles and HLA class Ialleles carrying the HLA-Bw4 motif. It was previously reported, that homozygosity for HLA-C1 group alleles in combination with KIR2DL3 is associated with a protective effect against HCV infection. In turn, HLA-Bw4 in combination with KIR3DL1 was associated with sustained treatment response of chronic HCV infection. Here, we addressed the impact of different KIR/KIR-ligand genotypes on the outcome of HCV-infection and on NK cell function in people who inject drugs (PWID). Methods: The KIR/KIR-ligand genotype was determined in 151 chronically HCV infected PWID and 64 PWID with resolved HCV infection. NK cells of 30 PWID with chronic HCV infection and 30 PWID with spontaneously resolved HCV infection were characterized by flow cytometry. Results: Multivariate logistic regression analysis revealed that the IL28B CC-genotype ( p = 0.012; OR = 2.63) as well as KIR3DL1/HLABw4-80T ( p = 0.007; OR = 2.90) but not KIR3DL1/HLA-Bw4-80I were associated with spontaneous resolution of HCV infection in PWID. Phenotypic analysis of NK cells from 60 PWID exposed to HCV showed superior functionality (IFN-γ, TNF-α, CD107a) of KIR3DL1+ NK cells from HLA-Bw4-80T-positive donors compared to HLA-Bw6positive and HLA-Bw4-80I-positive donors ( p < 0.05) irrespective of infection outcome. Interestingly, superior NK cell function was determined by KIR3DL1-alleles associated with high expression levels on NK cells whereas KIR3DL1-alleles associated with low expression-levels did not have an influence on NK cell function. Conclusions: KIR3DL1 in combination with HLA-Bw4-80T is associated with spontaneous immune control of HCV infection in PWIDs. Superior functionality of NK cells from KIR3DL1-positive donors carrying the HLA-Bw4-80T-motif irrespective of the HCV infection status suggests that this KIR/KIR-ligand genotype associates with distinct NK cell licensing that may be beneficial for early immune control of HCV infection. FRI-233 MODULATIONS IN VITAMIN D RECEPTOR (VDR) EXPRESSIONS IN LATE FIBROSIS MODEL OF BALB/C MICE INHIBITED 25-HYDROXYVITAMIN-D3 ALLEVIATION OF LIVER NK CYTOTOXICITY A. Salhab1, J. Amer1, R. Safadi1. 1Liver & Gastroenterology Units, Hadassah-Hebrew University Hospital, Jerusalem, Israel E-mail: [email protected] Background and Aims: CD8-subsets mediate hepatic-fibrosis and NK-cells exert anti-fibrotic effects via direct interactions with hepatic-stellate-cells (HSC); mediated by phagocytosis. Low 25Hydroxy-vitamin-D; “25(OH) D3” serum and vitamin D receptor (VDR) levels were recently correlated to advanced fibrosis in Chronic Hepatitis C (CHC). However, vitamin D receptor (VDR) mechanism in liver fibrosis modulations is not well understood. We aimed to evaluate associations of VDR with NK cell modulations during fibrogenesis. Methods: Carbon-tetrachloride (CCl4) hepatic-fibrosis was induced in Balb/c -mice for 4 weeks. Along 1th to 4th weeks; 25 (OH) D3 or sham solutions were i.p injected/2x week. Liver NK cells were isolated and were identified as resident (CD49a+/CD49b−) NK1.1. NK cells were further stained for CD107a (LAMP-1; Lysosomal-associated membrane protein 1) as a marker for activation and analyzed by flowcytometry. Liver proteins were quantified for VDR and aSMA expressions by western blot and RT PCR, respectively. Livers were also assessed histologically and serum ALT levels were estimated. Results: Hepatic fibrosis were gradually increased along 4 weeks injections of CCl4 as indicated by serum ALT levels and aSMA expressions ( p < 0.02) as well as H&E staining of liver necroinflammatory lesions. These results were associated with increased activations of NK1.1 CD107a. While 25(OH) D3 administrations did
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POSTER PRESENTATIONS not modulate fibrosis of early stages (weeks 2 & 3); 25(OH) D3 significantly worsen hepatic-fibrosis of late stage (week 4) as hepatic aSMA expressions and serum ALT levels increased. In week 4, no further activations of NK cells were seen following 25(OH)D3 injections and were associated with down expressions of VDR (Fold-1.7, p < 0.01) indicating the inability of 25(OH)D3 to ameliorate hepatic fibrosis were lost due to modulations in VDR. Conclusions: 25(OH) D3 alleviate liver NK cytotoxicity in early but not in late fibrosis model of Balb/c mice due to modulations in vitamin D receptor. More studies are needed to clarify the role of VDR in attenuation of liver fibrosis. FRI-234 INTERFERON SPARING DIRECTLY ACTING ANTIVIRAL (DAA) THERAPY FOR HCV IN DECOMPENSATED CIRRHOSIS: NATURAL KILLER CELL (NK) PHENOTYPE IS ASSOCIATED WITH NONRESPONSE K. Childs1, E. Merritt2, W. Swanson1, A. Sanchez-Fueyo2, M. MartinezLlordella1,2, K. Agarwal1, I. Carey1. 1Institute of Liver Studies, King’s College Hospital; 2Institute of Liver Studies, King’s College London, London, United Kingdom E-mail: [email protected] Background and Aims: HCV activates type 1 interferon (IFN), upregulating IFN stimulated genes (ISGs) and activating natural killer (NK) cells. Elevated ISG expression and higher expression of the NK activation marker NKp30 predict treatment (Rx) failure to pegIFN. Recent data indicate that nonSVR to DAAs have lower expression of hepatic ISGs on Rx than SVR, reflecting inability of the innate IFN system to control residual HCV. We hypothesised that NK cell phenotype and changes in IP-10, a marker of hepatic ISG, may be associated with SVR to IFN free DAAs.
Methods: The UK Early Access Program gave 12 weeks of DAAs for pts with Childs-Pugh B7 cirrhosis. We recruited those treated Aug-Nov 2014. Pts who were post OLT, HIV+ or transplanted, died or had infective complications were excluded. Of 44 pts, we included 32. EDTA plasma and PBMC were sampled at baseline, treatment week 4 (TW4) and end-of-therapy (TW12). IP-10 was quantified by ELISA, Cytokine profile by cytokine chip assay. NK profile was determined by FACS Results: 24/32 achieved SVR and 8/32 relapsed post Rx (nonSVR). HCV RNA was undetectable in all by week 10. Results are shown in Table 1. IP-10 levels were higher at baseline in nonSVR. In SVR pts, most showed a rise in IP-10 from baseline to TW4 whereas in nonSVR IP-10 decreased in 100%.(p = 0.002 IP-10 fold change). There was no difference in serum IFNγ, L1, 2, 3 or other cytokines except for TGFB which was higher at TW4 in nonSVR. NK phenotype differed between SVR and nonSVR. SVR had a higher proportion of NK and lower NKp30+ NK expression at all timepoints. The pre-Rx CD56-CD16+ subset was significantly higher in SVR compared to non-SVR. By TW12 the median CD56-CD16+ had decreased in SVR but increased in non-SVR ( p = 0.001 for fold change) Conclusions: In nonSVR we saw higher baseline IP-10 and NKp30+ NK. IP-10 fell at TW4 but increased in the majority of SVR. TGF B mediates HCV induced inhibition of NK function; we found lower TGFB and higher CD56br (IFNγ producing) and CD56dim (cytotoxic) in SVR pts on Rx. The CD56-/CD16+ subset was greater in SVR at baseline than nonSVR. This is a dysfunctional NK subset with a loss of polyfunctionality likened to exhaustion seen in T-cells. This population decreased in SVR by the end of Rx but increased in nonSVR. We show that response to IFN free therapy is associated with higher levels of NK cells, increased IFN signalling and a decrease in CD56- NK on Rx. These parameters could help to identify pts at risk of non-SVR, permitting an extended duration of therapy. FRI-235 VACCINE-INDUCED HCV-SPECIFIC CD8+ T CELL RESPONSE RESTRICTED BY THE PROTECTIVE HLA ALLELE B*27: BROAD CROSSRECOGNITION OF EVOLVING VIRAL VARIANTS K. Frohnmüller1, L. Swadling2, K. Nitschke1, P. Klenerman2, R. Thimme1, E. Barnes2, C. Neumann-Haefelin1. 1Department of Medicine II, University of Freiburg, Freiburg, Germany; 2Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom E-mail: [email protected] Background and Aims: The HLA allele B*27 provides protection from the development of persistent infection after hepatitis C virus (HCV) exposure. This protective effect has been linked to a broad crossrecognition of evolving viral variants by the HLA-B*27 restricted CD8+ T cell response targeting an immunodominant and conserved viral epitope. Vaccine-induced CD8+ T cell responses with the capability to cross-recognize evolving viral variants may mediate effective protective immunity against persistent HCV infection upon challenge. We thus aimed to characterize in detail the HLA-B*27 restricted immune response induced in a human phase-I prophylactic HCV T cell vaccine trial. Methods: HLA-B*27+ healthy volunteers (n = 5) were vaccinated with a simian adenovirus prime/modified vaccinia Ankara boost regimen expressing the HCV proteins NS3 to NS5B in a prime/boost strategy given 8 weeks apart (AdCh3-NSmut at 2.5 × 1010 viral particles and MVA-NSmut at 2 × 107 (n = 2) – 2 × 108 (n = 3) plaque forming units). Peptide/HLA multimer staining and multiparametric functional and phenotypic functional analysis staining including intracellular interferon-gamma staining was performed, using 14 peptides corresponding to viral epitope variants that had been observed in persistently HCV infected patients positive for HLA-B*27 previously. Results: 4 of the 5 HLA-B*27+ vaccinees displayed strong ex vivo multimer responses targeting the immunodominant HLA-B*27
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Journal of Hepatology 2016 vol. 64 | S425–S630