- Email: [email protected]
GRANZYME DEPENDENT CYTOLYSIS
POSTERS cells is regulated by both, soluble factors and cross-talk with other immunocompetent cells. Gradual loss of CD4+ T cells is a hallmark of HIV infection. Here, we analyzed the impact of CD4+ T cells on NK cell anti-fibrotic activity. Methods: A total of 60 individuals (HCV(+), n = 24; HIV(+)/HCV(+), n = 20; healthy controls, n = 16) were enrolled into this study. Purified NK cells were cultured in the presence or absence of supernatants from CD3/CD28-stimulated CD4+ T cells and than coincubated with activated primary human HSC (ScienCell) followed by flowcytometric analysis of HSC apoptosis (detection of active caspase-3), NK cell degranulation (CD107a), and IFN-g secretion. Results: Following incubation with supernatants from CD4+ T cells obtained from healthy controls, NK cells displayed a significantly increased activity against primary HSC as compared to unstimulated NK cells with higher CD107a expression (3.8±0.8% vs. 19.2±1.9%; p < 0.05) and IFN-g secretion (0.3±0.1% vs. 7.4±1.1%; p < 0.05). Moreover, we found a significant increase in NK cell-induced HSC apoptosis (6.1±1% vs. 46.1±4.7%). Blocking experiments suggested an IL-2 mediated mechanism. Anti-HSC activity of NK cells was positively correlated to the number of CD4+ T cells used in the experiments. Of note, supernatants of equivalent numbers of CD4+ T cells from HIV/HCV patients but not from HCV mono-infected patients displayed a significantly reduced ability to trigger anti-HSC activity of NK cells. Accordingly, we found that NK cells from HIV/HCV patients showed a significantly lower ex vivo activity against activated HSC as compared to NK cells from HCV mono-infected patients (10.4±1.5% vs. 17.4±1.8%; p = 0.006). Conclusion: CD4+ T cells can trigger anti-fibrotic NK cell activity via an IL-2 mediated mechanism. Both, HIV-induced loss of CD4+ T cells and impaired IL-2 secretion, may contribute to accelerated progression of liver fibrosis in HIV/HCV infected patients. 783 HBV-SPECIFIC CD8+ T CELLS AND NK CELLS MEDIATE THEIR ANTIVIRAL EFFECT PRIMARILY VIA PERFORIN/GRANZYME DEPENDENT CYTOLYSIS D. Grimm1 , M. Heeg1 , N. Kersting1 , H.E. Blum1 , A. Bertoletti2 , R. Thimme1 . 1 Dept. of Internal Medicine II, University Medical Center Freiburg, Freiburg im Breisgau, Germany; 2 Singapore Institute for Clinical Sciences, Singapore, Singapore E-mail: [email protected] Background and Aims: Hepatitis B virus (HBV) specific CD8+ T cells and NK cells are of major importance in resolution of HBV infection. Findings in HBV-transgenic mice revealed that noncytolytic effector mechanisms are crucial in this model system. However, studies in HBV-infected chimpanzees indicate that cytolytic functions are relevant as well. The relative contribution of cytolytic vs. non-cytolytic effector functions in humans is not fully understood. This study aimed to analyze HBV-specific CD8+ T cell and NK cell effector functions in a human cell culture model. Methods: The HBV producing human hepatoma cell line HepG2.117 was utilized in this study. Co-culture experiments were performed with T cell receptor transduced effector T cells and NK cells. The effect on viral replication was measured by quantitative PCR and verified by southern blot. Cell viability was assessed by ATP and AST quantification as well as by measuring caspase activation. To determine the killing pathways used by HBV-specific CD8+ T cells and NK cells, co-culture assays with respective blocking antibodies were performed. Results: Our results can be summarized as follows: (i) In coculture experiments between HepG2.117 cells and HBV-specific CD8+ T cells and NK cells, a significant suppression of HBV DNA was observed. (ii) The decline in HBV DNA occurred in concert with an increase in AST levels, suggesting the presence of cytolytic
effector functions. (iii) The antiviral effect was lost in transwell experiments, showing that direct cell-cell contact is required for the antiviral effect arguing against a dominant role of non-cytolytic effector functions. This is further supported by our finding that the addition of recombinant IFN-g and/or TNF-a to HepG2.117 cells did not lead to a significant reduction of HBV DNA. (iv) Blocking experiments showed that CD8+ T cells and NK cells mediate this cytolytic function primarily by the perforin/granzyme pathway. Conclusions: Using a novel HepG2.117 based model system of HBV infection we could show that HBV-specific CD8 T cells and NK cells mediate their antiviral effect primarily via perforin/granzyme dependent cytolysis. 784 UNRAVELING THE MECHANISMS OF HEPATITIS C VIRUS (HCV) INTERFERENCE WITH LIPID-RESTRICTED IMMUNITY E. Heeregrave1 , P. Riese2 , N. Bjorkstr ¨ om ¨ 1 , M. Moll1 . 1 Center for Infectious Medicine, Dept. of Medicine, Karolinska Institute, Stockholm, Sweden; 2 Dept. of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany E-mail: [email protected] Background and Aims: CD1d is a MHC-like molecule presenting endogenous and exogenous lipid antigens to lipid-specific natural killer T (NKT) cells. The precise role of lipid-restricted immunity in virus infections is not yet defined, as viral antigens presented by CD1d have not been identified so far. NKT cell activation may instead be triggered by recognition of CD1d-bound endogenous lipids in combination with virus-induced cytokines. HIV-1 and herpesviruses can interfere with CD1d expression levels, impairing potent NKT cell responses. HCV infection results in widespread CD1d upregulation in the liver through a yet obscure mechanism and with unknown consequences. Altered CD1d expression may influence the activation of NKT cells. It is unclear whether these cells are part of a successful antiviral response or contribute to the disease process associated with HCV infection. We aim to unravel the mechanism of HCV-induced CD1d upregulation and determine the consequences for NKT cell function. Methods: We infect the Huh7.5 liver cell line stably expressing CD1d with the recombinant infectious HCV-Jc1 strain. Using flow cytometry and confocal microscopy we will analyze changes in CD1d expression. The consequences for NKT cell activation will be determined in a co-culture assay of Huh7.5 cells with primary NKT cells. Results: Flow cytometry analysis of CD1d surface expression levels revealed a marked difference between uninfected and infected cells in the same culture. Whereas HCV-negative cells showed increased CD1d expression compared to mock-infected cells, HCV-positive cells demonstrated lower CD1d levels. We now aim to identify the responsible factors causing these differential changes in CD1d expression and reveal the consequences for NKT cell activation. Conclusion: The differential effect of HCV infection on CD1d expression in uninfected and infected liver cells may indicate both a role for CD1d-restricted immunity in the antiviral host defense and a novel strategy of HCV to evade immunity. We anticipate that the presented study will contribute considerably to improved insight into the role of lipid-restricted immunity in HCV infection.
Journal of Hepatology 2012 vol. 56 | S225–S388
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effector functions. (iii) The antiviral effect was lost in transwell experiments, showing that direct cell-cell contact is required for the antiviral effect arguing against a dominant role of non-cytolytic effector functions. This is further supported by our finding that the addition of recombinant IFN-g and/or TNF-a to HepG2.117 cells did not lead to a significant reduction of HBV DNA. (iv) Blocking experiments showed that CD8+ T cells and NK cells mediate this cytolytic function primarily by the perforin/granzyme pathway. Conclusions: Using a novel HepG2.117 based model system of HBV infection we could show that HBV-specific CD8 T cells and NK cells mediate their antiviral effect primarily via perforin/granzyme dependent cytolysis. 784 UNRAVELING THE MECHANISMS OF HEPATITIS C VIRUS (HCV) INTERFERENCE WITH LIPID-RESTRICTED IMMUNITY E. Heeregrave1 , P. Riese2 , N. Bjorkstr ¨ om ¨ 1 , M. Moll1 . 1 Center for Infectious Medicine, Dept. of Medicine, Karolinska Institute, Stockholm, Sweden; 2 Dept. of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany E-mail: [email protected] Background and Aims: CD1d is a MHC-like molecule presenting endogenous and exogenous lipid antigens to lipid-specific natural killer T (NKT) cells. The precise role of lipid-restricted immunity in virus infections is not yet defined, as viral antigens presented by CD1d have not been identified so far. NKT cell activation may instead be triggered by recognition of CD1d-bound endogenous lipids in combination with virus-induced cytokines. HIV-1 and herpesviruses can interfere with CD1d expression levels, impairing potent NKT cell responses. HCV infection results in widespread CD1d upregulation in the liver through a yet obscure mechanism and with unknown consequences. Altered CD1d expression may influence the activation of NKT cells. It is unclear whether these cells are part of a successful antiviral response or contribute to the disease process associated with HCV infection. We aim to unravel the mechanism of HCV-induced CD1d upregulation and determine the consequences for NKT cell function. Methods: We infect the Huh7.5 liver cell line stably expressing CD1d with the recombinant infectious HCV-Jc1 strain. Using flow cytometry and confocal microscopy we will analyze changes in CD1d expression. The consequences for NKT cell activation will be determined in a co-culture assay of Huh7.5 cells with primary NKT cells. Results: Flow cytometry analysis of CD1d surface expression levels revealed a marked difference between uninfected and infected cells in the same culture. Whereas HCV-negative cells showed increased CD1d expression compared to mock-infected cells, HCV-positive cells demonstrated lower CD1d levels. We now aim to identify the responsible factors causing these differential changes in CD1d expression and reveal the consequences for NKT cell activation. Conclusion: The differential effect of HCV infection on CD1d expression in uninfected and infected liver cells may indicate both a role for CD1d-restricted immunity in the antiviral host defense and a novel strategy of HCV to evade immunity. We anticipate that the presented study will contribute considerably to improved insight into the role of lipid-restricted immunity in HCV infection.
Journal of Hepatology 2012 vol. 56 | S225–S388
S307