Final Report Summary - LIVER IVM (Visualizing liver immunopathology progression during hepatitis B infection by liver Intravital Microscopy (IVM))
1.1. Summary description of the project objectives
HBV infection: epidemiology and therapy. HBV infection is one of the major medical scourges of our time. Over one third of the world's population has been infected by HBV at some point in their lives. While most of these people have successfully cleared the virus, more than 350 million people remain persistently infected. Since HBV can be naturally controlled by the immune system, there is a general consensus in the scientific community that new immune therapeutic strategies should be explored for the treatment of chronic HBV infection. Comprising the use of therapeutic T cell vaccines and the infusion of autologous, virus-specific effector CD8 T cells expanded ex vivo. Undoubtedly, the implementation of these strategies will greatly benefit from a clearer understanding of the mechanisms by which CD8 T cells exert their effector functions in vivo.
HBV replicates noncytopathically in the hepatocyte, so most of the clinical syndromes associated with this infection reflect the immune response. It is also apparent that the innate immune response does not contribute significantly to the pathogenesis of liver disease or viral clearance, while the adaptive immune response, especially the virus-specific CD8 T cell response, contributes to both. There is also clear evidence that CD8 T cell effector functions play a prominent role in the resolution of HBV infection and that viral persistence reflects the failure to induce or maintain these CD8 T cell-dependent events. Moreover, a quantitatively and qualitatively dysfunctional effector CD8 T cell response that fails to clear HBV infection creates a smoldering, chronic necroinflammatory process the end results of which are liver fibrosis/cirrhosis and HCC.
In spite of these accomplishments, however, a number of fundamental issues pertaining to the pathogenesis of HBV infection remain unanswered, to address them is the objective of this project. Up until now, the visualization of CD8 T cell- induced pathology has been limited to static imaging of sectioned tissues. These approaches are simply inadequate to achieve the main goals of this project:
A. Unravel the spatiotemporal dynamics by which HBV-specific effector CD8 cells recognize antigen within the liver
B. Understand how CD8 cells kill infected hepatocytes within the liver.
C. Learn how such processes are affected by the anatomical and hemodynamical changes that characterize fibrosis/cirrhosis.
To do so, this project took advantage of:
• Mouse effector CD8 T cells that are specific for the hepatitis B core antigen (HBcAg) or surface antigen (HBsAg), restricted by the H2b or H2d molecule
• HBV replicating competent transgenic mice.
• AAV vectors that specifically and efficiently transduce murine hepatocytes
• Mouse models of fibrosis/cirrosis
• Molecular and cellular techniques
• State-of-the-art multicolor intravital microscopy (IVM)
1.2 Description of the work performed since the beginning of the Project
To address the above mentioned aims, AAV vectors pseudoserotyped with capsid 8 were designed and constructed, containing hepatitis B (HBV)-derived antigens and fluorescent reporter genes, driven by the liver-specific promoter (alpha1 antitrypsin) and the albumin enhancer to establish efficient and long-term expression in hepatocytes in vivo. After in vitro and in vivo validation, these vectors were produced in large scale.
These novel vectors were injected at different doses in wild type mice, achieving different levels of transduced hepatocytes, and then adoptively transferred with TCR transgenic CD8 T cells specific for HBV antigens (HBV core or HBV surface Ag). Using state-of-the-art intravital microscopy we were able to visualize and characterize real-time interactions of HBV-specific effector CD8T cells with HBV-expressing hepatocytes.
1.3 Description of the main results achieved so far
In our study we coupled advanced imaging techniques and mouse models of HBV pathogenesis to reveal previously unappreciated determinants that regulate the migration and function of virus-specific CD8 TE within the liver. We found that, in contrast to what has been described for most other organs (where the arrest of CD8 TE is mainly restricted to postcapillary venules, it is promoted by inflammation and it involves various combinations of selectins, chemokines and integrins), CD8 TE circulating through the liver initially arrest within sinusoids, and they do so independently of inflammation-induced endothelial activation or the specific contribution of selectins, Gai-coupled chemokine receptors, b2- and a4-integrins, PECAM-1 and VAP-1or antigen (Ag) recognition; rather, they hijack platelets that have adhered to sinusoidal hyaluronan via CD44. Following the initial platelet-mediated homing, CD8 TE crawl along liver sinusoids in search of Ag; hepatocellular Ag recognition leading to cytokine production and target cell killing occurs in a diapedesis-independent manner via protrusions that intravascular CD8 TE extend through sinusoidal fenestrae. These findings reveal previously unappreciated determinants regulating the trafficking and function of CD8 TE within the liver and extend our understanding of the mechanisms whereby these cells contribute to the pathogenesis of viral hepatitis.
Importantly, our data show that the intrasinusoidal crawling behavior represents a form of immune surveillance, since it ceases following hepatocellular Ag recognition. Indeed, virus-specific CD8 TE were shown to recognize hepatocellular Ags (i.e. they produced IFN-γ) and perform pathogenic functions (i.e. they killed HBV-expressing hepatocytes) while remaining intravascular. These processes were mediated by cellular protrusions that intravascular CD8 TE extend through sinusoidal fenestrae, producing contact sites with the hepatocyte membrane that are compatible in size with the formation of an immunological synapse.
1.4 Expected final results and their potential impact and use
This work will have significant influence particularly within the field of viral hepatitis, thus constituting an important advance in key issues of immunity in liver infectious diseases. Altogether, the data described herein reveal novel dynamic determinants regulating the trafficking and the effector function of CD8 TE recognizing hepatocellular Ags. This is particularly relevant for the pathogenesis of HBV or HCV infection, as these highly noncytopathic viruses replicate almost exclusively in the hepatocyte and cause acute or chronic liver disease that are triggered by virus-specific CD8 TE. Moreover, these results also suggest a potential mechanism whereby advanced liver fibrosis (a condition where extracellular matrix gets deposited within the space of Disse causing a functional blockade of sinusoidal fenestrae) might reduce CD8 TE immune surveillance towards transformed hepatocytes and, thus, favor the development of hepatocellular carcinoma (HCC). This is a critical discovery, as HCC is diagnosed to more than half a million people worldwide. Most cases (85%) appear in developing countries, with highest incidence rates in regions endemic with hepatitis B or C virus (HBV or HCV).
We are convinced that our findings could be applied to the design of new immune therapeutic strategies for the treatment of chronic HBV infection and its complications. We also believe that these results will have far-reaching implications, also in other pathological conditions of the liver where effector CD8 T cells play crucial pathogenic/curative roles (autoimmunity, graft rejection, different cancers, metastasis).
Moreover, during this project we have also started to study the role of interleukin (IL)-10. Although IL-10 is generally regarded as a cytokine that represses proinflammatory responses and limits unnecessary tissue disruption caused by inflammation, the role that this cytokine plays in liver immunopathology is poorly understood. By using our model that is independent of T cell priming, i.e. the adoptive transfer of hepatitis B virus (HBV)-specific effector CD8+ T cells (CD8 TE) into HBV replication-competent transgenic mice, we are unraveling that IL-10 promotes, rather than suppresses, liver immunopathology. In this experimental setup, IL-10 is exclusively produced by the transferred CD8 TE upon in vivo hepatocellular antigen recognition and acts in an autocrine/paracrine fashion to prevent CD8 TE apoptosis. Altogether, these data will extend our understanding of the tissue- and context-specific role that IL-10 exerts in immune regulation.
HBV infection: epidemiology and therapy. HBV infection is one of the major medical scourges of our time. Over one third of the world's population has been infected by HBV at some point in their lives. While most of these people have successfully cleared the virus, more than 350 million people remain persistently infected. Since HBV can be naturally controlled by the immune system, there is a general consensus in the scientific community that new immune therapeutic strategies should be explored for the treatment of chronic HBV infection. Comprising the use of therapeutic T cell vaccines and the infusion of autologous, virus-specific effector CD8 T cells expanded ex vivo. Undoubtedly, the implementation of these strategies will greatly benefit from a clearer understanding of the mechanisms by which CD8 T cells exert their effector functions in vivo.
HBV replicates noncytopathically in the hepatocyte, so most of the clinical syndromes associated with this infection reflect the immune response. It is also apparent that the innate immune response does not contribute significantly to the pathogenesis of liver disease or viral clearance, while the adaptive immune response, especially the virus-specific CD8 T cell response, contributes to both. There is also clear evidence that CD8 T cell effector functions play a prominent role in the resolution of HBV infection and that viral persistence reflects the failure to induce or maintain these CD8 T cell-dependent events. Moreover, a quantitatively and qualitatively dysfunctional effector CD8 T cell response that fails to clear HBV infection creates a smoldering, chronic necroinflammatory process the end results of which are liver fibrosis/cirrhosis and HCC.
In spite of these accomplishments, however, a number of fundamental issues pertaining to the pathogenesis of HBV infection remain unanswered, to address them is the objective of this project. Up until now, the visualization of CD8 T cell- induced pathology has been limited to static imaging of sectioned tissues. These approaches are simply inadequate to achieve the main goals of this project:
A. Unravel the spatiotemporal dynamics by which HBV-specific effector CD8 cells recognize antigen within the liver
B. Understand how CD8 cells kill infected hepatocytes within the liver.
C. Learn how such processes are affected by the anatomical and hemodynamical changes that characterize fibrosis/cirrhosis.
To do so, this project took advantage of:
• Mouse effector CD8 T cells that are specific for the hepatitis B core antigen (HBcAg) or surface antigen (HBsAg), restricted by the H2b or H2d molecule
• HBV replicating competent transgenic mice.
• AAV vectors that specifically and efficiently transduce murine hepatocytes
• Mouse models of fibrosis/cirrosis
• Molecular and cellular techniques
• State-of-the-art multicolor intravital microscopy (IVM)
1.2 Description of the work performed since the beginning of the Project
To address the above mentioned aims, AAV vectors pseudoserotyped with capsid 8 were designed and constructed, containing hepatitis B (HBV)-derived antigens and fluorescent reporter genes, driven by the liver-specific promoter (alpha1 antitrypsin) and the albumin enhancer to establish efficient and long-term expression in hepatocytes in vivo. After in vitro and in vivo validation, these vectors were produced in large scale.
These novel vectors were injected at different doses in wild type mice, achieving different levels of transduced hepatocytes, and then adoptively transferred with TCR transgenic CD8 T cells specific for HBV antigens (HBV core or HBV surface Ag). Using state-of-the-art intravital microscopy we were able to visualize and characterize real-time interactions of HBV-specific effector CD8T cells with HBV-expressing hepatocytes.
1.3 Description of the main results achieved so far
In our study we coupled advanced imaging techniques and mouse models of HBV pathogenesis to reveal previously unappreciated determinants that regulate the migration and function of virus-specific CD8 TE within the liver. We found that, in contrast to what has been described for most other organs (where the arrest of CD8 TE is mainly restricted to postcapillary venules, it is promoted by inflammation and it involves various combinations of selectins, chemokines and integrins), CD8 TE circulating through the liver initially arrest within sinusoids, and they do so independently of inflammation-induced endothelial activation or the specific contribution of selectins, Gai-coupled chemokine receptors, b2- and a4-integrins, PECAM-1 and VAP-1or antigen (Ag) recognition; rather, they hijack platelets that have adhered to sinusoidal hyaluronan via CD44. Following the initial platelet-mediated homing, CD8 TE crawl along liver sinusoids in search of Ag; hepatocellular Ag recognition leading to cytokine production and target cell killing occurs in a diapedesis-independent manner via protrusions that intravascular CD8 TE extend through sinusoidal fenestrae. These findings reveal previously unappreciated determinants regulating the trafficking and function of CD8 TE within the liver and extend our understanding of the mechanisms whereby these cells contribute to the pathogenesis of viral hepatitis.
Importantly, our data show that the intrasinusoidal crawling behavior represents a form of immune surveillance, since it ceases following hepatocellular Ag recognition. Indeed, virus-specific CD8 TE were shown to recognize hepatocellular Ags (i.e. they produced IFN-γ) and perform pathogenic functions (i.e. they killed HBV-expressing hepatocytes) while remaining intravascular. These processes were mediated by cellular protrusions that intravascular CD8 TE extend through sinusoidal fenestrae, producing contact sites with the hepatocyte membrane that are compatible in size with the formation of an immunological synapse.
1.4 Expected final results and their potential impact and use
This work will have significant influence particularly within the field of viral hepatitis, thus constituting an important advance in key issues of immunity in liver infectious diseases. Altogether, the data described herein reveal novel dynamic determinants regulating the trafficking and the effector function of CD8 TE recognizing hepatocellular Ags. This is particularly relevant for the pathogenesis of HBV or HCV infection, as these highly noncytopathic viruses replicate almost exclusively in the hepatocyte and cause acute or chronic liver disease that are triggered by virus-specific CD8 TE. Moreover, these results also suggest a potential mechanism whereby advanced liver fibrosis (a condition where extracellular matrix gets deposited within the space of Disse causing a functional blockade of sinusoidal fenestrae) might reduce CD8 TE immune surveillance towards transformed hepatocytes and, thus, favor the development of hepatocellular carcinoma (HCC). This is a critical discovery, as HCC is diagnosed to more than half a million people worldwide. Most cases (85%) appear in developing countries, with highest incidence rates in regions endemic with hepatitis B or C virus (HBV or HCV).
We are convinced that our findings could be applied to the design of new immune therapeutic strategies for the treatment of chronic HBV infection and its complications. We also believe that these results will have far-reaching implications, also in other pathological conditions of the liver where effector CD8 T cells play crucial pathogenic/curative roles (autoimmunity, graft rejection, different cancers, metastasis).
Moreover, during this project we have also started to study the role of interleukin (IL)-10. Although IL-10 is generally regarded as a cytokine that represses proinflammatory responses and limits unnecessary tissue disruption caused by inflammation, the role that this cytokine plays in liver immunopathology is poorly understood. By using our model that is independent of T cell priming, i.e. the adoptive transfer of hepatitis B virus (HBV)-specific effector CD8+ T cells (CD8 TE) into HBV replication-competent transgenic mice, we are unraveling that IL-10 promotes, rather than suppresses, liver immunopathology. In this experimental setup, IL-10 is exclusively produced by the transferred CD8 TE upon in vivo hepatocellular antigen recognition and acts in an autocrine/paracrine fashion to prevent CD8 TE apoptosis. Altogether, these data will extend our understanding of the tissue- and context-specific role that IL-10 exerts in immune regulation.