Liver Immunology group
Lab head: Patrick Bertolino
Location: Liver Immunology Program, Centenary Institute
Liver Immunology: The liver has fascinating immunological properties. In animal models of transplantation, livers grafted across MHC barriers do not result in rejection and are able to prevent rejection of a different organ from the same donor, suggesting that the liver has induced donor-specific tolerance. This bias toward tolerance is advantageous in transplantation as it promotes graft acceptance. However, it can be detrimental when pathogens infecting the liver, such as malaria, Hepatitis B and C viruses, induce tolerance and evade immune responses, leading to chronic infection. Understanding how the liver induces tolerance will reveal new strategies to boost immune responses to clear liver pathogens, or to increase the survival of solid organ transplants.
Research in the group: Our group is recognized internationally for contributions to this field. Using a variety of transgenic mouse models, we were the first to demonstrate that naïve CD8 T cells can be directly activated in the liver. We have now evidence that the liver can also support activation of naïve CD4 T cells. These are unique findings, as it was thought that naïve T cells could only be activated in lymphoid organs. We showed that unlike CD8 T cells activated in lymphoid tissues, liver-activated T cells die by apoptosis, leading to tolerance. The fate of CD4 T cells activated in the liver is totally unknown. Recently, we discovered a different mechanism that the liver uses to delete liver-activated CD8 T cells. This mechanism was very efficient, resulting in rapid clearance of T cells within a few hours. T cells were found to invade hepatocytes (the main liver cells), and were subsequently degraded within lysosomal compartments. This is a novel cellular phenomenon, which will open up new ways to understand immune cell regulation by the liver. The two projects available in our group for 2015 will use a new generation of recombinant adeno-associated virus (AAV) vectors able to transduce 100% of mouse hepatocytes in vivo. These recently developed vectors represent new powerful tools to investigate the phenotype, function and fate of T cells activated intrahepatically.
Funding: approx 500K per year
Project 2 (2014): Preventing T cell degradation in hepatocytes by blocking the endosomal pathway
Primary supervisor: Patrick Bertolino
Background and aims: We have recently showed that following activation in the liver, CD8 T cells invade hepatocytes and are degraded in lysosomal compartments. This novel mechanism opens up new ways to explain the phenomenon of liver tolerance. Currently, we do not know how to inhibit this process and rescue T cells from degradation, a strategy that might be used to rescue T cells for fighting liver pathogens. In this project, you will use recombinant AAV to express dominant negative forms of Rab5 and Rab7, two molecules that interfere with formation of late endosomes and lysosomes in hepatocytes to test if T cell degradation can be prevented. The molecules are fluorescently-tagged and have been recently developed in our group. They represent new tools to visualize T cell fate within these hepatocyte vesicles. This is currently investigated and the detail of the project will depend on progress made when you will start.
1. Analyse whether purified hepatocytes from mice injected with rAAV vectors accumulate or exclude fluorescently tagged transgenic T cells in vitro. The formation of vesicles decorated with Rab5 and Rab7 will be assessed by imaging cells using confocal microscopy.
2. Adoptively transfer transgenic T cells in mice expressing the antigen recognized by the TCR and transduced with rAAV vectors expressing DN RAb5 and Rab7, then analyse the phenotype and fate of transgenic T cells. Measure and visualize liver damage by rescued T cells.
3. If transgenic T cell are rescued following rAAV transduction, it will be important to analyse whether rAAV-treated livers also accumulate endogenous CD8 T cells. This will be done by injecting mice with rAAV vectors in the absence of transgenic T cells and wait for a few weeks before analyzing liver leucocytes. Liver damage and onset of autoimmunity in the liver and other tissues will be tested.
Expected outcomes: We expect to inhibit formation of lysosomes within hepatocytes based on published findings on activities of the dominant negative mutants. We predict that inhibiting the ability of hepatocytes to degrade T cells will lead to T cell rescue. The rescued T cells might survive, expand and induce liver damage.
Why is this project important? This project investigates a newly described phenomenon by our group, published in 2011 in Proc. Nat. Acad. Sci. USA. As we are the first to describe this process in hepatocytes, much is still to be learnt about the underlying molecular pathway. Being able to inhibit this pathway, might (a) reveal a novel physiological role for the liver in peripheral tolerance; (b) prevent degradation of T cells specific for liver pathogens leading to enhanced immunity to these pathogens.
Techniques you will learn for the project: This project will give you experience in mouse handling and organ harvesting; multicolour flow cytometry; immunohistochemistry; and confocal microscopy. Experience in these techniques are invaluable for a career in immunology research. You will use recombinant AAV vectors that are currently in clinical trials for gene therapy in a range of conditions. There is potential for both projects to evolve into a PhD research project for the right candidate.
Discipline: Infectious diseases and Immunology
Co-supervisors: Frederic Sierro, David Bowen
Keywords: Liver, T-cells, tolerance, transplantation, AAV vectors, gene therapy