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Regulation of hepatic CD1d-restricted antigen presentation and Natural Killer T cell homeostasis by the microsomal triglyceride transfer protein

Final Report Summary - HEPATOCYTE MTP (Regulation of hepatic CD1d-restricted antigen presentation and Natural Killer T cell homeostasis by the microsomal triglyceride transfer protein)

Introduction and Aims
The presentation of antigens to T-Lymphocytes is a central feature of adaptive immunity. This process includes the presentation of peptides bound to major histocompatibility complex (MHC) class I or II to conventional CD8+ and CD4+ T cells. In addition to this classical pathway of peptide antigen presentation, cells can present lipids bound to atypical MHC class I molecules of the CD1 family including CD1a, CD1b, CD1c, and CD1d in humans as well as CD1d in mice. Interestingly, this mode of antigen presentation is not limited to professional antigen presenting cells (APCs). Thus, hepatocytes as parenchymal cells of the liver abundantly express the lipid antigen-presenting molecule CD1d, while T cells responding to lipids presented in the context of CD1d, which have been termed Natural Killer T (NKT) cells, constitute a major fraction of hepatic immune cells. We have previously made the observation that the microsomal triglyceride transfer protein (MTP), an endoplasmic reticulum-resident protein, is involved in the transfer of lipids onto CD1 molecules and is required for the function of CD1 (Kaser, Eur. J. Immunol. 2008). Based on these observations, we generated mice with an hepatocyte-specific deletion of MTP to characterize the regulation and biological function of CD1d-mediated lipid antigen presentation by hepatocytes.

Results and conclusions:
To study the role of hepatocyte CD1d and MTP in lipid antigen presentation, we developed mice with conditional, tissue-specific deletion of MTP within hepatocytes. Hepatocytes obtained from these mice exhibited severe defects in CD1d-restricted presentation of endogenous (self) and exogenous (microbial-derived) lipids to NKT cells. In accordance with an hepatocyte-specific deletion of MTP, other cell types including thymocytes and splenocytes showed unaltered CD1d-restricted lipid antigen presentation. Moreover, classical MHC class I-restricted presentation of peptide antigens by hepatocytes served as control and was unaffected by deletion of hepatocyte MTP. While the expression of CD1d and its intracellular trafficking seemed unimpaired at first glance in hepatocytes with genetic deletion of MTP, in-depth-analysis revealed that a subset of monoclonal anti-CD1d antibodies, which bind in the proximity of the CD1d antigen binding groove failed to recognize their epitopes, consistent with an altered tertiary structure of CD1d in the absence of MTP-mediated lipid transfer. Biochemical studies indeed revealed instability of the complex of CD1d and β2-microglobulin in the absence of MTP in accordance with structural CD1d defects. Moreover, functional studies revealed increased dissociation of lipids from CD1d in the absence of hepatocyte MTP as well as saturation of CD1d-restricted antigen presentation at lower concentrations of lipid antigen. Both results indicated an inability of proper antigen binding by a large fraction of CD1d molecules in MTP-deficient hepatocytes. Together, these studies have revealed, for the first time, a critical role of MTP in the regulation of hepatocyte CD1d-restricted antigen presentation and have offered unique insight into the mechanisms of CD1d biosynthesis. Importantly, some of these results were confirmed in a parallel study in human cells. As such, we characterized patients with abetalipoproteinemia (ABL), a metabolic disease caused by mutations in MTP. When obtained from patients with ABL, dendritic cells, an APC population expressing all antigen presenting CD1 molecules, exhibited increased proteasomal degradation of CD1a, CD1b, and CD1c, while CD1d – similar to observations in mouse hepatocytes – showed indirect signs of an altered structure including the inability to load lipid antigens (Zeissig et al., J. Clin. Investig. 2010). These results confirmed our studies in mouse hepatocytes and demonstrated that, across different cell types, MTP-mediated lipid transfer is critical for lipid antigen presentation by CD1.

In addition to defining the molecular and cellular regulation of CD1d-restricted lipid antigen presentation by MTP, we aimed to delineate the biological function of CD1d-mediated presentation of lipids by hepatocytes. We first addressed the role of hepatocyte CD1d lipid presentation for the homeostasis of liver NKT cells under constitutive conditions. We observed that genetic deletion of MTP in hepatocytes, which is associated with CD1d dysfunction, leads to increased relative and absolute numbers of hepatic invariant NKT (iNKT) cells, a subgroup of NKT cells expressing a Vα14/Jα18 T cell receptor. Increased hepatic iNKT cell numbers were the consequence of defects in iNKT cell apoptosis, while the proliferation of liver iNKT cells was not affected by hepatocyte-specific deletion of MTP. As such, hepatocyte MTP regulates the homeostasis of liver iNKT cells. In current studies, we are addressing the signaling pathways underlying MTP-dependent regulation of liver iNKT cell apoptosis.

We further studied the role of hepatocyte MTP and CD1d for the function of liver NKT cells in the context of infectious hepatitis. To this end, we investigated Hepatitis B virus (HBV) infection, one of the most common infectious diseases worldwide. These studies revealed an unanticipated crucial role of hepatocyte MTP- and CD1d-restricted lipid antigen presentation in the defense against HBV. Specifically, we could show that HBV infection and recruitment of host endoplasmic reticulum lipids for the viral envelope leads to alterations in the repertoire of endogenous hepatocyte lipid antigens. This is associated with an increased abundance of particular phospholipids including phosphatidylethanolamine (PE) in infected hepatocytes. PE, which binds to CD1d but does not activate NKT cells, is processed by secretory phospholipases induced by HBV infection, which leads to the generation of lysophosphatidylethanolamine (LPE), a lipid which also binds to CD1d but which – in contrast to PE – is highly stimulatory to NKT cells. As such, LPE leads to direct CD1d-dependent activation of NKT cells as well as NKT cell-dependent activation of HBV-specific conventional T and B lymphocytes. In accordance with pronounced defects in CD1d function in the absence of hepatocyte MTP, mice with hepatocyte-specific MTP deletion as well as mice with genetic deletion of CD1d exhibited impaired HBV-specific T- and B cell responses, delayed viral clearance and chronic hepatitis (Zeissig et al., Nat. Med. 2012). Together, these studies reveal an unanticipated crucial role of MTP-, CD1d- and hepatocyte-dependent lipid antigen presentation in the immune response against HBV with significant implications for potential therapeutic strategies for the treatment of chronic Hepatitis B.

Based on the observation of a critical role of MTP in CD1d-restricted lipid antigen presentation, we developed related mouse models with deletion of MTP specifically in intestinal epithelial cells. Using these mice, we could demonstrate that intestinal epithelial lipid antigen presentation plays a critical role in the homeostasis of intestinal NKT cells and the prevention of intestinal inflammation (Olszak et al., Nature 2014).

Socio-economic impact and wider societal implications
Our studies have revealed that the presentation of lipids by hepatocyte CD1d is critically regulated by the lipid transfer protein MTP and is required for the homeostasis of liver NKT cells under constitutive conditions. Moreover, we have uncovered a crucial role of hepatocyte CD1d-restricted lipid antigen presentation in the immune response against HBV infection, a disease that affects more than two billion people worldwide and which is associated with an estimated 400 million people with chronic HBV infection and an estimated one million people who die each year from hepatitis B and its complications. As such, these data revealed by our studies have major implications for the treatment of chronic HBV infection as one of the most frequent diseases worldwide and are likely to be of broad relevance for other viral infection associated with alterations in the repertoire of cellular lipids.