Periodic Reporting for period 2 - RegNK (Regulation of NK cell function)
Periodo di rendicontazione: 2022-06-01 al 2023-11-30
The immune system has evolved sophisticated mechanisms to recognize and eliminate infected, stressed or tumor cells. These processes have to be tightly regulated, balancing immune responsiveness to pathogens against excessive immune activation and pathological responses against self, that result in autoimmune diseases. Presentation of antigens by HLA class I (HLA-I) molecules enables the identification of diseased cells by diversified antigen-specific T cells receptors, while loss of HLA-I or changes in HLA-I expression levels are sensed by germline-encoded receptors expressed on NK cells. Many NK cell receptors recognize HLA-I; however whether and how NK cells might interact with HLA class II (HLA-II) molecules remained unknown. The projects builds on our recent discovery that a subset of commonly expressed HLA-II molecules, namely HLA-DP molecules, serve as ligands for the natural cytotoxicity receptor (NCR) NKp44, implicating HLA-II in the regulation of NK cells. The ability of NK cell receptors to bind HLA-II molecules represented a paradigm shift from previous models describing NK cell function largely in the context of HLA-I, and suggests a novel regulatory framework in which NK cells interact directly with HLA-II-expressing cells. HLA-DP allotypes have been associated with the incidence and outcome of several infectious and inflammatory human diseases, and the newly identified interactions between HLA-DP and NKp44 now provide a molecular mechanism implicating NK cells in the pathogenesis of these diseases. Within RegNK, we conduct an innovative and integrated research program, combining functional immunological and virological approaches with recently developed human organoid systems and proteomics technologies to determine the impact of interactions between NKp44 and HLA-DP for human diseases. The ultimate goal of these studies is to harness NK cells for immunotherapeutic interventions against infections and inflammatory diseases; diseases that represent an important burden for our society.
To accomplish this goal, three research objectives are proposed: (1) to characterize the mechanisms by which interactions between HLA-DP molecules and NKp44 enable NK cells to recognize HBV-infected cells; (2) to determine the role of NKp44+ NK cells in regulating immune responses during GvHD and autoimmune diseases; and (3) to identify novel peptide-dependent interactions between NK cell receptors and HLA-II that regulate NK cell function. Since the beginning of the project, we have made significant progress towards all three objectives.
To accomplish this goal, three research objectives are proposed: (1) to characterize the mechanisms by which interactions between HLA-DP molecules and NKp44 enable NK cells to recognize HBV-infected cells; (2) to determine the role of NKp44+ NK cells in regulating immune responses during GvHD and autoimmune diseases; and (3) to identify novel peptide-dependent interactions between NK cell receptors and HLA-II that regulate NK cell function. Since the beginning of the project, we have made significant progress towards all three objectives.
Since the beginning of the project, we have made significant progress towards all three objectives.
Objective 1: to characterize the mechanisms by which interactions between HLA-DP molecules and NKp44 enable NK cells to recognize HBV-infected cells
For this objective, we have successfully established hepatocyte organoid models, and co-culture systems between NK cells and hepatocytes. We furthermore established in vitro models to investigate the lysis of hepatocytes by NK cells, and the consequences of viral infections. We are currently in the process to optimize infection protocols for productive HBV infection of hepatocyte organoids.
Objective 2: to determine the role of NKp44+ NK cells in regulating immune responses during GvHD and autoimmune diseases
Within this objective, we have now completed two studies. One study investigated the role of interactions between NKp44 and HLA-DP molecules in ulcerative colitis, and demonstrated that HLA-DP expressed on epithelial cells enables tissue damage by NKp44+ NK cells. These studies combined patient cohorts, intestinal organoid systems and co-cultures with NK cells, and were recently published in Gastroenterology. The second study investigated the role of interactions between NKp44 and HLA-DP in primary sclerosing cholangitis (PSC), and autoimmune disease of the liver. In these studies we identified a novel HLA-DP risk allotype for PSC, and demonstrated using in vitro studies that HLA-DP is upregulated on cholangiocytes in individuals with PSC, and can activate NKp44+ NK cells. This study was recently published in Gut. A third project investigating the role of NKp44+ NK cells in GvHD is ongoing.
Objective 3: to identify novel peptide-dependent interactions between NK cell receptors and HLA-II that regulate NK cell function.
In objective 3, we have started to identify several novel peptides that are presented by HLA-DP, focusing initially on HLA-DP401, and started to determine their consequences for NKp44 binding and NKp44+ NK cell activation. Work on this objective will be to focus during the remaining funding period of RegNK.
Objective 1: to characterize the mechanisms by which interactions between HLA-DP molecules and NKp44 enable NK cells to recognize HBV-infected cells
For this objective, we have successfully established hepatocyte organoid models, and co-culture systems between NK cells and hepatocytes. We furthermore established in vitro models to investigate the lysis of hepatocytes by NK cells, and the consequences of viral infections. We are currently in the process to optimize infection protocols for productive HBV infection of hepatocyte organoids.
Objective 2: to determine the role of NKp44+ NK cells in regulating immune responses during GvHD and autoimmune diseases
Within this objective, we have now completed two studies. One study investigated the role of interactions between NKp44 and HLA-DP molecules in ulcerative colitis, and demonstrated that HLA-DP expressed on epithelial cells enables tissue damage by NKp44+ NK cells. These studies combined patient cohorts, intestinal organoid systems and co-cultures with NK cells, and were recently published in Gastroenterology. The second study investigated the role of interactions between NKp44 and HLA-DP in primary sclerosing cholangitis (PSC), and autoimmune disease of the liver. In these studies we identified a novel HLA-DP risk allotype for PSC, and demonstrated using in vitro studies that HLA-DP is upregulated on cholangiocytes in individuals with PSC, and can activate NKp44+ NK cells. This study was recently published in Gut. A third project investigating the role of NKp44+ NK cells in GvHD is ongoing.
Objective 3: to identify novel peptide-dependent interactions between NK cell receptors and HLA-II that regulate NK cell function.
In objective 3, we have started to identify several novel peptides that are presented by HLA-DP, focusing initially on HLA-DP401, and started to determine their consequences for NKp44 binding and NKp44+ NK cell activation. Work on this objective will be to focus during the remaining funding period of RegNK.
In particular the studies performed under objective 2 have resulted in very interesting and novel findings, in particular the identification of novel HLA-DP risk allotypes for autoimmune diseases (UC, PSC), and the demonstration that activation of NKp44+ NK cells by primary human cells, using intestinal and cholangiocyte organoid systems, can result in inflammation and tissue destruction. The establishment of rebust in vitro co-cultures of human organoid systems and immune cells, in particular NK cells, represents important progress beyond the state of the art. These studies have already resulted in one publication and a 2nd manuscript in revision, and we anticipate to complete a third project, focusing on GvHD, by the end of the project. By the end of the project we also expect to complete a comprehensive assessment of the role of HLA-II presented peptides on the interactions with NK cell receptors (objective 3). For objective 1, we need to overcome the current challenge to establish robust and reproducible HBV infections of hepatocyte organoids, and will focus on this during the remaining funding period.