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“Molecular dissection of cytotoxic lymphocyte exocytosis

Periodic Reporting for period 1 - CL_Exocytosis (“Molecular dissection of cytotoxic lymphocyte exocytosis)

Reporting period: 2019-01-01 to 2020-12-31

The Action CL Exocytosis 794830 was carried by the researcher Dr Donatella Galgano under the supervision of Prof. Yenan Bryceson at the Centre Hematology Regenerative Medicine (HERM), Karolinska Institutet, Stockholm, Sweden from January 1st 2019 to December 31st 2020. The project focused on studying the molecular mechanism underlying cytotoxic lymphocyte effector function. These cells are part of the immune system and are responsible for destroying virus-infected or tumor cells. Cytotoxic lymphocytes kill their targets through directed release of cytotoxic proteins which are stored in specialized organelles, termed cytotoxic granules (CGs). The exit of CGs from the cells towards the target is called exocytosis. Defects in CG exocytosis profoundly impair cytotoxic lymphocyte function, resulting in severe, often life-threatening hyperinflammatory syndromes, as well as increased susceptibility to cancer. Clinically, these syndromes are characterized by an uncontrolled growth of immune cells, including cytotoxic lymphocytes and macrophages which infiltrate other tissues, leading directly to necrosis and organ failure. Nowadays, the only curative treatment for these severe conditions is hematopoietic stem cell transplantation, often not without complication. Increasing knowledge about cytotoxic lymphocytes, their activation and molecular mechanisms regulating their effector functions is needed to study such diseases in detail. Using advanced molecular techniques, this research aimed to study how different proteins are required for the effector functions of cytotoxic lymphocytes as well as identify novel regulators that control CG exocytosis. A detailed dissection of lymphocyte cytotoxicity promises to guide new strategies for the diagnosis and therapy of patients suffering of hyperinflammatory syndromes associated with defects in lymphocyte cytotoxicity.
This research aimed to gain insight into the mechanisms underlying CG exocytosis and their perturbation in the context of immune disorders. To reach this overall objective, the project was carried out through two work packages (WPs):
WP1) Dissecting the function in lymphocyte cytotoxicity of two distinct Munc13-4isoforms
WP2) Gaining insights to the regulation of RE fusion required for subsequent CG release

WP1. Munc13-4 is a crucial regulator of CG exocytosis. Mutations in UNC13D, the gene encoding for Munc13-4, cause a life-threatening hyperinflammatory syndrome, namely familial hemophagocytic lymphocytosis (FHL) type 3. Mutation in other genes encoding for other key factors of lymphocyte cytotoxicity including Perforin, Syntaxin 11 and Munc18-2 account for FHL2, FHL4 and FHL5, respectively. FHL affects about 1 in 50,000 new borns, with 30% of cases depending on mutations affecting Munc13-4. A previous study from Prof. Bryceson’s laboratory based on genetic analysis of FHL3 patients identified a novel alternative isoform of Munc13-4. Interestingly, this isoform originates from a non-coding region of the gene. In this project, the role in the regulation of CG exocytosis of the novel isoform was compared to the conventional Munc13-4. This part of the research was performed in collaboration with the group of Prof. Jens Rettig, Saarland University, Homburg, Germany and Prof. Marina Cavazzana, Imagine Institute, Paris, France. We showed that these isoforms have a similar localization inside the cells and similarly contribute to lymphocyte cytotoxicity. These results have been included in an open access original research article published in June 2020.
WP2. Another key question of this research was to address the role in lymphocyte cytotoxicity of special intracellular organelles, namely recycling endosome (RE). They are small vesicles inside the cells which, as their name suggest, contribute to the transport and recycling of molecules between intracellular organelles and the plasma membrane. A recent study from Prof. Bryceson’s laboratory showed that in cytotoxic lymphocytes, REs contribute to transport towards the plasma membrane of Syntaxin 11, which is a crucial protein for CG exocytosis. The transport and the delivery of Syntaxin 11 to the plasma membrane through REs is regulated by the protein VAMP8. The goal was to identify other important regulators of CG exocytosis, which in addition to Syntaxin 11, are delivered to the plasma membrane through REs regulated by VAMP8 using a high-throughput Mass spectrometry approach. This analytic technique is often used to dissect different components of complex samples. As model of study, a NK-92 cell line that recapitulates the phenotype of the cytotoxic lymphocytes in our body was used. The proposed approach included to genetically manipulate NK-92 to overexpress VAMP8 coupled with a tag. The experimental condition to achieve RE purification from NK-92 were successfully optimized by the researcher. This part of the research is still ongoing. However, the experimental strategy as well as preliminary data were presented by the Researcher during the HERM seminar.
"Results from WP1 showed that a functional Munc13-4 isoform originates from a non-coding region of UNC13D. First, having demonstrated that both canonical and alternative isoforms can support lymphocyte cytotoxicity, this study provided mechanistic insight to molecular regulation of one of the main genes controlling CG exocytosis. Highlighting the impact of a non-coding region in the regulation of a gene involved in lymphocyte cytotoxicity, these data emphasize the need to develop novel diagnostic tools which should be able to detect mutations outside of the coding regions to provide an accurate diagnosis in a greater number of patients. This is not only limited to FHL, but also to other hyperinflammatory syndromes associated with defects in genes involved in lymphocyte cytotoxicity. Finally, this work also contributes to guide novel approaches for the treatment of FHL, including gene therapy. This strategies can be based on the genetic manipulation of a patient’s lymphocytes to correct the genetic defect and regenerate the patient’s immune capacities. Viral vectors are generally used to deliver the ""correct form of gene"" within the cells. This research work demonstrated that Munc13-4 isoform re-expression, through lentiviral delivery, in cytotoxic lymphocytes from FHL3 patients successfully restores their degranulation capacity. These findings confirmed that the lentiviral system is a successful in vitro strategy to correct defective hematopoietic exocytosis in patient cells. Furthermore, having demonstrated that both Munc13-4 isoforms equally contribute to CG degranulation, either isoform could potentially be used to design constructs for gene therapy. Data from WP2 are still preliminary. Identifying novel regulators of CG exocytosis will be crucial to guide new strategies for the diagnosis and therapy of immune disorders associated with defects in lymphocyte cytotoxicity, including cancer and viral infections. Finally, this MSCA allowed the researcher to strongly improve her scientific background and technical expertise, providing a strong platform for the development of her scientific carrier. She also gained a set of transferable skills, including lab management, grant writing, student mentoring, and teaching, definitely opening up tremendous opportunities for her next career steps."
CL exocytosis summary