AbstractsBiology & Animal Science

Natural killer (NK) cells are large granular lymphocytes of the innate immune system that spontaneously kill foreign, tumor and virus-infected cells without prior sensitization. In addition, NK cells act as immune regulators by secretion of chemokines and cytokines as well as direct interaction with other immune cells such as dendritic cells. NK cell function is regulated by a balance between inhibitory and activating signals that are transduced into the cell upon target cell interaction. One of the major activating NK cell receptors is the natural cytotoxicity receptor NKp30. Notably, NKp30 plays a unique role since it is the only NK cell receptor involved in triggering NK cell-mediated cytotoxicity as well as shaping the adaptive immune response. Reduced NKp30 expression has clinical implications in patients with acute myeloid leukemia, cervical cancer, and high grade squamous intraepithelial lesions as well as gastrointestinal sarcoma. Furthermore, downregulation of NKp30 expression resulted in an impaired natural cytotoxicity against leukemia cells and was directly correlated with reduced survival. NKp30 is a type I transmembrane protein of approximately 30 kDa comprised of an I-type Ig-like ligand binding domain (LBD), a flexible membrane proximal stalk domain, a single transmembrane helix, and a short cytosolic tail. For intracellular signal transduction, NKp30 associates with the immunoreceptor tyrosine-based activating motif (ITAM)-bearing adaptor molecule CD3zeta via oppositely charged amino acid residues within their transmembrane domains. In 2011, the 3D structure of the NKp30LBD was solved in an unbound and a ligand-bound form. However, so far, only few cellular ligands (BAG-6 and B7-H6) have been discovered, and the molecular details of ligand recognition by NKp30 are poorly understood. Recently, it was shown that the membrane proximal stalk domain of NKp30 is important for efficient ligand binding and signaling with respect to its length and amino acid composition. Additionally, it was demonstrated that proper N-linked glycosylation of the ligand binding domain is essential for ligand binding. But it is still vague, how this germline-encoded receptor is able to recognize multiple nonrelated ligands. Interestingly, a crystallographic dimer of the NKp30 ectodomain was observed arguing for potential intrinsic capability to self-assemble. Moreover, a fraction of NKp30 expressed in E. coli forms oligomers as detected by size exclusion chromatography. The aim of this thesis was to identify structural models and mechanisms, which enable variations of the ligand binding interface of NKp30 to recognize a multiplicity of diverse ligands. In this respect, soluble NKp30 ectodomain variants as well as an anti-NKp30 antibody specifically recognizing an epitope within the LBD of NKp30 were generated for molecular and cellular investigation to address the intrinsic ability of NKp30 to form oligomers, which might impact ligand binding affinity and the efficiency of target cell killing by NK cells. In this thesis,…