Homo-oligomerization of the Activating Natural Killer Cell Receptor NKp30 Ectodomain Increases Its Binding Affinity for Cellular Ligands

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, it was demonstrated that baculovirus-infected insect cells were a suitable expression system to produce correctly folded, post-translationally modified and ligand binding-receptive NKp30 proteins, which were functionally equivalent to those derived from human expression hosts. Furthermore, a polyclonal anti-NKp30 antibody, which is specific for an epitope located within the NKp30LBD, was purified from the blood serum of a peptide-immunized rabbit and validated for several molecular and cellular applications. Based on soluble NKp30 proteins comprising either the entire NKp30 ectodomain (LBD and stalk domain) or the ligand binding domain alone, a concentration-dependent formation of NKp30 ectodomain homo-oligomers was found, which was effected by two specific binding sites, one present within the Ig domain of NKp30 and one within its membrane proximal stalk domain. Moreover, both NKp30 ectodomain variant oligomers were functional in ligand binding and contributed to a highaffinity interaction with its cellular ligand B7-H6, which was strongly promoted by the stalk domain. Although both NKp30 ectodomain variant oligomers formed spherical particles of the same diameter in solution, in presence of the stalk domain one oligomer was composed of more monomers. Single particle electron microscopy analyses revealed that the number of calculated 2D classes increased tremendously for the particles of the longer NKp30 ectodomain variant. Therefore, a densest packing of spheres for the monomers within both NKp30 ectodomain variant oligomers is suggested, whereas the flexibility of the stalk domain might allow for a more variable orientation of these monomers within the particle, thereby increasing the heterogeneity of structural arrangements. However, based on decoration experiments with soluble NKp30 ectodomain proteins and primary NK cells facilitating more physiological conditions for NKp30 self-assembly, an ordered arrangement of several NKp30 receptors on the plasma membrane in head-to-head orientation is proposed. Based on these data, oligomerization of the NKp30 ectodomain represents a potent mechanism to modulate the ligand binding affinity of NKp30 for corresponding ligands by increased avidity. Because the degree of oligomerization is dependent on the local receptor concentration, which is upregulated by IL-2 upon NK cell activation, oligomerization of NKp30 might be a molecular transformer of NK cell activation into enhanced cytotoxicity. Future experiments are now focused to investigate NKp30 self-assembly on the plasma membrane of NK cells to enable modulation of the cytotoxicity of NK cell based therapeutics.