Natural killer cells (NK cells) are effector lymphocytes of the innate immune system, which are able to recognize and eliminate virus-infected and malignantly transformed cells. Therefore, they play an important role for the containment of pathophysiological processes. An understanding of the molecular mechanisms that lead to NK cell activation is crucial to enhance the effectivity of NK cell-based anti-cancer therapies. Effector functions are regulated by a variety of germline-encoded activating and inhibitory receptors on the surface of the NK cell. One of the major activating NK cell receptors is NKp30, belonging to the natural cytotoxicity receptors (NCRs). NKp30 is a functional receptor in humans and primates (macaques and chimpanzees) as well as on rat NK cell subsets. In contrast, it is only present as a pseudogene with two premature stop-codons in mouse. The only exception is the mouse strain Mus caroli, where two single nucleotide polymorphisms (SNPs) eliminate the premature stop-codons. The evolutionary reasons for the development of the murine NKp30 pseudogene are currently unknown.
For signaling, NKp30 associates with immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor proteins like CD3ζ or FcεRIγ. Until now, the mechanism how ligand binding at the ectodomain of NKp30 is communicated to the adaptor protein CD3ζ is still unknown. Therefore, the molecular details of receptor activation as well as the role of the murine NKp30 pseudogene were analyzed in this thesis.
Formerly, it was shown that the stalk domain of NKp30, a 15 amino acid sequence stretch between the immunoglobulin (Ig) domain and the transmembrane domain, is important for ligand binding and signaling. Therefore, in this thesis, mutated NKp30 variants were produced as full length receptors in A5-GFP reporter cells or NKp30::hIgG1-Fc (NKp30-Fc) fusion proteins in HEK 293T/17 cells and subsequently analyzed in binding studies (surface plasmon resonance, SPR) and signaling reporter assays. Surprisingly, analysis of NKp30/NKp46 tandem mutants showed that despite the existence of a conserved sequence motif in the membrane-proximal region, the stalk domains of NKp30 and NKp46 are not exchangeable without drastic deficiencies in folding, plasma membrane targeting and/or ligand-induced receptor signaling. Additionally, it was shown that the stalk domain of NKp30 is very sensitive to sequence alterations, as alanine substitution of any of the stalk amino acids led to impaired ligand binding and/or signaling capacity. Mutation of the arginine on amino acid position 143 to alanine (R143A) had the most drastic effect. Based on further mutational studies, N-glycosylation mapping and plasma membrane targeting studies, the existence of two interconvertible types of NKp30/CD3ζ complexes can be hypothesized: (1) a signaling incompetent structural NKp30/CD3ζ complex and (2) a ligand-induced signaling competent NKp30/CD3ζ complex. Furthermore, it can be proposed, that ligand binding at the Ig-fold of NKp30 triggers translocation of amino acid R143 of the stalk domain from the interface between membrane and extracellular region more deeply into the lipid bilayer to enable alignment with oppositely charged aspartate residues within CD3ζ and activation of CD3ζ signaling.
Although several cellular and pathogen-derived NKp30 ligands have been identified in the last years, there is evidence for the existence of further, yet unknown cellular ligands. This assumption is based on former studies that showed binding of NKp30-Fc fusion proteins to tumor cell lines that do not express the cellular NKp30 ligands B7-H6 and BAG-6 on their surface. Therefore, in the present thesis, a screening method was established, based on transduction of ligand-bearing cell lines with a genome-wide shRNA library. After shRNA knockdown of putative ligands, cells were decorated with NKp30-Fc fusion proteins and sorted for reduced NKp30 ligand expression (fluorescence activated cell sorting, FACS). shRNA sequences were amplified from genomic DNA of the cells by PCR and subsequently analyzed via deep sequencing. The same screening method was additionally implemented for the identification of ligands of the other two NCRs, NKp44 and NKp46. Interestingly, inspite of the high number of advantages in contrast to conventional screening strategies, the existence of further cellular proteinaceous NCR ligands could not be confirmed with this screening.
There are different suggestions about the evolutionary appearance of the NCRs. Divergence from a common ancestor (at least in case of NKp30 and NKp44) might have led to an increase in complexity and fine-tuning of the immune system. Different studies suggest development of the NKp46 gene from a common NCR ancestor or from a common ancestor with the KIR genes. Interestingly, murine NKp46 is a functional protein, while NKp30 is only present as a pseudogene and NKp44 is completely lost in mouse. To shed light on the evolutionary reasons for the development of the murine NKp30 pseudogene, the two premature stop codons in the extracellular domain of the M. musculus NKp30 gene sequence were repaired and the protein was expressed as full length receptor in A5-GFP reporter cells and as soluble mNKp30-Fc fusion protein in HEK 293T/17 cells. Interestingly, the full length receptor as well as the mNKp30-Fc fusion protein were intracellularly retained. Repair of the three N-linked glycosylation sites in the extracellular region of mNKp30 (mNKp30-glyco) led to the secretion of the Fc fusion protein, while the full length receptor stayed intracellularly retained. As shown previously, association with CD3ζ impacts plasma membrane targeting and retention of human NKp30. Therefore, failure of mNKp30 to assemble with CD3ζ might be the reason for intracellular retention of the full length receptor. Furthermore, the mNKp30-glyco-Fc fusion protein showed specific binding to P815 murine mastocytoma cells. This speaks for the existence of a cancer- or mast cell-related mNKp30-glyco ligand. Altogether, these were the first experiments to show expression and functional analysis of a putative mNKp30 on protein level.
Based on these data, the present thesis provides deeper insight into the function of the major activating NK cell receptor NKp30. This might contribute to a better understanding of the molecular mechanisms that lead to NK cell activation, and this knowledge is crucial to enhance the effectivity of related treatments like anti-cancer and anti-viral therapies
