NKp44: identification of new ligands and in vivo study employing a transgenic mouse model

BSF Grant 201123 - NKp44: identification of new ligands and in vivo study of function employing a transgenic mouse model Executive Summary of Final Science Report NKp44 (NCR2) is a Natural Cytotoxicity Receptor (NCR) that can stimulate cytokines and cytotoxicity in human NK cells. Heparan sulfate proteoglycans (HSPGs) are expressed on normal and cancerous tissues, including NK cells, and serve as ligands/co-ligands for NKp44 and other NCRs.

We showed that HSPGs are allosteric regulators of NKp44 function through interactions on NK cells themselves (-cis). Specifically, the interaction of the NK cell- associated syndecan-4 (SDC4) HSPG and NKp44 was found to directly affect NKp44 membrane distribution and receptor function.

We also identified a unique expression pattern of NKp44 that is detrimental to survival in patients with acute myeloid leukemia (AML), which is a tumor susceptible to NK cell-mediated immune attack. Three distinct splice variants of NKp44 are expressed in humans: NKp44-1, containing a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM), and NKp44-2 and -3, lacking the ITIM. We found that exclusive expression of the NKp44-1 in bone marrow is associated with poor survival of AML patients, and patients with high NKp44-1 expression had the worst survival.

Furthermore, retroviral expression of NKp44-1 in the NK-92 cells significantly suppressed IFN-γ production and F-actin accumulation at the immune synapse toward PCNA-expressing target cells.

We unfortunately failed to successfully engineer a mouse expressing human NKp44 exclusively in all mouse NK cells. Overall, after screening hundreds of progeny generated using both zinc finger nuclease and CRISPR/Cas9 techniques, we generated only one mouse with the NKp44 construct appropriately integrated, but that mouse had a mutation that prevented protein expression and could not be repaired.