Natural killer (NK)1 cells are large granular lymphocytes that provide a first innate immune defense. They are able to kill virus-infected and transformed cells and furthermore release cytokines and chemokines to activate adaptive immune cells (1, 2).The balance of signals from activating and inhibitory NK cell surface receptors tightly regulates NK cell activity. Activated NK cells release lytic granules through a process called degranulation. Therefore, NK cell cytotoxicity requires the formation of the F-actin-rich NK immune synapse (NKIS) and the transport of Perforin-containing lytic granules to the NKIS. Furthermore, this process requires granule-associated MYH9 protein (non-muscle Myosin IIa) mediating the interaction of granules with F-actin at the NKIS (3-5), leading to lytic granule exocytosis. Whereas related phenotypes and functional properties are well characterized, the underlying regulatory protein network mediating differentiation, cytokine release, and cytotoxicity, is still incomplete.NK cells are defined by the expression of the surface molecule CD56 (NCAM1) and the absence of the T cell receptor (TCR) associated protein CD3 and can be further subdivided into subsets (6, 7). CD56 expressing cells originate from CD34 ϩ HSCs. Notably, the commitment to the NK lineage includes discrete steps from HSC to cells, expressing high CD56 levels (CD56 bright ) (8, 9), which act immune regulatory by the release of various cytokines. NK cells with low CD56-expression (CD56 dim ) predominantly constitute cytotoxic responses (10, 11). Contact of CD56 (NCAM1) with fibroblasts (12) and neutrophils (13) supports the differentiation process from CD56 bright to CD56 dim NK cells. The progression of early differentiation steps is proven by telomere length investigation (14 ) and early presence in blood after HSC transplantation (HSCT) (14, 15). Indeed, CD56 dim NK cells are able to change their phenotypic properties, which can be correlated with continued differentiation throughout their whole lifespan (15-18). CD57 was determined to be a senescence marker in T cells (19 1 The abbreviations used are: NK, natural killer; CD56, NK cell marker; NCAM1, neural cell adhesion molecule; CD57, senescence marker in T and NK cells (HNK-1 or Leu-7); CMV, cytomegalovirus; CPDA-1, anticoagulant , containing citric acid, sodium citrate, monobasic sodium phosphate and dextrose; CTLs, cytotoxic T lymphocytes; FDR, false discovery rate; HLA, self-human leukocyte antigen; HSC, hematopoietic stem cell; iTRAQ, isobaric tags for relative and absolute quantification in mass spectrometry; KIR, killer immunoglobulin-related receptors in NK cells; LC-MS/MS, liquid-chromatography coupled with peptide sequencing (mass spectrometry); MAD, median absolute deviation from the median; NKIS, NK cell immune synapse; RF, regulation factor. Research

BackgroundNatural killer (NK) cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244) and DNAM-1 (CD226), act synergistically. After receptor engagement, protein kinases play a major role in signaling networks controlling NK cell effector functions. However, it has not been characterized systematically which of all kinases encoded by the human genome (kinome) are involved in NK cell activation.ResultsA kinase-selective phosphoproteome approach enabled the determination of 188 kinases expressed in human NK cells. Crosslinking of CD16 as well as 2B4 and DNAM-1 revealed a total of 313 distinct kinase phosphorylation sites on 109 different kinases. Phosphorylation sites on 21 kinases were similarly regulated after engagement of either CD16 or co-engagement of 2B4 and DNAM-1. Among those, increased phosphorylation of FYN, KCC2G (CAMK2), FES, and AAK1, as well as the reduced phosphorylation of MARK2, were reproducibly observed both after engagement of CD16 and co-engagement of 2B4 and DNAM-1. Notably, only one phosphorylation on PAK4 was differentally regulated.ConclusionsThe present study has identified a significant portion of the NK cell kinome and defined novel phosphorylation sites in primary lymphocytes. Regulated phosphorylations observed in the early phase of NK cell activation imply these kinases are involved in NK cell signaling. Taken together, this study suggests a largely shared signaling pathway downstream of distinct activation receptors and constitutes a valuable resource for further elucidating the regulation of NK cell effector responses.

Natural killer (NK) cells are part of the innate immune response and play a crucial role in the defense against tumors and virus-infected cells. Their effector functions include the specific killing of target cells, as well as the modulation of other immune cells by cytokine release. Kinases constitute a relevant part in signaling, are prime targets in drug research and the protein kinase inhibitor Dasatinib is already used for immune-modulatory therapies. In this study, we tested the effects of the kinase inhibitors CK59 and CID755673. These inhibitors are directed against calmodulin kinase II (CaMKII; CK59) and PKD family kinases (CID755673) that were previously suggested as novel components of NK activation pathways. Here, we use a multi-parameter, FACS-based assay to validate the influence of CK59 and CID755673 on the effector functions of primary NK cells. Treatment with CK59 and CID755673 indeed resulted in a significant dose-dependent reduction of NK cell degranulation markers and cytokine release in freshly isolated Peripheral blood mononuclear cell populations from healthy blood donors. These results underline the importance of CaMKII for NK cell signaling and suggest protein kinase D2 as a novel signaling component in NK cell activation. Notably, kinase inhibition studies on pure NK cell populations indicate significant donor variations.