CD56 regulates human NK cell cytotoxicity through Pyk2

Abstract

Natural killer (NK) cells are innate immune cells that control viral infection and tumorigenic cell growth through targeted cell lysis and cytokine secretion. Human NK cells are classically defined as CD56 + CD3 − in peripheral blood. CD56 is neural cell adhesion molecule (NCAM1), and despite its ubiquitous expression on human NK cells, the role of CD56 in human NK cell cytotoxic function has not been fully explored. In non-immune cells, NCAM can induce signaling, mediate adhesion, and promote exocytosis, in part through interactions with focal adhesion kinase (FAK). Here we describe the generation and use of CD56-deficient human NK cell lines to define a novel requirement for CD56 in target cell lysis. Namely, we demonstrate that deletion of CD56 on the NK92 cell line led to impaired cytotoxic function against multiple susceptible target cell lines. Deletion of CD56 in a second NK cell line, YTS cells, led to a less severe cytotoxicity defect but impairment in cytokine secretion. Confocal microscopy of wild-type and CD56-KO NK92 cells conjugated to susceptible targets revealed that CD56-KO cells failed to polarize during immunological synapse (IS) formation and had severely impaired exocytosis of lytic granules at the IS. Phosphorylation of the FAK family member Pyk2 at tyrosine 402 was decreased in NK92 CD56-KO cells, demonstrating a functional link between CD56 and IS formation and signaling in human NK cells. Cytotoxicity, lytic granule exocytosis, and the phosphorylation of Pyk2 were rescued by the reintroduction of NCAM140 (CD56), into NK92 CD56-KO cells. These data highlight a novel functional role for CD56 in stimulating exocytosis and promoting cytotoxicity in human NK cells. cytotoxicity | natural killer cell | NCAM | CD56 Correspondence: em3375@cumc.columbia.edu 2 | bioRχiv Mace et al. | CD56 and human NK cell cytotoxicity

Design and Implementation of NK Cell-Based Immunotherapy to Overcome the Solid Tumor Microenvironment

“…CAR-NK cells are a promising option as a therapeutic platform and CAR-induced activation has shown to result in heightened intracellular down-stream signaling which supports canonical activation pathways leading to CAR-mediated cytotoxicity [ 125 ]. Historically, primary NK cell cytotoxicity and pro-inflammatory cytokine secretion is thought to be tied to the intensity of surface CD56 expression (CD56bright and CD56dim) although that notion is now being challenged [ 126 , 127 , 128 ]. While CD56bright NK cells have been associated with survival in melanoma patients, their role in the efficacy of cellular therapies is unknown [ 129 ].…”

“…Recent reports suggest a role for CD56 in immune synapse formation between NK KIR and targets cells, suggesting CD56’s importance in NK cytotoxic function. However, this biology has not been confirmed in ex vivo manipulated NK cells [ 128 ]. Additionally, to improve in vivo persistence, CAR-NK cells have been engineered to express IL-15 which may prove useful in overcoming the suppressive TME [ 47 ].…”

Sialic Acids and Their Influence on Human NK Cell Function

“…Besides, a recent publication could show that CRISPR-Cas9-mediated deletion of CD56/NCAM in the NK cell line NK-92 leads to a reduced killing of CD56 negative tumor cells. In contrast, loss of CD56/NCAM on primary NK cells had no impact on cytotoxic activity [ 151 ]. With experiments on NK cell lines and primary cells, Mace and colleagues could show that CD56/NCAM is involved in migration during NK cell development [ 152 ].…”

“…Moreover, because CD56/NCAM on NK cells seems to bind to fungal mold Aspergillus fumigatus , a role as a pattern recognition receptor is also discussed [ 153 ]. Additionally, a role for CD56/NCAM in cell signaling was assumed, since phosphorylation of Pyk2, a tyrosine kinase was decreased in CD56/NCAM knockout NK cells and could be rescued by CD56/NCAM transduction [ 151 ].…”

High-dimensional mass cytometry analysis of NK cell alterations in Acute Myeloid Leukemia identifies a subgroup with adverse clinical outcome

“…19,20 Indeed, CD56 is an important adhesion molecule involved in NK cell development, motility and pathogen recognition. [22][23][24][25][26][27] CD56 is also required for the formation of the immunological synapse between NK cells and target cells, lytic functions, and cytokine production. 26,28 As a consequence, CD56 -CD16 + NK cells display lower degranulation capacities, decreased expression of triggering receptors, perforin, and granzyme B, dramatically reducing their cytotoxic potential, notably against tumor target cells.…”