NK cell detachment from target cells is regulated by successful cytotoxicity and influences cytokine production

Anft, Moritz; Netter, Petra; Urlaub, Doris; Prager, Isabel; Schaffner, Samantha L.; Watzl, Carsten

doi:10.1038/s41423-019-0277-2

Cited by 44 publications

(44 citation statements)

References 41 publications

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“…NK cell cytotoxic function includes serial killing of target cells, with target cell lysis followed by NK cell detachment and re-engagement with subsequent targets ( Anft et al, 2020 ; Bhat and Watzl, 2007 ; Jenkins et al, 2015 ; Vanherberghen et al, 2013 ). Given the demonstrated defect in NK cell migration in CD56-KO NK92 cells ( Mace et al, 2016 ) we sought to measure whether the defect in NK cell killing could be attributed to impaired serial killing.…”

Section: Resultsmentioning

confidence: 99%

CD56 regulates human NK cell cytotoxicity through Pyk2

Gunesch

Dixon

Ebrahim

et al. 2020

eLife

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Human natural killer (NK) cells are defined as CD56+CD3−. Despite its ubiquitous expression on human NK cells the role of CD56 (NCAM) in human NK cell cytotoxic function has not been defined. In non-immune cells, NCAM can induce signaling, mediate adhesion, and promote exocytosis through interactions with focal adhesion kinase (FAK). Here we demonstrate that deletion of CD56 on the NK92 cell line leads to impaired cytotoxic function. CD56-knockout (KO) cells fail to polarize during immunological synapse (IS) formation and have severely impaired exocytosis of lytic granules. Phosphorylation of the FAK family member Pyk2 at tyrosine 402 is decreased in NK92 CD56-KO cells, demonstrating a functional link between CD56 and signaling in human NK cells. Cytotoxicity, lytic granule exocytosis, and the phosphorylation of Pyk2 are rescued by the reintroduction of CD56. These data highlight a novel functional role for CD56 in stimulating exocytosis and promoting cytotoxicity in human NK cells.

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Section: Resultsmentioning

confidence: 99%

CD56 regulates human NK cell cytotoxicity through Pyk2

Gunesch

Dixon

Ebrahim

et al. 2020

eLife

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“…NK cell cytotoxic function includes serial killing of target cells, with target cell lysis followed by NK cell detachment and re-engagement with subsequent targets (43)(44)(45)(46). Given the demonstrated defect in NK cell migration in CD56-KO NK92 cells (35), we sought to measure whether the defect in NK cell killing could be attributed to impaired serial killing.…”

Section: Cd56-ko Nk Cells Have Impaired Lytic Function Towards Cd56-nmentioning

confidence: 99%

CD56 regulates human NK cell cytotoxicity through Pyk2

Gunesch

Dixon

Ebrahim

et al. 2020

Preprint

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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

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“…Assembly of the immune synapse has been widely studied ( 6 , 7 ), but how activating signals are terminated and how NK cells dissociate from target cells are understudied elements of the overall process ( 8 ). An inability for NK cells to detach from target cells leads to prolonged engagement and increased cytokine production ( 9 ). Detachment after lysis allows NK cells to move onto additional target cells ( 10 ) and serial killing by NK cells has been revealed by in-vitro live microscopy ( 11 , 12 ).…”

Section: Introductionmentioning

confidence: 99%

Antibody Afucosylation Augments CD16-Mediated Serial Killing and IFNγ Secretion by Human Natural Killer Cells

et al. 2021

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One mechanism by which monoclonal antibodies (mAb) help treat cancer or autoimmune disease is through triggering antibody-dependent cellular cytotoxicity (ADCC) via CD16 on Natural Killer (NK) cells. Afucosylation is known to increase the affinity of mAbs for CD16 on NK cells and here, we set out to assess how mAb afucosylation affects the dynamics of NK cell interactions, receptor expression and effector functions. An IgG1 version of a clinically important anti-CD20 mAb was compared to its afucosylated counterpart (anti-CD20-AF). Opsonization of CD20-expressing target cells, 721.221 or Daudi, with anti-CD20-AF increased NK cell cytotoxicity and IFNγ secretion, compared to anti-CD20. The afucosylated mAb also caused a more rapid and greater loss of CD16 from NK cell surfaces. Loss of CD16 has recently been shown to be important for NK cell detachment and sequential engagement of multiple target cells. Here, live-cell time-lapse microscopy of individual cell-cell interactions in an aqueous environment and a three-dimensional matrix, revealed that anti-CD20-AF induced more rapid killing of opsonized target cells. In addition, NK cells detached more quickly from target cells opsonized with anti-CD20-AF compared to anti-CD20, which increased engagement of multiple targets and enabled a greater proportion of NK cells to perform serial killing. Inhibition of CD16 shedding with TAPI-0 led to reduced detachment and serial killing. Thus, disassembly of the immune synapse caused by loss of cell surface CD16 is a factor determining the efficiency of ADCC and antibody afucosylation alters the dynamics of intercellular interactions to boost serial killing.

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NK cell detachment from target cells is regulated by successful cytotoxicity and influences cytokine production

Cited by 44 publications

References 41 publications

CD56 regulates human NK cell cytotoxicity through Pyk2

CD56 regulates human NK cell cytotoxicity through Pyk2

CD56 regulates human NK cell cytotoxicity through Pyk2

Antibody Afucosylation Augments CD16-Mediated Serial Killing and IFNγ Secretion by Human Natural Killer Cells

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