Abstract
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.
Highlights
Natural Killer (NK) cells are key players in immune defense against cancerous or virally infected cells
Opsonization of 221 target cells with anti-CD20 and anti-CD20AF partially reduced surface CD20 availability to fluorescently labeled anti-CD20 added after opsonization, indicating that loading was equivalent between the two antibodies but that not all surface CD20 was bound by the monoclonal antibodies (mAb) when opsonizing targets (Supplementary Figure 1B)
The Fc portion of mAbs is responsible for ligation to the activating Fc receptor CD16 on NK cells, and dictates their antibody-dependent cellular cytotoxicity (ADCC) response
Summary
Natural Killer (NK) cells are key players in immune defense against cancerous or virally infected cells. NK cells can directly kill diseased cells by secretion of lytic granules that typically contain pore-forming. NK cells augment the immune response by secreting immuno-stimulatory cytokines and chemokines including pro-inflammatory interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) [4, 5]. 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]. While it is clear that NK cell detachment is important for effective NK cell killing, very few specific mechanisms have been described
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