Background: Natural Killer (NK) cells express the Fc receptor CD16 (FcγRIIIa), which can trigger antibody-dependant cellular cytotoxicity (ADCC) against opsonized cells. ADCC is clinically important as one of the mechanisms by which therapeutic antibodies work. The anti-CD20 mAb rituximab is, for example, widely used for targeting B cells in treatments of autoimmune disease and non-Hodgkin’s lymphoma. Here, we set out to study individual NK cell-target cell interactions by microscopy to determine what happens as NK cells meet different target cells sequentially, some opsonised and some not.

In addition, genetic diversity in human NK cell receptors has been linked with resistance and susceptibility to many diseases, but underlying mechanisms remain unclear. The effect of this genetic diversity on the cell surface organisation and signalling of receptors is a major unknown. Thus, we set out to study the organisation and signalling of inhibitory Killer Ig-like receptors (KIR) encoded by different genes and alleles using super-resolution microscopy.

Results: We found that repeated activation via CD16 decreased the amount of perforin secreted. However, perforin secretion was restored upon subsequent activation via a different NK cell activating receptor, NKG2D. Repeated stimulation via NKG2D also decreased perforin secretion but this was not rescued by stimulation via CD16. These different outcomes of sequential stimulation could be accounted for by shedding of CD16 being triggered by cellular activation. Shedding of CD16 shedding also increased NK cell motility and allowed for detachment of NK cells from target cells. In turn, this aided NK cell survival and boosted serial engagement of target cells.

In addition, we report that inhibitory NK cell receptors encoded by different genes and alleles organise differently at the surface of primary human NK cells. In particular, expression level has major effects on receptor organisation. KIR that are expressed at a low level assemble in smaller clusters than KIR that are highly expressed. Unexpectedly, upon receptor triggering, low-expressed receptors in smaller clusters generate more phosphorylated Crk than highly expressed receptors.

Conclusions: (¹ Shedding of the Fc receptor CD16 has an unexpectedly complex impact on NK cell responses. Shedding this receptor renders NK cells less potent at CD16-mediated activation, as expected, but promotes the detachment from opsonized targets to aid sequential target cell surveillance. Thus, counter-intuitively, shedding of Fc receptor CD16 can positively impact immune responses.

(² Genetic variation modulates the nanoscale organisation of inhibitory NK cell receptors, which in turn impacts receptor signalling. This may be important in how genetic variation impacts immune responses and disease susceptibilities.

Disclosure of Interest: None declared

DOI: 10.1136/annrheumdis-2018-eular.7694