The mature activating natural killer cell immunologic synapse is formed in distinct stages

Abstract

Rationale Natural killer (NK) cells form a structure called the activating NK cell immunologic synapse (NKIS) at their interface with susceptible target cells. These studies were designed to further elucidate this structure as well as the cytoskeletal requirements for its formation. Methods Ex vivo NK cells from normal individuals or patients with Wiskott-Aldrich syndrome (WAS) were evaluated in conjugation with susceptible target cells by laser scanning confocal microscopy. An immortalized NK cell line transfected with a CD2-green fluorescent protein construct using a retroviral expression system was also studied. Results The mature activating NKIS contains a central (c) and peripheral (p) supramolecular activation cluster (SMAC) and includes polarized surface receptors, filamentous actin (F-actin) and perforin. Evaluation of the NKIS revealed CD2, CD11a, CD11b and F-actin in the pSMAC with perforin in the cSMAC. The accumulation of F-actin and surface receptors was rapid and depended upon WAS protein (WASp)-driven actin polymerization. These processes were impaired in the presence of an actin polymerization inhibitor (by >75%, p<0.05) or in cells from patients with WAS (by >50%, p<0.05). The accumulation at and arrangement of these molecules in the pSMAC was not affected by microtubule depolymerization. The polarization of perforin, however was slower than for F-actin or surface receptors (15 vs 2.5 min) and required intact actin, WASp and microtubule function (reduced by >50% by inhibitors or in patient cells, p<0.05). Conclusions The process of CD2, CD11a, CD11b and F-actin accumulation in the pSMAC and perforin accumulation in the cSMAC of the NKIS are sequential processes with distinct cytoskeletal requirements.