SKAP2 acts downstream of CD11b/CD18 and regulates neutrophil effector function.

Background: The importance of CD11b/CD18 expression in neutrophil effector functions is well known. Beyond KINDLIN3 and TALIN1, which are involved in the induction of the high-affinity binding CD11b/CD18 conformation, the signaling pathways that orchestrate this response remain incompletely understood. Method: We performed an unbiased screening method for protein selection by biotin identification (BioID) and investigated the KINDLIN3 interactome. We used liquid chromatography with tandem mass spectrometry as a powerful analytical tool. Generation of NB4 CD18, KINDLIN3, or SKAP2 knockout neutrophils was achieved using CRISPR-Cas9 technology, and the cells were examined for their effector function using flow cytometry, live cell imaging, microscopy, adhesion, or antibody-dependent cellular cytotoxicity (ADCC). Results: Among the 325 proteins significantly enriched, we identified Src kinase-associated phosphoprotein 2 (SKAP2), a protein involved in actin polymerization and integrin-mediated outside-in signaling. CD18 immunoprecipitation in primary or NB4 neutrophils demonstrated the presence of SKAP2 in the CD11b/CD18 complex at a steady state. Under this condition, adhesion to plastic, ICAM-1, or fibronectin was observed in the absence of SKAP2, which could be abrogated by blocking the actin rearrangements with latrunculin B. Upon stimulation of NB4 SKAP2-deficient neutrophils, adhesion to fibronectin was enhanced whereas CD18 clustering was strongly reduced. This response corresponded with significantly impaired CD11b/CD18-dependent NADPH oxidase activity, phagocytosis, and cytotoxicity against tumor cells. Conclusion: Our results suggest that SKAP2 has a dual role. It may restrict CD11b/CD18-mediated adhesion only under resting conditions, but its major contribution lies in the regulation of dynamic CD11b/CD18-mediated actin rearrangements and clustering as required for cellular effector functions of human neutrophils.

Cancer cells resist antibody-mediated destruction by neutrophils through activation of the exocyst complex.

BACKGROUND: Neutrophils kill antibody-opsonized tumor cells using trogocytosis, a unique mechanism of destruction of the target plasma. This previously unknown cytotoxic process of neutrophils is dependent on antibody opsonization, Fcγ receptors and CD11b/CD18 integrins. Here, we demonstrate that tumor cells can escape neutrophil-mediated cytotoxicity by calcium (Ca2+)-dependent and exocyst complex-dependent plasma membrane repair. METHODS: We knocked down EXOC7 or EXOC4, two exocyst components, to evaluate their involvement in tumor cell membrane repair after neutrophil-induced trogocytosis. We used live cell microscopy and flow cytometry for visualization of the host and tumor cell interaction and tumor cell membrane repair. Last, we reported the mRNA levels of exocyst in breast cancer tumors in correlation to the response in trastuzumab-treated patients. RESULTS: We found that tumor cells can evade neutrophil antibody-dependent cellular cytotoxicity (ADCC) by Ca2+-dependent cell membrane repair, a process induced upon neutrophil trogocytosis. Absence of exocyst components EXOC7 or EXOC4 rendered tumor cells vulnerable to neutrophil-mediated ADCC (but not natural killer cell-mediated killing), while neutrophil trogocytosis remained unaltered. Finally, mRNA levels of exocyst components in trastuzumab-treated patients were inversely correlated to complete response to therapy. CONCLUSIONS: Our results support that neutrophil attack towards antibody-opsonized cancer cells by trogocytosis induces an active repair process by the exocyst complex in vitro. Our findings provide insight to the possible contribution of neutrophils in current antibody therapies and the tolerance mechanism of tumor cells and support further studies for potential use of the exocyst components as clinical biomarkers.