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

Area of Science:

• Immunology
• Cell Biology
• Molecular Biology

Background:

• CD11b/CD18 expression is crucial for neutrophil effector functions.
• Signaling pathways regulating CD11b/CD18 high-affinity binding are not fully understood.
• KINDLIN3 and TALIN1 are known regulators, but other factors remain elusive.

Purpose of the Study:

• To identify novel proteins involved in CD11b/CD18 regulation using an unbiased screening approach.
• To investigate the role of identified proteins in neutrophil effector functions.
• To elucidate the signaling pathways orchestrating CD11b/CD18-mediated responses.

Main Methods:

• Biotin identification (BioID) for protein screening.
• Liquid chromatography with tandem mass spectrometry for analysis.
• CRISPR-Cas9 technology for generating knockout neutrophils (NB4 CD18, KINDLIN3, SKAP2).
• Flow cytometry, live cell imaging, microscopy, adhesion assays, and ADCC assays for functional examination.

Main Results:

• Src kinase-associated phosphoprotein 2 (SKAP2) was identified as a component of the CD11b/CD18 complex.
• SKAP2 influences CD11b/CD18-mediated actin rearrangements and clustering.
• SKAP2 deficiency impaired neutrophil effector functions, including NADPH oxidase activity, phagocytosis, and cytotoxicity.
• SKAP2 may restrict adhesion under resting conditions but is essential for dynamic responses.

Conclusions:

• SKAP2 has a dual role in regulating CD11b/CD18-mediated adhesion.
• SKAP2 is critical for dynamic actin rearrangements and clustering required for neutrophil effector functions.
• Understanding SKAP2's role provides insights into integrin signaling and neutrophil immunity.