Detection of Experimental and Clinical Immune Complexes by Measuring SHIP-1 Recruitment to the Inhibitory FcγRIIB

Area of Science:

• Immunology
• Biochemistry
• Cell Biology

Background:

• Fc gamma receptors (FcγR) are crucial for immune regulation, monoclonal antibody (mAb) therapeutics, and autoimmune disease pathology.
• Accurate measurement of FcγR interactions with IgG immune complexes (IC) is limited, hindering research and clinical applications.

Purpose of the Study:

• To develop a novel, sensitive assay for detecting IgG immune complexes (IC) and studying Fc gamma receptor (FcγR) interactions.
• To model the inhibitory FcγRIIB (CD32B) signaling pathway using NanoLuc Binary Interaction Technology.
• To demonstrate the assay's utility in detecting circulating ICs in autoimmune diseases.

Main Methods:

• Utilized NanoLuc Binary Interaction Technology to measure the recruitment of SHIP1 to the inhibitory FcγRIIB (CD32B) ITIM.
• Required prior cross-linking of an ITAM-containing activatory receptor to evoke luciferase activity, mimicking CD32B signaling.
• Tested the assay's sensitivity and specificity using various experimental ICs, including aggregated IgG and antibody-antigen complexes.

Main Results:

• The assay detected experimental ICs sensitively (≤1 microg/ml) and differentiated between complexes of varying size and isotype.
• Successfully modeled FcγRIIB (CD32B) signaling, showing luciferase activity in discrete cell surface clusters.
• Provided proof-of-concept for detecting circulating ICs in sera from patients with systemic lupus erythematosus and rheumatoid arthritis.

Conclusions:

• A rapid, robust, and sensitive assay for detecting IgG immune complexes (IC) has been developed.
• The assay facilitates the study of Fc gamma receptor (FcγR) biology and FcγRIIB (CD32B) signaling.
• Potential applications include monitoring IC levels in autoimmune diseases and advancing FcγR-targeted therapeutics.