Detection of Experimental and Clinical Immune Complexes by Measuring SHIP-1 Recruitment to the Inhibitory Fc{gamma}RIIB [NOVEL IMMUNOLOGICAL METHODS]

Fc receptors (FcR) are involved in multiple aspects of immune cell regulation, are central to the success of mAb therapeutics, and underpin the pathology of several autoimmune diseases. However, reliable assays capable of accurately measuring FcR interactions with their physiological ligands, IgG immune complexes (IC), are limited. A method to study and detect IC interactions with FcRs was therefore developed. This method, designed to model the signaling pathway of the inhibitory FcRIIB (CD32B), used NanoLuc Binary Interaction Technology to measure recruitment of the Src homology 2 domain–containing inositol phosphatase 1 to the ITIM of this receptor. Such recruitment required prior cross-linking of an ITAM-containing activatory receptor, and evoked luciferase activity in discrete clusters at the cell surface, recapitulating the known biology of CD32B signaling. The assay detected varying forms of experimental IC, including heat-aggregated IgG, rituximab–anti-idiotype complexes, and anti-trinitrophenol–trinitrophenol complexes in a sensitive manner (≤1 μg/ml), and discriminated between complexes of varying size and isotype. Proof-of-concept for the detection of circulating ICs in autoimmune disease was provided, as responses to sera from patients with systemic lupus erythematosus and rheumatoid arthritis were detected in small pilot studies. Finally, the method was translated to a stable cell line system. In conclusion, a rapid and robust method for the detection of IC was developed, which has numerous potential applications including the monitoring of IC in autoimmune diseases and the study of underlying FcR biology.

from #ORL-AlexandrosSfakianakis via ola Kala on Inoreader http://ift.tt/2ojEN6k