The Comprehensive Live Cell‐Based Cytotoxicity Assay for Monitoring Disease Activity and Guiding Rescue Therapy in Acute Attacks of NMOSD

Vollständiger Abstract

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ABSTRACT Objective Neuromyelitis optica spectrum disorder (NMOSD) is a devastating neurological disease that lacks serological biomarkers that can accurately reflect disease activity. We established a live cell‐based assay (LCBA) using serum with endogenous complement to quantify the overall cytotoxicity, offering a novel functional tool for monitoring disease activity. Methods 111 samples from 65 AQP4 ‐ IgG + NMOSD patients were enrolled in this prospective study for serological analysis of C1q, C5a, sC5b ‐9, CH50 , and AQP4 ‐ IgG titers. A novel live cell‐based complement‐dependent cytotoxicity ( NMO ‐ LCBA ‐ CDC ) assay was developed using AQP4 ‐transfected HEK293T cells exposed to test sera. Serum cytotoxicity was assessed by immunofluorescence and flow cytometry and quantified by calculating the cytotoxic index ( CI ). We also evaluated the cytotoxicity following intravenous methylprednisolone ( IVMP ) and subsequent rescue therapies. Results Serum C5a and sC5b‐9 levels were significantly elevated during NMOSD acute attacks ( p < 0.05). Following IVMP, the complement system including C5a, sC5b‐9, CH50, and C1q decreased ( p < 0.05), without change in AQP4‐IgG titers. The CI was higher in the acute phase than in the remission phase ( p = 0.0011). However, IVMP did not significantly reduce the CI during acute attacks. The NMO‐LCBA‐CDC assay demonstrated robust reproducibility in detecting serum cytotoxicity. Among IVMP non‐responders receiving rescue therapy, both PE/IA and C5 inhibitors appeared to be associated with favorable CI reduction profiles. Conclusion By integrating AQP4‐IgG and endogenous complement activity, the NMO‐LCBA‐CDC assay provides a comprehensive assessment of serum cytotoxicity in NMOSD. It holds significant potential for monitoring disease activity and evaluating therapeutic effects in NMOSD.