B-cell immune repertoire analysis of autoimmune neurological syndromes with anti-GAD65 antibodies

Background: Autoimmune neurological syndromes (AINS) associated with anti-GAD65 antibodies encompass a spectrum of disorders, including limbic encephalitis (LE), stiff-person syndrome (SPS), and cerebellar ataxia (CA). Despite the universal presence of anti-GAD65 antibodies across these syndromes, patients exhibit remarkable clinical phenotypic diversity. Exploring the overall B-cell receptor (BCR) profile of patients with anti-GAD65 AINS may provide clues to the molecular pathological mechanisms underlying this phenotypic diversity. Methods: We performed high-throughput B-cell receptor (BCR) heavy-chain sequencing (Illumina NovaSeq 6000) on peripheral blood samples from 9 anti-GAD65 AINS patients (7 LE, 1 SPS, 1 CA/SPS overlapping) and 11 healthy controls (HC). Analysis encompasses clonotype profiling, somatic hypermutation (SHM) rates, CDR3 physicochemical characterization, repertoire diversity, and machine learning-based VDJ feature identification. Results: ⁵), lower SHM rates (3.54% vs. 4.73%), and enrichment of hyperexpanded clones. Distinct IGHV/IGHJ usage was identified, notably decreased IGHV1-2 and IGHV1-46 with elevated IGHV3-7. A random forest classifier built on characteristic VDJ clonotypes achieved near-perfect diagnostic performance (AUC > 0.99). Cross-patient shared clonal clusters further highlighted disease-associated BCR convergence. Conclusions: BCR repertoire heterogeneity across anti-GAD65 AINS subtypes illuminates the immunological underpinnings of phenotypic diversity. The identified VDJ signatures and common clonotypes represent candidate biomarkers for precise diagnosis and rational targets for BCR-lineage-directed immunotherapy.