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BACKGROUND: The antigenic trigger that drives the expansion of circulating plasmablasts and CD4(+) cytotoxic T cells (CD4(+) CTLs) in patients with IgG4-Related Disease (IgG4-RD) is presently unknown. OBJECTIVE: We sought to sequence Ig genes from single cell clones of dominantly-expanded plasmablasts and generate recombinant human monoclonal antibodies to identify relevant antigens in IgG4-RD using mass spectrometry. METHODS: Paired heavy and light chain cDNAs from dominant plasmablast clones were expressed as monoclonal antibodies (mAbs) and used to purify antigens using immunoaffinity chromatography. Affinity-purified antigens were identified by mass spectrometry and validated by ELISA. Plasma levels of the antigen of interest were also determined using ELISA RESULTS: mAbs expressed from the two dominant plasmablast clones of a patient with multi-organ IgG4-RD stained human pancreatic tissue sections. Galectin-3 was identified as the antigen specifically recognized by both mAbs. Anti-galectin-3 autoantibody responses were predominantly of the IgG4 isotype (28% of the IgG4-RD cohort, p = 0.0001) and IgE isotype (11% of IgG4-RD cohort, p = 0.009). No significant responses were seen from the IgG1, IgG2 or IgG3 isotypes. IgG4 anti-galectin-3 autoantibodies correlated with elevated plasma galectin-3 levels (p=0.001), lymphadenopathy (p=0.04), total IgG elevation (p=0.05), and IgG4 elevation (p=0.03). CONCLUSION: Affinity chromatography using patient-derived monoclonal antibodies identifies relevant auto-antigens in IgG4-RD. IgG4 galectin-3 autoantibodies are present in a subset of IgG4-RD patients and correlate with galectin-3 plasma levels. The marked elevations of circulating IgG4 and IgE observed clinically are, at least in part, due to the development of IgG4 and IgE specific autoantibody responses.