Identification of galectin-3 as an autoantigen in patients with IgG4-related disease

Abstract

Background: The antigenic trigger that drives expansion of circulating plasmablasts and CD4⁺ cytotoxic T cells in patients with IgG₄-related disease (IgG₄-RD) is presently unknown.

Objective: We sought to sequence immunoglobulin genes from single-cell clones of dominantly expanded plasmablasts and generate recombinant human mAbs to identify relevant antigens in patients with IgG₄-RD by using mass spectrometry.

Methods: Paired heavy and light chain cDNAs from dominant plasmablast clones were expressed as mAbs and used to purify antigens by using immunoaffinity chromatography. Affinity-purified antigens were identified by using mass spectrometry and validated by means of ELISA. Plasma levels of the antigen of interest were also determined by using ELISA.

Results: mAbs expressed from the 2 dominant plasmablast clones of a patient with multiorgan 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 = .0001) and IgE isotype (11% of the IgG₄-RD cohort, P = .009). No significant responses were seen from the IgG₁, IgG₂, or IgG₃ isotypes. IgG₄ anti-galectin-3 autoantibodies correlated with increased plasma galectin-3 levels (P = .001), lymphadenopathy (P = .04), total IgG level increase (P = .05), and IgG₄ level increase (P = .03).

Conclusion: Affinity chromatography using patient-derived mAbs identifies relevant autoantigens in patients with IgG₄-RD. IgG₄ galectin-3 autoantibodies are present in a subset of patients with IgG₄-RD and correlate with galectin-3 plasma levels. The marked increases in levels of circulating IgG₄ and IgE observed clinically are, at least in part, caused by the development of IgG₄- and IgE-specific autoantibody responses.

Keywords: IgG(4)-related disease; autoantibody; autoantigen; galectin-3; plasmablast.

Figure 1

A) Bulk repertoire of sorted plasmablasts demonstrating IGH V gene usage on the x-axis, IGH J gene usage on the z-axis and number of sequencing reads on the y-axis. Each bar represents a clonal family. B) Pie-chart showing the frequency of dominantly expanded clones from single cell sequencing. The specific CDR3 amino acid sequence and percentage of wells from the single cell plate are displayed. The dominant clone, labeled “Clone 1” represented 22 of 34 sequenced individual cells.

Figure 2

Immunofluorescence of pancreatic tissue. A-B) Negative (anti-CD57) and positive (anti-actin) control antibodies. C-D) Patient-derived monoclonal antibodies from dominantly expanded plasmablasts demonstrating intense reactivity in a cytosolic pattern.

Figure 3

A) Intra-cellular flow cytometry revealed differential reactivity of mAbs with PaCa2 cells compared controls. Vertical cutoff lines drawn to exclude unstained cells. No difference was observed in HEK293T cells. B) Confocal microscopy of PaCa2 cells stained with biotinylated mAb clone 1 demonstrating cytosolic and cell surface staining patterns.

Figure 4

Heat map demonstrating differential protein binding of PaCa2 cell lysate by mAb clones #1 and #2. Scale reflects log2-fold change divided by isotype control. Background was determined using an isotype control. Galectin-3 was identified as the antigen with the highest binding affinity for both mAb clones.

Figure 5

IgG4 and IgE isotype-specific anti-galectin-3 antibodies were confirmed by ELISA with 34 and 12 of 121 IgG4-RD subjects demonstrating IgG4 and IgE antibody responses, respectively. Dashed lines represent two standard deviations above the healthy donor mean.

Figure 6

IgG subclass and IgE serum values of IgG4-RD cohort plotted as frequency of elevation among the cohort (y-axis), isotype (z-axis), and degree of elevation (x-axis). ULN indicates the upper limit of normal for the clinical reference laboratory value for each isotype.

Figure 7

A) Circulating Galectin-3 levels were quantified by ELISA and 15.5% of the IgG4-RD cohort were found to have elevated levels compared to age-matched healthy donors. Subjects with elevated galectin-3 levels are indicated by red color. B) Segregating the cohort by those with vs those without elevated galectin-3 levels showed a nearly 3-fold enrichment in IgG4 anti-galectin-3 autoantibody responses among the subset with elevated circulating Gal-3 levels.

Figure 8

Three subjects with positive anti-Galectin-3 antibody responses studied longitudinally by ELISA and plotted against IgG4-RD-Responder Index (RI) on the right Y-axis. Yellow symbols indicate dates of B cell depletion therapy with rituximab, after which, anti-galectin-3 antibody titers consistently declined. The black arrow in Subject 336 indicates the initiation of low-dose prednisone that successfully maintained remission in this subject with a subsequent rise in anti-galectin-3 response.