De novo oligoclonal expansions of circulating plasmablasts in active and relapsing IgG4-related disease

Abstract

Background: IgG4-related disease (IgG4-RD) is a poorly understood, multiorgan, chronic inflammatory disease characterized by tumefactive lesions, storiform fibrosis, obliterative phlebitis, and accumulation of IgG4-expressing plasma cells at disease sites.

Objective: The role of B cells and IgG4 antibodies in IgG4-RD pathogenesis is not well defined. We evaluated patients with IgG4-RD for activated B cells in both disease lesions and peripheral blood and investigated their role in disease pathogenesis.

Methods: B-cell populations from the peripheral blood of 84 patients with active IgG4-RD were analyzed by using flow cytometry. The repertoire of B-cell populations was analyzed in a subset of patients by using next-generation sequencing. Fourteen of these patients were longitudinally followed for 9 to 15 months after rituximab therapy.

Results: Numbers of CD19(+)CD27(+)CD20(-)CD38(hi) plasmablasts, which are largely IgG4(+), are increased in patients with active IgG4-RD. These expanded plasmablasts are oligoclonal and exhibit extensive somatic hypermutation, and their numbers decrease after rituximab-mediated B-cell depletion therapy; this loss correlates with disease remission. A subset of patients relapse after rituximab therapy, and circulating plasmablasts that re-emerge in these subjects are clonally distinct and exhibit enhanced somatic hypermutation. Cloning and expression of immunoglobulin heavy and light chain genes from expanded plasmablasts at the peak of disease reveals that disease-associated IgG4 antibodies are self-reactive.

Conclusions: Clonally expanded CD19(+)CD27(+)CD20(-)CD38(hi) plasmablasts are a hallmark of active IgG4-RD. Enhanced somatic mutation in activated B cells and plasmablasts and emergence of distinct plasmablast clones on relapse indicate that the disease pathogenesis is linked to de novo recruitment of naive B cells into T cell-dependent responses by CD4(+) T cells, likely driving a self-reactive disease process.

Keywords: CDR3; IgG(4)-related disease; autoreactivity; immunoglobulin heavy chain variable region repertoire; next-generation sequencing; plasmablasts; rituximab; somatic hypermutation.

Fig 1. Expansion of CD19⁺CD20^-IgG4⁺ plasmablasts in IgG4-related disease

A, H&E and immunohistochemical stains for CD19, CD20, and IgG4 performed on serial sections of a submandibular gland biopsy are shown. An area peripheral to a CD19⁺CD20⁺B-cell follicle that is enriched for IgG4⁺CD19⁺CD20^- cells is marked. B, Flow cytometry gating scheme used to identify circulating CD19⁺CD27⁺CD38^hi plasmablasts (red box). C, Surface staining of CD19⁺CD27⁺CD38^hi plasmablasts for IgG4, IgG, CD20 and SLAMF7 cells. Data from representative patients is shown as histograms overlaid on CD19⁺CD27^-CD38^- B cells as a negative control. D, The numbers of plasmablasts in the peripheral blood of IgG4-RD subjects (n = 84) and healthy controls (n = 16) are shown (mean ± S.E.M., p < 0.001).

Fig 2. Restricted repertoire of expanded plasmablast populations

A, The immunoglobulin heavy chain (IGH) repertoire of CD19⁺CD27⁺CD38^hi plasmablasts sorted from a representative patient (P26) is shown as a 3D histogram. The x- and y- axes represent IGH-V and IGH-J regions, respectively, and the z- axis indicates the number of productive IGH-rearrangements detected by next-generation sequencing. B, Comparison of read counts of expanded clones from patient P26 detected by next-generation sequencing (green bars) and SC-PCR sequencing (orange bars). The corresponding CDR3 sequences ordered by frequency as seen on next-generation sequencing are shown in the table inlay. C, Correlation between the absolute read counts of the most frequent IGH CDR3 sequences determined by next-generation sequencing and number of cells with the corresponding CDR3 sequence determined by SC-PCR (n = 80 cells) of flow-sorted IgG4⁺ plasmablasts from patient P26.

Fig 3. Rituximab-mediated depletion of plasmablasts improves clinical outcome

A, Measurement of the therapeutic response to rituximab using the IgG4-RD Responder Index. B, Decline in plasma-IgG4 concentrations after rituximab infusion. Colored lines represent individual patients followed upto 15 months after rituximab infusion. Dotted-horizontal line indicates the upper limit of normal (135 mg/dL). C and D, Rituximab-induced decline in the proportion of circulating B-cells (n =23, p < 0.001) (C) and plasmablasts (n=23, p < 0.001) (D) by day 30.

Fig 4. Repopulation of plasmablasts as a marker of disease relapse

A, Longitudinal follow-up of the CD19⁺ B cell compartment in 14 IgG4-RD patients following rituximab therapy, plotted as a percentage of the baseline B cell count. B and C, Longitudinal follow-up of CD19⁺CD27⁺CD38⁺ plasmablast counts following rituximab treatment in 6 IgG4-RD patients who experienced sustained remission (B) and 6 patients who relapsed (C).

Fig 5. Clonal divergence in relapsing pool of plasmablasts

The heat maps show the degree of expansion of plasmablast clones bearing specific IGH V and J combinations in two patients who relapsed, P11 (A) and P15 (B), using a blue-red color gradient with the most expanded clones shown in red. Unused V-J combinations are colored black. The tables show a comparison of the CDR3 amino acid sequences of the dominant clones sharing the same IGHV-J combinations before and after rituximab therapy. Highly expanded clones are colored red in the tables and the numbers in parentheses indicate CDR3 nucleotide sequence identity.

Fig 6. Expanded populations of plasmablasts exhibit enhanced somatic hypermutation and autoreactivity

A & B, Comparison of replacement-R and silent-S mutations and R/S ratio in FR3 and CDR3 regions of IGHV-sequences of plasmablasts from (A) 5 untreated IgG4-RD subjects and (B) 3 relapsing IgG4-RD patients following rituximab therapy with CDR3 sequences from naïve B cells from 3-control samples as analyzed from NGS-reads (mean ± S.E.M., * p < 0.05, ** p < 0.005). C, Immunofluorescence staining of Hep-2 coated slides with single plasmablast derived antibody clones. Clones with bright cytosolic staining are labeled in red.

Fig 7. A speculative model for the role of activated B cells and plasmablasts in a presumed CD4+ T cell mediated pathogenic process causing IgG4-RD

Activated somatically hypermutated B cells or plasmablasts may serve as effective antigen presenting cells for pathogenic effector CD4+ T cells in disease sites. In patients that relapse after rituximab-mediated B cell depletion therapy plasmablasts may be generated de novo from naïve B cells (solid arrow) or from CD20⁺ memory B cells that survive rituximab therapy (dashed arrow), facilitated by T-B cooperation in secondary lymphoid organs (not shown).