Accumulation of Antigen-Driven Lymphoproliferations in Complement Receptor 2/CD21-/low B Cells From Patients With Sjögren's Syndrome

Abstract

Objective: Patients with Sjögren's syndrome (SS) are prone to develop malignant lymphomas, and a correlation has been established between the lymphoproliferations occurring in these disorders and the presence in patients' blood of an unusual B cell population that down-regulates complement receptor 2/CD21. This study was undertaken to identify the B cell compartment from which these lymphoproliferations emerge and determine the mechanisms that promote clonal B cell expansion in patients with SS.

Methods: The reactivity of antibodies expressed by CD19+CD10-CD27-IgM+CD21^-/low cells isolated from the blood of patients with SS was tested using a polymerase chain reaction-based approach that allows us to clone and express, in vitro, recombinant antibodies produced by single B cells.

Results: Clonal expansions were identified in CD21^-/low B cells isolated from the peripheral blood of 3 patients with SS. These lymphoproliferations expressed B cell receptors (BCRs) that displayed somatic hypermutation lineage trees characteristic of a strong selection by antigens; one of these antigens was identified as a ribosomal self antigen. When the mutated BCR sequences expressed by the expanded CD21^-/low B cell clones from patients with SS were reverted in vitro to their germline counterparts, one clone remained autoreactive.

Conclusion: Clonal lymphoproliferations in patients with SS preferentially accumulate in the autoreactive CD21^-/low B cell compartment often expanded in these subjects, and recognition of self antigens may drive the clonal B cell expansion while further refining BCR self-reactivity.

Figure 1. SS monoclonal expansions accumulate in CD21^−/low B cells

(A) Dot plots represent CD19 and CD21 expression on CD19⁺CD27⁻ gated B cells from a representative healthy donor (Tables S3–4) and SS patients. (B) The frequencies of monoclonal expansion (in black) in CD19⁺CD27⁻CD21^−/low and mature naïve B cells are summarized in pie charts with the number of sequences analyzed indicated in the centers. ND, not done.

Figure 2. Clonal expansions in SS patients are driven by antigenic stimulation

(A) Sequence alignment for Ig heavy chains of the monoclonal expansions found in three SS patients. Replacements are in uppercase and silent mutations are in lowercase. Codon positions within framework regions (FWRs) and complementary determining regions (CDRs) are indicated. (B) Clonal trees of the monoclonal expansions found in the three SS patients are represented. Mutations and their codon position are written on the sides of branches, with affected regions in parenthesis. Amino acid changes are indicated. (C) Frequencies of mutations in VH genes from combined lymphoproliferations were calculated from the number of replacement and silent nucleotide exchanges per base pair in FWRs and CDRs. The replacement versus silent (R/S) mutation ratio for each region is indicated when the number of silent mutation is not zero.

Figure 3. Kappa chains from SS expanded clones also displayed intraclonal diversification with poorly branched lineage trees

(A) Sequence alignment for Ig kappa chains of the monoclonal expansions found in three SS patients. Replacements are in uppercase and silent mutations are in lowercase. Codon positions within FWRs and CDRs are indicated. (B) Clonal trees of the monoclonal expansions found in the three SS patients are represented. Mutations and their codon position are written on the sides of branches, with affected regions in parenthesis. Amino acid changes are indicated. (C) Frequencies of mutations in Vκ genes from combined lymphoproliferations were calculated from the number of replacement and silent nucleotide exchanges per base pair in FWRs and CDRs. The replacement versus silent (R/S) mutation ratio for each region is indicated when the number of silent mutation is not zero.

Figure 4. SS monoclonal expansions express autoreactive antibodies

(A) Antibodies from mutated (full diamonds) and revertant (open diamonds) clones from SS patients were tested by ELISA for reactivity against HEp-2 cell lysate. Dotted and green lines show ED38-positive and mnUNGdef05 λ50 low positive control, respectively. Horizontal lines show cutoff OD405 for positive reactivity. (B) Recombinant mutated antibody from SS59 shows anti-nuclear reactivity. (C) Antibodies from mutated and revertant clones from SS patients were tested by ELISA for polyreactivity against dsDNA, insulin, and LPS antigens. Dotted and red lines show ED38-positive and mnUNGdef05 κ69 low positive control, respectively. (D) Ro52/SSA and rheumatoid factor (RF) reactivity was assessed by ELISA for mutated and revertant antibodies.

Figure 5. SS59 lymphoproliferation binds ribosomal components

Antibodies from mutated and revertant clones from SS patients were used for immunoprecipitation from human keratinocyte lysates. RNAs within immunoprecipitates were end-labeled with [32P]pCp. The boxed region denotes the 5S and 5.8S ribosomal RNAs enriched after immunoprecipitation with recombinant mutated SS59 clones.