Expression of IgVH mRNAs in plasma cells derived from rheumatoid arthritis synovium detected by single-cell RT-PCR

Introduction

The synovial membrane in rheumatoid arthritis (RA) contains large lymphocytic infiltrates, sometimes organized in germinal-like centres. The presence of plasma cells within the synovium and the production of clonally related immunoglobulin transcripts have been observed. The exact nature of clonal expansion and its role in generation of specific, mutated antibodies in RA is not known. Somatic mutations and isotype switching in synovial B lymphocytes might suggest the antigen-driven process. This would be supported by the expression of recombination-activating genes 1 and 2 (Rag1 and Rag2).

Objectives

To calculate mutational frequencies of immunoglobulin heavy-chain transcripts in the single plasma cells generated from RA synovium. To analyze the frequency of isotype-switched plasma cells and Rag1 and Rag2 gene expression in inflamed RA synovial tissue.

Methods

Individual CD19⁺CD38⁺ plasma cells were isolated from digested synovium of two Caucasian RA patients using single-cell deposition. The cDNA from each single plasma cell was generated and a nested PCR specific for VH genes and Rag1 and Rag2 genes was performed. After sequencing, the VBASE database was used to assign VH, DH and JH gene segments and somatic mutations.

Results

Three different subsets of CD19⁺CD38⁺ plasma cells were detected. The first subset represents cells expressing only IgM transcripts (IgM⁺, 13.5%), cells in the second subset expressed only IgG transcripts (IgG⁺, 48.7%), and the cells in the third subset expressed both IgM and IgG mRNAs (IgM⁺IgG⁺, 37.8%). All of these detected IgVH mRNAs contained mutated sequences, indicating their memory cell origin. Significant differences in mutational frequencies were found between the subsets (IgM⁺ plasma cells, 3.8%; IgG⁺ plasma cells, 11.2%; and IgM⁺IgG⁺ cells, 6.3%). Interestingly, either Rag1 or Rag2 mRNA was observed in 83.3% of all analyzed CD19⁺CD38⁺ plasma cells, with the highest frequency in the IgM⁺IgG⁺ subset (71.4%).

Conclusions

The population of CD19⁺CD38⁺ plasma cells differentially expressing mutated IgVH mRNAs and reinducing Rag1 and Rag2 genes was observed in RA synovium. The IgM⁺IgG⁺ cells might represent cells switching from the IgM to the IgG isotype, and IgG⁺ plasma cells might correspond to post-switched cells producing high-affinity (auto)antibodies. The high mutational rate and reinduction of Rag genes suggest an antigen-driven process.