Date: Monday, November 9, 2015
Session Type: ACR Poster Session B
Session Time: 9:00AM-11:00AM
Background/Purpose: B cells play a central role in Sjogren’s syndrome (SS) pathogenesis whereby autoreactive B-cells populate ectopic germinal centres (eGC) in SS-salivary glands (SG) and undergo somatic hypermutation (SHM) and class-switch recombination of the immunoglobulin (Ig) genes. However, the capacity of specific B cell clones to seed eGC in different SG and undergo clonal diversification is unclear. Here we aimed to unravel the dynamics of B cell recirculation among adjacent minor SG (mSG) biopsies, by investigating immunoglobulin heavy chain (IgH) gene rearrangements and SHM using a high-throughput next-generation sequencing approach.
Methods: IgH gene usage and SHM were investigated in four pairs of mSG biopsies from 4 pSS patients with high B cell infiltration and eGC. Retro-transcription of 100 ng total RNA using constant domain Ig-l, -c and -a specific primers yielded Ig-specific cDNA for use as template in producing libraries representing B cell Ig-gene rearrangements in SS mSGs. Sequencing was performed using the Roche GS-FLX titanium platform.
Results: We generated ~166,000 reads >350 bp in total, and detected 1631 clonotypes (defined as reads with the same IgHV and IgHJ gene usage and equal CDR3 length) across all eight samples. Between 5 and 9 shared clonotypes were observed among paired SG biopsies in all four patients, demonstrating the same B cell can recirculate between different glands. Lineage tree analysis revealed three different patterns of B cell circulation across the biopsies: a) unidirectional circulation of B cell clones from one biopsy to another; b) circulation between the two biopsies – circulating in both directions; c) an undefined pattern with a less-mutated and unidentified precursor migrating from one site to the other.
Conclusion: We show that B cells recirculate between mSG in SS and undergo further rounds of SHM in adjacent glands. These findings demonstrate the dynamic nature of B cell affinity maturation in SS within eCG.
To cite this abstract in AMA style:Murray-Brown W, Carlotti E, Tappuni A, Sutcliffe N, Pitzalis C, Bombardieri M. Next-Generation Sequencing Demonstrates Dynamic Recirculation of B Cell Clones in Ectopic Lymphoid Structures of Sjögren’s Syndrome [abstract]. Arthritis Rheumatol. 2015; 67 (suppl 10). https://acrabstracts.org/abstract/next-generation-sequencing-demonstrates-dynamic-recirculation-of-b-cell-clones-in-ectopic-lymphoid-structures-of-sjgrens-syndrome/. Accessed July 3, 2020.
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