Background/Purpose:

The immunological hallmark of rheumatoid arthritis (RA) is autoreactivity to citrullinated proteins and rheumatoid factor activity. Hence, the adaptive immune system and B cells are postulated to be central in RA pathogenesis, yet possible underlying B cell baseline autoreactivity defects have not been studied. Here, we use next generation sequencing (NGS) to study overall B cell repertoire shifts in RA patients compared to healthy individuals, as well as in ACPA specific B-cell populations.

Methods:

Peripheral blood B cell receptor (BCR) repertoires were investigated in 13 seropositive RA patients and six age-matched healthy blood donors using PCR multiplex amplicon libraries with a molecular barcode strategy and the Illumina MiSeq platform to generate full variable region coverage. Citrullinated vimentin (Cit-Vim, aa 59-74) and citrullinated fibrinogen (Cit-Fib, aa a566-580) positive B cells were sorted using antigen-tetramers based on a scaffold with grafted RA peptides into the sunflower trypsin inhibitor-1, SFTI-1. Sequences were filtered using pRESTO, annotated by IMGT and Change-O. Filtering of 14×10^6 transcript reads generated 587 000 unique antibody V-regions. Genomic DNA was used to analyze recombination frequency in out-of-frame (OOF) sequences. Analysis of variable gene frequency was performed by Chi-square with Yates correction.

Results:

Several significant shifts in the RA B cell repertoire could be observed. RA-derived circulating B cells had increased class-switching to IgG3 and IgG4 but lower IgA. Strikingly, there was a significantly higher frequency of VH with low somatic hypermutation (SHM) level in RA-derived B cells (<5 mutations, p<0.0001 14.7% vs 8,7). This was seen in all sequences, IgM and class-switched, but especially prominent in IgG1 rearrangements (9.6% vs 18.8% low mutation, p<0.0001 OR=2.2 CI:2.0-2.35). Yet, IgG1 displayed evidence of stronger antigen selection pressure as compared to controls (5.5% vs 3.7%, p<0.0001 OR=1.5 CI: 1.3-1.7) suggesting that an increased IgG1 class-switching of unmutated BCR may initiate further B cells selection. Recombination of VH4 sequences were higher in RA and there was a general RA VH4 bias that was enriched in Cit-Vim and Cit-Fib positive cells, although class-switched Cit-Fib had a VH1, VH3 profile. Furthermore, VH N-linked glycosylation sites accumulated with SHM and were increased in RA (IgG > 15 mutations, p<0.0001, 17.2% vs 13.8%), primarily driven by IgG1, and further elevated in Cit-Vim and Cit-Fib populations (19.3%, 32.2%). BCR carrying the autoreactivity-associated VH4-34 gene with a germline-encoded N-glyc site were also enriched in RA and tetramer-sorted populations, especially in IgMs.

Conclusion:

We found significant distortions in the B cell populations in seropositive RA compared to controls, especially within IgG1. Many findings were further enriched in the citrulline specific B cells suggesting correlation with ACPA autoreactivity. Overall, the differences may partly be explained by a strong ongoing B cell response but could also reflect baseline shifts and elevated natural autoreactivity as an underlying mechanism in the RA pathogenesis.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/repertoire-studies-in-rheumatoid-arthritis-reveal-b-cell-distortions-and-baseline-shifts-in-unmutated-igg/