Background/Purpose: Autoantibodies is a hallmark of rheumatoid arthritis, placing the adaptive immune system and B-cells centrally in the pathogenesis. The anti-citrullinated autoantibodies (ACPA) detected in the serum can be produced either by circulating plasma blasts, locally residing plasma cells the joint, or long-lived plasma cells in the bone marrow (BM). Here we use different single cell methods to investigate the bone marrow niche.

Methods: Bone marrow samples from proximal femur in RA patients undergoing hip joint replacement was processed to isolate mononuclear cells. In total four CCP2+ and one CCP2- RA patients were investigated. All patients were HLA shared epitope positive. Firstly, CD138+ plasma cells were single cell sorted by flow cytometry followed by BCR sequencing. For two of the RA patients, MACS enriched BM B cell and peripheral B cells were further investigated by 10X Genomics Chromium methodology for single-cell RNAseq profiling and immunoglobulin sequencing. Selected paired heavy-light Ig-chains with N-glycosylation sites and high somatic hypermutation level were subsequently expressed as recombinant IgG and investigated for citrulline reactivity using an autoantigen array.

Results: For the single-cell sorted CD138+ cells, in total 509 complete IgG+ BCR sequences were generated and 44 monoclonal IgG were expressed (36 from CCP+ RA). Among the mAbs, two clones from two different patients were identified as CCP2+. One of these originated from an IgG4+ plasma cell and had strong reactivity and multi-specificity for different cit-peptides (e.g. cit-fibrinogen, cit-tenascin C, cit-hnRNP derived). 10X cell capture of 8391 BM B-cells from the same patient, generated 856 Ig with a RNAseq plasma cell profile (i.e. CD27+ XBP1+ MZB+ DERL3+ JCHAIN+ SSR4+) from 832 unique clones defined by paired HCDR3-LCDR3, occurring 1-6 times. Five 10X clones whereof two in plasma cell clusters were overlapping with the single cell sort (N=92). We observed a high frequency of both Fab glycosylation (31% of IgG+ plasma cells) and IgG4+ cells (6% of IgG+ plasma cells). Notably, also IgA+ plasma cells could be identified (37% IgA1+ 3% IgA2+). In comparison, by 10X, 24% BM IgG+ plasma cells from the CCP2- patient had Fab N-glyc sites and no IgG4+ were detected. We found no direct BCR overlap with the BM plasma cells in investigated paired peripheral B cells.

Conclusion: ACPA+ long lived plasma cells can be found in the bone marrow niche and BM-derived ACPA display multi-reactivity profiles as also seen in synovial ACPA+ plasma cells [1-2]. However, they may be relatively rare and no large clonal expansions were found. In the bone marrow, similar to blood, B-cell repertoire skewing with increased N-glycosylation and IgG4 frequencies can be observed. 

References:

[1] Steen, J., et al Recognition of amino acid motifs, rather than specific proteins, by human plasma cell-derived monoclonal antibodies to posttranslationally modified proteins in rheumatoid arthritis. Arthritis Rheumatol. 2019

[2] Sahlström, P., et al. Different hierarchies of anti-modified protein autoantibody reactivities in rheumatoid arthritis. Arthritis Rheumatol. 2020

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/exploring-the-ra-bone-marrow-niche-by-single-cell-technology-to-identify-long-lived-acpa-plasma-cells/