Date: Sunday, November 8, 2020
Session Type: Abstract Session
Session Time: 10:00AM-10:50AM
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.
 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
 Sahlström, P., et al. Different hierarchies of anti-modified protein autoantibody reactivities in rheumatoid arthritis. Arthritis Rheumatol. 2020
To cite this abstract in AMA style:Amara K, Hensvold A, Thyagarajan R, Israelsson L, Steen J, Wähämaa H, Hansson M, Engström M, van Vollenhoven A, Catrina A, Malmström V, Grönwall C. Exploring the RA Bone Marrow Niche by Single-cell Technology to Identify Long Lived ACPA+ Plasma Cells [abstract]. Arthritis Rheumatol. 2020; 72 (suppl 10). https://acrabstracts.org/abstract/exploring-the-ra-bone-marrow-niche-by-single-cell-technology-to-identify-long-lived-acpa-plasma-cells/. Accessed June 22, 2021.
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