¹University of Lübeck, Klinik für Rheumatologie & Klinische Immunologie, Lübeck
²University of Lübeck, single cell core facility, Lübeck
³University of Lübeck, Medizinische Klinik 3, Lübeck
⁴CellTrend GmbH, Luckenwalde

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Introduction: Circulating antibodies directed against G protein-coupled receptors (GPCR) are found in varying amounts in both healthy people and patients with autoimmune diseases such as systemic sclerosis (SSc) [1]. With respect to function, antibodies targeting the angiotensin II type 1 receptor (AT1R) have been associated with SSc-like signs of skin and lung inflammation as well as fibrosis [2]. Less is known about the frequency of GPCR antibody-producing B cells and structural properties of GPCR antibodies. The aim of this study was to characterize the Ig gene repertoire of individual peripheral blood plasma cells derived from SSc patients by high-throughput single cell sequencing and compare the results to known AT1R antibodies.

Methods: Plasma cells derived from peripheral blood of SSc patients (n=3) were isolated using magnetic beads-based sorting (MACS). This was followed by simultaneous single cell-sequencing of the transcriptome and the Ig gene repertoire. Data preprocessing and analysis were conducted in R using packages such as Seurat and scRepertoire. Quality control and cell type annotations were performed to identify plasma cells and match them to their corresponding Ig gene sequences. The Ig gene repertoire of these plasma cells was analyzed with respect to complementarity determining region (CDR) 3 length, clonotypes, V(D)J gene segment usage and isotype. The data was examined for the presence of Ig genes resembling those of AT1R antibodies using Levenshtein distance. In addition, serum level of AT1R antibodies were determined using commercial Elisa.

Results: Based on cell type annotation, across the three samples, approx. 85% of cells were designated as plasma cells following MACS and scRNA seq. The number of plasma cells matching with productive and paired Ig genes reached up to approx. 94% of all cells that matched with the scRNA seq data. While the CDR3 length of the IGHV genes ranged from 6 until 32 amino acids (aa), the most frequent ones were 16 or 17 aa, respectively. The most abundant IGHV gene segments were IGHV3-23 and IGHV4-59. In terms of clonotypes, defined as displaying a highly similar CDR3 nucleotide sequence and an identical V gene, most of the clonotypes were unique, but hyperexpanded clones (> 1%) were also observed in 2 out of 3 samples. Regarding the isotype distribution, IgA, was most frequent, followed by IgG, IgM and IgD. Based upon Levenshtein distance, some IgG were identified that were most similar to known CDR3 sequences of IGHV and IGLV genes of monoclonal antibodies recognizing AT1R.

Conclusion: Combining transcriptomic and Ig gene single cell sequencing allows to identify immunoglobulins genes expressed by individual plasma cells derived from peripheral blood in SSc. Based upon these findings, additional experimental and bioinformatic analyses could lead to the identification and generation of donor-derived monoclonal antibodies targeting GPCR such as AT1R.

References

[1] Akbarzadeh R, Müller A, Humrich JY, Riemekasten G. When natural antibodies become pathogenic: autoantibodies targeted against G protein-coupled receptors in the pathogenesis of systemic sclerosis. Front Immunol. 2023 Jun 8;14:1213804. DOI: 10.3389/fimmu.2023.1213804
[2] Yue X, Yin J, Wang X, Heidecke H, Hackel AM, Dong X, Kasper B, Wen L, Zhang L, Schulze-Forster K, Junker J, Grasshoff H, Müller A, Wallukat G, Schimke I, Zeiner J, Deckstein LM, Mertens N, Kerstein-Staehle A, Hundt JE, Kostenis E, Yu X, Riemekasten G, Petersen F. Induced antibodies directed to the angiotensin receptor type 1 provoke skin and lung inflammation, dermal fibrosis and act species overarching. Ann Rheum Dis. 2022 Aug 11;81(9):1281-9. DOI: 10.1136/annrheumdis-2021-222088