B lymphocyte autoimmunity in patients with mitral valve disease and secondary pulmonary hypertension

T. C. Funk-Hilsdorf (Berlin)¹, N. Dranishnikov (Berlin)², J. Klages (Berlin)³, F. F. Heinrich (Berlin)⁴, P. Durek (Berlin)⁴, G. Gabriela (Berlin)⁴, M. Kucherenko (Berlin)⁵, R. Rutenberg (Berlin)⁶, N. Merke (Berlin)², W. Karle (Berlin)¹, N. Krishnasamy (Berlin)⁷, M. Hommel (Berlin)⁶, T. Kaiser (Berlin)⁸, J. Kirsch (Berlin)⁹, H. Heidecke (Luckenwalde)¹⁰, K. Schulze-Forster (Luckenwalde)¹⁰, R. Dechend (Berlin)¹¹, C. Knosalla (Berlin)², H. Kuppe (Berlin)⁶, J. Grune (Berlin)¹², M. Mashreghi (Berlin)⁴, B. O'Brien (Berlin)⁶, W. M. Kuebler (Berlin)¹², S. Simmons (Berlin)^13
1Charité - Universitätsklinikum Berlin, Institut für Physiologie Berlin, Deutschland; ²Deutsches Herzzentrum der Charite (DHZC) Klinik für Herz-, Thorax- und Gefäßchirurgie Berlin, Deutschland; ³St. Joseph Krankenhaus Department of Anesthesia and Anesthesiological Intensive Care Medicine Berlin, Deutschland; ⁴German Rheumatism Research Centre Berlin (DRFZ) Berlin, Deutschland; ⁵Department of Cardiothoracic and Vascular Surgery Deutsches Herzzentrum der Charité Berlin, Deutschland; ⁶Deutsches Herzzentrum Berlin Klinik für Kardioanästhesiologie und Intensivmedizin Berlin, Deutschland; ⁷Charité - Universitätsmedizin Berlin Institut für Physiologie Berlin, Deutschland; ⁸German Rheumatism Research Centre Berlin (DRFZ) Flow Cytometry Core Facility Berlin, Deutschland; ⁹Deutsches Rheuma-Forschungszentrum Flow Cytometry Core Facility Berlin, Deutschland; ¹⁰CellTrend Gesellschaft mit beschränkter Haftung (GmbH) Luckenwalde, Deutschland; ¹¹HELIOS Klinikum Berlin-Buch Klinik und Poliklinik für Kardiologie und Nephrologie Berlin, Deutschland; ¹²Charité - Universitätsmedizin Berlin CC2: Institut für Physiologie, CCO Berlin, Deutschland; ¹³Institut für Physiologie, Charité - Universitätsmedizin Berlin Labor für Lungenkreislaufforschung, Nachwuchsgruppe Immunodynamik Berlin, Deutschland

Pulmonary hypertension (PH) due to left heart disease (LHD) is the most common form of PH. It is defined by a mean pulmonary arterial pressure >20 mmHg and a pulmonary arterial wedge pressure of >15 mmHg and is associated with increased morbidity and mortality. Emerging evidence suggests that immune dysregulation, particularly altered B cell homeostasis, may contribute to PH pathogenesis. Inflammatory conditions promote the escape of autoreactive B cells, which bypass central and peripheral tolerance mechanisms. A deeper insight into the contribution of B cell immunity to PH-LHD could lead to a better understanding of the underlying pathophysiological processes and thus facilitate the identification of novel cellular therapeutic targets.

We hypothesized that PH-LHD is associated with B cell dysregulation and increased autoreactivity, which drive disease progression. Blood samples were collected from patients with mitral valve (MV) disease undergoing MV repair or replacement and were stratified by echocardiography into low (n=35), intermediate (n=8), and high (n=26) PH probability groups.

Of note, integrated single-cell transcriptomic profiling and uniform manifold approximation and projection (UMAP) analysis of activated B cells and plasmablasts revealed a significant increase in the number of plasmablasts in MV-PH^high patients. Gene set enrichment analysis (GSEA) was performed using reference gene sets derived from previously published data on human IL-2/IL-21-activated B cells that had been differentiated into plasmablasts and then stimulated with transforming growth factor (TGF)-β in the presence of IL-6 and IL-21. This analysis revealed that the expansion of plasmablasts associated with the disease was driven by TGF-β signaling. This plasmablast response was accompanied by the upregulation of a dominant immunoglobulin (Ig)-A1 and -A2 class-switched response in MV-PH^high patients. Single-cell B cell receptor (BCR) repertoire analysis showed evidence of a seroconversion with the preferential use of the Ig heavy chain variable (IGHV) regions IGHV4-59 and IGHV4-61 paired with Ig kappa variable (IGKV) light chains IGKV1D-39 and IGKV3-20 in MV-PH^high patients, indicating clonal expansion. Organ-specific autoantibody testing revealed evidence of anti-liver, anti-kidney and anti-medulla/stomach autoreactivity in 58% of high-risk patients, 31% of low-risk patients and 7% of healthy donors. Elevated levels of anti-G protein-coupled receptor (GPCR) autoantibodies, particularly anti-protease-activated receptor 1 (PAR1) were also detected.

Our findings identify previously unrecognized B lineage-mediated autoimmunity in response to mechanistic volume overload induced by mitral valve insufficiency. In MV-PH^high patients, this response is characterized by the egress of plasmablasts into the circulation under TGF-β signaling control and predominant IgG- and IgA-mediated autoimmunity. These immune alterations likely contribute to the pathogenesis of PH-LHD and highlight novel pathways for diagnostic and therapeutic intervention. 

Funding: Teresa C. Funk-Hilsdorf reports a fellowship of the BIH and the DZHK. Wolfgang M. Kuebler reports grants from the Bundesministerium für Forschung, Technologie und Raumfahrt (BMFTR), the Deutsche Forschungsgemeinschaft (DFG) and the DZHK. Szandor Simmons reports grants from the DZHK and the German Foundation for Heart Research (F-09-19).