Background: Systemic lupus erythematosus (SLE) is a prototypic B-cell driven autoimmune disease in which the loss of B-cell tolerance and emergence of pathogenic autoreactive clones are accompanied by dysregulation of immune checkpoint molecules. Recent work from our group demonstrated that CD72, a C-type lectin and checkpoint molecule, is downregulated on B cells in active SLE, suggesting impaired regulation of B-cell activation. Nevertheless, the functional properties and molecular programmes defining disease-relevant B cells remain elusive, limiting the development of targeted immunomodulatory strategies. A better understanding of autoreactivity-associated B-cell subsets and their underlying functional and transcriptional programmes is therefore needed to advance targeted therapies in SLE.

Objectives: To identify autoreactivity-associated B-cell subsets defined by downregulation of CD72 and by the activation marker and death receptor CD95, and to comprehensively characterize the functional properties and transcriptional programmes of such CD72-negative B cells using functional analyses and bulk RNA sequencing.

Methods: Thirty patients with SLE, including 26 with active disease (SLEDAI-2K ≥ 4), and sixteen healthy controls (HCs) were evaluated. Among active SLE patients, thirteen had lupus nephritis (LN). B-cell phenotypes were analyzed using full-spectrum flow cytometry, and correlation analyses were performed. Functional analyses included B-cell receptor (BCR) signalling, cytokine (IL6, IL8 and TNF-α) expression and immunoglobulin (IgG and IgM) production. Transcriptomic profiling of expanded CD72-CD95+B cells in active SLE was performed using bulk RNA-sequencing.

Results: Extending our previous findings, we found that CD72-CD95+B cells were expanded in active SLE patients and especially enriched in plasmablast (20%), switched-memory (50%) and double negative 2 (30%) B-cell sub-populations. Transcriptomic profiling of lupus CD72-CD95+B cells revealed a distinct inflammatory transcriptional programme characterized by enrichment of cytokines- interferon- and NF-κB-associated pathways- as well as innate immune activation signatures. Consistent with these transcriptomic findings, enhanced NF-κB pathway activation aligned with increased phosphorylation of downstream BCR signalling molecules, SYK- and ERK-, following BCR stimulation. In parallel, enrichment of cytokine signalling pathways was reflected functionally by the capacity of SLE CD72-CD95+B cells to express IL6 and IL8. Upon in vitro stimulation, this cell subset produced high levels of IgG, indicating their capacity to mediate proinflammatory and antibody-secreting effector functions. Clinically, the frequency of CD72-CD95+B cells was increased in patients with lupus nephritis and in anti-dsDNA positive group and positively correlated with SLEDAI-2K ( r =0.6, p =0.02).

Conclusions: CD72-CD95+B cells are expanded in SLE and display an inflammatory phenotype defined by interferon- and NF-κB-associated transcriptional programmes, enhanced BCR signalling and increased cytokine and antibody production, suggestive of chronic immune activation and potential relevance to disease mechanisms. The enrichment of this B-cell phenotype in patients with lupus nephritis, together with its positive correlation with disease activity, underscores its association with relevant pathophysiological events.

REFERENCES: NIL.

Acknowledgments: NIL.

Disclosure of Interests: Kittikorn Wangriatisak ONO pharmaceuticals, Salim Ghannoum: None declared, Wenqi Huang: None declared, Maho Nakazawa: None declared, Giorgio Sechi: None declared, Sofia Zachari: None declared, Caroline Grönwall: None declared, Karine Chemin: None declared, Iva Gunnarsson: None declared, Vivianne Malmström: None declared, Francesca Faustini: None declared.