Background: Systemic sclerosis (SSc) is a heterogeneous autoimmune disease characterized by immune dysregulation, vasculopathy and fibrosis. Existing clinical stratification, based on clinical assessment and autoantibody profiles, is inadequate for distinguishing active and inactive disease states in SSc. This limitation underscores an unmet need for deeper mechanistic insights into the immunological processes underlying the active disease processes in SSc. Distinguishing active from inactive disease remains challenging, limiting timely therapeutic intervention. We hypothesized that active SSc is defined by distinct peripheral immune signatures linked to chemokine-driven tissue trafficking.

Objectives: To identify and characterize the composition and functional states of the peripheral blood-derived immune cells in patients with active SSc, inactive SSc and healthy controls.

Methods: Peripheral blood mononuclear cells from patients with active SSc (n=14), inactive SSc (n=26) and healthy controls (n=10) were profiled using high-dimensional mass cytometry (CyTOF), a single-cell proteomic technology that allowed simultaneous characterization of surface and intracellular proteins on individual cells. Disease activity was defined using the EUSTAR activity index. Unsupervised clustering (FlowSOM), dimensionality reduction, supervised gating and immune network analyses were performed. Publicly available transcriptomic datasets from SSc-affected skin, lung and gastrointestinal tissues were re-analysed to interrogate the expression of chemokine ligands corresponding to receptors identified in peripheral immune subsets from CyTOF analyses.

Results: Active SSc demonstrated a distinct peripheral immunome compared with inactive SSc and healthy controls, whereas inactive SSc more closely resembled healthy individuals. Active disease was characterized by expansion of naïve and transitional B-cell subsets expressing mucosal homing receptors including CCR6. The monocyte compartment showed enrichment of CD14 ⁺ monocytes with interferon-response signatures. NK-cell composition was altered, with a shift toward more cytotoxic subsets. T-cell alterations included reduced circulating T cells co-expressing specific chemokine receptors with potential chemotaxis towards inflamed tissues. Innate-like MAIT cells with mucosal homing receptor and pro-fibrotic effector were increased in active SSc. Immune network analysis revealed a denser, highly interconnected immunome in active SSc, with a disease-specific module enriched for CLA ⁺ and CCR4 ⁺ clusters displaying increased centrality. Re-analysis of SSc tissue transcriptomic datasets demonstrated upregulation of CXCL9, CXCL11, CXCL12 and CX3CL1, ligands for chemokine receptors expressed by immune cell subsets identified in our CyTOF analyses, supporting chemokine-mediated recruitment of peripheral immune cells into affected tissues.

Conclusions: Active systemic sclerosis is defined by coordinated peripheral immune dysregulation with chemokine-driven tissue trafficking. Integration of single-cell immune profiling with tissue transcriptomics sheds light into mechanistic links between circulating immune states and tissue inflammation. These findings serve as a foundation for future studies investigating immune-based stratification of disease activity and highlight potential therapeutic targets in active SSc.

REFERENCES: NIL.

Acknowledgments: NIL.

Disclosure of Interests: Maria Noviani Boehringer Ingelheim, Alfred Chia: None declared, Ahmad Lajam: None declared, Pavanish Kumar: None declared, Vasuki Ranjani Chellamuthu: None declared, Katherine Nay Yaung: None declared, Martin Wasser: None declared, Joo Guan Yeo: None declared, Jacques Behmoaras: None declared, Enrico Petretto: None declared, Andrea Low Novartis, Boehringer Ingelheim, Johnson & Johnson, Boehringer Ingelheim, Boehringer Ingelheim, Johnson & Johnson, Salvatore Albani: None declared.