Juvenile systemic sclerosis (jSSc) is a rare autoimmune disorder marked by skin fibrosis and involvement of multiple organs. For patients with severe, treatment-refractory disease, autologous stem cell transplantation (ASCT) is an emerging therapeutic option. Despite clinical interest, the cellular and molecular immune changes that follow ASCT in jSSc are not well characterized. This study used longitudinal single-cell profiling to define how immune cells, with an emphasis on monocytes, evolve after ASCT in jSSc.
Peripheral blood mononuclear cells (PBMCs) were obtained from three patients with jSSc at four timepoints: before ASCT (baseline) and at 6, 12, and 24 months post-ASCT. Healthy control samples were also profiled for baseline comparison. All samples were assayed using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), enabling integrated single-cell measurement of RNA and surface protein features across immune populations.
Analyses were directed primarily at monocytes because of their known contributions to fibrosis-promoting inflammation in systemic sclerosis. The single-cell dataset allowed investigation of both gene expression changes within monocytes over time and shifts in monocyte subpopulation composition that could underlie observed molecular trends.
To detect temporal trends across the longitudinal samples, the investigators aggregated single-cell data into pseudobulk profiles (log-transformed) and regressed gene expression against time since ASCT. This regression-based, longitudinal modeling framework was applied to identify genes and pathways whose expression changed progressively over the 24-month follow-up. The authors emphasize that this approach is broadly applicable to longitudinal single-cell datasets.
The longitudinal model identified widespread changes in monocyte gene expression after ASCT. Notably, expression of genes previously linked to systemic sclerosis decreased following transplantation. The report highlights reduced expression of SERPINE1 among these SSc-associated genes. Details on the full list of differentially regulated genes were reported in the source material; specific effect sizes and p values were not reproduced here beyond the directional findings reported in the study.
At the pathway level, monocytes from jSSc patients exhibited elevated NF-κB–associated inflammatory signaling at baseline when compared with healthy controls. This NF-κB–linked inflammatory program declined progressively after ASCT across the sampled timepoints. Concurrently, genes related to mitochondrial function and oxidative phosphorylation showed progressive increases in expression following transplantation. These coordinated changes suggest a shift in monocyte functional state from an inflammatory phenotype toward one with greater mitochondrial and metabolic activity after ASCT.
The study also detected compositional changes among monocyte subpopulations over time. The authors note that these shifts in subset frequencies may have contributed to the longitudinal gene expression patterns observed in the bulked analyses. Specific subpopulation identities and their individual longitudinal trajectories were examined in the source dataset; summary results emphasize that both cell-intrinsic transcriptional reprogramming and changes in monocyte composition accompany recovery after ASCT.
Taken together, the findings characterize dynamic immune reconfiguration in jSSc following ASCT, with evidence of reduced expression of disease-associated genes, attenuation of NF-κB inflammatory signaling, and increased mitochondrial/oxidative phosphorylation gene expression in monocytes over 24 months. These results are consistent with a model in which ASCT induces a progressive reprogramming of monocytes that could underlie clinical improvements in refractory jSSc, though the source report focuses on molecular and cellular readouts rather than clinical outcomes.
The authors made code available via a GitHub repository referenced in the source. Funding sources reported in the preprint include support from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), the Department of Defense, and institutional awards; competing interests were declared as none. The source preprint provides supplementary material and data/code links for further inspection.