Background: Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrotic manifestations, which cause high mortality and severe impact on quality of life. The presence of autoantibodies is associated with disease progression, and reported to induce fibroblast activation in vitro. Recent studies from several centers have demonstrated beneficial effects upon deep B cell depletion in tissue upon CD19-CAR T-cell therapy. However, the molecular changes and activation states of fibroblast following CD19-CAR T-cell therapy remain poorly understood.

Objectives: We aimed to monitor the compositional shifts of fibroblast subpopulations and their functional states in SSc skin tissue upon CD19-CAR T-cell therapy by employing cyclic in situ hybridization (cISH)-based spatial transcriptomic assay.

Methods: We analyzed a longitudinal collection of skin tissue obtain from ten SSc patients underwent CD19 CAR T-cell therapy by cISH using Xenium Prime 5K Gene Expression assay (10x Genomics). Cell segmentation was conducted using transcript-based tool Baysor. The cell identity and fibroblast populations were identified by the expression of published marker genes. The compositional analysis was performed by using sccomp R package, accounting for the donor-specific effects through random effect modeling. The functional transitions of fibroblasts were evaluated by FGSEA.

Results: We first aimed to identify which cell type was affected the most by CD19-CAR T-cell therapy by computing a response score for each cell using the differential expressed genes obtained from bulk RNA-Seq. Fibroblasts demonstrated the highest response scores among other cell types, indicating a substantial contribution of fibroblasts to the overall transcriptional changes in skin following CAR T-cell therapy. Compositional analysis revealed the loss of previously reported pathogenic populations, including FAP ⁺ COL8A1 ⁺ myofibroblasts and PU.1 ⁺ S100A4 ⁺ fibroblasts. We also observed increased proportion of TNN ⁺ COCH ⁺ fibroblasts, especially in the reticular dermis. Although the overall abundance of PI16 ⁺ progenitor population remained unchanged, we detected decreased amount of PI16 ⁺ fibroblast in reticular dermis but increased number in the papillary dermis, suggesting a spatial redistribution following CAR T-cell therapy. In addition to the compositional changes, FGSEA identified functional changes within the fibroblast populations, including reduced collagen formation and inflammatory pathways.

Conclusions: We report the first spatially resolved transcriptomic data on fibroblasts in the skin tissue following CD19-CAR T-cell therapy. Our findings demonstrate that deep B cell depletion in tissue via CAR T-cell triggers profound phenotypic transitions in the dermal fibroblast populations. These results may provide mechanistic insights on the resolution of tissue fibrosis and facilitate the development of future therapeutics against SSc.

REFERENCES: NIL.

Acknowledgments: NIL.

Disclosure of Interests: Meilin Xu: None declared, Aleix Rius Rigau: None declared, Sara Chenguiti Fakhouri: None declared, Janina Auth: None declared, Panagiotis Garantziotis: None declared, Ziyuan Liu: None declared, Manoj Kumar Selvaraju: None declared, Maria Gabriella Raimondo: None declared, Carlo Tur: None declared, Tim Filla: None declared, Fabian Müller: None declared, Andreas Wirsching: None declared, Melanie Hagen: None declared, Pooja Gupta: None declared, Aline Bozec: None declared, Andreas Mackensen: None declared, Jörg Distler AbbVie, Active Biotech, Anamar, ARXX, AstraZeneca, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, Genentech, GSK, Inventiva, Janssen, Novartis, Pfizer, Roche and UCB, AbbVie, Anamar, ARXX, AstraZeneca, Bayer Pharma, Boehringer Ingelheim, Cantargia, Celgene, Galapagos, Genentech, GSK, Inventiva, Janssen, Novartis, Pfizer, Roche and UCB, 4D Science; FibroCure, AbbVie, Active Biotech, Anamar, ARXX, AstraZeneca, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, Genentech, GSK, Inventiva, Janssen, Novartis, Pfizer, Roche and UCB, AbbVie, Anamar, Argenx, ARXX, BMS, Bayer Pharma, Boehringer Ingelheim, Cantargia, Celgene, CSL Behring, ExoTherapeutics, Galapagos, GSK, Inventiva, Kiniksa, Lassen, Novartis, Sanofi-Aventis, RedX and UCB, Georg Schett BMS, Cabaletta, Janssen, Kyverna, Miltenyi, and Novartis, Yi-Nan Li: None declared, Christina Bergmann Novartis Argentina, Boehringer-Ingelheim and Horizon PPD.