Immune-Stromal Co-culture System to Interrogate Cytokine Responses in Rheumatoid Arthritis 2328589

Abstract Introduction A critical component of rheumatoid arthritis (RA) pathogenesis is the complex communication between immune and stromal cells. While this has been historically challenging to model in vitro, we set out to build a robust transwell culture system using human PBMCs and RA fibroblast-like-synoviocytes (FLS). Utilizing a disease relevant inflammatory stimulus (anti-CD3/CD28), this platform enables us to profile multiple cytokine responses and identify downstream pathway activation with translational relevance. Methods PBMCs and immortalized RA FLS were cultured for 48 h following the addition of anti-CD3/CD28 to the PBMC compartment. Readouts include multiplex cytokine profiling and single cell RNA-sequencing of both RA FLS and PBMCs. The assay was validated by inhibiting clinically relevant mechanisms (IL-6, TNFa, JAK/STAT) and comparing against clinical datasets. Results This co-culture system exhibited dynamic immune—stromal crosstalk, resulting in emergent properties that are more physiologically relevant than single pathway activation. Inhibition of clinically validated pathways (IL-6, TNFa, JAK/STAT) revealed robust transcriptomic effects unique to the individual pathways. These results were aligned with gene expression changes observed in clinical trial responder datasets. Single cell RNA sequencing allowed us to project transcriptomic profiles of individual cell types in vitro onto an internally compiled human RA Single Cell Atlas. This analysis revealed shared pathways and gene modules between our in vitro assay and human disease tissue, highlighting the translatability of this co-culture system. Conclusion This transwell co culture provides a scalable, robust platform for RA research. It supports parallel evaluation of cytokine targets and identification of orthogonal downstream mechanisms in both immune and stromal compartments. Future studies will further elucidate translatable pathways relevant to RA and novel therapeutic targets to explore. Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)