Engineered macroporous gelatin scaffolds enhance lymph node fibroblastic reticular cell identity and enable...

Current treatments for autoimmune diseases like Type 1 Diabetes (T1D) carry significant risks because they lack tissue specificity. A promising strategy is to achieve persistent presentation of relevant antigens (Ags) in non-inflamed sites by tolerogenic Ag-presenting cells (APCs) like fibroblastic reticular cells (FRCs). FRCs build lymph node (LN) reticula and act as immunomodulatory non-professional APCs. However, their therapeutic potential for Ag-specific immunomodulation for T1D remains unexplored. We engineered 3D FRC-based reticula using freeze-dried macroporous gelatin scaffolds with customizable pore diameters (small: <50 μm, medium: <200 μm, large: <300 μm) to evaluate FRC phenotype and FRC-T cell interactions, leveraging FRCs' ability to build dynamic LN reticula that expand and contract during inflammation. Our scaffolds promoted FRC viability, reticular formation, FRC phenotypic marker expression, and extracellular matrix secretion compared to 2D culture. GFP-Luciferase fusion (GLF)-expressing FRCs subcutaneously implanted in our scaffolds survived for at least 21 days regardless of pore size. Implantation in the vascularized fat pad led to graft rejection by day 14 in pre-diabetic NOD mice but not in immunodeficient NODscid. Our scaffolds outperformed clinically-used biologic gels, demonstrated the limitations of the NOD mouse model in longitudinal imaging of GLF⁺ cell survival, and validated our scaffold-based FRC delivery approach for future therapeutic applications. Co-culture of T cells in scaffolds with FRCs presenting T1D Ags enabled Ag-specific T cell engagement with reduced cytotoxic and increased anergic and regulatory phenotypes. Our results validate the use of macroporous gelatin scaffolds to enhance FRC phenotypic markers, in vivo survival, and diabetogenic cell immunomodulation in vitro.