Abstract
Restoring antigen (Ag)-specific immune tolerance represents a critical unmet need in Type 1 diabetes (T1D). Fibroblastic reticular cells (FRCs; CD45-gp38+CD31-), stromal cells that form interconnected reticula in lymph nodes (LNs), contribute to peripheral tolerance by presenting self-Ags, including β cell Ags, to autoreactive T cells. Here, two biomaterial platforms were developed to study FRC-mediated immune regulation in T1D. First, macroporous gelatin scaffolds were engineered to create three-dimensional FRC reticula that recapitulate LN architecture for localized FRC delivery. Non-obese diabetic (NOD) FRCs seeded onto scaffolds formed reticula and remained viable in vitro and in vivo. Ag-presenting FRC scaffolds interacted with CD4⁺ (BDC2.5) and CD8⁺ (NY8.3, G9C8) T cells, promoting engagement with reduced Granzyme B expression and increased regulation. Endogenous Ag presentation by FRCs was needed for regulatory T cell induction. In vivo, FRC-scaffolds enabled Ag-specific T cell engagement with distinct kinetics: exogenous Ag induced early retention while endogenous Ag supported sustained interactions that resolved into regulatory outcomes. Second, to investigate how the LN microenvironment shapes FRC phenotype, decellularized LNs (dLN) from T1D-prone (NOD) and T1D-resistant (non-obese resistant, NOR) mice were developed. Decellularization preserved ECM and supported FRC culture. Transcriptomic profiling revealed that NOD FRCs cultured on NOR dLNs partially restored gp38 and upregulated regulatory and ECM-associated pathways, whereas NOR FRCs cultured on NOD dLNs adopted a stress-associated inflammatory phenotype. FRCs cultured in NOD dLNs also induced higher Ag-specific CD8⁺ T cell proliferation and cytotoxic phenotypes. Together, this work demonstrates that FRCs can be leveraged for Ag-specific immune modulation and that LN ECM cues shape disease-associated FRC phenotypes, supporting future FRC-targeted strategies to restore tolerance in T1D.