Abstract
Type 1 Diabetes (T1D) results in β cell destruction from impaired T cell tolerance. Exploiting therapeutic tolerance induction mechanisms using fibroblastic reticular cells (FRC) could protect against the disease. FRCs contribute to peripheral tolerance in lymph nodes (LN) and create supportive, interconnected reticula and allow expansion during inflammation. FRCs also express and present self-antigens, including β cell antigens, to autoreactive T cells. However, unlike professional antigen-presenting cells, FRCs do so with limited co-stimulation, thereby inducing T cell tolerance. In T1D LNs, the FRC frequency, expression of T1D-relevant antigens, and reticular organization decrease, likely reducing FRC tolerogenic engagement of autoreactive T cells. Our goal is to tissue-engineer FRC reticula that recapitulate these organizational changes in inflamed and healthy LNs to determine tolerance mechanisms and develop T1D FRC-based therapies. We previously showed that genetically-engineered FRCs overexpressing T1D-specific autoantigens seeded in collagen scaffolds with 400μm-diameter pores generated reticula with pores recapitulating inflamed LNs (100-200 μm) and engaged antigen-specific T cells. We then fabricated freeze-dried gelatin scaffolds with tunable pore sizes to generate FRC reticula resembling non-inflamed LN pores (15-20µm). The reticular pores of scaffolds frozen in liquid nitrogen had smaller pore sizes (<50µm) compared to -80ºC (<150µm) when characterized by SEM. We confirmed that FRCs seeded in each scaffold form reticula with pore sizes mimicking healthy (37um) and inflamed (89um) LNs via confocal microscopy and remain viable for at least 21 days via CellTiter-Glow assay. We are currently investigating the effects of FRC reticula pore sizes on the engagement of T1D antigen-specific T cells with T1D antigen-expressing FRCs from a T1D mouse model. Our tissue-engineering approach will allow us to study FRC-T cell interactions in an environment that recapitulates the T1D FRC reticula. We ultimately aim to test FRC tolerogenic mechanisms and develop novel FRC-based therapies for peripheral tolerance induction in T1D.