Tu200 - Achieving Allograft Protection Through Co-implantation with Donor Antigen-primed Lymph Node Stromal Cells

Abstract Text: Objective/

Background: Pancreatic islet transplantation for type 1 diabetes requires lifelong immunosuppression, reducing its applicability. We aim to reduce the need for immunosuppression by co-transplanting lymph node fibroblastic reticular cells (FRCs). FRCs are unconventional antigen-presenting cells that contribute to peripheral tolerance and could be used in transplantation to regulate alloreactive T cells. We hypothesize that autologous FRCs co-transplanted with allogeneic donor islets can uptake and present alloantigen to induce alloreactive T cell anergy and/or regulation.

Methods: C57BL/6 FRCs were pre-conditioned with 10ng/mL IFNγ for 72h. FRCs were incubated with CellTrace-labeled NIT1 (fully mismatched to B6) cell lysate, 50µM chicken ovalbumin257-264 (SIINFEKL) peptide, or 1mg/mL ovalbumin (OVA) overnight. For kinetics, FRCs were cultured in regular media after overnight treatment. Flow cytometry was used to detect labeled lysate uptake and antigen presentation with anti-SIINFEKL-H2Kb. SIINFEKL or OVA antigen-primed FRCs were co-cultured with SIINFEKL-reactive cells and activation was measured with β-galactosidase reporter quantification or flow cytometry.

Results: FRC uptake of fluorescent NIT1 lysate was dose-dependent, and 96.3±2.6% of FRCs took up lysate at high dose. Confocal microscopy demonstrated lysate localization within lysosomes. Donor antigen was cleared from FRCs by 72h. IFNγ potentiates SIINFEKL presentation and 99.9±0.1% of IFNγ-treated FRCs presented SIINFEKL. SIIFNEKL-reactive cells were activated by SIINFEKL- and OVA-primed FRCs.

Conclusion: In vitro, FRCs can acquire exogenous peptide and protein antigen, process and present them in MHC-I, and activate CD8+ cells. These results predict that FRCs can uptake allogeneic donor cell antigens and present them to alloreactive recipient T cells in in vivo co-transplantation.