2016-LB: Uptake Kinetics and Presentation of Donor Alloantigen by Lymph Node Stromal Cells for Attenuating the Alloimmune Response in Pancreatic Islet Transplantation

Objective & Background: Pancreatic islet transplantation requires lifelong immunosuppression, reducing its applicability. We aim to eliminate the need for immunosuppression by co-transplanting fibroblastic reticular cells (FRCs), a lymph node stromal cell subtype. FRCs are unconventional antigen-presenting cells that contribute to restraining autoreactive T cells and could be used in transplantation to prevent alloreactive T cell activation. We hypothesize that autologous FRCs co-transplanted with allogeneic donor islets can uptake alloantigen and reprogram the recipient’s alloimmune response toward inactivation/anergy.

Methods: In all experiments, recipient-matched C57BL/6 FRCs were pre-conditioned with or without 10ng/mL interferon-γ (IFNγ) for 72h. FRCs were incubated with CellTrace-labeled NIT1 (fully mismatched to B6) cell lysate or 50µM OVA^257-264 (SIINFEKL) peptide overnight. No, low (7.5:1 NIT1:FRC cells), or high (75:1) doses of NIT1 cell lysate were administered. For kinetics, cells were cultured in regular media after overnight treatment. Flow cytometry was used to detect labeled lysate uptake and anti-SIINFEKL-H2K^b for antigen presentation. Data was analyzed by two-way ANOVA. Three biologically distinct cell lines were tested.

Results: With NIT1 lysate, FRC viability was not affected. Uptake of fluorescent lysate was dose-dependent, and 96.3±2.6% of FRCs took up NIT1 lysate at high dose. Confocal microscopy demonstrated donor antigen 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.

Conclusion: In vitro, FRCs can acquire exogenous antigen and present in MHC I. These results predict that FRCs can uptake allogeneic donor cell antigen and can present to alloreactive recipient T cells in in vitro co-culture studies and in vivo co-transplantation.