The Effect of Co-stimulation Blockade on Survival of Xenogeneic Pancreatic Beta-cells

© 2014 Zeng Zeng YapDiabetes mellitus is a chronic disease affecting millions of people worldwide. Currently, the only potential cure is through pancreatic transplantation, either as whole organ or with pancreatic islet cells. However, the morbidity associated with immunosuppression and the scarcity of donor organs do not support the common practice of pancreatic transplantation. Both these factors can potentially be addressed by xenotransplantation of genetically modified cells that are capable of attenuating the immune system. The costimulation pathway of the immune system was the focus of this project, in particular blockade of the ICOS (inducible costimulation molecule) and CTLA-4 (cytotoxic T lymphocyte associated antigen 4) pathways to prolong xenograft survival. To address the problem of diabetes as well as investigate the efficacy of costimulation blockade on xenograft survival, rat insulinoma (beta) cell lines (INS-1E) stably expressing either ICOS-Ig or CTLA4-Ig were generated. The secreted proteins were demonstrated to be biologically active in xenogeneic mixed lymphocyte reactions by their ability to inhibit lymphocyte proliferation. Unfortunately, the in vivo effect of these transgenic INS-1E cells on xenograft survival was unable to be determined because of their failure to establish as tumours due to the slow growth rate of these cells following subcutaneous injection into BALB/c mice. The aim of the second part of this project was to assess the effect of rationally mutated ICOS-Ig on xenograft survival. PIEC (pig iliac endothelial cells) stably expressing ICOS-Ig with single (K52/S and S76/E) and combined (K52/S + S76/E) amino acid mutations were generated. In vitro, compared to wild type ICOS-Ig, the mutants with single amino acid substitutions showed stronger binding avidity to ICOS ligand. This increase in binding avidity however did not translate into superior inhibition of lymphocyte proliferation compared to wild type ICOS-Ig in xenogeneic mixed lymphocyte reactions. Similarly in vivo, the PIECs secreting mutated forms of ICOS-Ig did not prolong xenograft survival after subcutaneous injection of these cells into BALB/c mice. These data suggest that despite their higher avidity, the ICOS-Ig mutants are not biologically more superior than wild type ICOS-Ig both in vitro and in vivo