Direct stimulation of human T-cells by highly purified porcine islets in vitro
Introduction Severe tissue rejection is probably the biggest obstacle for clinical xenogeneic porcine islet transplantation. Since antibody-mediated hyperacute rejection is of minor importance to xenogeneic islet transplantation, we focussed on the T-cell mediated acute rejection. In an vitro system we analyzed xenogeneic T-cell activation by purified porcine pancreatic islets.
Materials and methods Human peripheral blood leukocytes (PBL) or human T-cells (purified by magnetic beads, >99% pure) were cocultured with irradiated, highly purified porcine pancreatic islets (Ricordi-method, >90% pure) or porcine PBL for 6 days. Cell proliferation was measured by incorporation of 3H-thymidin during the last day of culture. The amount of human PBL in the assay was adjusted to contain the same number of T-cells as the purified T-cells preparation (5 × 104). Pancreatic islets were titrated from 20 to 160 IEQ.
Results Stimulation of human T-cells by porcine pancreatic islets is about 10fold weaker than by porcine PBL, but still clearly above background (approx. 10-fold). The absolute strength of T-cell stimulation is dependent on the human donor and can vary significantly between donors. Preliminarily, we can distinguish "low responders" and "high responders" occurring with about the same probability (3 "low responder" of 7 tested). T-cells stimulation also varies depending on the donor pig, but not as clearly as with the human responders. Furthermore, we could not find any influence of the age of the donor pig on the T-cell stimulation. The response of purified T-cells accounted for 20% of the response of whole human PBL containing the same number of T-cells. Since the purified T-cells did not contain human antigen presenting cells (APCs) (<0.1% HLA-DR + cells), 20% of the T-cell stimulation was caused by direct antigen-presentation by porcine APCs in the pancreatic islets. Accordingly indirect antigen presentation is responsible for 80% of the response of human PBL containing APCs. Direct antigen presentation could be blocked by adding an anti-SLA II monoclonal antibody (MSA-3). Adding CTLA4-Ig blocked stimulation completely, indicating the T-cell specificity of the response (CTLA4-Ig blocks human and porcine B7).
Conclusions Although there are only very few APCs in purified porcine pancreatic islets, they are still sufficient to cause a detectable stimulation of human T-cells in an in vitro system. However, the main stimulus comes from human APCs presenting porcine antigens to human T-cells. Interestingly, the ability to respond to porcine antigens varies among different individuals, making "low responders" more suitable for a clinical islet xenotransplantation. Nevertheless, they would require clinically not desirable immunosuppression. To avoid direct as well as indirect T-cell stimulation in xenogeneic islet transplantation, encapsulation of the islets is probably the best way to go.