Assessment of a cellular immune response from peripheral blood of non-human primate after porcine islet xenotransplantation

Abstract

Introduction: Outstanding results from nonhuman primate study put islet xenotransplantation with immunosuppression closer to the clinical application. To establish successful immune suppressive protocols, immune monitoring by which the fate of graft could be predictable would be critical. However, there are few reports showing predictive immune parameters associated with the fate of the graft in pig to nonhuman primate islet xenotransplantation model. Implementation of an appropriate monitoring method to detect the development of detrimental porcine antigen-specific cellular immune responses is also necessary. In addition, clarifying the causes of islet death in the chronic phase after islet transplantation is important.

Methods: Porcine islets were transplanted to diabetic nonhuman primate under immunosuppression. During the observation period, the number and ratio of T cell subsets were analyzed by flow cytometry from peripheral blood of seven-teen transplanted monkeys to find out graft-fate predictive immune parameters. To find out the optimal ELISpot assay conditions, the numbers of responder and stimulator cells were determined. Then, ELISpot assays were conducted on serial stocks of the peripheral blood mononuclear cell (PBMC) samples previously isolated from four NHP recipients transplanted with porcine islets to validate the essays utility to monitor the porcine antigen-specific cellular immune responses. Either splenocytes from donor pigs or pancreatic islets from third-party pigs were also used for antigen stimulation. Furthermore, I performed RNA sequencing with peripheral blood and bioinformatics analysis in two monkeys as a way to find out or predict potential cause(s) of graft failure after 100 days after transplantation.

Results: After the depletion of CD3+ T cells with rATG with immunosuppression, the mean recovery time of CD3+ T cells was 38.2 ± 47.7 days. CD4+ T cells were the dominant populations in CD3+ T cells before the anti-thymocyte globulin treatment. However, CD8+CD28-CD95+ effector memory T cells rapid expansion reversed the ratio of CD4+ versus CD8+ T cells. T lymphocyte subtype analysis with graft survival day revealed that CD4+/CD8+ T cell ratio was significantly associated with early graft failure. The optimal conditions for the ELISpot assay were defined as 2.5 × 105 responder cells incubated with 5.0 × 105 stimulator cells in 96-well, flat-bottom plates without further co-stimulation. Using donor splenocytes as stimulators, a serial interferon-gamma (IFN-γ) ELISpot assay with PBMCs from the monkeys with prolonged porcine islet grafts (>180 days) demonstrated that the number of donor antigen (not islet-specific)-specific IFN-γ-producing cells significantly increased upon overt graft rejection. By using novel bioinformatics tool, I found that highly relevant activated immunologic pathways were indeed manifest in graft failed animal compared with control one in chronic phase. In line with this notion, I further confirmed that the porcine islets were heavily infiltrated with CD3+ T cells by immunohistochemistry on biopsied liver samples. Conclusions: CD4+/CD8+ T cell ratio could be used as a surrogate marker to predict early graft failure in porcine islet xenotransplantation in NHPs with immunosuppression. I also showed that the use of recipient PBMCs in a porcine antigen-specific IFN-γ ELISpot assay is a reliable method for monitoring T-cell-mediated rejection in pig-to-NHP islet xenotransplantation. I further demonstrated that a new bioinformatics analysis combined with peripheral RNA sequencing could unveil insidious immune rejection in the chronic phase after pig-to-NHP islet xenotransplantation.