To explore the immunosuppressive protocol and its effect in the gene-edited pig-rhesus monkey xenotransplantation experiment, and to provide a reference for the formulation of immunosuppressive protocol for xenogeneic organ transplantation.

Through preoperative matching experiments including phenotypic identification, complement-dependent cytotoxicity (CDC) experiment, and IgG and IgM binding experiments and so on, a double-gene knockout donor pig (lot number 4207) and a rhesus monkey (experimental animal registration number 382350) were selected as experimental animals. An ectopic heart transplantation surgery was chosen for transplantation. The immunosuppressive protocol was divided into the induction period and the maintenance period of immunosuppression.

On December 16, 2023, the gene-edited pig-rhesus monkey ectopic heart transplantation surgery was performed successfully. The surgical process went on smoothly, and both the donor and recipient hearts successfully resumed beating. The general condition of the recipient was good after the surgery. On the 9th day after the surgery (T+ 9), due to ischemic necrosis of the left lower limb, a left lower limb amputation surgery was performed. The final survival time of the recipient monkey was 46 d. The possible cause of death was chronic rejection-induced decline in donor heart function and excessive dosage of anesthetic drugs administered to the recipient monkey before the myocardial tissue biopsy at T+ 46. After immunosuppression, the white blood cells and lymphocytes of the recipient were in a stable and low level, and the CDC reached steady state, settling below 4%. The flow cytometry analysis results of the recipient monkey showed that compared with 1 d before transplantation, the B cells with CD19 as the surface marker and T cells with CD3 as the surface marker were significantly reduced in T+ 46. The pathological examination results of the pig heart and rhesus monkey heart after the recipient′s death indicated that the staining of B cell surface marker CD19 and T cell surface marker CD3 was almost invisible, and the staining of complement activation marker C4D was relatively shallow; the staining frequency of macrophage surface marker CD68, IgG, and IgM was relatively higher.

The immunosuppressive protocol used in this experiment effectively inhibited hyperacute and acute rejection reactions, providing a feasible immunosuppressive protocol reference for future xenogeneic organ transplantation.