Progress in xenotransplantation: overcoming immune barriers

Abstract

A major limitation of organ allotransplantation is the insufficient supply of donor organs. Consequently, thousands of patients die every year while waiting for a transplant. Progress in xenotransplantation that has permitted pig organ graft survivals of years in non-human primates has led to renewed excitement about the potential of this approach to alleviate the organ shortage. In 2022, the first pig-to-human heart transplant was performed on a compassionate use basis, and xenotransplantation experiments using pig kidneys in deceased human recipients provided encouraging data. Many advances in xenotransplantation have resulted from improvements in the ability to genetically modify pigs using CRISPR-Cas9 and other methodologies. Gene editing has the capacity to generate pig organs that more closely resemble those of humans and are hence more physiologically compatible and less prone to rejection. Despite such modifications, immune responses to xenografts remain powerful and multi-faceted, involving innate immune components that do not attack allografts. Thus, the induction of innate and adaptive immune tolerance to prevent rejection while preserving the capacity of the immune system to protect the recipient and the graft from infection is desirable to enable clinical xenotransplantation.

Fig. 1 ∣. The growing organ shortage.

The disparity between the number of patients on waiting lists and the number of transplants performed in the USA has grown markedly over the past three decades. Data obtained from the OPTN database.

Fig. 2 ∣. Tolerance induction strategies.

Two strategies are currently being developed as a means of inducing tolerance across the pig-to-primate barrier. a ∣ In mixed haematopoietic chimerism, bone marrow from the donor pig is injected into a T cell-depleted baboon. Donor bone marrow-derived dendritic cells migrate to the host thymus, where they negatively select developing donor-reactive host T cells, resulting in tolerance to donor cells. b ∣ In thymus transplantation, the donor pig thymus is transplanted into a T cell-depleted, thymectomized baboon, either as a vascularized thymic lobe (not shown) or as part of a composite thymokidney in which autologous pig thymic tissue is grafted under the renal capsule. T cells develop from host T cell precursors in the donor thymus in which tolerance to the donor may be induced both by negative selection and by the generation of regulatory T (T_reg) cells. Thus, both methods of tolerance induction are dependent on the thymus.

Fig. 3 ∣. Tolerance and immune function with transplantation of swine (or hybrid) thymus and mixed xenogeneic chimerism.

a ∣ Human thymocyte selection in the normal human thymus, swine thymus transplanted into a human and hybrid swine thymus containing human cortical thymic epithelial cells (cTECs) transplanted into the human who provided the TECs. The hybrid thymus is generated by injecting TECs obtained from the recipient’s thymus at the time of thymectomy or generated de novo from induced pluripotent stem cells of the recipient into the swine thymus prior to transplantation. In the human thymus, human cTECs mediate positive selection so that T cell responses in the periphery are largely restricted by the human leukocyte antigen (HLA) of the recipient, resulting in immune protection against infection. In the transplanted swine thymus, swine cTECs mediate positive selection, so that T cell responses in the periphery are restricted by swine leukocyte antigen (SLA), resulting in immune protection against infection of a swine xenograft but not of the recipient. In the hybrid thymus, both human and swine cTECs participate in positive selection. The peripheral immune system is therefore able to recognize foreign peptides presented by recipient HLA and by donor SLA, resulting in good immune protection of the recipient and of a swine xenograft. In the transplanted swine thymus and the hybrid swine thymus, positive selection on swine cTECs will enable selection of swine donor-specific regulatory T (T_reg) cells and the presence of swine antigen-presenting cells (APCs) in the graft will result in negative selection of swine-reactive T cells, resulting in tolerance to the donor (not shown). b ∣ Advantages of combined mixed chimerism and swine thymus transplantation. In the transplanted swine thymus, swine haematopoietic antigen-reactive T cells would be deleted, as mixed chimerism ensures a constant supply of swine APCs. In addition, swine tissue-restricted antigen (TRA)-specific T cells would be deleted and swine TRA-specific T_reg cells positively selected by swine medullary thymic epithelial cells (mTECs), resulting in robust tolerance to the swine donor. However, human TRA-specific T cells would be released to the periphery owing to the absence of human mTECs, potentially predisposing the recipient to autoimmunity. The presence of a human thymus (in addition to the swine thymus) may be permissible in the setting of mixed chimerism, as many swine haematopoietic antigen-reactive T cells would be deleted by the presence of swine APCs in the human thymus. Swine TRA-specific T cells that escape the human thymus (owing to the absence of swine mTECs) would be suppressed by T_reg cells emigrating from the swine thymus graft, where swine mTECs are abundant. The presence of a human thymus would enhance human TRA-specific tolerance and HLA-restricted protective immunity owing to the participation of human mTECs in selection.

Fig. 4 ∣. Breeding for multiple transgenes in inbred miniature swine.

Breeding of genetically modified inbred miniature swine permits combination of multiple different modifications into pigs with the same inbred genetic background. Breeding of cloned outbred animals leads to random assortment of background genes (not shown), whereas a cross and intercross of edits made on the same inbred background can produce offspring with both edits still on the same background. dd denotes the homozygous SLA genotype of the pig.