Delayed Non-Myeloablative Irradiation to Induce Allograft Acceptance in a Large Animal Lung Transplantation Model

Objectives: Previously, we induced long-term allograft acceptance in our allogeneic lung transplantation model in miniature swine. The respective animals underwent perioperative irradiation combined with a donor-specific alloantigen infusion. Here, we delayed induction to improve its clinical applicability.

Methods: Left-sided single lung transplantation from MHC-mismatched male donors was performed in 14 outbred female minipigs. Group 1 (n = 5) received non-myeloablative irradiation (7 Gy thymus and 1,5 Gy whole body, IRR) 12 hours before transplantation and a perioperative donor-specific splenocyte infusion (SpTx). Group 2 (n = 3) also received a perioperative SpTx, but underwent delayed irradiation 3 days after transplantation. Group 3 (n = 6) was also exposed to delayed IRR but did not receive a donor-specific SpTx. Immunosuppression was maintained for 28 days with tacrolimus and methylprednisolone. Peripheral blood leukocyte chimerism was monitored by flow cytometry and real-time polymerase chain reaction.

Results: In group 1, three animals never rejected their grafts until elective sacrifice at postoperative day (POD) 800+, the other two showed late rejections on PODs 239 and 360. In group 2, median allograft survival was 105 days. Out of the third group, three animals rejected their grafts until POD 226, whereas three animals are currently alive at POD 100, 500 and 700, resulting in a preliminary median survival of 226 days. In all groups, chimerism was detectable 1 hour after reperfusion of the lung, and peaked 1 hour after SpTx in groups 1 and 2. Group 1 showed early leukocyte chimerism levels of up to 30%. Analysis of putative regulatory CD4⁺CD25^high T cells revealed increasing frequencies in group 1, whereas groups 2 and 3 had decreasing levels of this T cell subpopulation after withdrawal of immunosuppression.

Conclusion: Preoperative irradiation and perioperative SpTx had the potential to upregulate the frequency of putative regulatory CD4⁺CD25^high T cells. SpTx without preceding IRR seemed to rather upregulate the effector arm, than inducing Treg proliferation. Delayed IRR without perioperative SpTx lead to prolonged allograft survival in 50% of the animals. This success might be explained by unresponsiveness due to induced anergy.

No conflict of interest has been declared by the author(s).