Background: Previously, we induced longterm allograft acceptance in our allogeneic lung transplantation (LTx) model in miniature swine. Animals underwent perioperative non-myeloablative irradiation combined with the infusion of donor specific alloantigen. To improve clinical applicability, we delayed induction with irradiation in this study.
Methods: Left sided single LTx from MHC mismatched male donors was performed in 22 outbred female minipigs. Group 1 (n=7) received non-myeloablative irradiation (7 Gy thymus and 1.5 Gy whole body IRR) 12 hours before LTx and a perioperative donor specific splenocyte infusion (SpTx). Group 2 (n=3) received perioperative SpTx and delayed IRR three days after LTx. Group 3 (n=8) was exposed to delayed IRR without SpTx. Group 4 (n=4) underwent no induction therapy at all. Immunosuppression was maintained for 28 days with tacrolimus and methylprednisolone in all groups. Thereafter, allograft survival was monitored by bronchoscopy and sequential chest x-rays. Frequencies of CD4+CD25high+Foxp3+ putative Treg were monitored by flow cytometric analysis. Peripheral blood leukocyte chimerism was quantified by qPCR.
Results: Whereas 4 out of 7 animals from group 1 never rejected their grafts and were electively sacrificed on postoperative day (POD) 600+, 3 out of 8 animals from group 3 turned into longterm survivors with 1 animal currently alive at POD 210. All animals from group 2 rejected their grafts until POD 108 (p**=0.0012 vs group 1). In group 4, only 1 out of 4 animals did survive longterm (p*=0.0474 vs. group 1). In all groups, donor cell chimerism peaked up to 20% instantly after reperfusion of the lung. Whereas levels in groups 2-4 constantly decreased thereafter, chimerism in group 1 started rising again from POD 7 onwards (p**=0.0041). Relative putative Treg cell counts were significantly higher in group 3 compared to group 4 (p**=0.0077).
Conclusion: IRR in a delayed manner has the potential to improve longterm graft survival compared to untreated controls, as long as no SpTx is administered before IRR in our experimental protocol. Even though survival in group 1 is superior to group 3, we could again prove that IRR spares the proregulatory CD4+CD25high+Foxp3+ T cell subtype, resulting in a favorable immunological state. The lacking establishment of longterm donor cell coexistence in group 3 indicates, however, that long term stability of tolerance might be compromised.