Donor alloantigen infusion induces T cell regulation and transplant tolerance in small animals. Here, we study donor splenocyte infusion in a large animal model of pulmonary transplantation. Major histocompatibility complex-mismatched single lung transplantation was performed in 28 minipigs followed by a 28-day course of methylprednisolone and tacrolimus. Some animals received a perioperative donor or third party splenocyte infusion, with or without low-dose irradiation (IRR) before surgery. Graft survival was significantly prolonged in animals receiving both donor splenocytes and IRR compared with controls with either donor splenocytes or IRR only. In animals with donor splenocytes and IRR, increased donor cell chimerism and CD4(+) CD25(high+) T cell frequencies were detected in peripheral blood associated with decreased interferon-γ production of leukocytes. Secondary third-party kidney transplants more than 2 years after pulmonary transplantation were acutely rejected despite maintained tolerance of the lung allografts. As a cellular control, additional animals received third-party splenocytes or donor splenocyte protein extracts. While animals treated with third-party splenocytes showed significant graft survival prolongation, the subcellular antigen infusion showed no such effect. In conclusion, minipigs conditioned with preoperative IRR and donor, or third-party, splenocyte infusions may develop long-term donor-specific pulmonary allograft survival in the presence of high levels of circulating regulatory T cells.

Introduction: Bronchiolitis obliterans syndrome (BOS) limits long term survival after lung transplantation. CD4+CD25+ regulatory T cells are known to counteract acute rejection and may as well inhibit chronic rejection, i.e. BOS. We have developed a humanized mouse model in immune deficient NRG mice that are transplanted with allogeneic human small bronchus segments heterotopically. In this model, BOS-like pathology develops within 28 days. Here, we hypothesized regulatory T cells (Treg) would be important regulators of the BOS-like lesions in this humanized model.

Purpose: Heterotopic retroperitoneal cardiac transplantation combines the advantages of monitoring the allograft rejection by non invasive methods like palpation with facilitation of biopsying the transplant. Previously, our group developed a protocol for inducing stable tolerance in a lung transplantation model. In this study, we investigated if this established regimen would also work for inducing tolerance in a heterotopic cardiac transplantation model. Methods: Heterotopic cardiac transplantation was performed in five, and left-sided lung transplantation in six minipigs. Both groups received their grafts from gender and MHC mismatched donors. Furthermore, all recipients were administered non-myeloablative irradiation before transplantation, a splenocyte infusion on the day of transplantation and intravenous pharmacologic immunosuppression for 28 postoperative days (POD). Allograft survival was monitored by chest radiographs and bronchoscopy in lung-transplanted and by palpation, ultrasound examination and myocardial biopsies in heart-transplanted animals. Peripheral blood leukocyte chimerism was analyzed by Y-chromosomal quantitative real time PCR. Results: Four out of five heart transplants were rejected within POD 100. The median allograft survival was of 70 days. In contrast, three animals of the lung transplanted group developed a stable tolerance surviving beyond POD 500, two minipigs rejected chronically on POD 239 and POD 360 and one animal (POD 305) is currently alive without signs of rejection. In both groups peripheral blood leukocyte chimerism peaked on the day of transplantation in a sample drawn 1 hour after reperfusion of the allograft. Importantly, the early chimerism level in the lung-transplanted group was significantly higher than in the heart-transplanted group (>7 days) and tend to remained elevated (POD 7 to POD 28). Conclusion: We conclude lungs and hearts differ in their potential to induce transplantation tolerance. When other components of the conditioning protocol like preoperative irradiation and perioperative splenocyte infusion are standardized, the variable of the transplanted organ determines the success of a tolerance induction protocol. This effect may be due to the higher magnitude of early passenger leukocyte chimerism after lung as compared to heart transplantation.

Purpose: The role of naturally occurring versus alloantigen-primed regulatory T cells (Treg) in tolerized lung transplants is not fully understood yet. We reported successful induction of peripheral tolerance in a porcine lung transplantation model in the presence of high levels of circulating Treg. Here, we introduce an adoptive transfer model in immune deficient mice to study function of naïve T cells and naturally occurring porcine Treg in vivo. Methods: Left-sided lung transplantation from MHC mismatched donors was performed in 12 adult minipigs. To induce tolerance various protocols were conducted including pharmacologic immunosuppression, non-myeloablative irradiation and infusion of donor specific and non specific alloantigens. Before transplantation porcine bronchi from the lung donors were transplanted under the skin of NODrag−/−gammac−/− mice. Subsequently, 5 to 10 × 106 naive or Treg-depleted porcine PBMCs from the lung recipients were injected into these mice. Histologic alloinjury of the bronchus graft was assessed after 28 days. Results: Surviving beyond day 380 five out of 12 lung recipients developed stable tolerance of the graft. In the adoptive transfer model the damage score of the bronchi showed no significant differences between mice injected with PBMC derived from eventual long term survivors and eventually rejecting recipients. Depletion of CD4+CD25high T cells from the PBMC transferred led to more severe damage without differences between groups Conclusion: Naive PBMC from all porcine lung recipients induced rejection of the donor bronchus in mice. This rejection was reliably regulated by naturally occurring Treg in depletion experiments. These results had no impact on eventual development of stable peripheral tolerance in porcine lung recipients following the respective induction protocols. We therefore conclude that peripheral tolerance in our model is more likely a result of alloantigen priming, than of pre-existing naturally occurring T cell regulation.

Objectives: Donor splenocyte infusion has been shown to induce long term allograft acceptance in a model of porcine lung transplantation. Allograft survival correlated with the frequency of CD4+CD25high+ regulatory T cells (Treg). However, it is unknown, if this type of T cell regulation is the cause for, or merely an epiphenomenon of long term allograft survival. To study this type of immune regulation in vivo, we implanted porcine donor arteries and adoptively transferred porcine recipient PBMC into NODrag–/–gammac–/– (NRG) mice.

Aims: Whole body irradiation (WBI) in combination with donor splenocyte infusion has led a maintenance of peripheral immune tolerance in porcine lung transplant recipients. In order to reduce the toxicity of irradiation, we used a total lymphoid irradiation (TLI) instead. Therefore, the clinical feasibility of the preconditioning protocol potentially improves.

Warnecke, G.1; Dreckmann, K.1; Avsar, M.2; Knöfel, A.3; Madrahimov, N.1; Sommer, W.1; Thissen, S.1; Kruse, B.1; Ziehme, P.1; Gottlieb, J.1; Simon, A. R.1; Karstens, J.1; Haverich, A.4; Strüber, M.1 Author Information

Warnecke, G.; Dreckmann, K.; Avsar, M.; Knöfel, A.; Madrahimov, N.; Sommer, W.; Kruse, B.; Thissen, S.; Karstens, J.; Simon, A. R.; Haverich, A.; Strüber, M. Author Information

Dreckmann, K.; Avsar, M.; Knoefel, A.; Madrahimov, N.; Ziehme, P.; Sommer, W.; Gottlieb, J.; Karstens, J.; Haverich, A.; Strueber, M.; Warnecke, G. Author Information

Purpose: Low dose irradiation and donor splenocyte infusion led to the maintenance of peripheral tolerance in a porcine lung transplantation model. Here, we wished to study modifications of the protocol for improved clinical feasibility.