A.-K. Knöfel

Allogeneic CD4+CD25high T cells regulate obliterative bronchiolitis of heterotopic bronchus allografts in both porcinized and humanized mouse models.

Background Bronchiolitis obliterans syndrome is caused by a fibroproliferative process in lung allografts resulting in irreversible damage. In this study, we induced obliterative bronchiolitis and studied the contribution of regulatory T cells to its development in immune-deficient mice receiving heterotopic porcine bronchus transplants, and major histocompatibility complex-mismatched porcine peripheral blood mononuclear cell. Furthermore, we aimed to corroborate our findings in a humanized mouse model. Methods Heterotopic bronchus transplantation was performed in 33 NOD.rag−/−&ggr;c−/− mice, using miniature pigs as tissue donors. The recipient mice then either received saline (negative control), unsorted MHC-mismatched PBMC (positive control), PBMC enriched with CD4+CD25high cells or PBMC depleted of CD4+CD25high cells for reconstitution. The results were validated in 28 NOD.rag−/−&ggr;c−/− mice undergoing heterotopic human bronchus transplantation and reconstitution with allogeneic human PBMC. Results Histological lesions similar to those typical for obliterative bronchiolitis developed in vivo after reconstitution with allogeneic PBMC and were more severe in animals engrafted with PBMC depleted of CD4+CD25high cells. In contrast, the group reconstituted with PBMC enriched with CD4+CD25high cells showed well-preserved histology. The results of the humanized model confirmed those obtained in the porcinized model. Conclusions In conclusion, both porcinized and humanized mouse models of heterotopic subcutaneous bronchus transplantation imitate the in vivo development of bronchiolitis obliterans syndrome–like lesions and reveal its sensitivity to T-cell regulation.

Splenocyte Infusion and Whole-Body Irradiation for Induction of Peripheral Tolerance in Porcine Lung Transplantation: Modifications of the Preconditioning Regime for Improved Clinical Feasibility

Background Preoperative low-dose whole-body irradiation (IRR) with 1.5 and 7 Gy thymic IRR of the recipient, combined with a perioperative donor splenocyte infusion lead to reliable donor specific peripheral tolerance in our allogeneic porcine lung transplantation model. To reduce the toxicity of this preconditioning regime, modifications of the IRR protocol and their impact on allograft survival were assessed. Methods Left-sided single lung transplantation from major histocompatibility complex and sex mismatched donors was performed in 14 adult female minipigs. Recipient animals were exposed to 3 different protocols of nonmyeloablative IRR within 12 hours before transplantation. All animals were administered a donor splenocyte infusion on the day of lung transplantation. Intravenous pharmacologic immunosuppression was withdrawn after 28 postoperative days. Allograft survival was monitored by chest radiographs and bronchoscopy. Results IRR prolonged transplant survival in a dose- and field-dependent manner. Shielding of the bone marrow from IRR (total lymphoid IRR at 1.5 and 7 Gy thymic IRR) significantly reduced protocol toxicity defined as thrombocytopenia and consecutive increased bleeding propensity, but had a less effective impact on graft survival. Whole-body IRR at 0.5 and 7 Gy thymic IRR proved to be ineffective for reliable tolerance induction. Eventually, high levels of circulating CD4+CD25high regulatory T cells were present in long-term survivors. Conclusions These data show that the infusion of donor-specific alloantigen in combination with IRR is efficient once a threshold dose is exceeded.

Irradiation before and donor splenocyte infusion immediately after transplantation induce tolerance to lung, but not heart allografts in miniature swine

Solid organs may differ in their potential to induce and maintain a state of donor‐specific tolerance. Previously, we induced stable immunological tolerance in a lung transplantation model in miniature swine. Here, we wished to transfer this established protocol into a heart transplantation model in miniature swine. Heterotopic heart transplantation (HTX) was performed in four and left‐sided lung transplantation (LTX) in seven minipigs from gender‐ and SLA‐mismatched donors. All recipients received nonmyeloablative irradiation, donor splenocyte infusion and intravenous pharmacologic immunosuppression for 28 postoperative days. All transplanted hearts were rejected within 95 days. In contrast, four animals of the LTX group developed stable tolerance surviving beyond 500 days, and three further animals rejected 119, 239 and 360 days post‐transplantation. In both groups, peripheral blood donor leucocyte chimerism peaked 1 h after reperfusion of the allograft. Importantly, the early chimerism level in the LTX group was significantly higher compared to the HTX group and remained detectable throughout the entire observation period. In conclusion, lungs and hearts vary in their potential to induce a state of tolerance after transplantation in a protocol with pre‐operative recipient irradiation and donor splenocyte co‐transplantation. This could be due to differential early levels of passenger leucocyte chimerism.

Novel mouse model of cardiopulmonary bypass

OBJECTIVES Cardiopulmonary bypass (CPB) is an essential component of many cardiac interventions, and therefore, there is an increasing critical demand to minimize organ damage resulting from prolonged extracorporeal circulation. Our goal was to develop the first clinically relevant mouse model of CPB and to examine the course of extracorporeal circulation by closely monitoring haemodynamic and oxygenation parameters. METHODS Here, we report the optimization of device design, perfusion circuit and microsurgical techniques as well as validation of physiological functions during CPB in mice after circulatory arrest and reperfusion. Validation of the model required multiple blood gas analyses, and therefore, this initial report describes an acute model that is incompatible with survival due to the need of repetitive blood draws. RESULTS Biochemical and histopathological assessment of organ damage revealed only mild changes in the heart and lungs and signs of the beginning of acute organ failure in the liver and kidneys. CONCLUSIONS This new CPB mouse model will facilitate preclinical testing of therapeutic strategies in cardiovascular diseases and investigation of CPB in relation to different insults and pre-existing comorbidities. In combination with genetically modified mice, this model will be an important tool to dissect the molecular mechanisms involved in organ damage related to extracorporeal circulation.

In Vivo Development of Transplant Arteriosclerosis in Humanized Mice Reflects Alloantigen Recognition and Peripheral Treg Phenotype of Lung Transplant Recipients.

Experimentally, regulatory T cells inhibit rejection. In clinical transplantations, however, it is not known whether T cell regulation is the cause for, or an epiphenomenon of, long‐term allograft survival. Here, we study naïve and alloantigen‐primed T cell responses of clinical lung transplant recipients in humanized mice. The pericardiophrenic artery procured from human lung grafts was implanted into the aorta of NODrag−/−/IL‐2rγc−/− mice reconstituted with peripheral blood mononuclear cells (PBMCs) from the respective lung recipient. Naïve or primed allogeneic PBMCs procured 21 days post–lung transplantation with or without enriching for CD4+CD25high T cells were used. Transplant arteriosclerosis was assessed 28 days later by histology. Mice reconstituted with alloantigen‐primed PBMCs showed significantly more severe transplant arteriosclerosis than did mice with naïve PBMCs (p = 0.005). Transplant arteriosclerosis was equally suppressed by enriching for autologous naïve (p = 0.012) or alloantigen‐primed regulatory T cells (Tregs) (p = 0.009). Alloantigen priming in clinical lung recipients can be adoptively transferred into a humanized mouse model. Transplant arteriosclerosis elicited by naïve or alloantigen‐primed PBMCs can be similarly controlled by potent autologous Tregs. Cellular therapy with expanded autologous Tregs in lung transplantation might be a promising future strategy.

Cardiopulmonary Bypass in a Mouse Model: A Novel Approach

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization and for minimizing organ damage resulting from prolonged extracorporal circulation. The goal of this paper was to demonstrate a fully functional and clinically relevant model of cardiopulmonary bypass in a mouse. We report on the device design, perfusion circuit optimization, and microsurgical techniques. This model is an acute model, which is not compatible with survival due to the need for multiple blood drawings. Because of the range of tools available for mice (e.g., markers, knockouts, etc.), this model will facilitate investigation into the molecular mechanisms of organ damage and the effect of cardiopulmonary bypass in relation to other comorbidities.

T Cell Responses in Lung Transplantion – Role of Alloantigen Priming and Regulation on Development of Transplant Arteriosclerosis in a Humanized Mouse Model

Purpose In animal models, both naive and alloantigen-primed Treg inhibit rejection. In clinical recipients of transplants, however, not much is known with regard to the actual function of those Treg that can be found in vivo. Here, we studied the functional importance of both naive and alloantigen-primed T cell responses in a humanized mouse model, utilizing transplant arteriosclerosis as the experimental readout. Methods and Materials The pericardiophrenic artery was procured from leftover tissue of lung grafts transplanted within our clinical program and was implanted into the abdominal aorta of NRG mice. Group A received no human leukocyte and served as negative controls. Group B received 5x106 allogeneic human PBMC from the respective lung recipient harvested at the time of transplant and group C received the same naive allogeneic PBMC, depleted of CD4+CD25+ putative Treg. Group D received naive allogeneic PBMC enriched of these CD4+CD25+ cells. Group E received PBMC harvested 21 days after transplantation, containing the alloantigen-primed T cells. Human leukocyte engraftment was monitored and transplant arteriosclerosis was histologically assessed 28 days later. Results The control group A showed mild thickening of the intima. In group B intimal thickening resulting in severe obliteration of the vessel lumen was evident. In group C obliteration of the lumen was even more severe. By contrast, in group D intimal thickening was less severe. In group E, reconstituted with alloantigen-primed total PBMC, only very mild transplant arteriosclerosis developed, comparable to group D enriched of naive Treg. Conclusions We show the importance of T cell regulation for controlling alloresponses in vivo. The finding of reduced transplant arteriosclerosis elicited by alloantigen-primed PBMC is intriguing and may indicate enhanced T cell regulation in these clinical transplant recipients. Ongoing studies aim at correlating clinical transplant outcomes to T cell function assessed using humanized mouse.

Preoperative irradiation and donor splenocyte infusion induce tolerance in lung, but not heart allografts in a minipig model – role of passenger leukocytes

respectively. Mean PA pressure was 49 11.7 mmHg and 39.8 9.8 mmHg in the PAH and D-PH groups, respectively. Median ET-1 levels were significantly different across all groups (p 0.001) with higher levels in the PAH (1.545 fmol/ml, range 0.31-100) and D-PH (1.30 fmol/ml, range 0.14-1000) groups as compared to controls (0.44 fmol/ml, range 0.02-3.88; p 0.0001 and p 0.0005, respectively). There was no significant difference in median ET-1 levels between D-PH and PAH groups (p 0.25). Conclusions: This study is the first to compare ET-1 levels in PAH, D-PH, and normal controls and suggests a role of ET-1 in the pathogenesis and perhaps treatment of D-PH.

ALLOANTIGEN REQUIREMENTS FOR THE INDUCTION AND THE MAINTENANCE OF PERIPHERAL TOLERANCE FOLLOWING PORCINE LUNG TRANSPLANTATION: 1938

G. Warnecke1, K. Dreckmann2, M. Avsar2, A. Knöfel3, N. Madrahimov1, W. Sommer1, B. Kruse1, S. Thissen1, J. Karstens1, A.R. Simon1, A. Haverich4, M. Strüber1 1, Hannover Medical School, Hannover/GERMANY, 2Department Of Cardiothoracic, Transplantation And Vascular Surgery, Hannover Medical School, Hannover/GERMANY, 3Httg, Hannover Medical School, Hannover/GERMANY, 4Heart Transplant Surgery, Medical School Hannover, Hannover/GERMANY