K. Jansson

Augmentation of Transient Donor Cell Chimerism and Alloantigen‐Specific Regulation of Lung Transplants in Miniature Swine

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+CD25high+ 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.

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.

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.

Leukocytes from Lung Transplant Recipients with Severe Primary Graft Dysfunction Adoptively Transfer Alloreactivity to Vessel Grafts Trans Vivo

Introduction Primary graft dysfunction (PGD) is the main cause of early mortality after lung transplantation (LTx). Multiple mechanisms have been described, but the link between PGD and T cell priming and chronic rejection has not been defined. In humanized mice, we studied leukocytes from human lung recipients undergoing PGD with different kinetics and their impact on rejection of artery grafts. Methods Segments of the pericardiophrenic arteries were procured from surplus donor lung tissue and implanted into the abdominal aorta of immunodeficient mice. Development of transplant arteriosclerosis after reconstitution with PBMC of the respective human recipient was determined at day 28. Experiments were retrospectively devided; 4 patients developing transient PGD grade ≥2 at T0. 5 patients developing PGD grade ≥2 at T24 and/or T48. Mice from groups A-C were reconstituted with PBMC from PGD+ T0 patients. Group A mice were reconstituted with PBMC from the respective allogeneic human recipient. Group B mice additionally received CD4+CD25high Treg cells with their PBMC. In Group C, administered PBMC were depleted of CD4+CD25high Treg cells. Mice from groups D-F were reconstituted with PBMC from PGD+ T24/T48 patients, with group D receiving unmodified PBMC, group E receiving Treg enriched PBMC and group F receiving PBMC depleted of Tregs. Results Luminal occlusion of aortic vessels was higher in group D mice compared to group A. The addition of CD4+CD25high Treg cells in group B & E mice had a suppressive effect on luminal occlusion (see Figure). Conclusion We conclude that leukocytes from lung transplantation recipients undergoing PGD 2 or 3 transfer enhanced alloreactivity to humanized mice already immediately perioperatively. Therein, PGD 2 or 3 at T24 and/or T48 hours after lung transplantation translates to more severe rejection in humanized mice than transient PGD 2 or 3 at T0 only. This inflammatory response is suppressed by CD4+CD25high Treg cells indicating a potential target for future interventions in lung transplantation. Figure. No caption available.

Immunoengineering of the vascular endothelium to silence MHC expression during normothermic ex vivo lung perfusion

Disparities at the major histocompatibility complex (MHC) antigens and associated minor antigens trigger harmful immune responses, leading to graft rejection after transplantation. We showed that MHC-silenced cells and tissues are efficiently protected against rejection. In complex vascularized organs, the endothelium is the major interface between donor and recipient. This study therefore aimed to reduce the immunogenicity of the lung by silencing MHC expression on the endothelium. In porcine lungs, short-hairpin RNAs targeting beta-2-microglobulin and class II-transactivator transcripts were delivered by lentiviral vectors during normothermic ex vivo perfusion to silence swine leukocyte antigen (SLA) I and II expression permanently. The results demonstrated the feasibility of genetically engineering all lung regions, achieving a targeted silencing effect for SLA I and II of 67% and 52%, respectively, without affecting cell viability or tissue integrity. This decrease in immunogenicity carries the potential to generate immunologically invisible organs to counteract the burden of rejection and immunosuppression.

Prediction of transplant outcome after 24-hour ex vivo lung perfusion using the Organ Care System in a porcine lung transplantation model

Ex vivo lung perfusion (EVLP) has become routine practice in lung transplantation. Still, running periods exceeding 12 hours have not been undertaken clinically to date, and it remains unclear how the perfusion solution for extended running periods should be composed and which parameters may predict outcomes. Twenty‐four porcine lungs underwent EVLP for 24 hours using the Organ Care System (OCS). Lungs were ventilated and perfused with STEENs solution enriched with erythrocytes (n = 8), acellular STEENs solution (n = 8), or low‐potassium dextran (LPD) solution enriched with erythrocytes (n = 8). After 24 hours, the left lungs were transplanted into recipient pigs. After clamping of the contralateral lung, the recipients were observed for 6 hours. The most favorable outcome was observed in organs utilizing STEEN solution enriched with erythrocytes as perfusate, whereas the least favorable outcome was seen with LPD solution enriched with erythrocytes for perfusion. Animals surviving the observation period showed lower peak airway pressure (PAWP) and pulmonary vascular resistance (PVR) during OCS preservation. The results suggest that transplantation of lungs following 24 hours of EVLP is feasible but dependent on the composition of the perfusate. PAWP and PVR during EVLP are early and late predictors of transplant outcome, respectively.

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.