Lung Transplant Immunomodulation with Genetically Engineered Mesenchymal Stromal Cells-Therapeutic Window for Interleukin-10.

Lung transplantation results are compromised by ischemia-reperfusion injury and alloimmune responses. Ex vivo lung perfusion (EVLP) is used to assess marginal donor lungs before transplantation but is also an excellent platform to apply novel therapeutics. We investigated donor lung immunomodulation using genetically engineered mesenchymal stromal cells with augmented production of human anti-inflammatory hIL-10 (MSCsIL-10). Pig lungs were placed on EVLP for 6 h and randomized to control (n = 7), intravascular delivery of 20 × 106 (n = 5, low dose) or 40 × 106 human MSCs IL-10 (n = 6, high dose). Subsequently, single-lung transplantation was performed, and recipient pigs were monitored for 3 days. hIL-10 secretion was measured during EVLP and after transplantation, and immunological effects were assessed by cytokine profile, T and myeloid cell characterization and mixed lymphocyte reaction. MSCIL-10 therapy rapidly increased hIL-10 during EVLP and resulted in transient hIL-10 elevation after lung transplantation. MSCIL-10 delivery did not affect lung function but was associated with dose-related immunomodulatory effects, with the low dose resulting in a beneficial decrease in apoptosis and lower macrophage activation, but the high MSCIL-10 dose resulting in inflammation and cytotoxic CD8+ T cell activation. MSCIL-10 therapy during EVLP results in a rapid and transient perioperative hIL-10 increase and has a therapeutic window for its immunomodulatory effects.

L-alanyl-L-glutamine modified perfusate improves human lung cell functions and extend porcine ex vivo lung perfusion.

BACKGROUND: The clinical application of normothermic ex vivo lung perfusion (EVLP) has increased donor lung utilization for transplantation through functional assessment. To develop it as a platform for donor lung repair, reconditioning and regeneration, the perfusate should be modified to support the lung during extended EVLP. METHODS: Human lung epithelial cells and pulmonary microvascular endothelial cells were cultured, and the effects of Steen solution (commonly used EVLP perfusate) on basic cellular function were tested. Steen solution was modified based on screening tests in cell culture, and further tested with an EVLP cell culture model, on apoptosis, GSH, HSP70, and IL-8 expression. Finally, a modified formula was tested on porcine EVLP. Physiological parameters of lung function, histology of lung tissue, and amino acid concentrations in EVLP perfusate were measured. RESULTS: Steen solution reduced cell confluence, induced apoptosis, and inhibited cell migration, compared to regular cell culture media. Adding L-alanyl-L-glutamine to Steen solution improved cell migration and decreased apoptosis. It also reduced cold preservation and warm perfusion-induced apoptosis, enhanced GSH and HSP70 production, and inhibited IL-8 expression on an EVLP cell culture model. L-alanyl-L-glutamine modified Steen solution supported porcine lungs on EVLP with significantly improved lung function, well-preserved histological structure, and significantly higher levels of multiple amino acids in EVLP perfusate. CONCLUSIONS: Adding L-alanyl-L-glutamine to perfusate may provide additional energy support, antioxidant, and cytoprotective effects to lung tissue. The pipeline developed herein, with cell culture, cell EVLP, and porcine EVLP models, can be used to further optimize perfusates to improve EVLP outcomes.

Ex vivo enzymatic treatment converts blood type A donor lungs into universal blood type lungs.

Donor organ allocation is dependent on ABO matching, restricting the opportunity for some patients to receive a life-saving transplant. The enzymes FpGalNAc deacetylase and FpGalactosaminidase, used in combination, have been described to effectively convert group A (ABO-A) red blood cells (RBCs) to group O (ABO-O). Here, we study the safety and preclinical efficacy of using these enzymes to remove A antigen (A-Ag) from human donor lungs using ex vivo lung perfusion (EVLP). First, the ability of these enzymes to remove A-Ag in organ perfusate solutions was examined on five human ABO-A1 RBC samples and three human aortae after static incubation. The enzymes removed greater than 99 and 90% A-Ag from RBCs and aortae, respectively, at concentrations as low as 1 µg/ml. Eight ABO-A1 human lungs were then treated by EVLP. Baseline analyses of A-Ag in lungs revealed expression predominantly in the endothelial and epithelial cells. EVLP of lungs with enzyme-containing perfusate removed over 97% of endothelial A-Ag within 4 hours. No treatment-related acute lung toxicity was observed. An ABO-incompatible transplant was then simulated with an ex vivo model of antibody-mediated rejection using ABO-O plasma as the surrogate for the recipient circulation using three donor lungs. The treatment of donor lungs minimized antibody binding, complement deposition, and antibody-mediated injury as compared with control lungs. These results show that depletion of donor lung A-Ag can be achieved with EVLP treatment. This strategy has the potential to expand ABO-incompatible lung transplantation and lead to improvements in fairness of organ allocation.

Engineered mesenchymal stromal cell therapy during human lung ex vivo lung perfusion is compromised by acidic lung microenvironment.

Ex vivo lung perfusion (EVLP) is an excellent platform to apply novel therapeutics, such as gene and cell therapies, before lung transplantation. We investigated the concept of human donor lung engineering during EVLP by combining gene and cell therapies. Premodified cryopreserved mesenchymal stromal cells with augmented anti-inflammatory interleukin-10 production (MSCIL-10) were administered during EVLP to human lungs that had various degrees of underlying lung injury. Cryopreserved MSCIL-10 had excellent viability, and they immediately and efficiently elevated perfusate and lung tissue IL-10 levels during EVLP. However, MSCIL-10 function was compromised by the poor metabolic conditions present in the most damaged lungs. Similarly, exposing cultured MSCIL-10 to poor metabolic, and especially acidic, conditions decreased their IL-10 production. In conclusion, we found that "off-the-shelf" MSCIL-10 therapy of human lungs during EVLP is safe and feasible, and results in rapid IL-10 elevation, and that the acidic target-tissue microenvironment may compromise the efficacy of cell-based therapies.

An extracellular oxygen carrier during prolonged pulmonary preservation improves post-transplant lung function.

BACKGROUND: The use of a novel extracellular oxygen carrier (EOC) preservation additive known as HEMO2Life has recently been shown to lead to a superior preservation of different types of solid organs. Our study aimed to investigate the effect of this EOC on extending lung preservation time and its mechanism of action. METHODS: Donor pigs were randomly allocated to either of the following 2 groups (n = 6 per group): (1) 36 hours cold preservation or (2) 36 hours cold preservation with 1 g/liter of EOC. The lungs were evaluated through 12 hours of normothermic ex vivo lung perfusion (EVLP) followed by a left-single lung transplant into a recipient pig. Grafts were reperfused for 4 hours, followed by right pulmonary artery clamping to assess graft oxygenation function. RESULTS: During EVLP assessment, EOC-treated lungs showed improvements in physiologic parameters, whereas the control lungs deteriorated. After a total of 48 hours of preservation (36 hours cold + 12 hours normothermic EVLP), transplanted grafts in the treatment group displayed significantly better oxygenation than in the controls (PaO2/FiO2: 437 ± 36 mm Hg vs 343 ± 27 mm Hg, p = 0.041). In addition, the use of EOC led to significantly less edema formation (wet-to-dry ratio: 4.95 ± 0.29 vs 6.05 ± 0.33, p = 0.026), less apoptotic cell death (p = 0.041), improved tight junction preservation (p = 0.002), and lower levels of circulating IL-6 within recipient plasma (p = 0.004) compared with non-use of EOC in the control group after transplantation. CONCLUSION: The use of an EOC during an extended pulmonary preservation period led to significantly superior early post-transplant lung function.