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Background: T cells drive acute cellular rejection (ACR) and its progression to chronic lung allograft dysfunction (CLAD) following lung transplantation. International Society for Heart and Lung Transplantation grade A1 ACR without associated allograft dysfunction is often untreated, yet some patients develop progressive graft dysfunction. T-cell composition of A1 ACR lesions may have prognostic value; therefore, protein-level and epigenetic techniques were applied to transbronchial biopsy tissue to determine whether differential T-cell infiltration in recipients experiencing a first episode of stable grade A1 ACR (StA1R) is associated with early CLAD., Methods: Sixty-two patients experiencing a first episode of StA1R were divided into those experiencing CLAD within 2 years (n = 13) and those remaining CLAD-free for 5 or more years (n = 49). Imaging mass cytometry (IMC) was used to profile the spectrum and distribution of intragraft T cell phenotypes on a subcohort (n = 16; 8 early-CLAD and 8 no early-CLAD). Immunofluorescence was used to quantify CD4 + , CD8 + , and FOXP3 + cells. Separately, CD3 + cells were fluorescently labeled, micro-dissected, and the degree of Treg-specific demethylated region methylation was determined., Results: PhenoGraph unsupervised clustering on IMC revealed 50 unique immune cell subpopulations. Methylation and immunofluorescence analyses demonstrated no significant differences in Tregs between early-CLAD and no early-CLAD groups. Immunofluorescence revealed that patients who developed CLAD within 2 years of lung transplantation showed greater CD8 + T cell infiltration compared to those who remained CLAD-free for 5 or more years., Conclusions: In asymptomatic patients with a first episode of A1 rejection, greater CD8 + T cell content may be indicative of worse long-term outlook., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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 (MSCs IL-10 ). Pig lungs were placed on EVLP for 6 h and randomized to control ( n = 7), intravascular delivery of 20 × 10 6 ( n = 5, low dose) or 40 × 10 6 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. MSC IL-10 therapy rapidly increased hIL-10 during EVLP and resulted in transient hIL-10 elevation after lung transplantation. MSC IL-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 MSC IL-10 dose resulting in inflammation and cytotoxic CD8 + T cell activation. MSC IL-10 therapy during EVLP results in a rapid and transient perioperative hIL-10 increase and has a therapeutic window for its immunomodulatory effects.

Chronic lung allograft dysfunction (CLAD) is the major cause of death after lung transplantation. Angiotensin II (AngII), the main effector of the renin-angiotensin system, elicits fibrosis in both kidney and lung. We identified six AngII-regulated proteins (Ras homolog family member B (RHOB), bone marrow stromal cell antigen 1 (BST1), lysophospholipase 1 (LYPA1), glutamine synthetase (GLNA), thrombospondin 1 (TSP1) and laminin subunit β2 (LAMB2)) that were increased in urine of patients with kidney allograft fibrosis. We hypothesised that the renin-angiotensin system is active in CLAD and that AngII-regulated proteins are increased in bronchoalveolar lavage fluid (BAL) of CLAD patients.We performed immunostaining of AngII receptors (AGTR1 and AGTR2), TSP1 and GLNA in 10 CLAD lungs and five controls. Using mass spectrometry, we quantified peptides corresponding to AngII-regulated proteins in BAL of 40 lung transplant recipients (stable, acute lung allograft dysfunction (ALAD) and CLAD). Machine learning algorithms were developed to predict CLAD based on BAL peptide concentrations.Immunostaining demonstrated significantly more AGTR1 + cells in CLAD versus control lungs (p=0.02). TSP1 and GLNA immunostaining positively correlated with the degree of lung fibrosis (R 2 =0.42 and 0.57, respectively). In BAL, we noted a trend towards higher concentrations of AngII-regulated peptides in patients with CLAD at the time of bronchoscopy, and significantly higher concentrations of BST1, GLNA and RHOB peptides in patients that developed CLAD at follow-up (p<0.05). The support vector machine classifier discriminated CLAD from stable and ALAD patients at the time of bronchoscopy (area under the curve (AUC) 0.86) and accurately predicted subsequent CLAD development (AUC 0.97).Proteins involved in the renin-angiotensin system are increased in CLAD lungs and BAL. AngII-regulated peptides measured in BAL may accurately identify patients with CLAD and predict subsequent CLAD development., Competing Interests: Conflict of interest: G. Berra has nothing to disclose. Conflict of interest: S. Farkona has nothing to disclose. Conflict of interest: Z. Mohammed-Ali has nothing to disclose. Conflict of interest: M. Kotlyar has nothing to disclose. Conflict of interest: L. Levy has nothing to disclose. Conflict of interest: S. Clotet-Freixas has nothing to disclose. Conflict of interest: P. Ly has nothing to disclose. Conflict of interest: B. Renaud-Picard has nothing to disclose. Conflict of interest: G. Zehong has nothing to disclose. Conflict of interest: T. Daigneault has nothing to disclose. Conflict of interest: A. Duong has nothing to disclose. Conflict of interest: I. Batruch has nothing to disclose. Conflict of interest: I. Jurisica reports grants and nonfinancial support (in-kind contribution to grants) from IBM, personal fees for lectures and other (reimbursement of expenses) from Novartis and Canadian Rheumatology Association, outside the submitted work. Conflict of interest: A. Konvalinka has nothing to disclose. Conflict of interest: T. Martinu reports grants from Canadian Donation and Transplantation Research Program and Di Pochi funds, during the conduct of the study; grants from Sanofi, nonfinancial support from APCBio, outside the submitted work., (Copyright ©The authors 2021. For reproduction rights and permissions contact permissions@ersnet.org.)

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 (MSC IL-10 ) were administered during EVLP to human lungs that had various degrees of underlying lung injury. Cryopreserved MSC IL-10 had excellent viability, and they immediately and efficiently elevated perfusate and lung tissue IL-10 levels during EVLP. However, MSC IL-10 function was compromised by the poor metabolic conditions present in the most damaged lungs. Similarly, exposing cultured MSC IL-10 to poor metabolic, and especially acidic, conditions decreased their IL-10 production. In conclusion, we found that "off-the-shelf" MSC IL-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., Competing Interests: C.E. is an employee of Tissue Regeneration Therapeutics Inc.; J.E.D is the CEO of Tissue Regeneration Therapeutics Inc.; S.J. and T.M. have received research support from Sanofi; T.W., M.C., M.L., and S.K. are owners of Perfusix Canada and XOR Labs Toronto. T.W., M.C., and S.K. are scientific medical advisors for Lung Bioengineering. The remaining authors declare no competing interests., (© 2021 The Author(s).)

Adenocarcinoma of the esophagus is increasing in frequency and is the 6th most common cause of cancer death in North America. In adenocarcinoma cell lines, we have previously demonstrated that expression of miR-145, leads to enhanced invasion, resistance to anoikis and better attachment to fibronectin in esophageal adenocarcinoma. In contrast, expression of miR-145 acts as a tumor suppressor in squamous cell carcinoma. The molecular mechanisms responsible for the oncogenic effects of miR-145 were investigated. In this report, we demonstrate that we can partially recreate the miR-145 effects in EAC by knock down of the expression of c-Myc, which is one of the targets of miR-145. Knocking down of c-Myc expression resulted in upregulation of integrin subunits α5 and β3. Finally, we demonstrated that integrin α5 expression correlates to fibronectin attachment potential whereas integrin β3 expression correlates with resistance to anoikis and invasion potential. Finally, we demonstrate that expression of miR-145 in esophageal adenocarcinoma cell line (SK-GT-4) enhances tumor growth and metastasis in a NOD/SCID xenograft model. Overall, the oncogenic potential of miR-145 in EAC appears to be mediated by downregulation of c-Myc leading to the expression of integrins subunits α5 and β3., Competing Interests: CONFLICTS OF INTEREST No conflicts of interest to disclose.