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Solid organ transplant represents a potentially lifesaving procedure for patients suffering from end-stage heart, lung, liver, and kidney failure. However, rejection remains a significant source of morbidity and immunosuppressive medications have significant toxicities. Janus kinase (JAK) inhibitors are effective immunosuppressants in autoimmune diseases and graft versus host disease after allogeneic hematopoietic cell transplantation. Here we examine the role of JAK inhibition in preclinical fully major histocompatibility mismatched skin and heart allograft models. Baricitinib combined with cyclosporine A (CsA) preserved fully major histocompatibility mismatched skin grafts for the entirety of a 111-day experimental period. In baricitinib plus CsA treated mice, circulating CD4 + T-bet + T cells, CD8 + T-bet + T cells, and CD4 + FOXP3 + regulatory T cells were reduced. Single cell RNA sequencing revealed a unique expression profile in immune cells in the skin of baricitinib plus CsA treated mice, including decreased inflammatory neutrophils and increased CCR2 - macrophages. In a fully major histocompatibility mismatched mismatched heart allograft model, baricitinib plus CsA prevented graft rejection for the entire 28-day treatment period compared with 9 days in controls. Our findings establish that the combination of baricitinib and CsA prevents rejection in allogeneic skin and heart graft models and supports the study of JAK inhibitors in human solid organ transplantation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision, (Copyright © 2023 Abboud, Kim, Staser, Jayasinghe, Lim, Amatya, Frye, Kopecky, Ritchey, Gao, Lavine, Kreisel, DiPersio and Choi.)

Background: Cellular rejection after heart transplantation imparts significant morbidity and mortality. Current immunosuppressive strategies are imperfect, target recipient T cells, and have adverse effects. The innate immune response plays an essential role in the recruitment and activation of T cells. Targeting the donor innate immune response would represent the earliest interventional opportunity within the immune response cascade. There is limited knowledge about donor immune cell types and functions in the setting of cardiac transplantation, and no current therapeutics exist for targeting these cell populations., Methods: Using genetic lineage tracing, cell ablation, and conditional gene deletion, we examined donor mononuclear phagocyte diversity and macrophage function during acute cellular rejection of transplanted hearts in mice. We performed single-cell RNA sequencing on donor and recipient macrophages and monocytes at multiple time points after transplantation. On the basis of our imaging and single-cell RNA sequencing data, we evaluated the functional relevance of donor CCR2 + (C-C chemokine receptor 2) and CCR2 - macrophages using selective cell ablation strategies in donor grafts before transplant. Last, we performed functional validation that donor macrophages signal through MYD88 (myeloid differentiation primary response protein 88) to facilitate cellular rejection., Results: Donor macrophages persisted in the rejecting transplanted heart and coexisted with recipient monocyte-derived macrophages. Single-cell RNA sequencing identified donor CCR2 + and CCR2 - macrophage populations and revealed remarkable diversity among recipient monocytes, macrophages, and dendritic cells. Temporal analysis demonstrated that donor CCR2 + and CCR2 - macrophages were transcriptionally distinct, underwent significant morphologic changes, and displayed unique activation signatures after transplantation. Although selective depletion of donor CCR2 - macrophages reduced allograft survival, depletion of donor CCR2 + macrophages prolonged allograft survival. Pathway analysis revealed that donor CCR2 + macrophages are activated through MYD88/nuclear factor kappa light chain enhancer of activated B cells signaling. Deletion of MYD88 in donor macrophages resulted in reduced antigen-presenting cell recruitment, reduced ability of antigen-presenting cells to present antigen to T cells, decreased emergence of allograft-reactive T cells, and extended allograft survival., Conclusions: Distinct populations of donor and recipient macrophages coexist within the transplanted heart. Donor CCR2 + macrophages are key mediators of allograft rejection, and deletion of MYD88 signaling in donor macrophages is sufficient to suppress rejection and extend allograft survival. This highlights the therapeutic potential of donor heart-based interventions.

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after transplantation, and the impact of immunomodulatory therapies on graft rejection. Heterotopic cervical heart transplantation in mice was first described in 1991 using sutured anastomoses and subsequently modified to include cuff techniques. This modification allowed for improved success rates, and since then, there have been multiple reports that have proposed further technical improvements. However, translation into more widespread utilization remains limited due to the technical difficulty associated with graft anastomoses, which requires precision to achieve adequate length and caliber of the cuffs to avoid vascular anastomotic twisting or excessive tension, which can result in damage to the graft. The present protocol describes a modified technique for performing heterotopic cervical cardiac transplantation in mice which involves cuff placement on the recipient's common carotid artery and the donor's pulmonary artery in alignment with the direction of the blood flow.

Background: Donor hearts and lungs are more susceptible to the inflammatory physiologic changes that occur after brain death. Prior investigations have shown that protocolized management of potential organ donors can rehabilitate donor organs that are initially deemed unacceptable. In this review we discuss advances in donor management models with particular attention to the specialized donor care facility model. In addition we review specific strategies to optimize donor thoracic organs and improve organ yield in thoracic transplantation., Methods: We performed a literature review by searching the PubMed database for medical subject heading terms associated with organ donor management models. We also communicated with our local organ procurement organization to gather published and unpublished information first-hand., Results: The specialized donor care facility model has been shown to improve the efficiency of organ donor management and procurement while reducing costs and minimizing travel and its associated risks. Lung protective ventilation, recruitment of atelectatic lung, and hormone therapy (eg, glucocorticoids and triiodothyronine/thyroxine) are associated with improved lung utilization rates. Stroke volume-based resuscitation is associated with improved heart utilization rates, whereas studies evaluating hormone therapy (eg, glucocorticoids and triiodothyronine/thyroxine) have shown variable results., Conclusions: Lack of high-quality prospective evidence results in conflicting practices across organ procurement organizations, and best practices remain controversial. Future studies should focus on prospective, randomized investigations to evaluate donor management strategies. The specialized donor care facility model fosters a collaborative environment that encourages academic inquiry and is an ideal setting for these investigations., (Copyright © 2022 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Purpose of Review: Transplantation continues to be the only treatment option for end-stage organ failure when other interventions have failed. Although short-term outcomes have improved due to advances in perioperative care, long-term outcomes continue to be adversely affected by chronic rejection. Little is known about the role microbiota play in modulating alloimmune responses and potentially contributing to graft failure. Initial data have identified a correlation between specific changes of the recipient and/or donor microbiota and transplant outcomes. In this review, we will focus on recent findings concerning the complex interplay between microbiota and the innate immune system after heart and lung transplantation., Recent Findings: Gut microbiome derangements in heart failure promote an inflammatory state and have lasting effects on the innate immune system, with an observed association between increased levels of microbiota-dependent metabolites and acute rejection after cardiac transplantation. The lung allograft microbiome interacts with components of the innate immune system, such as toll-like receptor signalling pathways, NKG2C+ natural killer cells and the NLRP3 inflammasome, to alter posttransplant outcomes, which may result in the development of chronic rejection., Summary: The innate immune system is influenced by alterations in the microbiome before and after heart and lung transplantation, thereby offering potential therapeutic targets for prolonging allograft survival., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

The life-saving benefits of organ transplantation can be thwarted by allograft dysfunction due to both infectious and sterile inflammation post-surgery. Sterile inflammation can occur after necrotic cell death due to the release of endogenous ligands [such as damage-associated molecular patterns (DAMPs) and alarmins], which perpetuate inflammation and ongoing cellular injury via various signaling cascades. Ischemia-reperfusion injury (IRI) is a significant contributor to sterile inflammation after organ transplantation and is associated with detrimental short- and long-term outcomes. While the vicious cycle of sterile inflammation and cellular injury is remarkably consistent amongst different organs and even species, we have begun understanding its mechanistic basis only over the last few decades. This understanding has resulted in the developments of novel, yet non-specific therapies for mitigating IRI-induced graft damage, albeit with moderate results. Thus, further understanding of the mechanisms underlying sterile inflammation after transplantation is critical for identifying personalized therapies to prevent or interrupt this vicious cycle and mitigating allograft dysfunction. In this review, we identify common and distinct pathways of post-transplant sterile inflammation across both heart and lung transplantation that can potentially be targeted.

Since the 1960s, heart and lung transplantation has remained the optimal therapy for patients with end-stage disease, extending and improving quality of life for thousands of individuals annually. Expanding donor organ availability and immunologic compatibility is a priority to help meet the clinical demand for organ transplant. While effective, current immunosuppression is imperfect as it lacks specificity and imposes unintended adverse effects such as opportunistic infections and malignancy that limit the health and longevity of transplant recipients. In this review, we focus on donor macrophages as a new target to achieve allograft tolerance. Donor organ-directed therapies have the potential to improve allograft survival while minimizing patient harm related to global suppression of recipient immune responses. Topics highlighted include the role of ontogenically distinct donor macrophage populations in ischemia-reperfusion injury and rejection, including their interaction with allograft-infiltrating recipient immune cells and potential therapeutic approaches. Ultimately, a better understanding of how donor intrinsic immunity influences allograft acceptance and survival will provide new opportunities to improve the outcomes of transplant recipients., (© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Primary graft dysfunction (PGD) and acute cellular rejection (ACR) are important causes of early morbidity and mortality following lung and heart transplantation. While many studies have elucidated donor-related risk factors of PGD and ACR, these complications often occur even with "ideal" donors. Therefore, we investigated potential associations of PGD and ACR between bilateral lung and heart transplant recipients from the same multiorgan donor, respectively., Methods: Between 2011 and 2017, 100 donors contributed 100 bilateral lung transplants and 100 heart transplants performed. Logistic regression analysis for PGD and Cox proportional hazards regression analysis for ACR were used to estimate the relationship of heart and lung transplants., Results: The incidence of PGD was 33% among lung and 17% among heart transplant recipients. Similarly, the incidence of ACR grade ≥ A2 for lung recipients was 38% (30/80), and the incidence of ACR grade ≥ 2R for heart recipients was 19% (15/80). There was no association between the development of PGD and ACR in lung and heart transplant recipients from the same donor, respectively., Conclusions: These findings suggest that inherent donor factors are not critical to the development of PGD and ACR after lung and heart transplantation., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Non-apoptotic forms of cell death can trigger sterile inflammation through the release of danger-associated molecular patterns, which are recognized by innate immune receptors. However, despite years of investigation the mechanisms which initiate inflammatory responses after heart transplantation remain elusive. Here, we demonstrate that ferrostatin-1 (Fer-1), a specific inhibitor of ferroptosis, decreases the level of pro-ferroptotic hydroperoxy-arachidonoyl-phosphatidylethanolamine, reduces cardiomyocyte cell death and blocks neutrophil recruitment following heart transplantation. Inhibition of necroptosis had no effect on neutrophil trafficking in cardiac grafts. We extend these observations to a model of coronary artery ligation-induced myocardial ischemia reperfusion injury where inhibition of ferroptosis resulted in reduced infarct size, improved left ventricular systolic function, and reduced left ventricular remodeling. Using intravital imaging of cardiac transplants, we uncover that ferroptosis orchestrates neutrophil recruitment to injured myocardium by promoting adhesion of neutrophils to coronary vascular endothelial cells through a TLR4/TRIF/type I IFN signaling pathway. Thus, we have discovered that inflammatory responses after cardiac transplantation are initiated through ferroptotic cell death and TLR4/Trif-dependent signaling in graft endothelial cells. These findings provide a platform for the development of therapeutic strategies for heart transplant recipients and patients, who are vulnerable to ischemia reperfusion injury following restoration of coronary blood flow.

Rationale: Early graft inflammation enhances both acute and chronic rejection of heart transplants, but it is unclear how this inflammation is initiated., Objective: To identify specific inflammatory modulators and determine their underlying molecular mechanisms after cardiac transplantation., Methods and Results: We used a murine heterotopic cardiac transplant model to identify inflammatory modulators of early graft inflammation. Unbiased mass spectrometric analysis of cardiac tissue before and ≤72 hours after transplantation revealed that 22 proteins including haptoglobin, a known antioxidant, are significantly upregulated in our grafts. Through the use of haptoglobin-deficient mice, we show that 80% of haptoglobin-deficient recipients treated with perioperative administration of the costimulatory blocking agent CTLA4 immunoglobulin exhibited >100-day survival of full major histocompatibility complex mismatched allografts, whereas all similarly treated wild-type recipients rejected their transplants by 21 days after transplantation. We found that haptoglobin modifies the intra-allograft inflammatory milieu by enhancing levels of the inflammatory cytokine interleukin-6 and the chemokine MIP-2 (macrophage inflammatory protein 2) but impair levels of the immunosuppressive cytokine interleukin-10. Haptoglobin also enhances dendritic cell graft recruitment and augments antidonor T-cell responses. Moreover, we confirmed that the protein is present in human cardiac allograft specimens undergoing acute graft rejection., Conclusions: Our findings provide new insights into the mechanisms of inflammation after cardiac transplantation and suggest that, in contrast to its prior reported antioxidant function in vascular inflammation, haptoglobin is an enhancer of inflammation after cardiac transplantation. Haptoglobin may also be a key component in other sterile inflammatory conditions., (© 2015 American Heart Association, Inc.)

Background: Antithymocyte globulin is frequently used as a component of induction therapy in thoracic organ transplantation. This study evaluates the utility of monitoring peripheral CD3 lymphocytes to rationally adjust antithymocyte globulin therapy in this patient population., Methods: A total of 17 heart and 19 lung transplant recipients received antithymocyte globulin (ATGAM or thymoglobulin) as induction therapy or to treat steroid-resistant acute or chronic rejection. Absolute CD3 counts were maintained between 50 and 100 cells/microl., Results: With CD3 monitoring, the doses of antithymocyte globulin were reduced from 10-15 mg/kg to 1-5 mg/kg during the course of therapy. The total amount of antithymocyte globulin given to each CD3 monitored patient was reduced by 48%. Dose reduction did not alter the number of acute rejection or infectious episodes, and hematological side effects were infrequent., Conclusion: CD3 monitoring of antithymocyte globulin therapy in thoracic organ recipients reduced the amount of drug received by each patient, while maintaining CD3 counts less than 100 cells/microl.

Background: We have described that chimeric rat hearts bearing recipient-type antigen-presenting cells (APCs) do not reject acutely, but develop cardiac allograft vasculopathy (CAV) in untreated recipients. This suggests that CAV is triggered either by CD8+ direct allorecognition or by CD4+ indirect allorecognition. To determine the allorecognition pathway responsible for CAV in this model, recipients of chimeric hearts underwent either CD8+ or CD4+ T cell depletion., Methods: Chimeric hearts were created via bone marrow transplantation in two fully major histocompatibility-mismatched rat strain combinations. DA recipients were thymectomized and treated with Ox8 and Ox38 murine monoclonal antibodies, which deplete CD8+ and CD4+ T cells, respectively. Chimeric PVG hearts bearing DA APCs, abbreviated PVG(DA), were heterotopically transplanted into recipients undergoing thymectomy alone or recipients undergoing thymectomy plus either CD4+ or CD8+ T cell depletion., Results: PVG(DA) allografts survived 100 days, but developed CAV in thymectomized recipients and in those permanently depleted of CD8+ T cells. In contrast, chimeric hearts transplanted into permanently CD4+ T cell-depleted recipients survived 100 days and demonstrated no evidence of CAV., Conclusions: In this specific strain combination, recipient CD8+ T cells are neither necessary nor sufficient for the development of CAV, whereas recipient CD4+ T cells are required for the development of CAV. These findings suggest that CAV is dependent on CD4+ indirect allorecognition and that CD8+ direct allorecognition stimulated by nonprofessional APCs plays a minor role.

Despite evidence that human non-hematopoietic cells, such as vascular endothelium, can activate allogeneic T lymphocytes in vitro, the prevailing view has been that hematopoietic antigen-presenting cells are required to trigger alloimmune responses in vivo. Here we report that mouse non-hematopoietic cells activate alloreactive CD8+ T lymphocytes in vitro and in vivo. We also show that vascularized cardiac allografts are acutely rejected via CD8+ direct allorecognition even if the alloantigen is not presented by hematopoietic professional antigen-presenting cells. Because activation of alloreactive CD8+ T cells by donor-type non-hematopoietic cells can continue for the life of the allograft, these findings present a new clinically relevant mechanism of allorecognition and should be taken into consideration when developing strategies to prevent allograft vasculopathy or to induce tolerance.

With advancements in basic science and clinical medicine, lung transplantation (LT) has evolved rapidly over the last three decades. However, it is unclear if significant regional variations exist in long-term outcomes after LT. To investigate potential differences, we performed a retrospective, comparative cohort analysis of adult patients undergoing deceased donor single or double LT in North America (NA) or Europe between January 2006 and December 2016. Data up to April 2019 were abstracted from the International Society for Heart and Lung Transplantation (ISHLT) Thoracic Organ Registry. We compared overall survival (OS) between North American and European LT centers in a propensity score matched analysis. In 3,115 well-matched pairs, though 30-day survival was similar between groups (NA 96.2% vs Europe 95.4%, p = 0.116), 5-year survival was significantly higher in European patients (NA 60.1% vs Europe 70.3%, p < 0.001). This survival difference persisted in a sensitivity analysis excluding Canadian patients. Prior observations suggest that these disparities are at least partly related to better access to care via universal healthcare models prevalent in Europe. Future studies are warranted to confirm our findings and explore other causal mechanisms. It is likely that potential solutions will require concerted efforts from healthcare providers and policymakers. [ABSTRACT FROM AUTHOR]

Improved understanding of lung transplant disease states is essential because failure rates are high, often due to chronic lung allograft dysfunction. However, histologic assessment of lung transplant transbronchial biopsies (TBBs) is difficult and often uninterpretable even with 10 pieces. We prospectively studied whether microarray assessment of single TBB pieces could identify disease states and reduce the amount of tissue required for diagnosis. By following strategies successful for heart transplants, we used expression of rejection-associated transcripts (annotated in kidney transplant biopsies) in unsupervised machine learning to identify disease states. All 242 single-piece TBBs produced reliable transcript measurements. Paired TBB pieces available from 12 patients showed significant similarity but also showed some sampling variance. Alveolar content, as estimated by surfactant transcript expression, was a source of sampling variance. To offset sampling variation, for analysis, we selected 152 single-piece TBBs with high surfactant transcripts. Unsupervised archetypal analysis identified 4 idealized phenotypes (archetypes) and scored biopsies for their similarity to each: normal; T-cell‒mediated rejection (TCMR; T-cell transcripts); antibody-mediated rejection (ABMR)-like (endothelial transcripts); and injury (macrophage transcripts). Molecular TCMR correlated with histologic TCMR. The relationship of molecular scores to histologic ABMR could not be assessed because of the paucity of ABMR in this population. Molecular assessment of single-piece TBBs can be used to classify lung transplant biopsies and correlated with rejection histology. Two or 3 pieces for each TBB will probably be needed to offset sampling variance. [ABSTRACT FROM AUTHOR]

BACKGROUND Lung transplant (LTx) recipients have low long-term survival and a high incidence of bronchiolitis obliterans syndrome (BOS). However, few long-term, multicenter, and precise estimates of BOS-free survival (a composite outcome of death or BOS) incidence exist. METHODS This retrospective cohort study of primary LTx recipients (1994–2011) reported to the International Society of Heart and Lung Transplantation Thoracic Transplant Registry assessed outcomes through 2012. For the composite primary outcome of BOS-free survival, we used Kaplan-Meier survival and Cox proportional hazards regression, censoring for loss to follow-up, end of study, and re-LTx. Although standard Thoracic Transplant Registry analyses censor at the last consecutive annual complete BOS status report, our analyses allowed for partially missing BOS data. RESULTS Due to BOS reporting standards, 99.1% of the cohort received LTx in North America. During 79,896 person-years of follow-up, single LTx (6,599 of 15,268 [43%]) and bilateral LTx (8,699 of 15,268 [57%]) recipients had a median BOS-free survival of 3.16 years (95% confidence interval [CI], 2.99–3.30 years) and 3.58 years (95% CI, 3.53–3.72 years), respectively. Almost 90% of the single and bilateral LTx recipients developed the composite outcome within 10 years of transplantation. Standard Registry analyses "overestimated" median BOS-free survival by 0.42 years and "underestimated" the median survival after BOS by about a half-year for both single and bilateral LTx (p < 0.05). CONCLUSIONS Most LTx recipients die or develop BOS within 4 years, and very few remain alive and free from BOS at 10 years post-LTx. Less inclusive Thoracic Transplant Registry analytic methods tend to overestimate BOS-free survival. The Registry would benefit from improved international reporting of BOS and other chronic lung allograft dysfunction (CLAD) events. [ABSTRACT FROM AUTHOR]

Two-photon intravital microscopy has substantially broadened our understanding of tissue- and organ-specific differences in the regulation of inflammatory responses. However, little is known about the dynamic regulation of leukocyte recruitment into inflamed heart tissue, largely due to technical difficulties inherent in imaging moving tissue. Here, we report a method for imaging beating murine hearts using intravital 2-photon microscopy. Using this method, we visualized neutrophil trafficking at baseline and during inflammation. Ischemia reperfusion injury induced by transplantation or transient coronary artery ligation led to recruitment of neutrophils to the heart, their extravasation from coronary veins, and infiltration of the myocardium where they formed large clusters. Grafting hearts containing mutant ICAM-1, a ligand important for neutrophil recruitment, reduced the crawling velocities of neutrophils within vessels, and markedly inhibited their extravasation. Similar impairment was seen with the inhibition of Mac-1, a receptor for ICAM-1. Blockade of LFA-1, another ICAM-1 receptor, prevented neutrophil adherence to endothelium and extravasation in heart grafts. As inflammatory responses in the heart are of great relevance to public health, this imaging approach holds promise for studying cardiac-specific mechanisms of leukocyte recruitment and identifying novel therapeutic targets for treating heart disease. [ABSTRACT FROM AUTHOR]