Ischemia-reperfusion injury (IRI) contributes to deleterious outcomes after lung transplantation. Although we have shown a unique protective role for eosinophils in both establishing and maintaining lung allograft tolerance, their role in IRI remains unclear. Based on previous research demonstrating a protective role for eosinophils in liver IRI, we hypothesized that they might play a similar function in the lung. In this study, we show that donor-, but not recipient-derived, eosinophils worsen injury of syngeneic and allogeneic lung grafts rather than protect from it. Eosinophils in the lungs stored in a low potassium dextran-based extracellular preservation solution become activated, degranulate, and die during rewarming. This damage correlates with decreased oxygenation and increased tissue injury upon reperfusion. In vitro studies confirm that eosinophils exposed to a clinically used low-potassium dextran-based preservation solution, but not to other preservation solutions, experience oxidative stress. Supplementing such a solution with the antioxidant glutathione, which is present in solutions used to preserve other solid organs, reduces activation and injury. While IRI has been attributed solely to graft damage mediated by recipient-derived cells entering the graft upon reperfusion, our new findings uncover a previously unrecognized role for donor-derived leukocytes in this process and open unexplored avenues to improve graft function.

How pathogens inhibit transplant tolerance remains unclear. Here, we found that Pseudomonas aeruginosa infection, but not other common bacterial respiratory infections, increases antibody-mediated rejection (AMR) risk in recipients of lung transplants. To explore this relationship, we performed orthotopic lung transplants in mice, infected recipients with P. aeruginosa, and observed for the development of AMR. Intravital two-photon microscopy showed that P. aeruginosa rapidly invaded bronchial-associated lymphoid tissues, which resulted in acute lymphocytotoxicity, including the death of forkhead box P3 (Foxp3)+CD4+ T cells that are required to suppress AMR. P. aeruginosa-mediated AMR required expression of the type III secretion system (T3SS), which injects exotoxins into the cell cytoplasm. Through a combination of mutagenesis and epitope tagging experiments, we revealed that T3SS exotoxin T ADP ribosyl-transferase activity was sufficient for graft-resident Foxp3+CD4+ T cell apoptosis, leading to myeloid differentiation primary response 88 (Myd88)-dependent generation of T-box expressed in T cells (T-bet)- and C-X-C motif chemokine receptor 3 (CXCR3)-positive germinal center and memory B cells with high donor antigen avidity. We also found that T-bet+ and CXCR3+ B cells were elevated in biopsies from recipients of lung transplants who were diagnosed with AMR. In mice, CXCR3 deficiency restricted to B cells or CXCR3 blockade prevented AMR despite P. aeruginosa infection. Our work has identified a previously unrecognized role of bacterial virulence in lung allograft rejection and suggests potential strategies to prevent AMR for those at high risk of P. aeruginosa infection after transplant.

Tolerance after lung transplantation is associated with the induction of Foxp3+ regulatory T cell-enriched bronchus-associated lymphoid tissue, which suppresses local and systemic alloimmune responses. How this tolerogenic graft environment shapes responses to respiratory viral infections, a known contributor to adverse outcomes after lung transplantation, remains unknown. Using a mouse model of a seasonally circulating parainfluenza virus, we found that acute infection of tolerant lung allografts results in temporary reductions of both bronchus-associated lymphoid tissue size and abundance of graft-resident Foxp3+ cells but does not trigger rejection. At late time points, pathologic chronic type 2 inflammatory responses characteristic of severe parainfluenza virus infection decreased and Krt5+ and Krt8+ lesions were reduced in tolerant allografts when compared with infected native lungs or syngeneic grafts. This reduction in dysplastic alveolar regeneration in tolerant allografts was associated with an increase in amphiregulin-expressing Foxp3+ cells. Furthermore, type II alveolar epithelial cells in lung allografts upregulated genes related to injury when recipient Foxp3+ cells were deficient in amphiregulin. These findings shed new light on how immune pathways that are established in tolerant lung allografts, in addition to preventing rejection, protect against aberrant tissue repair after a clinically relevant respiratory viral infection.

Because the lung is a mucosal barrier organ with a unique immunologic environment, mechanisms of immunoregulation in lung cancer may differ from those of other malignancies. Consistent with this notion, we found that CD8+ T cells played a paradoxical role in facilitating, rather than ameliorating, the growth of multiple lung adenocarcinoma models. These included spontaneous, carcinogen-induced, and transplantable tumor cell line models. Specifically, we found that CD8+ T cells promoted homing of CD4+Foxp3+ Tregs to the tumor bed by increasing the levels of CCR5 chemokines in the tumor microenvironment in an IFN-γ- and TNF-α-dependent manner. Contrary to their canonical role, these Th1 cytokines contributed to accelerated growth of murine lung adenocarcinomas, while suppressing the growth of other malignancies. Surprisingly, lung cancer cells themselves can serve as a dominant source of IFN-γ, and deletion of this cytokine from cancer cells using CRISPR/Cas9 decreases tumor growth. Importantly for translational applications, in patients with lung cancer, a high level of IFN-γ was also found at both the mRNA and protein levels. Our data outline what we deem a novel and previously undefined lung cancer-specific immunoregulatory pathway that may be harnessed to tailor immune-based therapy specifically for this malignancy.

Being a barrier mucosal organ, the lung provides a unique immunoregulatory environment. We hypothesized that immunoregulation of lung cancer differs from that of other malignancies. Contrary to the accepted dogma, we found that the presence of CD8+ T cells accelerated the growth of genetically engineered, carcinogen induced, and transplantable lung tumor models, while ameliorating the growth of non-lung tumor models. Specifically, we found that CD8+ T cells promote homing of CD4+Foxp3+ T regulatory cells to the tumor bed by increasing levels of CCR5 chemokines, CCL3/4/5. Furthermore, Maraviroc, an FDA-approved CCR5 antagonist, significantly reduces lung tumor growth. Further cytokine analysis of the tumor beds revealed a significant increase of Th-1 polarizing cytokines, IFN-γ and TNF-α, in LLC lung cancer-bearing but not B16 melanoma-bearing mice in the presence of CD8+ T cells. Importantly for translational applications, high levels of IFN-γ are also seen at mRNA and protein levels in human lung cancer patients. Neutralization of these Th1 cytokines resulted in accelerated tumor growth in B16-bearing mice but counterintuitively decreased tumor growth in LLC-bearing mice. Surprisingly, we identified lung tumor cells themselves as prominent producers of IFN-γ, and knockout of IFN-γ from LLC cells significantly reduced their growth in vivo. Our data suggests that immunoregulation of lung cancer is unique and may require novel immunomodulating strategies for future treatments. Supported by T32CA154274; I01-BX002299 Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Technological innovations have improved many barriers in lung transplantation, but high rates of acute and chronic rejection still limit lung allograft survival. This may be explained by the unique environment of the lung. As a mucosal barrier organ, the lung is constantly exposed to the external environment, leading to unique immunological features that are not seen in other transplantable solid organ allografts such as hearts, kidneys, and livers. Thus, the higher rates of rejection and poor long-term survival of lung transplant recipients may stem from the global immunosuppression strategies that are indiscriminately used for all solid organ grafts. Data from our laboratory, and others, have demonstrated that the unique immunoregulatory pathways of the lung may require different strategies for long-term graft survival. For example, depletion of CD8 + T cells typically contributes to the acceptance of transplanted organs. However, these cells facilitate lung allograft acceptance through interferon gamma mediated nitric oxide production. Interestingly CD8 + T cells modify and polarize eosinophils to produce nitric oxide as a means of tolerance induction. Such eosinophils also function to maintain long-term lung allograft acceptance by interfering with humoral alloimmunity. For most other organs eosinophils are suspected to contribute to graft rejection. In this review, we aim to describe the cytokine pathways involved in lung allograft rejection or tolerance, contrast such pathways to those evident in other solid organs, and discuss the need for further studies that can be used to design rational methods for altering the cytokine environment to improve lung allograft survival.

Abstract Introduction: Despite the theoretic potential of immunostimulatory therapy in the treatment of cancer, the disruption of negative checkpoints in the tumor microenvironment remains the dominant clinical strategy. The failure of immunostimulation, utilizing agonistic cytokines or activating antibodies, can be attributed to off target side effects, the failure to preferentially activate cytotoxic lymphocytes (CTLs) over regulatory T cells (Tregs), and the development of T cell exhaustion resulting from Jak/STAT/PI3K signaling in T cells. Methods: To address these issues we designed a first in class immunostimulatory fusion protein (RB101) that utilizes NKG2D-targeting (without activation of the NKG2D signaling) to exclusively activate CTLs (CD8+ T, NK, γδ T cells). Previous approaches to target CD8+ T cells relied upon activation of the Jak/STAT/PI3K signal transduction pathway. In contrast, RB101 treatment results in selective activation of the nuclear factor of activated T cell (NFAT) signaling pathway. NFAT activation results in increased mitochondrial biogenesis, increased T cell receptor recognition of tumor antigens, accelerated proliferation, and augmented generation of memory T cells when compared to canonical cytokines and alternative methods of immunostimulation. Results: We observed that the unique combination of NFAT signaling and NKG2D-targeting led to CTL priming and expansion without systematic toxicity in a non-human primate (NHP) model. Subcutaneous RB101 administration to healthy cynomolgus macaques at or below the No Observed Adverse Event Level (NOAEL) increases the number of interferon gamma and granzyme B positive CD8+ T and NK cells 10-100-fold over vehicle treated animals. In addition, such administration resulted in >6-fold increase in CD8+ T/Treg and NK/Treg ratios, two leading indicators of clinical efficacy. Similar results were evident in human PBMC cultures in vitro. Subcutaneous RB101 administration results in partial and/or complete responses in mice bearing multiple syngeneic murine tumors. RB101 treatment also drives improved tumor control when combined with checkpoint inhibitors or radiation. Conclusion: The powerful immune response generated by signaling through the NFAT pathway has long been evident in organ allograft rejection. Clinical organ transplantation only became possible with clinical therapies targeting this signaling pathway (such as with tacrolimus). To our knowledge, RB101 is the first therapeutic designed to stimulate the NFAT pathway of T cell activation. The precise targeting of this stimulus to CTLs allows for the safe, subcutaneous administration of RB101 and results in a compelling preclinical profile of safety and efficacy in human cells and across multiple animal models. Citation Format: Eric Lazear, Dan Watkins, Alexander Krupnick, John Westwick. A first in class immunotherapy that selectively activates the NFAT pathway in CD8+ T cells [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A112.

The main barrier to long-term survival of the lung allograft is the development of a fibrotic form of chronic rejection, known as chronic lung allograft dysfunction (CLAD). Human observational studies have identified high numbers of eosinophils in chronically rejected lungs. This association has led to the presumption that eosinophils may contribute to CLAD. To study this, we transplanted B10 minor antigen mismatched lungs into B6 recipients or mice rendered deficient in eosinophils though the deletion of GATA1. Unexpectedly, we noted that markers of CLAD; airway obliteration (17.3±2.3), peri-airway fibrosis (1.8±0.7), and lung parenchymal fibrosis (9.8±4), were substantially lower in the presence of eosinophils. Such data suggested that eosinophils ameliorate, rather than potentiate CLAD. Gene expression analysis revealed upregulation of pathways associated with Th17 polarization (IL-17a, IL-17Rα, ROR-gt, IL-6) in the absence of eosinophils. Further cytokine analysis demonstrated that eosinophils are the dominant source of IFNg in the chronically rejected lung allograft. Th17 has previously been linked to chronic rejection in multiple transplant models. Due to the antagonistic nature of Th1 and Th17, along with our current data, eosinophil-production of IFNg- may mediate Th1 rather than Th17 polarization to ameliorate CLAD. Our murine data suggests that augmenting or targeting eosinophils may be one method for improving clinical outcomes of chronic lung rejection. P01AI116501 NIH/NIAAD R01AI145108 NIH/NIAAD I01BX002299 VA/Merit R01HL166402 NIH/NHLBI Transplantation Immunology (TRAN)

Despite advances in preservation techniques, approximately 15% of lung transplant recipients experience complications due to ischemia-reperfusion injury (IRI). IRI is a complex inflammatory condition involving rapid oxidative stress, which ultimately leads to lung damage and dysfunction, potentiating both acute and chronic rejection. It is known that reactive oxygen species produced by inducible nitric oxide synthase (iNOS) can damage lung parenchymal tissue. Thus, we evaluated the phenotype of the iNOS-expressing cells in resting lungs and those subject to storage in Perfadex® preservation solution. In resting lungs, roughly 2/3 of the iNOS-expressing cells consisted of myeloid cells, with 1/3 expressing markers of eosinophils. After cold storage in Perfadex® preservation solution, eosinophils became the dominant iNOS-producing cell in the lung (>75% of iNOS cells). To explore the involvement of eosinophil-derived iNOS in lung IRI, eosinophil-sufficient and deficient grafts were preserved in Perfadex® storage, followed by implantation. Comparable to human data, donor-derived eosinophils demonstrated signs of activation as measured by CD69, CD63 and CD107a and expressed high levels of iNOS. Depleting donor eosinophils or inhibiting donor-derived iNOS by adding L-N6-(1-Iminoethyl) lysine dihydrochloride (LNIL) to the preservation solution improved oxygenation. Thus, targeting eosinophil-derived iNOS could significantly enhance transplant outcomes. NIH R01AI145108-01 NIH PO1 AI116501 Transplantation Immunology (TRAN)

Previous studies have suggested that activation of the IL-33/ST2 axis as well as elevated expression of the full-length IL-33 precursor acting in an ST2-independent fashion both contribute to pulmonary fibrosis. The protective effect of genetic ST2 deficiency on pulmonary fibrosis is known to be partial, with unclear mechanisms preventing a more complete protection. Here, we report that ST2 deficiency failed to fully protect the lungs from excess collagen accumulation after the profibrotic bleomycin injury and simultaneously facilitated elevations in pulmonary levels of a previously suggested profibrotic mediator, IL-9, as well as a known activator of IL-9 expression, TSLP. Pulmonary CD4+ T cells were the main producers of IL-9. Neutralizing antibody-mediated in vivo blockade of TSLP potently attenuated pulmonary levels of both IL-9 and collagen in the bleomycin injury model in wild-type and particularly ST2-deficient mice. All these observations were markedly pronounced in mice with single deficiency of ST2 and the overall pattern of findings was also preserved in mice with dual deficiency of ST2 and IL-33. It was concluded that the antifibrotic effect of ST2 deficiency is hindered by the simultaneous activation of the TSLP-IL-9 axis in experimental bleomycin-induced pulmonary fibrosis. These findings inform further development of antifibrotic therapies.

Mechanisms that mediate allograft tolerance differ between organs. We have previously shown that Foxp3+ T cell-enriched bronchus-associated lymphoid tissue (BALT) is induced in tolerant murine lung allografts and that these Foxp3+ cells suppress alloimmune responses locally and systemically. Here, we demonstrated that Foxp3+ cells that reside in tolerant lung allografts differed phenotypically and transcriptionally from those in the periphery and were clonally expanded. Using a mouse lung retransplant model, we showed that recipient Foxp3+ cells were continuously recruited to the BALT within tolerant allografts. We identified distinguishing features of graft-resident and newly recruited Foxp3+ cells and showed that graft-infiltrating Foxp3+ cells acquired transcriptional profiles resembling those of graft-resident Foxp3+ cells over time. Allografts underwent combined antibody-mediated rejection and acute cellular rejection when recruitment of recipient Foxp3+ cells was prevented. Finally, we showed that local administration of IL-33 could expand and activate allograft-resident Foxp3+ cells, providing a platform for the design of tolerogenic therapies for lung transplant recipients. Our findings establish graft-resident Foxp3+ cells as critical orchestrators of lung transplant tolerance and highlight the need to develop lung-specific immunosuppression.

Lung transplantation (LTx) offers a last resort for patients battling end-stage lung disease. Even though short-term survival has improved, these patients still face several long-term challenges, such as chronic rejection and ischemic bronchial anastomosis. In lung transplant recipients, the bronchial anastomosis is prone to complications—such as poor wound healing, necrosis, stenosis, and dehiscence—due to the marginal blood supply at this site. During peri-LTx, hypoxia and ischemia stimulate fibrotic and inflammatory cytokines at anastomotic sites, leading to abnormal collagen production and excessive granulation, which impair wound healing. Despite meticulous techniques, bronchial anastomosis remains a major cause of morbidity and mortality among lung transplant recipients. After LTx, most bronchial complications are attributed to ischemic insult since normal bronchial blood flow is disrupted, and bronchial revascularization usually takes two to four weeks, making the anastomotic bronchial vessels dependent on pulmonary artery circulation. It is clear that hypoxia, inflammation, oxidative stress, and extracellular matrix remodeling play critical roles in bronchial complications, but there is no small animal model to study them. In the context of LTx, mouse tracheal models are essential tools for studying bronchial complications, particularly ischemia, fibrosis, and stenosis, as well as evaluating potential therapeutic interventions. A well-established mouse model of orthotopic tracheal transplantation (OTT) mimics the anastomosis of the bronchi and the subsequent microvascular injury, providing a pathological correlation with anastomotic complications. A series of previous studies using the OTT model explored the microvascularization, ischemia-reperfusion, airway epithelial injury, and fibrotic remodeling effects after airway anastomosis. This review describes OTT as a model of airway anastomotic complications, which is crucial for understanding the immunological and molecular pathways as seen in clinical bronchial anastomoses, as well as improving anastomotic healing and reducing complications through targeted therapeutic strategies.

Neutrophils exacerbate pulmonary ischemia-reperfusion injury (IRI) resulting in poor short and long-term outcomes for lung transplant recipients. Glycolysis powers neutrophil activation, but it remains unclear if neutrophil-specific targeting of this pathway will inhibit IRI. Lipid nanoparticles containing the glycolysis flux inhibitor 2-deoxyglucose (2-DG) were conjugated to neutrophil-specific Ly6G antibodies (NP-Ly6G(2-DG)). Intravenously administered NP-Ly6G(2-DG) to mice exhibited high specificity for circulating neutrophils. NP-Ly6G(2-DG)-treated neutrophils were unable to adapt to hypoglycemic conditions of the lung airspace environment as evident by the loss of demand-induced glycolysis, reductions in glycogen and ATP content and an increased vulnerability to apoptosis. NP-Ly6G(2-DG) treatment inhibited pulmonary IRI following hilar occlusion and orthotopic lung transplantation. IRI protection was associated with less airspace neutrophil extracellular trap generation, reduced intragraft neutrophilia and enhanced alveolar macrophage efferocytotic clearance of neutrophils. Collectively, our data show that pharmacologically targeting glycolysis in neutrophils inhibits their activation and survival leading to reduced pulmonary IRI.

Over the past decade, our laboratory has made significant progress in developing and refining vascularized mouse lung transplantation models using an efficient and highly reliable "cuff technique" of transplantation. This article describes a sophisticated and comprehensive method for orthotopic lung transplantation in a vascularized orthotopic lung model, representing the most physiologic and clinically relevant model of mouse lung transplantation to date. The transplantation process consists of two distinct stages: donor harvest and subsequent implantation into the recipient. The method has been successfully mastered, and with several months of sufficient training, a skilled practitioner can perform the procedure in approximately 90 min from skin-to-skin. Surprisingly, once individuals overcome the initial learning curve, the survival rate during the perioperative period approaches nearly 100%. The mouse model allows for the use of multiple commercially available transgenic and mutant strains of mice, enabling the study of tolerance and rejection. Additionally, the unique features of this model make it a valuable tool for investigating tumor biology and immunology.

The presence of bronchus-associated lymphoid tissue (BALT) in donor lungs has been suggested to accelerate graft rejection after lung transplantation. Although chronic smoke exposure can induce BALT formation, the impact of donor cigarette use on alloimmune responses after lung transplantation is not well understood. Here, we show that smoking-induced BALT in mouse donor lungs contains Foxp3+ T cells and undergoes dynamic restructuring after transplantation, including recruitment of recipient-derived leukocytes to areas of pre-existing lymphoid follicles and replacement of graft-resident donor cells. Our findings from mouse and human lung transplant data support the notion that a donor's smoking history does not predispose to acute cellular rejection or prevent the establishment of allograft acceptance with comparable outcomes to nonsmoking donors. Thus, our work indicates that BALT in donor lungs is plastic in nature and may have important implications for modulating proinflammatory or tolerogenic immune responses following transplantation.