Research Highlights

Dysregulated Treg Repair Responses Leads to Chronic Rejection After Heart Transplantation Warunek JJP, Fan L, Zhang X, et al. J Clin Invest. 2024;134(23):e173593. doi:10.1172/JCI173593 Organ transplantation, the frontier therapy of the 20th century, has progressed from an experimental procedure in identical twins available at a handful of leading academic centers to standard-of-care treatment performed routinely in >100 countries. Despite substantial advances in immunosuppression and prophylactic antiviral medication leading to dramatic improvements in 1 y graft and patient survival, long-term results have progressed only marginally. The inexorable decline in allograft function, even with the most judiciously selected donors, is one of the most feared consequences of transplantation.1 Specific leukocyte subsets, particularly cells from the monocyte/macrophage lineage, sit at the junction of an innate immune response to ischemia/reperfusion injury and alloantigen recognition. Regulatory T cells (Treg), initially defined for suppressive function,2 also demonstrate distinct reparative action.3 Interleukin (IL)-33, a member of the IL-1 cytokine family, is recognized as a danger-associated molecular pattern ligating ST2 (also known as IL-1 receptor-like 1) as the primary receptor. IL-33 expands ST2+ Treg (through IL-2 secretion from CD11c+ dendritic cells)4 and induces the production of amphiregulin (an epidermal growth factor) and IL-13, which stimulates arginase-1+ macrophages, initiating tissue repair. Warunek et al5 (Figure 1) exploited a mouse model of heart transplantation to understand the potentially detrimental effects of Treg in cardiac repair. Initially, the team used a standard transplant model of vasculopathy (chronic rejection), transplanting IL-33+ or IL-33KO Bm12 hearts into C57BL/6 mice. Single-cell RNA sequencing from postoperative day 14 demonstrated altered recipient macrophage profiles, with IL-33 promoting a reparative phenotype. Pseudotime analysis, which represents cellular progression through differentiation or development without a chronological requirement, showed IL-33 restrained state 1 macrophages (monocytes, with proinflammatory and oxidative profiles) and favored state 4 cells (reparative and alternately activated, with upregulated complement). Grafts expressing IL-33 upregulated ST2, amphiregulin, and IL-13 in Treg. This was complemented by increased T-cell receptor transcripts.FIGURE 1.: The role of regulatory T cells in allogeneic transplantation mediated by IL-33. Created with BioRender.com. FoxP3, forkhead box P3; GvHD, graft-vs-host disease; ICOS, inducible T-cell costimulator; IFN, interferon; IL, interleukin; MAPK, mitogen-activated protein kinase.The IL-33+/+Bm12 hearts were transplanted into B6 FoxP3YFP-Cre or Foxp3YFP-CreAregfl/fl mice (which have no amphiregulin secretion in response to IL-33) to understand the role of amphiregulin. The latter combination led to a significant reduction in overall fibrosis and vasculopathy at 50 or 100 d posttransplant, with no changes in CD4+ or CD8+ T-cell activation, memory, or differentiation. Interestingly, Foxp3YFP-CreAregfl/fl mice showed an increase in myocardial CD3+ T-cell infiltrates, which eventually cleared thought due to migration to the perivascular space in FoxP3YFP-Cre controls, in conjunction with CD11b+ macrophages. Total Treg numbers were not different between the groups. Single-cell RNA sequencing on human samples confirmed high amphiregulin expression in Treg, which was only present in chronic allograft vasculopathy and in the context of substantial inflammation/rejection. Fibrosis is characterized by the elaboration of extracellular matrix, which most cells are capable of synthesizing. This phenomenon is accentuated by the differentiation of fibroblasts into myofibroblasts and epithelial-to-mesenchymal transition. FoxP3YFP-Cre or Foxp3YFP-Cre-Aregfl/fl recipient mice were assessed for α-smooth muscle actin expression, with amphiregulin expression in recipient Treg demonstrating increased α-smooth muscle actin. Indeed, Treg isolated from either mice and cocultured with fibroblasts (with IL-2 and IL-33) increased proliferation (Ki67+ cells), which was amphiregulin dependent. The authors then further analyzed the initial preclinical experiments, showing increased fibroblasts in IL-33+ grafts and gene set enrichment associated with mesenchymal cell proliferation, collagen synthesis, and vascular growth factor signaling. In final experiments, it appeared that T-cell receptor stimulation, but not CD28 costimulation, decreased amphiregulin levels, as did inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells, signal transducer and activator of transcription 3, or mammalian target of rapamycin, the latter identifying a therapeutic mechanism for the current clinical premise of mammalian target of rapamycin inhibitor use to reduce cardiac vasculopathy. The authors clearly show the potential for opposing Treg roles, with a detrimental/fibrotic phenotype related to amphiregulin production. Treg cellular therapy in solid organ transplantation may be a therapeutic option in the near future. As a persistently active cell in vivo that is required for therapeutic benefit, to date, much of the focus has been on the maintenance of immunosuppressive function; however, the potential consequences of inducing pathological repair processes need further consideration. A Proof-of-Concept Study in Small and Large Animal Models for Coupling Liver Normothermic Machine Perfusion With Mesenchymal Stromal Cell Bioreactors Cillo U, Lonati C, Bertacco A, et al. Nat Commun. 2025;16(1):283. doi:10.1038/s41467-024-55217-7 Mesenchymal stem cells (MSCs) are multipotent progenitor stromal cells that are immediate precursors of bone, cartilage, and adipose tissue and can also undergo transdifferentiation to unrelated cell lineages. Their primary function is to support hematopoietic stem cells within bone marrow niches, but MSCs also play pivotal roles in tissue homeostasis and repair and regulate the effector function of immune subsets. These versatile aspects of MSC phenotype and function represent a promising approach to modulating allograft function. However, more recent evidence suggests that their beneficial effect is mediated by the release of soluble factors (secretome) and extracellular vesicles. This indirect mechanism of effect has the added advantage of avoiding the deleterious effects of cellular therapy, including death of effector cells and loss of efficacy, microvascular thrombosis associated with endothelial activation from cellular debris, and unwarranted differentiation based on microenvironmental cues. Normothermic machine perfusion (NMP) represents a transformative advance in organ preservation, assessment, and optimization,1 with applications in the liver,2 kidney,3,4 lungs,5 and heart,6 although there are recognized limitations in its capacity to mitigate ischemia/reperfusion injury. NMP also provides a platform for additional therapeutic interventions7 that may further enhance allograft quality before implantation. Cillo et al8 combined the clinical utility of NMP and MSCs, establishing a preclinical platform for liver perfusion coupled with adipose tissue–derived MSCs seeded into a bioreactor that is intrinsic to the circuit. Technical challenges were clearly overcome—bioreactor preparation occurred in a different location from perfusion with no apparent impact on MSC viability, and system adaptation from rat to porcine livers was undertaken according to circuit volume and organ weight. The addition of the organ significantly changed the MSC secretome, lowering interleukin (IL)-6, IL-8, and indoleamine deoxygenase and increasing IL-4, IL-10, IL-1 receptor antagonist, and galectin-3 production. Furthermore, improvements in perfusate biochemical parameters (aspartate transaminase, alanine transaminase, lactate dehydrogenase), factors involved in cellular regeneration (adiponectin, hepatocyte growth factor), and proinflammatory cytokine/chemokine profile (RANTES, VEGF, IL-6), and hepatic mitochondrial function (ATP content, succinate measurements) were demonstrated. MSCs enzymatically detached from the bioreactors showed characteristic flow cytometric markers (CD73, CD90, CD105) and remained viable. These findings provide exciting progress for NMP technology, with evident feasibility for adaptation to human livers combined with bespoke (a)cellular therapy. Furthermore, in vivo studies are needed to know whether such investment translates to delayed graft function rates and prolonged allograft function.