Hedgehog costimulation during ischemia-reperfusion injury potentiates cytokine and homing responses of CD4+ T cells.

Introduction: Ischemia reperfusion injury (IRI) confers worsened outcomes and is an increasing clinical problem in solid organ transplantation. Previously, we identified a "PtchHi" T-cell subset that selectively received costimulatory signals from endothelial cell-derived Hedgehog (Hh) morphogens to mediate IRI-induced vascular inflammation. Methods: Here, we used multi-omics approaches and developed a humanized mouse model to resolve functional and migratory heterogeneity within the PtchHi population. Results: Hh-mediated costimulation induced oligoclonal and polyclonal expansion of clones within the PtchHi population, and we visualized three distinct subsets within inflamed, IRI-treated human skin xenografts exhibiting polyfunctional cytokine responses. One of these PtchHi subsets displayed features resembling recently described T peripheral helper cells, including elaboration of IFN-y and IL-21, expression of ICOS and PD-1, and upregulation of positioning molecules conferring recruitment and retention within peripheral but not lymphoid tissues. PtchHi T cells selectively homed to IRI-treated human skin xenografts to cause accelerated allograft loss, and Hh signaling was sufficient for this process to occur. Discussion: Our studies define functional heterogeneity among a PtchHi T-cell population implicated in IRI.

A ZFYVE21-Rubicon-RNF34 signaling complex promotes endosome-associated inflammasome activity in endothelial cells.

Internalization of complement membrane attack complexes (MACs) assembles NLRP3 inflammasomes in endothelial cells (EC) and promotes IL-ß-mediated tissue inflammation. Informed by proteomics analyses of FACS-sorted inflammasomes, we identify a protein complex modulating inflammasome activity on endosomes. ZFVYE21, a Rab5 effector, partners with Rubicon and RNF34, forming a "ZRR" complex that is stabilized in a Rab5- and ZFYVE21-dependent manner on early endosomes. There, Rubicon competitively disrupts inhibitory associations between caspase-1 and its pseudosubstrate, Flightless I (FliI), while RNF34 ubiquitinylates and degradatively removes FliI from the signaling endosome. The concerted actions of the ZRR complex increase pools of endosome-associated caspase-1 available for activation. The ZRR complex is assembled in human tissues, its associated signaling responses occur in three mouse models in vivo, and the ZRR complex promotes inflammation in a skin model of chronic rejection. The ZRR signaling complex reflects a potential therapeutic target for attenuating inflammasome-mediated tissue injury.

Hedgehog-induced ZFYVE21 promotes chronic vascular inflammation by activating NLRP3 inflammasomes in T cells.

The zinc finger protein ZFYVE21 is involved in immune signaling. Using humanized mouse models, primary human cells, and patient samples, we identified a T cell-autonomous role for ZFYVE21 in promoting chronic vascular inflammation associated with allograft vasculopathy. Ischemia-reperfusion injury (IRI) stimulated endothelial cells to produce Hedgehog (Hh) ligands, which in turn induced the production of ZFYVE21 in a population of T memory cells with high amounts of the Hh receptor PTCH1 (PTCHhi cells, CD3+CD4+CD45RO+PTCH1hiPD-1hi), vigorous recruitment to injured endothelia, and increased effector responses in vivo. After priming by interferon-γ (IFN-γ), Hh-induced ZFYVE21 activated NLRP3 inflammasome activity in T cells, which potentiated IFN-γ responses. Hh-induced NLRP3 inflammasomes and T cell-specific ZFYVE21 augmented the vascular sequelae of chronic inflammation in mice engrafted with human endothelial cells or coronary arteries that had been subjected to IRI before engraftment. Moreover, the population of PTCHhi T cells producing high amounts of ZFYVE21 was expanded in patients with renal transplant-associated IRI, and sera from these patients expanded this population in control T cells in a manner that depended on Hh signaling. We conclude that Hh-induced ZFYVE21 activates NLRP3 inflammasomes in T cells, thereby promoting chronic inflammation.

Membrane attack complexes, endothelial cell activation, and direct allorecognition.

Endothelial cells (ECs) form a critical immune interface regulating both the activation and trafficking of alloreactive T cells. In the setting of solid organ transplantation, donor-derived ECs represent sites where alloreactive T cells encounter major and minor tissue-derived alloantigens. During this initial encounter, ECs may formatively modulate effector responses of these T cells through expression of inflammatory mediators. Direct allorecognition is a process whereby recipient T cells recognize alloantigen in the context of donor EC-derived HLA molecules. Direct alloresponses are strongly modulated by human ECs and are galvanized by EC-derived inflammatory mediators. Complement are immune proteins that mark damaged or foreign surfaces for immune cell activation. Following labeling by natural IgM during ischemia reperfusion injury (IRI) or IgG during antibody-mediated rejection (ABMR), the complement cascade is terminally activated in the vicinity of donor-derived ECs to locally generate the solid-phase inflammatory mediator, the membrane attack complex (MAC). Via upregulation of leukocyte adhesion molecules, costimulatory molecules, and cytokine trans-presentation, MAC strengthen EC:T cell direct alloresponses and qualitatively shape the alloimmune T cell response. These processes together promote T cell-mediated inflammation during solid organ transplant rejection. In this review we describe molecular pathways downstream of IgM- and IgG-mediated MAC assembly on ECs in the setting of IRI and ABMR of tissue allografts, respectively. We describe work demonstrating that MAC deposition on ECs generates 'signaling endosomes' that sequester and post-translationally enhance the stability of inflammatory signaling molecules to promote EC activation, a process potentiating EC-mediated direct allorecognition. Additionally, with consideration to first-in-human xenotransplantation procedures, we describe clinical therapeutics based on inhibition of the complement pathway. The complement cascade critically mediates EC activation and improved understanding of relevant effector pathways will uncover druggable targets to obviate dysregulated alloimmune T cell infiltration into tissue allografts.

Coronavirus Disease 2019 (COVID-19) Coronary Vascular Thrombosis: Correlation with Neutrophil but Not Endothelial Activation.

Severe coronavirus disease 2019 (COVID-19) increases the risk of myocardial injury that contributes to mortality. This study used multiparameter immunofluorescence to extensively examine heart autopsy tissue of 7 patients who died of COVID-19 compared to 12 control specimens, with or without cardiovascular disease. Consistent with prior reports, no evidence of viral infection or lymphocytic infiltration indicative of myocarditis was found. However, frequent and extensive thrombosis was observed in large and small vessels in the hearts of the COVID-19 cohort, findings that were infrequent in controls. The endothelial lining of thrombosed vessels typically lacked evidence of cytokine-mediated endothelial activation, assessed as nuclear expression of transcription factors p65 (RelA), pSTAT1, or pSTAT3, or evidence of inflammatory activation assessed by expression of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), tissue factor, or von Willebrand factor (VWF). Intimal EC lining was also generally preserved with little evidence of cell death or desquamation. In contrast, there were frequent markers of neutrophil activation within myocardial thrombi in patients with COVID-19, including neutrophil-platelet aggregates, neutrophil-rich clusters within macrothrombi, and evidence of neutrophil extracellular trap (NET) formation. These findings point to alterations in circulating neutrophils rather than in the endothelium as contributors to the increased thrombotic diathesis in the hearts of COVID-19 patients.

Complement Membrane Attack Complex: New Roles, Mechanisms of Action, and Therapeutic Targets.

The complement membrane attack complex (MAC) is classically known as a cytolytic effector of innate and adaptive immunity that forms pores in the plasma membrane of pathogens or targeted cells, leading to osmolysis. Nucleated cells resist MAC-mediated cytolysis by expression of inhibitors that block MAC assembly or by rapid removal of MAC through endocytosis or shedding. In the absence of lysis, MAC may induce intracellular signaling and cell activation, responses implicated in a variety of autoimmune, inflammatory, and transplant disease settings. New discoveries into the structure and biophysical properties of MAC revealed heterogeneous MAC precursors and conformations that provide insights into MAC function. In addition, new mechanisms of MAC-mediated signaling and its contribution to disease pathogenesis have recently come to light. MAC-activated cells have been found to express proinflammatory proteins-often through NF-κB-dependent transcription, assemble inflammasomes, enabling processing, and facilitate secretion of IL-1ß and IL-18, as well as other signaling pathways. These recent insights into the mechanisms of action of MAC provide an updated framework to therapeutic approaches that can target MAC assembly, signaling, and proinflammatory effects in various complement-mediated diseases.

Complement-activated interferon-γ-primed human endothelium transpresents interleukin-15 to CD8+ T cells.

Alloantibodies in presensitized transplant candidates deposit complement membrane attack complexes (MACs) on graft endothelial cells (ECs), increasing risk of CD8+ T cell-mediated acute rejection. We recently showed that human ECs endocytose MACs into Rab5+ endosomes, creating a signaling platform that stabilizes NF-κB-inducing kinase (NIK) protein. Endosomal NIK activates both noncanonical NF-κB signaling to synthesize pro-IL-1ß and an NLRP3 inflammasome to process and secrete active IL-1ß. IL-1ß activates ECs, increasing recruitment and activation of alloreactive effector memory CD4+ T (Tem) cells. Here, we report that IFN-γ priming induced nuclear expression of IL-15/IL-15Rα complexes in cultured human ECs and that MAC-induced IL-1ß stimulated translocation of IL-15/IL-15Rα complexes to the EC surface in a canonical NF-κB-dependent process in which IL-15/IL-15Rα transpresentation increased activation and maturation of alloreactive CD8+ Tem cells. Blocking NLRP3 inflammasome assembly, IL-1 receptor, or IL-15 on ECs inhibited the augmented CD8+ Tem cell responses, indicating that this pathway is not redundant. Adoptively transferred alloantibody and mouse complement deposition induced IL-15/IL-15Rα expression by human ECs lining human coronary artery grafts in immunodeficient mice, and enhanced intimal CD8+ T cell infiltration, which was markedly reduced by inflammasome inhibition, linking alloantibody to acute rejection. Inhibiting MAC signaling may similarly limit other complement-mediated pathologies.

Endothelial Cell-Derived Interleukin-18 Released During Ischemia Reperfusion Injury Selectively Expands T Peripheral Helper Cells to Promote Alloantibody Production.

BACKGROUND: Ischemia reperfusion injury (IRI) predisposes to the formation of donor-specific antibodies, a factor contributing to chronic rejection and late allograft loss. METHODS: We describe a mechanism underlying the correlative association between IRI and donor-specific antibodies by using humanized models and patient specimens. RESULTS: IRI induces immunoglobulin M-dependent complement activation on endothelial cells that assembles an NLRP3 (NOD-like receptor pyrin domain-containing protein 3) inflammasome via a Rab5-ZFYVE21-NIK axis and upregulates ICOS-L (inducible costimulator ligand) and PD-L2 (programmed death ligand 2). Endothelial cell-derived interleukin-18 (IL-18) selectively expands a T-cell population (CD4+CD45RO+PD-1hiICOS+CCR2+CXCR5-) displaying features of recently described T peripheral helper cells. This population highly expressed IL-18R1 and promoted donor-specific antibodies in response to IL-18 in vivo. In patients with delayed graft function, a clinical manifestation of IRI, these cells were Ki-67+IL-18R1+ and could be expanded ex vivo in response to IL-18. CONCLUSIONS: IRI promotes elaboration of IL-18 from endothelial cells to selectively expand alloreactive IL-18R1+ T peripheral helper cells in allograft tissues to promote donor-specific antibody formation.

Complement Membrane Attack Complexes Assemble NLRP3 Inflammasomes Triggering IL-1 Activation of IFN-γ-Primed Human Endothelium.

RATIONALE: Complement activation contributes to multiple immune-mediated pathologies. In late allograft failure, donor-specific antibody deposits complement membrane attack complexes (MAC) on graft endothelial cells (ECs), substantially increasing their immunogenicity without causing lysis. Internalized MAC stabilize NIK (NF-κB [nuclear factor kappa-light-chain-enhancer of activated B cells]-inducing kinase) protein on Rab5+MAC+ endosomes, activating noncanonical NF-κB signaling. However, the link to increased immunogenicity is unclear. OBJECTIVE: To identify mechanisms by which alloantibody and internalized MAC activate ECs to enhance their ability to increase T-cell responses. METHODS AND RESULTS: In human EC cultures, internalized MAC also causes NLRP3 (NOD-like receptor family pyrin domain containing 3) translocation from endoplasmic reticulum to Rab5+MAC+NIK+ endosomes followed by endosomal NIK-dependent inflammasome assembly. Cytosolic NIK, stabilized by LIGHT (lymphotoxin-like inducible protein that competes with glycoprotein D for herpesvirus entry on T cells), does not trigger inflammasome assembly, and ATP-triggered inflammasome assembly does not require NIK. IFN-γ (interferon-γ) primes EC responsiveness to MAC by increasing NLRP3, pro-caspase 1, and gasdermin D expression. NIK-activated noncanonical NF-κB signaling induces pro-IL (interleukin)-1ß expression. Inflammasome processed pro-IL-1ß, and gasdermin D results in IL-1ß secretion that increases EC immunogenicity through IL-1 receptor signaling. Activation of human ECs lining human coronary artery grafts in immunodeficient mouse hosts by alloantibody and complement similarly depends on assembly of an NLRP3 inflammasome. Finally, in renal allograft biopsies showing chronic rejection, caspase-1 is activated in C4d+ ECs of interstitial microvessels, supporting the relevance of the cell culture findings. CONCLUSIONS: In response to antibody-mediated complement activation, IFN-γ-primed human ECs internalize MAC, triggering both endosomal-associated NIK-dependent NLRP3 inflammasome assembly and IL-1 synthesis, resulting in autocrine/paracrine IL-1ß-mediated increases in EC immunogenicity. Similar responses may underlie other complement-mediated pathologies.

ZFYVE21 is a complement-induced Rab5 effector that activates non-canonical NF-κB via phosphoinosotide remodeling of endosomes.

Complement promotes vascular inflammation in transplant organ rejection and connective tissue diseases. Here we identify ZFYVE21 as a complement-induced Rab5 effector that induces non-canonical NF-κB in endothelial cells (EC). In response to membrane attack complexes (MAC), ZFYVE21 is post-translationally stabilized on MAC+Rab5+ endosomes in a Rab5- and PI(3)P-dependent manner. ZFYVE21 promotes SMURF2-mediated polyubiquitinylation and proteasome-dependent degradation of endosome-associated PTEN to induce vesicular enrichment of PI(3,4,5)P3 and sequential recruitment of activated Akt and NF-κB-inducing kinase (NIK). Pharmacologic alteration of cellular phosphoinositide content with miltefosine reduces ZFYVE21 induction, EC activation, and allograft vasculopathy in a humanized mouse model. ZFYVE21 induction distinctly occurs in response to MAC and is detected in human renal and synovial tissues. Our data identifies ZFYVE21 as a Rab5 effector, defines a Rab5-ZFYVE21-SMURF2-pAkt axis by which it mediates EC activation, and demonstrates a role for this pathway in complement-mediated conditions.

Interferon-γ converts human microvascular pericytes into negative regulators of alloimmunity through induction of indoleamine 2,3-dioxygenase 1.

Early acute rejection of human allografts is mediated by circulating alloreactive host effector memory T cells (TEM). TEM infiltration typically occurs across graft postcapillary venules and involves sequential interactions with graft-derived endothelial cells (ECs) and pericytes (PCs). While the role of ECs in allograft rejection has been extensively studied, contributions of PCs to this process are largely unknown. This study aimed to characterize the effects and mechanisms of interactions between human PCs and allogeneic TEM. We report that unstimulated PCs, like ECs, can directly present alloantigen to TEM, but while IFN-γ-activated ECs (γ-ECs) show increased ability to stimulate alloreactive T cells, IFN-γ-activated PCs (γ-PCs) instead suppress TEM proliferation but not cytokine production or signaling. RNA sequencing analysis of PCs, γ-PCs, ECs, and γ-ECs reveal induction of indoleamine 2,3-dioxygenase 1 (IDO1) in γ-PCs to significantly higher levels than in γ-ECs that correlates with tryptophan depletion in vitro. Consistently, shRNA knockdown of IDO1 markedly reduces γ-PC-mediated immunoregulatory effects. Furthermore, human PCs express IDO1 in a skin allograft rejection humanized mouse model and in human renal allografts with acute T cell-mediated rejection. We conclude that immunosuppressive properties of human PCs are not intrinsic but instead result from IFN-γ-induced IDO1-mediated tryptophan depletion.

IL-17 Promotes Neutrophil-Mediated Immunity by Activating Microvascular Pericytes and Not Endothelium.

A classical hallmark of acute inflammation is neutrophil infiltration of tissues, a multistep process that involves sequential cell-cell interactions of circulating leukocytes with IL-1- or TNF-activated microvascular endothelial cells (ECs) and pericytes (PCs) that form the wall of the postcapillary venules. The initial infiltrating cells accumulate perivascularly in close proximity to PCs. IL-17, a proinflammatory cytokine that acts on target cells via a heterodimeric receptor formed by IL-17RA and IL-17RC subunits, also promotes neutrophilic inflammation but its effects on vascular cells are less clear. We report that both cultured human ECs and PCs strongly express IL-17RC and, although neither cell type expresses much IL-17RA, PCs express significantly more than ECs. IL-17, alone or synergistically with TNF, significantly alters inflammatory gene expression in cultured human PCs but not ECs. RNA sequencing analysis identifies many IL-17-induced transcripts in PCs encoding proteins known to stimulate neutrophil-mediated immunity. Conditioned media from IL-17-activated PCs, but not ECs, induce pertussis toxin-sensitive neutrophil polarization, likely mediated by PC-secreted chemokines, and they also stimulate neutrophil production of proinflammatory molecules, including TNF, IL-1α, IL-1ß, and IL-8. Furthermore, IL-17-activated PCs, but not ECs, can prolong neutrophil survival by producing G-CSF and GM-CSF, delaying the mitochondrial outer membrane permeabilization and caspase-9 activation. Importantly, neutrophils exhibit enhanced phagocytic capacity after activation by conditioned media from IL-17-treated PCs. We conclude that PCs, not ECs, are the major target of IL-17 within the microvessel wall and that IL-17-activated PCs can modulate neutrophil functions within the perivascular tissue space.

Innate immune mechanisms in transplant allograft vasculopathy.

PURPOSE OF REVIEW: Allograft vasculopathy is the leading cause of late allograft loss following solid organ transplantation. Ischemia reperfusion injury and donor-specific antibody-induced complement activation confer heightened risk for allograft vasculopathy via numerous innate immune mechanisms, including MyD88, high-mobility group box 1 (HMGB1), and complement-induced noncanonical nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling. RECENT FINDINGS: The role of MyD88, a signal adaptor downstream of the Toll-like receptors (TLR), has been defined in an experimental heart transplant model, which demonstrated that recipient MyD88 enhanced allograft vasculopathy. Importantly, triggering receptor on myeloid receptor 1, a MyD88 amplifying signal, was present in rejecting human cardiac transplant biopsies and enhanced the development of allograft vasculopathy in mice. HMGB1, a nuclear protein that activates Toll-like receptors, also enhanced the development of allograft vasculopathy. Complement activation elicits assembly of membrane attack complexes on endothelial cells which activate noncanonical NF-κB signaling, a novel complement effector pathway that induces proinflammatory genes and potentiates endothelial cell-mediated alloimmune T-cell activation, processes which enhance allograft vasculopathy. SUMMARY: Innate immune mediators, including HMGB1, MyD88, and noncanonical NF-κB signaling via complement activation contribute to allograft vasculopathy. These pathways represent potential therapeutic targets to reduce allograft vasculopathy after solid organ transplantation.

Complement membrane attack complexes activate noncanonical NF-κB by forming an Akt+ NIK+ signalosome on Rab5+ endosomes.

Complement membrane attack complexes (MACs) promote inflammatory functions in endothelial cells (ECs) by stabilizing NF-κB-inducing kinase (NIK) and activating noncanonical NF-κB signaling. Here we report a novel endosome-based signaling complex induced by MACs to stabilize NIK. We found that, in contrast to cytokine-mediated activation, NIK stabilization by MACs did not involve cIAP2 or TRAF3. Informed by a genome-wide siRNA screen, instead this response required internalization of MACs in a clathrin-, AP2-, and dynamin-dependent manner into Rab5(+)endosomes, which recruited activated Akt, stabilized NIK, and led to phosphorylation of IκB kinase (IKK)-α. Active Rab5 was required for recruitment of activated Akt to MAC(+) endosomes, but not for MAC internalization or for Akt activation. Consistent with these in vitro observations, MAC internalization occurred in human coronary ECs in vivo and was similarly required for NIK stabilization and EC activation. We conclude that MACs activate noncanonical NF-κB by forming a novel Akt(+)NIK(+) signalosome on Rab5(+) endosomes.

Interacting mechanisms in the pathogenesis of cardiac allograft vasculopathy.

Cardiac allograft vasculopathy is the major cause of late graft loss in heart transplant recipients. Histological studies of characteristic end-stage lesions reveal arterial changes consisting of a diffuse, confluent, and concentric intimal expansion containing graft-derived cells expressing smooth muscle markers, extracellular matrix, penetrating microvessels, and a host mononuclear cell infiltrate concentrated subjacent to an intact graft-derived luminal endothelial cell lining with little evidence of acute injury. This intimal expansion combined with inadequate compensatory outward remodeling produces severe generalized stenosis extending throughout the epicardial and intramyocardial arterial tree that causes ischemic graft failure. Cardiac allograft vasculopathy lesions affect ≥50% of transplant recipients and are both progressive and refractory to treatment, resulting in ≈5% graft loss per year through the first 10 years after transplant. Lesions typically stop at the suture line, implicating alloimmunity as the primary driver, but pathogenesis may be multifactorial. Here, we will discuss 6 potential contributors to lesion formation (1) conventional risk factors of atherosclerosis; (2) pre- or peritransplant injuries; (3) infection; (4) innate immunity; (5) T-cell-mediated immunity; and (6) B-cell-mediated immunity through production of donor-specific antibody. Finally, we will consider how these various mechanisms may interact with each other.

Sustained delivery of proangiogenic microRNA-132 by nanoparticle transfection improves endothelial cell transplantation.

Transplantation of endothelial cells (ECs) for therapeutic vascularization or tissue engineering is a promising method for increasing tissue perfusion. Here, we report on a new approach for enhanced EC transplantation using targeted nanoparticle transfection to deliver proangiogenic microRNA-132 (miR-132) to cultured ECs before their transplantation, thereby sensitizing cells to the effects of endogenous growth factors. We synthesized biodegradable PLGA polymer nanoparticles (NPs) that were loaded with miR-132 and coated with cyclic RGD (cRGD) peptides that target integrin αvß3 expressed on cultured human umbilical vein ECs (HUVECs), increasing NP uptake through clathrin-coated pits. Unlike previously reported NPs for miR delivery, these NPs slowly release RNA for several weeks. The endocytosed NPs remain in clathrin-coated vesicles from which they mediate intracellular delivery of siRNA or miRNA. Transfection of HUVECs with miR-132 enhances growth factor-induced proliferation and migration in 2D culture, producing a 1.8- and 5-fold increase, respectively. However, while the effects of conventional transfection were short-lived, NP transfection produced protein knockdown and biological effects that were significantly longer in duration (≥ 6 d). Transfection of HUVECs with miR-132 NP resulted in a 2-fold increase in the number of microvessels per square millimeter compared to lipid after transplantation into immunodeficient mice and led to a higher number of mural cell-invested vessels than control transfection. These data suggest that sustained delivery of miR-132 encapsulated in a targeted biodegradable polymer NP is a safe and efficient strategy to improve EC transplantation and vascularization.

Alloantibody and complement promote T cell-mediated cardiac allograft vasculopathy through noncanonical nuclear factor-κB signaling in endothelial cells.

BACKGROUND: Cardiac allograft vasculopathy is the major cause of late allograft loss after heart transplantation. Cardiac allograft vasculopathy lesions contain alloreactive T cells that secrete interferon-γ, a vasculopathic cytokine, and occur more frequently in patients with donor-specific antibody. Pathological interactions between these immune effectors, representing cellular and humoral immunity, respectively, remain largely unexplored. METHODS AND RESULTS: We used human panel reactive antibody to form membrane attack complexes on allogeneic endothelial cells in vitro and in vivo. Rather than inducing cytolysis, membrane attack complexes upregulated inflammatory genes, enhancing the capacity of endothelial cells to recruit and activate allogeneic interferon-γ--producing CD4(+) T cells in a manner dependent on the activation of noncanonical nuclear factor-κB signaling. Noncanonical nuclear factor-κB signaling was detected in situ within endothelial cells both in renal biopsies from transplantation patients with chronic antibody-mediated rejection and in panel-reactive antibody--treated human coronary artery xenografts in immunodeficient mice. On retransplantation into immunodeficient hosts engrafted with human T cells, panel-reactive antibody--treated grafts recruited more interferon-γ--producing T cells and enhanced cardiac allograft vasculopathy lesion formation. CONCLUSIONS: Alloantibody and complement deposition on graft endothelial cells activates noncanonical nuclear factor-κB signaling, initiating a proinflammatory gene program that enhances alloreactive T cell activation and development of cardiac allograft vasculopathy. Noncanonical nuclear factor-κB signaling in endothelial cells, observed in human allograft specimens and implicated in lesion pathogenesis, may represent a target for new pharmacotherapies to halt the progression of cardiac allograft vasculopathy.