Transendothelial Migration of Human B Cells: Chemokine versus Antigen.

B cells, like T cells, can infiltrate sites of inflammation, but the processes and B cell subsets involved are poorly understood. Using human cells and in vitro assays, we find only a very small number of B cells will adhere to TNF-activated (but not to resting) human microvascular endothelial cells (ECs) under conditions of venular flow and do so by binding to ICAM-1 and VCAM-1. CXCL13 and, to a lesser extent, CXCL10 bound to the ECs can increase adhesion and induce transendothelial migration (TEM) of adherent naive and memory B cells in 10-15 min through a process involving cell spreading, translocation of the microtubule organizing center (MTOC) into a trailing uropod, and interacting with EC activated leukocyte cell adhesion molecule. Engagement of the BCR by EC-bound anti-κ L chain Ab also increases adhesion and TEM of κ+ but not λ+ B cells. BCR-induced TEM takes 30-60 min, requires Syk activation, is initiated by B cell rounding up and translocation of the microtubule organizing center to the region of the B cell adjacent to the EC, and also uses EC activated leukocyte cell adhesion molecule for TEM. BCR engagement reduces the number of B cells responding to chemokines and preferentially stimulates TEM of CD27+ B cells that coexpress IgD, with or without IgM, as well as CD43. RNA-sequencing analysis suggests that peripheral blood CD19+CD27+CD43+IgD+ cells have increased expression of genes that support BCR activation as well as innate immune properties in comparison with total peripheral blood CD19+ cells.

Divergent TCR-Initiated Calcium Signals Govern Recruitment versus Activation of Human Alloreactive Effector Memory T Cells by Endothelial Cells.

Early human allograft rejection can be initiated when circulating human host versus graft Ag-specific CD8 and CD4 effector memory T cells directly recognize MHC class I and II, respectively, expressed on the luminal surface by endothelium lining graft blood vessels. TCR engagement triggers both graft entry (TCR-driven transendothelial migration or TEM) and production of proinflammatory cytokines. Both TCR-driven TEM and cytokine expression are known to depend on T cell enzymes, myosin L chain kinase, and calcineurin, respectively, that are activated by cytoplasmic calcium and calmodulin, but whether the sources of calcium that control these enzymes are the same or different is unknown. Using superantigen or anti-CD3 Ab presented by cultured human dermal microvascular cells to freshly isolated peripheral blood human effector memory T cells under conditions of flow (models of alloantigen recognition in a vascularized graft), we tested the effects of pharmacological inhibitors of TCR-activated calcium signaling pathways on TCR-driven TEM and cytokine expression. We report that extracellular calcium entry via CRAC channels is the dominant contributor to cytokine expression, but paradoxically these same inhibitors potentiate TEM. Instead, calcium entry via TRPV1, L-Type Cav, and pannexin-1/P2X receptors appear to control TCR-driven TEM. These data reveal new therapeutic targets for immunosuppression.

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.

Significant Differences in Antigen-Induced Transendothelial Migration of Human CD8 and CD4 T Effector Memory Cells.

OBJECTIVE: Circulating human T effector memory cell (TEM) recognition of nonself MHC (major histocompatibility complex) molecules on allograft endothelial cells can initiate graft rejection despite elimination of professional antigen-presenting cells necessary for naive T-cell activation. Our previous studies of CD4 TEM have established that engagement of the T-cell receptor not only activates T cells but also triggers transendothelial migration (TEM) by a process that is distinct from that induced by activating chemokine receptors on T cells, being slower, requiring microtubule-organizing center-directed cytolytic granule polarization to and release from the leading edge of the T cell, and requiring engagement of proteins of the endothelial cell lateral border recycling compartment. Although CD4 TEM may contribute to acute allograft rejection, the primary effectors are alloreactive CD8 TEM. Whether and how T-cell receptor engagement affects TEM of human CD8 TEM is unknown. APPROACH AND RESULTS: We modeled TEM of CD8 TEM across cultured human microvascular endothelial cells engineered to present superantigen under conditions of venular shear stress in vitro in a flow chamber. Here, we report that T-cell receptor engagement can also induce TEM of this population that similarly differs from chemokine receptor-driven TEM with regard to kinetics, morphological manifestations, and microtubule-organizing center dynamics as with CD4 TEM. However, CD8 TEM do not require either cytolytic granule release or interactions with proteins of the lateral border recycling compartment. CONCLUSIONS: These results imply that therapeutic strategies designed to inhibit T-cell receptor-driven recruitment based on targeting granule release or components of the lateral border recycling compartment will not affect CD8 TEM and are unlikely to block acute rejection in the clinic.

Polarized granzyme release is required for antigen-driven transendothelial migration of human effector memory CD4 T cells.

Human effector memory CD4 T cells may transmigrate across endothelial cell (EC) monolayers either in response to inflammatory chemokines or in response to TCR recognition of Ag presented on the surface of the EC. The kinetics, morphologic manifestations, and molecular requirements of chemokine- and TCR-driven transendothelial migration (TEM) differ significantly. In this study, we report that, whereas the microtubule organizing center (MTOC) and cytosolic granules follow the nucleus across the endothelium in a uropod during chemokine-driven TEM, MTOC reorientation to the contact region between the T cell and the EC, accompanied by dynein-driven transport of granzyme-containing granules to and exocytosis at the contact region, are early events in TCR-driven, but not chemokine-driven TEM. Inhibitors of either granule function or granzyme proteolytic activity can arrest TCR-driven TEM, implying a requirement for granule discharge in the process. In the final stages of TCR-driven TEM, the MTOC precedes, rather than follows, the nucleus across the endothelium. Thus, TCR-driven TEM of effector memory CD4 T cells appears to be a novel process that more closely resembles immune synapse formation than it does conventional chemotaxis.

αvß6 integrin is required for TGFß1-mediated matrix metalloproteinase2 expression.

Transforming growth factor (TGF) ß1 activity depends on a complex signalling cascade that controls expression of several genes. Among others, TGFß1 regulates expression of matrix metalloproteinases (MMPs) through activation of Smads. In the present study, we demonstrate for the first time that the αvß6 integrin interacts with TGFß receptor II (TßRII) through the ß6 cytoplasmic domain and promotes Smad3 activation in prostate cancer (PrCa) cells. Another related αv integrin, αvß5, as well as the αvß6/3 integrin, which contains a chimeric form of ß6 with a ß3 cytoplasmic domain, do not associate with TßRII and fail to show similar responses. We provide evidence that αvß6 is required for up-regulation of MMP2 by TGFß1 through a Smad3-mediated transcriptional programme in PrCa cells. The functional relevance of these results is underscored by the finding that αvß6 modulates cell migration in an MMP2-dependent manner on an αvß6-specific ligand, latency-associated peptide (LAP)-TGFß. Overall, these mechanistic studies establish that expression of a single integrin, αvß6, is sufficient to promote activation of Smad3, regulation of MMP2 levels and consequent catalytic activity, as well as cell migration. Our study describes a new TGFß1-αvß6-MMP2 signalling pathway that, given TGFß1 pro-metastatic activity, may have profound implications for PrCa therapy.

TCR-driven transendothelial migration of human effector memory CD4 T cells involves Vav, Rac, and myosin IIA.

Human effector memory (EM) CD4 T cells may be recruited from the blood into a site of inflammation in response either to inflammatory chemokines displayed on or specific Ag presented by venular endothelial cells (ECs), designated as chemokine-driven or TCR-driven transendothelial migration (TEM), respectively. We have previously described differences in the morphological appearance of transmigrating T cells as well as in the molecules that mediate T cell-EC interactions distinguishing these two pathways. In this study, we report that TCR-driven TEM requires ZAP-70-dependent activation of a pathway involving Vav, Rac, and myosin IIA. Chemokine-driven TEM also uses ZAP-70, albeit in a quantitatively and spatially different manner of activation, and is independent of Vav, Rac, and mysosin IIA, depending instead on an as-yet unidentified GTP exchange factor that activates Cdc42. The differential use of small Rho family GTPases to activate the cytoskeleton is consistent with the morphological differences observed in T cells that undergo TEM in response to these distinct recruitment signals.

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.

Endothelial reticulon-4B (Nogo-B) regulates ICAM-1-mediated leukocyte transmigration and acute inflammation.

The reticulon (Rtn) family of proteins are localized primarily to the endoplasmic reticulum (ER) of most cells. The Rtn-4 family, (aka Nogo) consists of 3 splice variants of a common gene called Rtn-4A, Rtn-4B, and Rtn-4C. Recently, we identified the Rtn-4B (Nogo-B) protein in endothelial and smooth muscle cells of the vessel wall, and showed that Nogo-B is a regulator of cell migration in vitro and vascular remodeling and angiogenesis in vivo. However, the role of Nogo-B in inflammation is still largely unknown. In the present study, we use 2 models of inflammation to show that endothelial Nogo-B regulates leukocyte transmigration and intercellular adhesion molecule-1 (ICAM-1)-dependent signaling. Mice lacking Nogo-A/B have a marked reduction in neutrophil and monocyte recruitment to sites of inflammation, while Nogo-A/B(-/-) mice engrafted with wild-type (WT) bone marrow still exhibit impaired inflammation compared with WT mice engrafted with Nogo-A/B(-/-) bone marrow, arguing for a critical role of host Nogo in this response. Using human leukocytes and endothelial cells, we show mechanistically that the silencing of Nogo-B with small interfering RNA (siRNA) impairs the transmigration of neutrophils and reduces ICAM-1-stimulated phosphorylation of vascular endothelial-cell cadherin (VE-cadherin). Our results reveal a novel role of endothelial Nogo-B in basic immune functions and provide a key link in the molecular network governing endothelial-cell regulation of diapedesis.

Identification of endothelial cell junctional proteins and lymphocyte receptors involved in transendothelial migration of human effector memory CD4+ T cells.

Human effector memory (EM) CD4(+) T cells can rapidly transmigrate across an endothelial cell (EC) monolayer in response either to chemokine or to TCR-activating signals displayed by human dermal microvascular EC under conditions of venular shear stress. We previously reported that the TCR-stimulated transendothelial migration (TEM) depends on fractalkine (CX3CL1), PECAM-1 (CD31), and ICAM-1 (CD54) expression by the EC, whereas chemokine-stimulated TEM does not. In this study, we further analyze these responses using blocking mAb and small interfering RNA knockdown to show that TCR-stimulated TEM depends on CD99 on EC as well as on PECAM-1 and depends on nectin-2 (CD112) and poliovirus receptor (CD155) as well as EC ICAM-1. ICAM-1 is engaged by EM CD4(+) T cell LFA-1 (CD11a/CD18) but not Mac-1 (CD11b/CD18); nectin-2 and poliovirus receptor are engaged by both DNAX accessory molecule-1 (CD226) and Tactile (CD96). EC junctional adhesion molecule-1 (JAM-1), an alternative ligand for LFA-1, contributes exclusively to chemokine-stimulated TEM and ICAM-2 appears to be uninvolved in either pathway. These data further define and further highlight the differences in the two pathways of EM CD4(+) T cell recruitment into sites of peripheral inflammation.

Multiscale genetic architecture of donor-recipient differences reveals intronic LIMS1 mismatches associated with kidney transplant survival.

Donor-recipient (D-R) mismatches outside of human leukocyte antigens (HLAs) contribute to kidney allograft loss, but the mechanisms remain unclear, specifically for intronic mismatches. We quantified non-HLA mismatches at variant-, gene-, and genome-wide scales from single nucleotide polymorphism (SNP) data of D-Rs from 2 well-phenotyped transplant cohorts: Genomics of Chronic Allograft Rejection (GoCAR; n = 385) and Clinical Trials in Organ Transplantation-01/17 (CTOT-01/17; n = 146). Unbiased gene-level screening in GoCAR uncovered the LIMS1 locus as the top-ranked gene where D-R mismatches associated with death-censored graft loss (DCGL). A previously unreported, intronic, LIMS1 haplotype of 30 SNPs independently associated with DCGL in both cohorts. Haplotype mismatches showed a dosage effect, and minor-allele introduction to major-allele-carrying recipients showed greater hazard of DCGL. The LIMS1 haplotype and the previously reported LIMS1 SNP rs893403 are expression quantitative trait loci (eQTL) in immune cells for GCC2 (not LIMS1), which encodes a protein involved in mannose-6-phosphase receptor (M6PR) recycling. Peripheral blood and T cell transcriptome analyses associated the GCC2 gene and LIMS1 SNPs with the TGF-ß1/SMAD pathway, suggesting a regulatory effect. In vitro GCC2 modulation impacted M6PR-dependent regulation of active TGF-ß1 and downstream signaling in T cells. Together, our data link LIMS1 locus D-R mismatches to DCGL via GCC2 eQTLs that modulate TGF-ß1-dependent effects on T cells.

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.

Peroxisome proliferator-activated receptor-γ agonists prevent in vivo remodeling of human artery induced by alloreactive T cells.

BACKGROUND: Ligands activating the transcription factor peroxisome proliferator-activated receptor-γ (PPARγ) have antiinflammatory effects. Vascular rejection induced by allogeneic T cells can be responsible for acute and chronic graft loss. Studies in rodents suggest that PPARγ agonists may inhibit graft vascular rejection, but human T-cell responses to allogeneic vascular cells differ from those in rodents, and the effects of PPARγ in human transplantation are unknown. METHODS AND RESULTS: We tested the effects of PPARγ agonists on human vascular graft rejection using a model in which human artery is interposed into the abdominal aorta of immunodeficient mice, followed by adoptive transfer of allogeneic (to the artery donor) human peripheral blood mononuclear cells. Interferon-γ-dependent rejection ensues within 4 weeks, characterized by intimal thickening, T-cell infiltrates, and vascular cell activation, a response resembling clinical intimal arteritis. The PPARγ agonists 15-deoxy-prostaglandin-J(2), ciglitazone, and pioglitazone reduced intimal expansion, intimal infiltration of CD45RO(+) memory T cells, and plasma levels of inflammatory cytokines. The PPARγ antagonist GW9662 reversed the protective effects of PPARγ agonists, confirming the involvement of PPARγ-mediated pathways. In vitro, pioglitazone inhibited both alloantigen-induced proliferation and superantigen-induced transendothelial migration of memory T cells, indicating the potential mechanisms of PPARγ effects. CONCLUSION: Our results suggest that PPARγ agonists inhibit allogeneic human memory T cell responses and may be useful for the treatment of vascular graft rejection.

VEGF blockade inhibits lymphocyte recruitment and ameliorates immune-mediated vascular remodeling.

RATIONALE: There are conflicting data on the effects of vascular endothelial growth factor (VEGF) in vascular remodeling. Furthermore, there are species-specific differences in leukocyte and vascular cell biology and little is known about the role of VEGF in remodeling of human arteries. OBJECTIVE: We sought to address the role of VEGF blockade on remodeling of human arteries in vivo. METHODS AND RESULTS: We used an anti-VEGF antibody, bevacizumab, to study the effect of VEGF blockade on remodeling of human coronary artery transplants in severe combined immunodeficient mice. Bevacizumab ameliorated peripheral blood mononuclear cell-induced but not interferon-gamma-induced neointimal formation. This inhibitory effect was associated with a reduction in graft T-cell accumulation without affecting T-cell activation. VEGF enhanced T-cell capture by activated endothelium under flow conditions. The VEGF effect could be recapitulated when a combination of recombinant intercellular adhesion molecule 1 and vascular cell adhesion molecule-1 rather than endothelial cells was used to capture T cells. A subpopulation of CD3+ T cells expressed VEGF receptor (VEGFR)-1 by immunostaining and FACS analysis. VEGFR-1 mRNA was also detectable in purified CD4+ T cells and Jurkat and HSB-2 T-cell lines. Stimulation of HSB-2 and T cells with VEGF triggered downstream ERK phosphorylation, demonstrating the functionality of VEGFR-1 in human T cells. CONCLUSIONS: VEGF contributes to vascular remodeling in human arteries through a direct effect on human T cells that enhances their recruitment to the vessel. These findings raise the possibility of novel therapeutic approaches to vascular remodeling based on inhibition of VEGF signaling.

Cutting edge: TNF-induced microRNAs regulate TNF-induced expression of E-selectin and intercellular adhesion molecule-1 on human endothelial cells: feedback control of inflammation.

MicroRNAs (miRNAs) pair with target sequences in the 3' untranslated region of mRNAs to posttranscriptionally repress gene expression. In this study, we report that TNF-mediated induction of endothelial adhesion molecules can be regulated by miRNAs that are induced by TNF. Specifically, E-selectin and ICAM-1 are targets of TNF-induced miRNAs miR-31 and miR-17-3p, respectively. Specific antagonism of these TNF-induced miRNAs increased neutrophil adhesion to cultured endothelial cells. Conversely, transfections with mimics of these miRNAs decreased neutrophil adhesion to endothelial cells. These data suggest that miRNAs provide negative feedback control of inflammation.

Kaposi's sarcoma-associated herpesvirus K3 and K5 proteins block distinct steps in transendothelial migration of effector memory CD4+ T cells by targeting different endothelial proteins.

ORFK3 (K3) and ORFK5 (K5) are Kaposi's sarcoma-associated herpesvirus-encoded E3 ubiquitin ligases that differentially reduce surface expression of various proteins in infected cells. In this study, we describe their effects on human dermal microvascular endothelial cells (ECs), a natural target of Kaposi's sarcoma-associated herpesvirus infection. TNF-treated human dermal microvascular ECs transduced to express K5 show reduced capacity to capture effector memory (EM) CD4+ T cells under conditions of venular shear stress. K5 but not K3 transduction significantly reduces ICAM-1 expression and the inhibition of T cell capture was phenocopied by small interfering RNA knockdown of ICAM-1 and by anti-ICAM-1 Ab blocking. Cotransduction with an ICAM-1 truncated construct not subject to K5 ubiquitylation restored EM CD4+ T cell capture. K3 transductants effectively capture EM CD4+ T cells, but fail to support their transendothelial migration (TEM) in response to TCR engagement by superantigen presented by the ECs, leaving intact chemokine-dependent TEM. K3 but not K5 transduction significantly reduces PECAM-1 expression, and the effect on TCR-induced TEM is phenocopied by small interfering RNA knockdown of PECAM-1 and by anti-PECAM-1 Ab blocking. TCR-dependent TEM was restored in K3 transductants cotransduced to express a mutant of PECAM-1 not subject to K3-induced ubiquitylation. EM CD4+ T cells lack any known PECAM-1 counter receptor, but heterophilic engagement of PECAM-1 can involve glycosaminoglycans. In addition, TCR-induced TEM, but not chemokine-induced TEM, appears to involve a heparan- or chondroitin-like molecule on T cells. These results both identify specific roles of K5 and K3 in immune evasion and further differentiate the processes of inflammatory chemokine- versus TCR-dependent recruitment of human EM CD4+ T cells.

Costimulators expressed on human endothelial cells modulate antigen-dependent recruitment of circulating T lymphocytes.

Endothelial cells (ECs) can present antigens to circulating effector memory T cells (TEM) and to regulatory T cells (T regs), triggering antigen-specific extravasation at specific sites where foreign antigens are introduced, e.g. by infection or transplantation. We model human antigen-induced transendothelial migration (TEM) using presentation of superantigen by cultured human dermal microvascular (HDM)ECs to isolated resting human peripheral blood T cell subpopulations or to T effector cells activated in vitro. T cell receptor (TCR)-mediated cytokine synthesis, a common assay of T cell activation by antigen, is modulated by antigen-independent signals provided by various positive or negative costimulator proteins (the latter known as checkpoint inhibitors) expressed by antigen presenting cells, including ECs. We report here that some EC-expressed costimulators also modulate TCR-TEM, but effects differ between TEM and cytokine production and among some T cell types. Blocking EC LFA-3 interactions with TEM CD2 boosts TEM but reduces cytokine production. Blocking EC ICOS-L interactions with TEM CD28 (but not ICOS) reduces both responses but these involve distinct CD28-induced signals. Activated CD4+ T effector cells no longer undergo TCR-TEM. Engagement of T cell CD28 by EC ICOS-L increases TCR-TEM by activated CD8 effectors while engagement of OX40 promotes TCR-TEM by activated CD4 T regs. B7-H3 mostly affects TEM of resting TEM and some checkpoint inhibitors affect cytokine synthesis or TEM depending upon subtype. Our data suggest that blockade or mimicry of costimulators/checkpoint inhibitors in vivo, clinically used to modulate immune responses, may act in part by modulating T cell homing.

Human vascular smooth muscle cells lack essential costimulatory molecules to activate allogeneic memory T cells.

OBJECTIVE: The arterial media, populated by vascular smooth muscle cells (VSMC), is an immunoprivileged compartment and, in contrast to the intima or adventitia containing endothelial cells, is generally spared by inflammatory processes, such as arteriosclerosis. To determine mechanisms of medial immunoprivilege, we investigated the ability of human VSMC versus endothelial cells to activate allogeneic T cells in vitro. METHODS AND RESULTS: Unlike cultured endothelial cells, cultured VSMC do not activate allogeneic memory CD4 or CD8 T cells and fail to effectively support T-cell proliferation to the polyclonal activator, phytohemagglutinin, consistent with a defect in costimulation function. Although many costimulators are comparably expressed on both cell types, endothelial cells but not VSMC basally express OX40 ligand and upregulate inducible costimulator ligand in response to proinflammatory cytokines. OX40 ligand-transduced, but not control- or inducible costimulator ligand-transduced, VSMC acquire the capacity to stimulate allogeneic memory CD4 T cells to produce cytokines and to proliferate in the presence of supplemental l-tryptophan. OX40 ligand overexpression, although not essential, also enhances allogeneic memory CD8 T-cell responses to VSMC after l-tryptophan supplementation. CONCLUSIONS: The inability of cultured VSMC to activate memory T cells results from a lack of essential costimulators, particularly OX40 ligand, in addition to indoleamine 2,3-dioxygenase-mediated tryptophan depletion.

Progenitor-derived human endothelial cells evade alloimmunity by CRISPR/Cas9-mediated complete ablation of MHC expression.

Tissue engineering may address organ shortages currently limiting clinical transplantation. Off-the-shelf engineered vascularized organs will likely use allogeneic endothelial cells (ECs) to construct microvessels required for graft perfusion. Vasculogenic ECs can be differentiated from committed progenitors (human endothelial colony-forming cells or HECFCs) without risk of mutation or teratoma formation associated with reprogrammed stem cells. Like other ECs, these cells can express both class I and class II major histocompatibility complex (MHC) molecules, bind donor-specific antibody (DSA), activate alloreactive T effector memory cells, and initiate rejection in the absence of donor leukocytes. CRISPR/Cas9-mediated dual ablation of ß2-microglobulin and class II transactivator (CIITA) in HECFC-derived ECs eliminates both class I and II MHC expression while retaining EC functions and vasculogenic potential. Importantly, dually ablated ECs no longer bind human DSA or activate allogeneic CD4+ effector memory T cells and are resistant to killing by CD8+ alloreactive cytotoxic T lymphocytes in vitro and in vivo. Despite absent class I MHC molecules, these ECs do not activate or elicit cytotoxic activity from allogeneic natural killer cells. These data suggest that HECFC-derived ECs lacking MHC molecule expression can be utilized for engineering vascularized grafts that evade allorejection.

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.