DEPTOR at the Nexus of Cancer, Metabolism, and Immunity.

DEP domain-containing mechanistic target of rapamycin (mTOR)-interacting protein (DEPTOR) is an important modulator of mTOR, a kinase at the center of two important protein complexes named mTORC1 and mTORC2. These highly studied complexes play essential roles in regulating growth, metabolism, and immunity in response to mitogens, nutrients, and cytokines. Defects in mTOR signaling have been associated with the development of many diseases, including cancer and diabetes, and approaches aiming at modulating mTOR activity are envisioned as an attractive strategy to improve human health. DEPTOR interaction with mTOR represses its kinase activity and rewires the mTOR signaling pathway. Over the last years, several studies have revealed key roles for DEPTOR in numerous biological and pathological processes. Here, we provide the current state of the knowledge regarding the cellular and physiological functions of DEPTOR by focusing on its impact on the mTOR pathway and its role in promoting health and disease.

A Combination therapy using an mTOR inhibitor and Honokiol effectively induces autophagy through the modulation of AXL and Rubicon in renal cancer cells and restricts renal tumor growth following organ transplantation.

Development of cancer, including renal cancer, is a major problem in immunosuppressed patients. The mTOR inhibitor Rapamycin (RAPA) is used as an immunosuppressive agent in patients with organ transplants and other immunological disorders; and it also has antitumorigenic potential. However, long-term use of RAPA causes reactivation of Akt, and ultimately leads to enhanced tumor growth. Honokiol (HNK) is a natural compound, which possesses both anti-inflammatory and antitumorigenic properties. In this study, we investigated the effect of a novel combination therapy using RAPA + HNK on allograft survival and post-transplantation renal tumor growth. We observed that it effectively modulated the expression of some key regulatory molecules (like Carabin, an endogenous Ras inhibitor; and Rubicon, a negative regulator of autophagy) that play important roles in tumor cell growth and survival. This combination induced toxic autophagy and apoptosis to promote cancer cell death; and was associated with a reduced expression of the tumor-promoting receptor tyrosine kinase AXL. Finally, we utilized a novel murine model to examine the effect of RAPA + HNK on post-transplantation renal tumor growth. The combination treatment prolonged the allograft survival and significantly inhibited post-transplantation tumor growth. It was associated with reduced tumor expression of Rubicon and the cytoprotective/antioxidant heme oxygenase-1 to overcome therapeutic resistance. It also downregulated the coinhibitory programmed death-1 ligand, which plays major role(s) in the immune escape of tumor cells. Together, this combination treatment has a great potential to restrict renal tumor growth in transplant recipients as well as other immunosuppressed patients.

Inhibition of mevalonate metabolism by statins augments the immunoregulatory phenotype of vascular endothelial cells and inhibits the costimulation of CD4+ T cells.

The statin family of therapeutics is widely used clinically as cholesterol lowering agents, and their effects to target intracellular mevalonate production is a key mechanism of action. In this study, we performed full transcriptomic RNA sequencing and qPCR to evaluate the effects of mevalonate on the immunoregulatory phenotype of endothelial cells (EC). We find that mevalonate-dependent gene regulation includes a reduction in the expression of multiple pro-inflammatory genes including TNFSF4 (OX40-L) and TNFSF18 (GITR-L) and a co-incident induction of immunoregulatory genes including LGALS3 (Galectin-3) and LGALS9 (Galectin-9). In functional assays, pretreatment of EC with simvastatin to inhibit mevalonate metabolism resulted in a dose-dependent reduction in the costimulation of CD45RO+ CD4+ T cell proliferation as well as IL-2, IFNγ and IL-6 production versus vehicle-treated EC. In contrast, pre-treatment of EC with L-mevalonate in combination with simvastatin reversed phenotypic and functional responses. Collectively, these results indicate that relative mevalonate metabolism by EC is critical to sustain EC-dependent mechanisms of immunity. Our findings have broad relevance for the repurposing of statins as therapeutics to augment immunoregulation and/or to inhibit local tissue pro-inflammatory cytokine production following transplantation.

T Cell-Specific Adaptor Protein Regulates Mitochondrial Function and CD4+ T Regulatory Cell Activity In Vivo following Transplantation.

The T cell-specific adaptor protein (TSAd), encoded by the SH2D2A gene, is an intracellular molecule that binds Lck to elicit signals that result in cytokine production in CD4+ T effector cells (Teff). Nevertheless, using Sh2d2a knockout (KO; also called TSAd-/-) mice, we find that alloimmune CD4+ Teff responses are fully competent in vivo. Furthermore, and contrary to expectations, we find that allograft rejection is accelerated in KO recipients of MHC class II-mismatched B6.C-H-2bm12 heart transplants versus wild-type (WT) recipients. Also, KO recipients of fully MHC-mismatched cardiac allografts are resistant to the graft-prolonging effects of costimulatory blockade. Using adoptive transfer models, we find that KO T regulatory cells (Tregs) are less efficient in suppressing Teff function and they produce IFN-γ following mitogenic activation. In addition, pyrosequencing demonstrated higher levels of methylation of CpG regions within the Treg-specific demethylated region of KO versus WT Tregs, suggesting that TSAd, in part, promotes Treg stability. By Western blot, Lck is absent in the mitochondria of KO Tregs, and reactive oxygen species production by mitochondria is reduced in KO versus WT Tregs. Full transcriptomic analysis demonstrated that the key mechanism of TSAd function in Tregs relates to its effects on cellular activation rather than intrinsic effects on mitochondria/metabolism. Nevertheless, KO Tregs compensate for a lack of activation by increasing the number of mitochondria per cell. Thus, TSAd serves as a critical cell-intrinsic molecule in CD4+Foxp3+ Tregs to regulate the translocation of Lck to mitochondria, cellular activation responses, and the development of immunoregulation following solid organ transplantation.

DEPTOR modulates activation responses in CD4+ T cells and enhances immunoregulation following transplantation.

DEPTOR is an evolutionarily conserved cell-intrinsic binding partner of mTOR that functions as a negative regulator of signaling responses. In this study, we show that DEPTOR is expressed within CD4+ T cells, and we observed that its relative level of expression modulates differentiation as well as glucose utilization within CD4+ T effectors in vitro. Using knock-in mice, we also find that induced expression of DEPTOR within CD4+ T regulatory cells stabilizes Foxp3 expression, shifts metabolism toward oxidative phosphorylation, and increases survival and suppressive function. In vivo, fully MHC mismatched cardiac allograft survival is significantly prolonged in knock-in recipients and sustained recipient expression of DEPTOR in combination with costimulatory blockade induces long-term graft survival. Furthermore, we show that the induced expression of DEPTOR in CD4+ T effectors fails to inhibit acute allograft rejection. Rather, prolonged survival is dominantly mediated via induced expression and function of DEPTOR within recipient CD4+ T regulatory cells. These collective findings identify DEPTOR as a novel protein that functions in CD4+ T cells to augment immunoregulation in vitro and in vivo.

Calcineurin inhibitors augment endothelial-to-mesenchymal transition by enhancing proliferation in association with cytokine-mediated activation.

Calcineurin Inhibitors (CNIs) are routinely used for immunosuppression following solid organ transplantation. However, the prolonged use of these agents lead to organ fibrosis which limits their efficacy. CNIs induce TGFß expression, which is reported to augment endothelial-to-mesenchymal transition (EndMT), but their role in this process is not known. In these studies, we find that the CNIs FK506 and cyclosporine (CsA) are potent to increase endothelial cell (EC) proliferation using established in vitro assays (P < 0.05). Furthermore, using phosphokinase arrays, we find that each CNI activates the MAPK and Akt/mTOR signaling pathways, and that pharmacological inhibition of each pathway targets CNI-induced proliferative responses (P < 0.001). EndMT was evaluated by FACS for N-cadherin and CD31 expression and by qPCR for the expression of α-smooth muscle actin, N-cadherin and Snail. We find that CNIs do not directly induce dedifferentiation, while TGFß and hypoxia induce EndMT in small numbers of EC. In contrast, the treatment of EC with the inflammatory cytokine TNFα was potent to elicit an EndMT response, and its effects were most notably in EC following proliferation/doubling. Taken together, these observations suggest that CNIs elicit proliferative responses, which enhance EndMT in association with local inflammation. The clinical implications of these findings are that anti-proliferative therapeutics have high potential to target the initiation of this EndMT response.

Study rationale, design, and pretransplantation alloantibody status: A first report of Clinical Trials in Organ Transplantation in Children-04 (CTOTC-04) in pediatric heart transplantation.

Anti-HLA donor-specific antibodies are associated with worse outcomes after organ transplantation. Among sensitized pediatric heart candidates, requirement for negative donor-specific cytotoxicity crossmatch increases wait times and mortality. However, transplantation with positive crossmatch may increase posttransplantation morbidity and mortality. We address this clinical challenge in a prospective, multicenter, observational cohort study of children listed for heart transplantation (Clinical Trials in Organ Transplantation in Children-04 [CTOTC-04]). Outcomes were compared among sensitized recipients who underwent transplantation with positive crossmatch, nonsensitized recipients, and sensitized recipients without positive crossmatch. Positive crossmatch recipients received antibody removal and augmented immunosuppression, while other recipients received standard immunosuppression with corticosteroid avoidance. This first CTOTC-04 report summarizes study rationale and design and relates pretransplantation sensitization status using solid-phase technology. Risk factors for sensitization were explored. Of 317 screened patients, 290 were enrolled and 240 underwent transplantation. Core laboratory evaluation demonstrated that more than half of patients were anti-HLA sensitized. Greater than 80% of sensitized patients had class I (with or without class II) HLA antibodies, and one-third of sensitized patients had at least 1 HLA antibody with median fluorescence intensity of ≥8000. Logistic regression models demonstrated male sex, weight, congenital heart disease history, prior allograft, and ventricular assist device are independent risk factors for sensitization.

Netrin-1 Augments Chemokinesis in CD4+ T Cells In Vitro and Elicits a Proinflammatory Response In Vivo.

Netrin-1 is a neuronal guidance cue that regulates cellular activation, migration, and cytoskeleton rearrangement in multiple cell types. It is a chemotropic protein that is expressed within tissues and elicits both attractive and repulsive migratory responses. Netrin-1 has recently been found to modulate the immune response via the inhibition of neutrophil and macrophage migration. However, the ability of Netrin-1 to interact with lymphocytes and its in-depth effects on leukocyte migration are poorly understood. In this study, we profiled the mRNA and protein expression of known Netrin-1 receptors on human CD4(+) T cells. Neogenin, uncoordinated-5 (UNC5)A, and UNC5B were expressed at low levels in unstimulated cells, but they increased following mitogen-dependent activation. By immunofluorescence, we observed a cytoplasmic staining pattern of neogenin and UNC5A/B that also increased following activation. Using a novel microfluidic assay, we found that Netrin-1 stimulated bidirectional migration and enhanced the size of migratory subpopulations of mitogen-activated CD4(+) T cells, but it had no demonstrable effects on the migration of purified CD4(+)CD25(+)CD127(dim) T regulatory cells. Furthermore, using a short hairpin RNA knockdown approach, we observed that the promigratory effects of Netrin-1 on T effectors is dependent on its interactions with neogenin. In the humanized SCID mouse, local injection of Netrin-1 into skin enhanced inflammation and the number of neogenin-expressing CD3(+) T cell infiltrates. Neogenin was also observed on CD3(+) T cell infiltrates within human cardiac allograft biopsies with evidence of rejection. Collectively, our findings demonstrate that Netrin-1/neogenin interactions augment CD4(+) T cell chemokinesis and promote cellular infiltration in association with acute inflammation in vivo.

The intragraft microenvironment as a central determinant of chronic rejection or local immunoregulation/tolerance.

PURPOSE OF REVIEW: Chronic rejection is associated with persistent mononuclear cell recruitment, endothelial activation and proliferation, local tissue hypoxia and related biology that enhance effector immune responses. In contrast, the tumor microenvironment elicits signals/factors that inhibit effector T cell responses and rather promote immunoregulation locally within the tissue itself. The identification of immunoregulatory check points and/or secreted factors that are deficient within allografts is of great importance in the understanding and prevention of chronic rejection. RECENT FINDINGS: The relative deficiency of immunomodulatory molecules (cell surface and secreted) on microvascular endothelial cells within the intragraft microenvironment, is of functional importance in shaping the phenotype of rejection. These regulatory molecules include coinhibitory and/or intracellular regulatory signals/factors that enhance local activation of T regulatory cells. For example, semaphorins may interact with endothelial cells and CD4 T cells to promote local tolerance. Additionally, metabolites and electrolytes within the allograft microenvironment may regulate local effector and regulatory cell responses. SUMMARY: Multiple factors within allografts shape the microenvironment either towards local immunoregulation or proinflammation. Promoting the expression of intragraft cell surface or secreted molecules that support immunoregulation will be critical for long-term graft survival and/or alloimmune tolerance.

Translational implications of endothelial cell dysfunction in association with chronic allograft rejection.

Advances in therapeutics have dramatically improved short-term graft survival, but the incidence of chronic rejection has not changed in the past 20 years. New insights into mechanism are sorely needed at this time and it is hoped that the development of predictive biomarkers will pave the way for the emergence of preventative therapeutics. In this review, we discuss a paradigm suggesting that sequential changes within graft endothelial cells (EC) lead to an intragraft microenvironment that favors the development of chronic rejection. Key initial events include EC injury, activation and uncontrolled leukocyte-induced angiogenesis. We propose that all of these early changes in the microvasculature lead to abnormal blood flow patterns, local tissue hypoxia, and an associated overexpression of HIF-1α-inducible genes, including vascular endothelial growth factor. We also discuss how cell intrinsic regulators of mTOR-mediated signaling within EC are of critical importance in microvascular stability and may thus have a role in the inhibition of chronic rejection. Finally, we discuss recent findings indicating that miRNAs may regulate EC stability, and we review their potential as novel non-invasive biomarkers of allograft rejection. Overall, this review provides insights into molecular events, genes, and signals that promote chronic rejection and their potential as biomarkers that serve to support the future development of interruption therapeutics.

DEPTOR regulates vascular endothelial cell activation and proinflammatory and angiogenic responses.

The maintenance of normal tissue homeostasis and the prevention of chronic inflammatory disease are dependent on the active process of inflammation resolution. In endothelial cells (ECs), proinflammation results from the activation of intracellular signaling responses and/or the inhibition of endogenous regulatory/pro-resolution signaling networks that, to date, are poorly defined. In this study, we find that DEP domain containing mTOR interacting protein (DEPTOR) is expressed in different microvascular ECs in vitro and in vivo, and using a small interfering RNA (siRNA) knockdown approach, we find that it regulates mammalian target of rapamycin complex 1 (mTORC1), extracellular signal-regulated kinase 1/2, and signal transducer and activator of transcription 1 activation in part through independent mechanisms. Moreover, using limited gene arrays, we observed that DEPTOR regulates EC activation including mRNA expression of the T-cell chemoattractant chemokines CXCL9, CXCL10, CXCL11, CX3CL1, CCL5, and CCL20 and the adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 (P < .05). DEPTOR siRNA-transfected ECs also bound increased numbers of peripheral blood mononuclear cells (P < .005) and CD3+ T cells (P < .005) in adhesion assays in vitro and had increased migration and angiogenic responses in spheroid sprouting (P < .01) and wound healing (P < .01) assays. Collectively, these findings define DEPTOR as a critical upstream regulator of EC activation responses and suggest that it plays an important role in endogenous mechanisms of anti-inflammation and pro-resolution.

Chronic allograft rejection: a fresh look.

PURPOSE OF REVIEW: New developments suggest that the graft itself and molecules expressed within the graft microenvironment dictate the phenotype and evolution of chronic rejection. RECENT FINDINGS: Once ischemia-reperfusion injury, cellular and humoral immune responses target the microvasculature, the associated local tissue hypoxia results in hypoxia-inducible factor 1α-dependent expression of pro-inflammatory and proangiogenic growth factors including vascular endothelial growth factor (VEGF) as a physiological response to injury. Local expression of VEGF can promote the recruitment of alloimune T cells into the graft. mTOR/Akt signaling within endothelial cells regulates cytokine- and alloantibody-induced activation and proliferation and their proinflammatory phenotype. Inhibition of mTOR and/or Akt results in an anti-inflammatory phenotype and enables the expression of coinhibitory molecules that limit local T cell reactivation and promotes immunoregulation. Semaphorin family molecules may bind to neuropilin-1 on regulatory T cell subsets to stabilize functional responses. Ligation of neuropilin-1 on Tregs also inhibits Akt-induced responses suggesting common theme for enhancing local immunoregulation and long-term graft survival. SUMMARY: Events within the graft initiated by mTOR/Akt-induced signaling promote the development of chronic rejection. Semaphorin-neuropilin biology represents a novel avenue for targeting this biology and warrants further investigation.

Pathological angiogenesis is induced by sustained Akt signaling and inhibited by rapamycin.

Endothelial cells in growing tumors express activated Akt, which when modeled by transgenic endothelial expression of myrAkt1 was sufficient to recapitulate the abnormal structural and functional features of tumor blood vessels in nontumor tissues. Sustained endothelial Akt activation caused increased blood vessel size and generalized edema from chronic vascular permeability, while acute permeability in response to VEGF-A was unaffected. These changes were reversible, demonstrating an ongoing requirement for Akt signaling for the maintenance of these phenotypes. Furthermore, rapamycin inhibited endothelial Akt signaling, vascular changes from myrAkt1, tumor growth, and tumor vascular permeability. Akt signaling in the tumor vascular stroma was sensitive to rapamycin, suggesting that rapamycin may affect tumor growth in part by acting as a vascular Akt inhibitor.

Assessing Vulnerability to COVID-19 in High-Risk Populations: The Role of SARS-CoV-2 Spike-Targeted Serology.

Individuals at increased risk for severe coronavirus disease-2019 (COVID-19) outcomes, due to compromised immunity or other risk factors, would benefit from objective measures of vulnerability to infection based on vaccination or prior infection. The authors reviewed published data to identify a specific role and interpretation of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike-targeted serology testing. Specific recommendations are provided for an evidence-based and clinically-useful interpretation of SARS-CoV-2 spike-targeted serology to identify vulnerability to infection and potential subsequent adverse outcomes. Decreased vaccine effectiveness among immunocompromised individuals is linked to correspondingly high rates of breakthrough infections. Negative results on SARS-CoV-2 antibody tests are associated with increased risk for subsequent infection. "Low-positive" results on semiquantitative SARS-CoV-2 spike-targeted antibody tests may help identify persons at increased risk as well. Standardized SARS-CoV-2 spike-targeted antibody tests may provide objective information on the risk of SARS-CoV-2 infection and associated adverse outcomes. This holds especially for high-risk populations that demonstrate a relatively high rate of seronegativity. The widespread availability of such tests presents an opportunity to refine risk assessment for individuals with suboptimal SARS-CoV-2 antibody levels and to promote effective interventions. Interim federal guidance would support physicians and patients while additional investigations are pursued.

Subsets of human CD4(+) regulatory T cells express the peripheral homing receptor CXCR3.

Regulatory T cells (Tregs) migrate into peripheral sites of inflammation such as allografts undergoing rejection, where they serve to suppress the immune response. In this study, we find that ∼30-40% of human CD25(hi) FOXP3(+) CD4(+) Tregs express the peripheral CXC chemokine receptor 3 (CXCR3) and that this subset has potent immunoregulatory properties. Consistently, we observed that proliferative responses as well as IFN-γ production were significantly higher using CXCR3-depleted versus undepleted responders in the mixed lymphocyte reaction, as well as following mitogen-dependent activation of T cells. Using microfluidics, we also found that CXCR3 was functional on CXCR3(pos) Tregs, in as much as chemotaxis and directional persistence towards interferon-γ-inducible protein of 10 kDa (IP-10) was significantly greater for CXCR3(pos) than CXCR3(neg) Tregs. Following activation, CXCR3-expressing CD4(+) Tregs were maintained in vitro in cell culture in the presence of the mammalian target of rapamycin (mTOR) inhibitor rapamycin, and we detected higher numbers of circulating CXCR3(+) FOXP3(+) T cells in adult and pediatric recipients of renal transplants who were treated with mTOR-inhibitor immunosuppressive therapy. Collectively, these results demonstrate that the peripheral homing receptor CXCR3 is expressed on subset(s) of circulating human Tregs and suggest a role for CXCR3 in their recruitment into peripheral sites of inflammation.

TRAF6 inhibits proangiogenic signals in endothelial cells and regulates the expression of vascular endothelial growth factor.

TNF-family molecules induce the expression Vascular Endothelial Growth Factor (VEGF) in endothelial cells (EC) and elicit signaling responses that result in angiogenesis. However, the role of TNF-receptor associated factors (TRAFs) as upstream regulators of VEGF expression or as mediators of angiogenesis is not known. In this study, HUVEC were cotransfected with a full-length VEGF promoter-luciferase construct and siRNAs to TRAF 1, -2, -3, -5, -6, and promoter activity was measured. Paradoxically, rather than inhibiting VEGF expression, we found that knockdown of TRAF6 resulted in a 4-6-fold increase in basal VEGF promoter activity compared to control siRNA-transfected EC (P<0.0001). In addition, knockdown of TRAF 1, -2, -3 or -5 resulted in a slight increase or no change in VEGF promoter activation. Using [(3)H]thymidine incorporation assays as well as the in vitro wound healing assay, we also found that basal rates of EC proliferation and migration were increased following TRAF6 knockdown; and this response was inhibited by the addition of a blocking anti-VEGF antibody into cell cultures. Using a limited protein array to gain insight into TRAF6-dependent intermediary signaling responses, we observed that TRAF6 knockdown resulted in an increase in the activity of Src family kinases. In addition, we found that treatment with AZD-0530, a pharmacological Src inhibitor, reduced the regulatory effect of TRAF6 knockdown on VEGF promoter activity. Collectively, these findings define a novel pro-angiogenic signaling response in EC that is regulated by TRAF6.

Constitutive activation of the mTOR signaling pathway within the normal glomerulus.

Agents that target the activity of the mammalian target of rapamycin (mTOR) kinase in humans are associated with proteinuria. However, the mechanisms underlying mTOR activity and signaling within the kidney are poorly understood. In this study, we developed a sensitive immunofluorescence technique for the evaluation of activated pmTOR and its associated signals in situ. While we find that pmTOR is rarely expressed in normal non-renal tissues, we consistently find intense expression in glomeruli within normal mouse and human kidneys. Using double staining, we find that the expression of pmTOR co-localizes with nephrin in podocytes and expression appears minimal within other cell types in the glomerulus. In addition, we found that pmTOR was expressed on occasional renal tubular cells within mouse and human kidney specimens. We also evaluated mTOR signaling in magnetic bead-isolated glomeruli from normal mice and, by Western blot analysis, we confirmed function of the pathway in glomerular cells vs. interstitial cells. Furthermore, we found that the activity of the pathway as well as the expression of VEGF, a target of mTOR-induced signaling, were reduced within glomeruli of mice following treatment with rapamycin. Collectively, these findings demonstrate that the mTOR signaling pathway is constitutively hyperactive within podocytes. We suggest that pmTOR signaling functions to regulate glomerular homeostasis in part via the inducible expression of VEGF.

Effect of vascular endothelial growth factor and its receptor KDR on the transendothelial migration and local trafficking of human T cells in vitro and in vivo.

In these studies, we find that the vascular endothelial growth factor (VEGF) receptor KDR is expressed on subsets of mitogen-activated CD4(+) and CD8(+) T cells in vitro. We also found that KDR colocalizes with CD3 on mitogen-activated T cells in vitro and on infiltrates within rejecting human allografts in vivo. To evaluate whether VEGF and KDR mediate lymphocyte migration across endothelial cells (ECs), we used an in vitro live-time transmigration model and observed that both anti-VEGF and anti-KDR antibodies inhibit the transmigration of both CD4(+) and CD8(+) T cells across tumor necrosis factor α (TNFα)-activated, but not unactivated ECs. In addition, we found that interactions among CD4(+) or CD8(+) T cells and TNFα-activated ECs result in the induction of KDR on each T cell subset, and that KDR-expressing lymphocytes preferentially transmigrate across TNFα-activated ECs. Finally, using a humanized severe combined immunodeficient mouse model of lymphocyte trafficking, we found that KDR-expressing lymphocytes migrate into human skin in vivo, and that migration is reduced in mice treated with a blocking anti-VEGF antibody. These observations demonstrate that induced expression of KDR on subsets of T cells, and locally expressed VEGF, facilitate EC-dependent lymphocyte chemotaxis, and thus, the localization of T cells at sites of inflammation.

Cutting edge: Vascular endothelial growth factor-mediated signaling in human CD45RO+ CD4+ T cells promotes Akt and ERK activation and costimulates IFN-gamma production.

In this study, we find that CD45RO+ memory populations of CD4+ T lymphocytes express the vascular endothelial growth factor (VEGF) receptors KDR and Flt-1 at both the mRNA and protein levels. Furthermore, by Western blot analysis, we find that VEGF increases the phosphorylation and activation of ERK and Akt within CD4+CD45RO+ T cells. These VEGF-mediated signaling responses were inhibited by a KDR-specific small interfering RNA in a VEGF receptor-expressing Jurkat T cell line and by SU5416, a pharmacological KDR inhibitor, in CD4+CD45RO+ T cells. We also find that VEGF augments mitogen-induced production of IFN-gamma in a dose-dependent manner (p < 0.001) and significantly (p < 0.05) increases directed chemotaxis of this T cell subset. Collectively, our results for the first time define a novel function for VEGF and KDR in CD45RO+ memory T cell responses that are likely of great pathophysiological importance in immunity.

Every allograft needs a silver lining.

The development of chronic allograft rejection is based on the hypothesis that cumulative, time-dependent tissue injury eventually leads to a fibrotic response. In this issue of the JCI, Babu and colleagues found that alloimmune-mediated microvascular loss precedes tissue damage in murine orthotopic tracheal allografts (see the related article beginning on page 3774). The concept that injury to the endothelium may precede airway fibrosis suggests that interventions to maintain vascular integrity may be important, especially in the case of lung transplantation. Further, for all solid organ allografts, it is possible that the key to long-term allograft survival is physiological vascular repair at early times following transplantation.