Nir Grabie

Clonal deletion of thymocytes by circulating dendritic cells homing to the thymus

Dendritic cell (DC) presentation of self antigen to thymocytes is essential to the establishment of central tolerance. We show here that circulating DCs were recruited to the thymic medulla through a three-step adhesion cascade involving P-selectin, interactions of the integrin VLA-4 with its ligand VCAM-1, and pertussis toxin–sensitive chemoattractant signaling. Ovalbumin-specific OT-II thymocytes were selectively deleted after intravenous injection of antigen-loaded exogenous DCs. We documented migration of endogenous DCs to the thymus in parabiotic mice and after painting mouse skin with fluorescein isothiocyanate. Antibody to VLA-4 blocked the accumulation of peripheral tissue–derived DCs in the thymus and also inhibited the deletion of OT-II thymocytes in mice expressing membrane-bound ovalbumin in cardiac myocytes. These findings identify a migratory route by which peripheral DCs may contribute to central tolerance.

Endothelial expression of PD-L1 and PD-L2 down-regulates CD8+ T cell activation and cytolysis.

Interactions between CD8+ T cells and endothelial cells are important in both protective and pathologic immune responses. Endothelial cells regulate the recruitment of CD8+ Tcells into tissues, and the activation of CD8+ T cells by antigen presentation and costimulatory signals. PD‐L1 and PD‐L2 are recently described B7‐family molecules which bind to PD‐1 on activated lymphocytes and down‐regulate T cell activation. We found that PD‐L1 is expressed on interferon‐γ stimulated cultured human and mouse endothelial cells, while PD‐L2 was found on stimulated human but not mouse endothelial cells. Expression was further up‐regulated by TNF‐α. Antibody blockade of endothelial cell PD‐L1 and PD‐L2 enhanced endothelial cell costimulation of PHA‐activated human CD8+ T cells. Antibody blockade of mouse endothelial cell PD‐L1 enhanced both IFN‐γ secretion and cytolytic activity of CD8+ T cells in response to endothelial cellantigen presentation. These results show that IFN‐γ activated endothelial cells can inhibit T cell activation via expression of the immunoinhibitory PD‐L1 and PD‐L2 molecules. Endothelial expression of PD‐ligands would allow activation and extravasation of T cells without excessive vessel damage. Our findings highlight a potentially important pathway by which endothelial cells down‐regulate CD8+ T cell‐mediated immune responses.

Impaired Regulatory T-Cell Response and Enhanced Atherosclerosis in the Absence of Inducible Costimulatory Molecule

Background— T-cell–mediated immunity contributes to the pathogenesis of atherosclerosis, but little is known about how these responses are regulated. We explored the influence of the inducible costimulatory molecule (ICOS) on atherosclerosis and associated immune responses. Methods and Results— Bone morrow chimeras were generated by transplanting ICOS-deficient or wild-type bone marrow into irradiated atherosclerosis-prone, LDR receptor–deficient mice, and the chimeric mice were fed a high-cholesterol diet for 8 weeks. Compared with controls, mice transplanted with ICOS-deficient marrow had a 43% increase in the atherosclerotic burden, and importantly, their lesions had a 3-fold increase in CD4+ T cells, as well as increased macrophage, smooth muscle cell, and collagen content. CD4+ T cells from ICOS-deficient chimeras proliferated more and secreted more interferon-γ and tumor necrosis factor-α than T cells from control mice, which suggests a lack of regulation. FoxP3+ regulatory T cells (Treg) were found to constitutively express high ICOS levels, which suggests a role for ICOS in Treg function. ICOS-deficient mice had decreased numbers of FoxP3+ Treg and impaired in vitro Treg suppressive function compared with control mice. Conclusions— ICOS has a key role in regulation of atherosclerosis, through its effect on regulatory T-cell responses.

IL-17 and TNF-α Sustain Neutrophil Recruitment during Inflammation through Synergistic Effects on Endothelial Activation

IL-17A (IL-17) is the signature cytokine produced by Th17 cells and has been implicated in host defense against infection and the pathophysiology of autoimmunity and cardiovascular disease. Little is known, however, about the influence of IL-17 on endothelial activation and leukocyte influx to sites of inflammation. We hypothesized that IL-17 would induce a distinct pattern of endothelial activation and leukocyte recruitment when compared with the Th1 cytokine IFN-γ. We found that IL-17 alone had minimal activating effects on cultured endothelium, whereas the combination of TNF-α and IL-17 produced a synergistic increase in the expression of both P-selectin and E-selectin. Using intravital microscopy of the mouse cremaster muscle, we found that TNF-α and IL-17 also led to a synergistic increase in E-selectin–dependent leukocyte rolling on microvascular endothelium in vivo. In addition, TNF-α and IL-17 enhanced endothelial expression of the neutrophilic chemokines CXCL1, CXCL2, and CXCL5 and led to a functional increase in leukocyte transmigration in vivo and CXCR2-dependent neutrophil but not T cell transmigration in a parallel-plate flow chamber system. By contrast, endothelial activation with TNF-α and IFN-γ preferentially induced the expression of the integrin ligands ICAM-1 and VCAM-1, as well as the T cell chemokines CXCL9, CXCL10, and CCL5. These effects were further associated with a functional increase in T cell but not neutrophil transmigration under laminar shear flow. Overall, these data show that IL-17 and TNF-α act in a synergistic manner to induce a distinct pattern of endothelial activation that sustains and enhances neutrophil influx to sites of inflammation.

C-C chemokine–encoding DNA vaccines enhance breakdown of tolerance to their gene products and treat ongoing adjuvant arthritis

Depending on the method of immunization, a single administration of CFA may result in the development of a local inflammatory process or chronic polyadjuvant-induced arthritis (AA). We administered naked DNA vaccines encoding MIP-1 alpha, MCP-1, MIP-1 beta, and RANTES to Lewis rats and confirmed that each of these vaccines induced immunological memory to the corresponding gene product. Upon induction of disease, this memory effectively inhibited the development of the autoimmune condition. Self-specific Abs developed in DNA-vaccinated animals were neutralizing in vitro and could adoptively transfer the beneficial effect of each vaccine. Repeated administration of the constructs encoding MCP-1, MIP-1 alpha, or RANTES inhibited the development and progression of AA, even when each vaccine was administered only after the onset of disease. This suggests a highly effective way by which the immune system could be re-educated to generate protective immunity against its own harmful activities.

Endothelial Programmed Death-1 Ligand 1 (PD-L1) Regulates CD8+ T-Cell–Mediated Injury in the Heart

Background— PD-L1 and PD-L2 are ligands for the inhibitory receptor programmed death-1 (PD-1), which is an important regulator of immune responses. PD-L1 is induced on cardiac endothelial cells under inflammatory conditions, but little is known about its role in regulating immune injury in the heart. Methods and Results— Cytotoxic T-lymphocyte–mediated myocarditis was induced in mice, and the influence of PD-L1 signaling was studied with PD-L1/L2–deficient mice and blocking antibodies. During cytotoxic T-lymphocyte–induced myocarditis, the upregulation of PD-L1 on cardiac endothelia was dependent on T-cell–derived interferon-γ, and blocking of interferon-γ signaling worsened disease. Genetic deletion of both PD-1 ligands [PD-L1/2(−/−)], as well as treatment with PD-L1 blocking antibody, transformed transient myocarditis to lethal disease, in association with widespread polymorphonuclear leukocyte–rich microabscesses but without change in cytotoxic T-lymphocyte recruitment. PD-L1/2(−/−) mice reconstituted with bone marrow from wild-type mice remained susceptible to severe disease, which demonstrates that PD-L1 on non–bone marrow–derived cells confers the protective effect. Finally, depletion of polymorphonuclear leukocytes reversed the enhanced susceptibility to lethal myocarditis attributable to PD-L1 deficiency. Conclusions— Myocardial PD-L1, mainly localized on endothelium, is critical for control of immune-mediated cardiac injury and polymorphonuclear leukocyte inflammation.

Dynamic Changes in Regulatory T Cells Are Linked to Levels of Diet-Induced Hypercholesterolemia

Background— Regulatory T cells (Treg) are present in atherosclerotic lesions and can modulate disease. In this study we characterized changes in Treg responses associated with prolonged hypercholesterolemia and lesion progression. Methods and Results— Low-density lipoprotein receptor null mice in which Treg express green fluorescent protein were fed a control or cholesterol-rich diet, and green fluorescent protein–positive cells were enumerated in lymphoid tissues and in aorta. Splenic Treg numbers increased after 4, 8, and 20 weeks in cholesterol-diet–fed mice. However, the number of circulating and lesional Treg peaked at 4 weeks and decreased significantly at 8 and 20 weeks, concomitant with increased numbers of CD4+ effector T cells and increased lesion size over this period. Treg expression of selectin ligands and their ability to bind to aortic endothelium decreased after prolonged hypercholesterolemia, and apoptosis of lesional Treg increased. After 4 weeks of cholesterol-rich diet, a switch to a control diet for 4 weeks reduced serum cholesterol and stopped lesion growth, and the high aortic Treg content was maintained, compared with mice fed a cholesterol diet for 8 weeks. After the diet reversal, the splenic Treg retained the phenotype of Treg after 4 weeks of cholesterol diet. Conclusions— Prolonged hypercholesterolemia impairs Treg but not effector T cell accumulation in lesions, but reversal of hypercholesterolemia can prevent loss of lesional Treg. Therefore, cholesterol-lowering therapies may induce dynamic and beneficial changes in Treg:effector T cell ratios in atherosclerotic lesions.

IL-12 is required for differentiation of pathogenic CD8+ T cell effectors that cause myocarditis

Cardiac antigen-specific CD8(+) T cells are involved in the autoimmune component of human myocarditis. Here, we studied the differentiation and migration of pathogenic CD8(+) T cell effector cells in a new mouse model of autoimmune myocarditis. A transgenic mouse line was derived that expresses cardiac myocyte restricted membrane-bound ovalbumin (CMy-mOva). The endogenous adaptive immune system of CMy-mOva mice displays tolerance to ovalbumin. Adoptive transfer of naive CD8(+) T cells from the ovalbumin-specific T cell receptor-transgenic (TCR-transgenic) OT-I strain induces myocarditis in CMy-mOva mice only after subsequent inoculation with ovalbumin-expressing vesicular stomatitis virus (VSV-Ova). OT-I effector T cells derived in vitro in the presence or absence of IL-12 were adoptively transferred into CMy-mOva mice, and the consequences were compared. Although IL-12 was not required for the generation of cytolytic and IFN-gamma-producing effector T cells, only effectors primed in the presence of IL-12 infiltrated CMy-mOva hearts in significant numbers, causing lethal myocarditis. Furthermore, analysis of OT-I effectors collected from a mediastinal draining lymph node indicated that only effectors primed in vitro in the presence of IL-12 proliferated in vivo. These data demonstrate the importance of IL-12 in the differentiation of pathogenic CD8(+) T cells that can cause myocarditis.

Proatherogenic immune responses are regulated by the PD-1/PD-L pathway in mice

T lymphocyte responses promote proatherogenic inflammatory events, which are influenced by costimulatory molecules of the B7 family. Effects of negative regulatory members of the B7 family on atherosclerosis have not been described. Programmed death-ligand 1 (PD-L1) and PD-L2 are B7 family members expressed on several cell types, which inhibit T cell activation via binding to programmed death-1 (PD-1) on T cells. In order to test whether the PD-1/PD-L pathway regulates proatherogenic T cell responses, we compared atherosclerotic lesion burden and phenotype in hypercholesterolemic PD-L1/2(-/-)LDLR(-/-) mice and LDLR(-/-) controls. PD-L1/2 deficiency led to significantly increased atherosclerotic burden throughout the aorta and increased numbers of lesional CD4(+) and CD8(+) T cells. Compared with controls, PD-L1/2(-/-)LDLR(-/-) mice had iliac lymphadenopathy and increased numbers of activated CD4(+) T cells. Serum levels of TNF-alpha were higher in PD-L1/2(-/-)LDLR(-/-) mice than in controls. PD-L1/2-deficient APCs were more effective than control APCs in activating CD4(+) T cells in vitro, with or without cholesterol loading. Freshly isolated APCs from hypercholesterolemic PD-L1/2(-/-)LDLR(-/-) mice stimulated greater T cell responses than did APCs from hypercholesterolemic controls. Our findings indicate that the PD-1/PD-L pathway has an important role in downregulating proatherogenic T cell response and atherosclerosis by limiting APC-dependent T cell activation.

BLT1-mediated T cell trafficking is critical for rejection and obliterative bronchiolitis after lung transplantation

Leukotriene B4 is a lipid mediator that recently has been shown to have potent chemotactic activity for effector T lymphocytes mediated through its receptor, BLT1. Here, we developed a novel murine model of acute lung rejection to demonstrate that BLT1 controls effector CD8+ T cell trafficking into the lung and that disruption of BLT1 signaling in CD8+ T cells reduces lung inflammation and mortality in the model. In addition, we used BLT1-deficient mice and a BLT1 antagonist in two tracheal transplant models of lung transplantation to demonstrate the importance of BLT1 for the recruitment of T cells into tracheal allografts. We also show that BLT1-mediated CD8+ T cell recruitment plays an important role in the development of airway fibroproliferation and obliteration. Finally, in human studies of lung transplant recipients, we found that BLT1 is up-regulated on T lymphocytes isolated from the airways of patients with obliterative bronchiolitis. These data demonstrate that BLT1 contributes to the development of lung rejection and obliterative bronchiolitis by mediating effector T lymphocyte trafficking into the lung. This is the first report that describes a pathologic role for BLT1-mediated T lymphocyte recruitment in disease and identifies BLT1 as a potential therapeutic target after lung transplantation.