T cell checkpoint regulators in the heart.

T lymphocyte-mediated immune responses in the heart are potentially dangerous because they can interfere with the electromechanical function. Furthermore, the myocardium has limited regenerative capacity to repair damage caused by effector T cells. Myocardial T cell responses are normally suppressed by multiple mechanisms of central and peripheral tolerance. T cell inhibitory molecules, so called immune checkpoints, limit the activation and effector function of heart antigen-reactive T cells that escape deletion during development in the thymus. Programmed cell protein death-1 (PD-1) and cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) are checkpoint molecules homologous to the costimulatory receptor CD28, and they work to block activating signals from the T cell antigen receptor and CD28. Nonetheless, PD-1 and CTLA-4 function in different ways and at different steps in a T cell response to antigen. Studies in mice have established that genetic deficiencies of checkpoint molecules, including PD-1, PD-L1, CTLA-4, and lymphocyte activation gene-3, result in enhanced risk of autoimmune T cell-mediated myocarditis and increased pathogenicity of heart antigen-specific effector T cells. The PD-1/PD-L1 pathway appears to be particularly important in cardiac protection from T cells. PD-L1 is markedly up-regulated on myocardial cells by interferon-gamma secreted by T cells and PD-1 or PD-L1 deficiency synergizes with other defects in immune regulation in promoting myocarditis. Consistent with these studies, myocarditis has emerged as a serious adverse reaction of cancer therapies that target checkpoint molecules to enhance anti-tumour T cell responses. Histopathology and immunohistochemical analyses of myocardial tissue from immune checkpoint blockade (ICB)-treated patients echoes findings in checkpoint-deficient mice. Many questions about myocarditis in the setting of cancer immunotherapy still need to be answered, including the nature of the target antigens, genetic risk factors, and variations in the disease with combined therapies. Addressing these questions will require further immunological analyses of blood and heart tissue from patients treated with ICB.

Dendritic Cell KLF2 Expression Regulates T Cell Activation and Proatherogenic Immune Responses.

Dendritic cells (DCs) have been implicated as important regulators of innate and adaptive inflammation in many diseases, including atherosclerosis. However, the molecular mechanisms by which DCs mitigate or promote inflammatory pathogenesis are only partially understood. Previous studies have shown an important anti-inflammatory role for the transcription factor Krüppel-like factor 2 (KLF2) in regulating activation of various cell types that participate in atherosclerotic lesion development, including endothelial cells, macrophages, and T cells. We used a pan-DC, CD11c-specific cre-lox gene knockout mouse model to assess the role of KLF2 in DC activation, function, and control of inflammation in the context of hypercholesterolemia and atherosclerosis. We found that KLF2 deficiency enhanced surface expression of costimulatory molecules CD40 and CD86 in DCs and promoted increased T cell proliferation and apoptosis. Transplant of bone marrow from mice with KLF2-deficient DCs into Ldlr-/- mice aggravated atherosclerosis compared with control mice, most likely due to heightened vascular inflammation evidenced by increased DC presence within lesions, enhanced T cell activation and cytokine production, and increased cell death in atherosclerotic lesions. Taken together, these data indicate that KLF2 governs the degree of DC activation and hence the intensity of proatherogenic T cell responses.

AKAP9 regulates activation-induced retention of T lymphocytes at sites of inflammation.

The mechanisms driving T cell homing to lymph nodes and migration to tissue are well described but little is known about factors that affect T cell egress from tissues. Here, we generate mice with a T cell-specific deletion of the scaffold protein A kinase anchoring protein 9 (AKAP9) and use models of inflammatory disease to demonstrate that AKAP9 is dispensable for T cell priming and migration into tissues and lymph nodes, but is required for T cell retention in tissues. AKAP9 deficiency results in increased T cell egress to draining lymph nodes, which is associated with impaired T cell re-activation in tissues and protection from organ damage. AKAP9-deficient T cells exhibit reduced microtubule-dependent recycling of TCRs back to the cell surface and this affects antigen-dependent activation, primarily by non-classical antigen-presenting cells. Thus, AKAP9-dependent TCR trafficking drives efficient T cell re-activation and extends their retention at sites of inflammation with implications for disease pathogenesis.

Expansion of CD25+ Innate Lymphoid Cells Reduces Atherosclerosis.

OBJECTIVE: Innate lymphoid cells (ILCs) are a newly discovered subset of immune cells that promote tissue homeostasis and protect against pathogens. ILCs produce cytokines also produced by T lymphocytes that have been shown to affect atherosclerosis, but the influence of ILCs on atherosclerosis has not been explored. APPROACH AND RESULTS: We demonstrate that CD25(+) ILCs that produce type 2 cytokines (ILC2s) are present in the aorta of atherosclerotic immunodeficient ldlr(-/-)rag1(-/-) mice. To investigate the role of ILCs in atherosclerosis, ldlr(-/-)rag1(-/-) mice were concurrently fed an atherogenic diet and treated with either ILC-depleting anti-CD90.2 antibodies or IL-2/anti-IL-2 complexes that expand CD25(+) ILCs. Lesion development was not affected by anti-CD90.2 treatment, but was reduced in IL-2/anti-IL-2-treated mice. These IL-2-treated mice had reduced very low-density lipoprotein cholesterol and increased triglycerides compared with controls and reduced apolipoprotein B100 gene expression in the liver. IL-2/anti-IL-2 treatment caused expansion of ILC2s in aorta and other tissues, elevated levels of IL-5, systemic eosinophila, and hepatic eosinophilic inflammation. Blockade of IL-5 reversed the IL-2 complex-induced eosinophilia but did not change lesion size. CONCLUSIONS: This study demonstrates that expansion of CD25-expressing ILCs by IL-2/anti-IL-2 complexes leads to a reduction in very low-density lipoprotein cholesterol and atherosclerosis. Global depletion of ILCs by anti-CD90.2 did not significantly affect lesion size indicating that different ILC subsets may have divergent effects on atherosclerosis.

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.

PD-1 protects against inflammation and myocyte damage in T cell-mediated myocarditis.

PD-1, a member of the CD28 family of immune regulatory molecules, is expressed on activated T cells, interacts with its ligands, PD-L1/B7-H1 and PD-L2/B7-DC, on other cells, and delivers inhibitory signals to the T cell. We studied the role of this pathway in modulating autoreactive T cell responses in two models of myocarditis. In a CD8(+) T cell-mediated adoptive transfer model, we found that compared with Pd1(+/+) CD8(+) T cells, Pd1(-/-) CD8(+) T cells cause enhanced disease, with increased inflammatory infiltrate, particularly rich in neutrophils. Additionally, we show enhanced proliferation in vivo and enhanced cytotoxic activity of PD-1-deficient T lymphocytes against myocardial endothelial cells in vitro. In experimental autoimmune myocarditis, a disease model dependent on CD4(+) T cells, we show that mice lacking PD-1 develop enhanced disease compared with wild-type mice. PD-1-deficient mice displayed increased inflammation, enhanced serum markers of myocardial damage, and an increased infiltration of inflammatory cells, including CD8(+) T cells. Together, these studies show that PD-1 plays an important role in limiting T cell responses in the heart.

Loss of myeloid related protein-8/14 exacerbates cardiac allograft rejection.

BACKGROUND: The calcium-binding proteins myeloid-related protein (MRP)-8 (S100A8) and MRP-14 (S100A9) form MRP-8/14 heterodimers (S100A8/A9, calprotectin) that regulate myeloid cell function and inflammatory responses and serve as early serum markers for monitoring acute allograft rejection. Despite functioning as a proinflammatory mediator, the pathophysiological role of MRP-8/14 complexes in cardiovascular disease is incompletely defined. This study investigated the role of MRP-8/14 in cardiac allograft rejection using MRP-14(-/-) mice that lack MRP-8/14 complexes. METHODS AND RESULTS: We examined parenchymal rejection after major histocompatibility complex class II allomismatched cardiac transplantation (bm12 donor heart and B6 recipients) in wild-type (WT) and MRP-14(-/-) recipients. Allograft survival averaged 5.9±2.9 weeks (n=10) in MRP-14(-/-) recipients compared with >12 weeks (n=15; P<0.0001) in WT recipients. Two weeks after transplantation, allografts in MRP-14(-/-) recipients had significantly higher parenchymal rejection scores (2.8±0.8; n=8) than did WT recipients (0.8±0.8; n=12; P<0.0001). Compared with WT recipients, allografts in MRP-14(-/-) recipients had significantly increased T-cell and macrophage infiltration and increased mRNA levels of interferon-γ and interferon-γ-associated chemokines (CXCL9, CXCL10, and CXCL11), interleukin-6, and interleukin-17 with significantly higher levels of Th17 cells. MRP-14(-/-) recipients also had significantly more lymphocytes in the adjacent para-aortic lymph nodes than did WT recipients (cells per lymph node: 23.7±0.7×10(5) for MRP-14(-/-) versus 6.0±0.2×10(5) for WT; P<0.0001). The dendritic cells (DCs) of the MRP-14(-/-) recipients of bm12 hearts expressed significantly higher levels of the costimulatory molecules CD80 and CD86 than did those of WT recipients 2 weeks after transplantation. Mixed leukocyte reactions with allo-endothelial cell-primed MRP-14(-/-) DCs resulted in significantly higher antigen-presenting function than reactions using WT DCs. Ovalbumin-primed MRP-14(-/-) DCs augmented proliferation of OT-II (ovalbumin-specific T cell receptor transgenic) CD4(+) T cells with increased interleukin-2 and interferon-γ production. Cardiac allografts of B6 major histocompatibility complex class II(-/-) hosts and of B6 WT hosts receiving MRP-14(-/-) DCs had significantly augmented inflammatory cell infiltration and accelerated allograft rejection compared with WT DCs from transferred recipient allografts. Bone marrow-derived MRP-14(-/-) DCs infected with MRP-8 and MRP-14 retroviral vectors showed significantly decreased CD80 and CD86 expression compared with controls, indicating that MRP-8/14 regulates B7-costimulatory molecule expression. CONCLUSIONS: Our results indicate that MRP-14 regulates B7 molecule expression and reduces antigen presentation by DCs and subsequent T-cell priming. The absence of MRP-14 markedly increased T-cell activation and exacerbated allograft rejection, indicating a previously unrecognized role for MRP-14 in immune cell biology.

Foxp3+-inducible regulatory T cells suppress endothelial activation and leukocyte recruitment.

The ability of regulatory T cells (Treg) to traffic to sites of inflammation supports their role in controlling immune responses. This feature supports the idea that adoptive transfer of in vitro expanded human Treg could be used for treatment of immune/inflammatory diseases. However, the migratory behavior of Treg, as well as their direct influence at the site of inflammation, remains poorly understood. To explore the possibility that Treg may have direct anti-inflammatory influences on tissues, independent of their well-established suppressive effects on lymphocytes, we studied the adhesive interactions between mouse Treg and endothelial cells, as well as their influence on endothelial function during acute inflammation. We show that Foxp3(+) adaptive/inducible Treg (iTreg), but not naturally occurring Treg, efficiently interact with endothelial selectins and transmigrate through endothelial monolayers in vitro. In response to activation by endothelial Ag presentation or immobilized anti-CD3ε, Foxp3(+) iTreg suppressed TNF-α- and IL-1ß-mediated endothelial selectin expression and adhesiveness to effector T cells. This suppression was contact independent, rapid acting, and mediated by TGF-ß-induced activin receptor-like kinase 5 signaling in endothelial cells. In addition, Foxp3(+) iTreg adhered to inflamed endothelium in vivo, and their secretion products blocked acute inflammation in a model of peritonitis. These data support the concept that Foxp3(+) iTreg help to regulate inflammation independently of their influence on effector T cells by direct suppression of endothelial activation and leukocyte recruitment.

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.

Statin-induced Krüppel-like factor 2 expression in human and mouse T cells reduces inflammatory and pathogenic responses.

The transcription factor Krüppel-like factor 2 (KLF2) is required for the quiescent and migratory properties of naive T cells. Statins, a class of HMG-CoA reductase inhibitors, display pleiotropic immunomodulatory effects that are independent of their lipid-lowering capacity and may be beneficial as therapeutic agents for T cell-mediated inflammatory diseases. Statins upregulate KLF2 expression in endothelial cells, and this activity is associated with an antiinflammatory phenotype. We therefore hypothesized that the immunomodulatory effects of statins are due, in part, to their direct effects on T cell KLF2 gene expression. Here we report that lipophilic statin treatment of mouse and human T cells increased expression of KLF2 through a HMG-CoA/prenylation-dependent pathway. Statins also diminished T cell proliferation and IFN-gamma expression. shRNA blockade of KLF2 expression in human T cells increased IFN-gamma expression and prevented statin-induced IFN-gamma reduction. In a mouse model of myocarditis induced by heart antigen-specific CD8+ T cells, both statin treatment of the T cells and retrovirally mediated overexpression of KLF2 in the T cells had similar ameliorating effects on disease induction. We conclude that statins reduce inflammatory functions and pathogenic activity of T cells through KLF2-dependent mechanisms, and this pathway may be a potential therapeutic target for cardiovascular diseases.

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-gamma, and blocking of interferon-gamma 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.

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.

CTLA-4 ablation and interleukin-12 driven differentiation synergistically augment cardiac pathogenicity of cytotoxic T lymphocytes.

CD8+ cytotoxic T lymphocytes contribute to viral and autoimmune myocarditis and cardiac allograft rejection. The role of cytotoxic T-lymphocyte-associated antigen (CTLA)-4 as a negative regulator of CD4+ T cells is well defined, yet CTLA-4 regulation of CD8+ T cells is less clear. We studied CTLA-4 regulation of cytotoxic T lymphocytes in a transgenic model of CD8+ T-cell-mediated myocarditis. We generated CTLA-4(-/-) Rag 2(-/-) OT-1 mice, the CD8+ T cells of which express an ovalbumin (OVA) peptide-specific, class I major histocompatibility complex-restricted T-cell receptor. CTLA-4(-/-Tc12) OT-1 effectors, differentiated with interleukin-12 present, are hyperproliferative in vitro, compared with CTLA-4(+/+)Tc12 OT-1 controls. Transfer of low doses of CTLA-4(-/-Tc12) OT-1 cells to cMy-mOVA mice, which express OVA on cardiac myocytes, causes severe myocarditis, with 99% mortality, compared with no mortality after transfer of low doses of CTLA-4(+/+)Tc12 OT-1 cells. High doses of CTLA-4(+/+)Tc12 cells cause lethal myocarditis in cMy-mOVA mice, but high doses of CTLA-4(+/+)Tc0 CTL, generated without interleukin-12, are hypoproliferative within the cardiac-draining lymph node and do not significantly infiltrate the heart. In contrast, CTLA-4(-/-Tc0) cytotoxic T lymphocytes do proliferate in the cardiac-draining lymph node and diffusely infiltrate the heart. Nonetheless, high doses of CTLA-4(-/-Tc0) cells cause only limited tissue damage, and the disease is not lethal. These data show that CTLA-4 regulates myocarditic CD8+ T cell responses and that CTLA-4 deficiency partly overcomes a differentiation block that exists when naïve CD8+ T cells are stimulated without interleukin-12. Therefore, targeting CTLA-4 solely or in conjunction with interleukin-12 could influence effector CD8+ T cell responses in therapeutically beneficial ways.

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 marrow 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-gamma and tumor necrosis factor-alpha 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.

T-bet controls pathogenicity of CTLs in the heart by separable effects on migration and effector activity.

CD8+ CTL contribute to the pathogenesis of myocarditis and cardiac allograft rejection. Using a transgenic model of myocarditis, we examined the role of the transcription factor T-bet in the differentiation of pathogenic cardiac Ag-specific CTL. We demonstrate that T-bet-deficient CTL are significantly impaired in their ability to cause disease, despite intact proliferation and activation phenotypes. In the absence of T-bet, there is markedly reduced expression of the chemokine receptor CXCR3, and CXCR3-gene knockout CTL are significantly less pathogenic than control CTL. Retroviral-mediated CXCR3 expression in T-bet-deficient CD8+ T cells reconstitutes their ability to infiltrate but not to damage the heart, establishing that CD8+ T cell pathogenicity is related to T-bet-dependent CXCR3 expression, reduced cytotoxicity, and enhanced regulation. These findings highlight the potential therapeutic benefit of targeting T-bet-regulated gene expression and CXCR3-dependent migration in immune-mediated heart disease.

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.

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.

Neutrophils sustain pathogenic CD8+ T cell responses in the heart.

This study explores the influence of innate immunity on CD8(+) T-cell responses against heart tissue. Adoptive transfer of ovalbumin-specific CD8(+) effector T cells into CMy-mOva mice, which express ovalbumin in cardiac myocytes, results in a lethal acute myocarditis. The inflammatory infiltrate in the heart includes neutrophils as well as T cells. We used anti-Ly6G antibody to transiently deplete neutrophils at the time of onset of disease. By day 7 after receiving 5 x 10(5) CD8(+) effector T cells, 100% of control Ig-treated CMy-mOva mice had died, while 85% of anti-Ly6G-treated mice survived indefinitely. CD8(+) T-cell infiltration and tissue damage were present in both groups, but the disease was limited in the anti-Ly6G-treated mice, with a rapid disappearance of the adoptively transferred CD8(+) T cells within 11 days. Recovery occurred even though blood neutrophil counts began to rise 48 hours after the last anti-Ly6G treatment. Recovery was associated with a chronic CD4(+) cell infiltrate, and a rapid decline in expression of IFN-gamma and IP-10 mRNA in the myocardium. Neutrophil depletion did not effect survival of CMy-mOva mice that received 3 x 10(6) CD8(+) T cells. These data show that granulocytic inflammation sustains CD8(+) T-cell-mediated heart disease, which has important implications for the pathogenesis and treatment of acute myocarditis and allograft rejection.

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+ T cells 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-gamma 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-alpha. 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-gamma secretion and cytolytic activity of CD8+ T cells in response to endothelial cell antigen presentation. These results show that IFN-gamma 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.

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.