Evolution of recombinant lymphocytic choriomeningitis virus/Lassa virus in vivo highlights the importance of the GPC cytosolic tail in viral fitness.

UNLABELLED: A key characteristic of arenaviruses is their ability to establish persistent infection in their natural host. Different factors like host age, viral dose strain, and route of infection may contribute to the establishment of persistence. However, the molecular mechanisms governing persistence are not fully understood. Here, we describe gain-of-function mutations of lymphocytic choriomeningitis virus (LCMV) expressing Lassa virus (LASV) GP, which can prolong viremia in mice depending on the sequences in the GP-2 cytoplasmic tail. The initial mutant variant (rLCMV/LASV mut GP) carried a point mutation in the cytosolic tail of the LASV glycoprotein GP corresponding to a K461G substitution. Unlike what occurred with the original rLCMV/LASV wild-type (wt) GP, infection of C57BL/6 mice with the mutated recombinant virus led to a detectable viremia of 2 weeks' duration. Further replacement of the entire sequence of the cytosolic tail from LASV to LCMV GP resulted in increased viral titers and delayed clearance of the viruses. Biosynthesis and cell surface localization of LASV wt and mut GPs were comparable. IMPORTANCE: Starting from an emerging virus in a wild-type mouse, we engineered a panel of chimeric Lassa/lymphocytic choriomeningitis viruses. Mutants carrying a viral envelope with the cytosolic tail from the closely related mouse-adapted LCMV were able to achieve a productive viral infection lasting up to 27 days in wild-type mice. Biochemical assays showed a comparable biosynthesis and cell surface localization of LASV wt and mut GPs. These recombinant chimeric viruses could allow the study of immune responses and antivirals targeting the LASV GP.

Adaptive immune response in JAM-C-deficient mice: normal initiation but reduced IgG memory.

We have recently shown that junctional adhesion molecule (JAM)-C-deficient mice have leukocytic pulmonary infiltrates, disturbed neutrophil homeostasis, and increased postnatal mortality. This phenotype was partially rescued when mice were housed in ventilated isolators, suggesting an inability to cope with opportunistic infections. In the present study, we further examined the adaptive immune responses in JAM-C(-/-) mice. We found that murine conventional dendritic cells express in addition to Mac-1 and CD11c also JAM-B as ligand for JAM-C. By in vitro adhesion assay, we show that murine DCs can interact with recombinant JAM-C via Mac-1. However, this interaction does not seem to be necessary for dendritic cell migration and function in vivo, even though JAM-C is highly expressed by lymphatic sinuses of lymph nodes. Nevertheless, upon immunization and boosting with a protein Ag, JAM-C-deficient mice showed decreased persistence of specific circulating Abs although the initial response was normal. Such a phenotype has also been observed in a model of Ag-induced arthritis, showing that specific IgG2a Ab titers are reduced in the serum of JAM-C(-/-) compared with wild-type mice. Taken together, these data suggest that JAM-C deficiency affects the adaptive humoral immune response against pathogens, in addition to the innate immune system.

JAM-C induces endothelial cell permeability through its association and regulation of {beta}3 integrins.

OBJECTIVE: The molecular mechanisms regulating vascular permeability are only now being elucidated. The junctional adhesion molecule (JAM) JAM-C has been linked to the induction of vascular permeability. We sought to understand the mechanism whereby JAM-C may disrupt junctional integrity in endothelial cells (ECs). METHODS AND RESULTS: We show here that JAM-C alters permeability through modulation of integrin activity. JAM-C overexpression results in an increase in JAM-C at junctions and an increase in permeability. Conversely, knockdown of JAM-C by siRNA results in a reduction in permeability. JAM-C associates with alphavbeta3 integrin and regulates its localization and activity. JAM-C also inhibits the activation state of the beta(1) integrin although it does not associate with this integrin. These changes induced on the integrins are mediated through regulation of the small GTPase, Rap1b but not Rap1a. Thrombin, a powerful inductor of vascular leak, causes localization of JAM-C into the junctions, whereas angiopoietin-1, an inhibitor of permeability, prevents JAM-C translocation. CONCLUSIONS: The regulation of EC junctional integrity involves the coordinated and dynamic modification of localization and activity of junctional stabilizers such as the integrin beta(3) and the destabilizer, JAM-C.

Double-negative regulatory T cells: non-conventional regulators.

The crucial role of regulatory T (Treg) cells in self-tolerance and downregulating immune responses has been clearly established. Numerous different Treg subsets have been identified that possess distinct phenotypes and functions in various disease models. Among these subsets, alphabeta-TCR+CD3+CD4-CD8- double-negative (DN) Treg cells have been shown to be able to inhibit a variety of immune responses in part via direct killing of effector T cells in an antigenspecific manner in both mice and humans. This was shown to occur at least partially by acquisition of MHC-peptide complexes from antigen-presenting cells (APCs) and subsequent Fas/Fas-ligand interactions. In addition, DN Treg cells have been shown to express several molecules uncommon to other Treg cell subsets, such as IFN-gamma, TNF-alpha, Ly6A, FcRgamma, and CXCR5, which may contribute to their unique regulatory ability. Understanding the development and regulatory functions of DN Treg cells may elucidate the etiology for loss of self-tolerance and serve as a therapeutic modality for various diseases. This review will summarize the characteristics, developmental pathways, and mechanisms of action of DN Treg cells, as well as their role in transplant tolerance, autoimmunity, and anticancer immunity.

Divergent JAM-C Expression Accelerates Monocyte-Derived Cell Exit from Atherosclerotic Plaques.

Atherosclerosis, caused in part by monocytes in plaques, continues to be a disease that afflicts the modern world. Whilst significant steps have been made in treating this chronic inflammatory disease, questions remain on how to prevent monocyte and macrophage accumulation in atherosclerotic plaques. Junctional Adhesion Molecule C (JAM-C) expressed by vascular endothelium directs monocyte transendothelial migration in a unidirectional manner leading to increased inflammation. Here we show that interfering with JAM-C allows reverse-transendothelial migration of monocyte-derived cells, opening the way back out of the inflamed environment. To study the role of JAM-C in plaque regression we used a mouse model of atherosclerosis, and tested the impact of vascular JAM-C expression levels on monocyte reverse transendothelial migration using human cells. Studies in-vitro under inflammatory conditions revealed that overexpression or gene silencing of JAM-C in human endothelium exposed to flow resulted in higher rates of monocyte reverse-transendothelial migration, similar to antibody blockade. We then transplanted atherosclerotic, plaque-containing aortic arches from hyperlipidemic ApoE-/- mice into wild-type normolipidemic recipient mice. JAM-C blockade in the recipients induced greater emigration of monocyte-derived cells and further diminished the size of atherosclerotic plaques. Our findings have shown that JAM-C forms a one-way vascular barrier for leukocyte transendothelial migration only when present at homeostatic copy numbers. We have also shown that blocking JAM-C can reduce the number of atherogenic monocytes/macrophages in plaques by emigration, providing a novel therapeutic strategy for chronic inflammatory pathologies.

Targeting olfactomedin-like 3 inhibits tumor growth by impairing angiogenesis and pericyte coverage.

Antiangiogenic drugs have been used as anticancer agents to target tumor endothelial cells or pericytes. Because of limited efficacy of the current monotherapies, there is a strong demand for the dual targeting of endothelial cells and pericytes. Here, we identify Olfactomedin-like 3 (Olfml3) as a novel proangiogenic cue within the tumor microenvironment. Tumor-derived Olfml3 is produced by both tumor endothelial cells and accompanying pericytes and deposited in the perivascular compartment. Blockade of Olfml3 by anti-Olfml3 antibodies is highly effective in reducing tumor vascularization, pericyte coverage, and tumor growth. In vitro, Olfml3 targeting is sufficient to inhibit endothelioma cell migration and sprouting. Olfml3 alone or through binding to BMP4 enhances the canonical SMAD1/5/8 signaling pathway required for BMP4-induced angiogenesis. Therefore, Olfml3 blockade provides a novel strategy to control tumor growth by targeting two distinct cell types within the tumor microenvironment using a single molecule.

Prostaglandin E2 signaling through E prostanoid receptor 2 impairs proliferative response of double negative regulatory T cells.

Limited data are available on the mechanisms that constrain the function of regulatory populations of T cells. Prostaglandin E2 (PGE2) is an endogenous membrane phospholipid metabolite that has important immunomodulatory effects on T cell function. Our previous microarray data indicated that E prostanoid receptor 2 (EP2), a receptor for PGE2, is expressed by regulatory alphabetaTCR(+) CD4(-) CD8(-) NK1.1(-) double negative T (DN Treg) cell clones but not by their non-regulatory natural mutants. Hence, the hypothesis that PGE2 may influence DN Treg cell proliferation and/or regulatory function was tested in this study. Our data indicate that PGE2 acts via the EP2 receptor on DN Treg cells to inhibit their proliferation, an effect reproduced by the EP2-specific agonist butaprost and abrogated by the EP2 antagonist AH6809. In contrast, PGE2 did not affect the ability of DN Treg cells to kill syngeneic CD8(+) T cells activated by allogeneic stimulation. Together, these findings suggest a role for PGE2 in limiting the expansion of DN Treg cells.

Vascular and epithelial junctions: a barrier for leucocyte migration.

Rapid mobilization of leucocytes through endothelial and epithelial barriers is key in immune system reactivity. The underlying mechanisms that regulate these processes have been the basis for many recent studies. Traditionally, leucocyte extravasation had been believed to occur through a paracellular route, which involves localized disruption of endothelial cell junctions. However, more recently, a transcellular route has been described involving the passage through the endothelial cell body. Leucocytes are also able to migrate through epithelium to monitor mucosal tissues and microenvironments. A number of adhesion molecules are known to regulate transmigration of leucocytes through epithelial and endothelial layers. Paracellular and transcellular leucocyte transmigration are regulated by adhesion molecules such as PECAM-1 (platelet-endothelial cell adhesion molecule 1), CD99, VE-cadherin (vascular endothelial cadherin) and JAM (junctional adhesion molecule) proteins. The purpose of this review is to discuss the role of these molecules in leucocyte transmigration and how they contribute to the different mechanisms that regulate leucocyte trafficking.

FcR gamma presence in TCR complex of double-negative T cells is critical for their regulatory function.

TCRalphabeta+CD4-CD8- double-negative (DN) T regulatory (Treg) cells have recently been shown to suppress Ag-specific immune responses mediated by CD8+ and CD4+ T cells in humans and mice. Our previous study using cDNA microarray analysis of global gene expression showed that FcRgamma was the most highly overexpressed gene in functional DN Treg cell clones compared with nonfunctional mutant clones. In this study, we demonstrate that FcRgamma-deficient DN T cells display markedly reduced suppressive activity in vitro. In addition, unlike FcRgamma-sufficient DN T cells, FcRgamma-deficient DN T cells were unable to prolong donor-specific allograft survival when adoptively transferred to recipient mice. Protein analyses indicate that in addition to FcRgamma, DN Treg cell clones also express higher levels of TCRbeta, while mutant clones expressed higher levels of Zap70 and Lck. Within DN Treg cells, we found that FcRgamma associates with the TCR complex and that both FcRgamma and Syk are phosphorylated in response to TCR cross-linking. Inhibition of Syk signaling and FcRgamma expression were both found to reduce the suppressive function of DN Treg cells in vitro. These results indicate that FcRgamma deficiency significantly impairs the ability of DN Treg cells to down-regulate allogeneic immune responses both in vitro and in vivo, and that FcRgamma plays a role in mediating TCR signaling in DN Treg cells.

CXCR5/CXCL13 interaction is important for double-negative regulatory T cell homing to cardiac allografts.

Accumulating evidence indicates that regulatory T (Treg) cells control development of various diseases both systemically and locally. However, molecular mechanisms involved in Treg cell homing remain elusive. We have shown previously that alphabetaTCR(+)CD3(+)CD4(-)CD8(-) double-negative (DN) Treg cells selectively accumulate in tolerant allografts to maintain localized immune regulation. However, the molecular mechanism leading to the accumulation of DN Treg cells in tolerant grafts was not known. Our cDNA microarray analysis revealed significant up-regulation of chemokine receptor CXCR5 mRNA in DN Treg clones compared with nonregulatory clones. In this study, we examined the importance of CXCR5 in mediating DN Treg migration. Compared with CD4 and CD8 T cells, both primary DN Treg cells and clones constitutively express high levels of CXCR5 protein, enabling them to migrate toward increasing CXCL13 gradients in vitro. After infusion into recipient mice, CXCR5(+) DN Treg clones, but not their CXCR5(-) mutants, preferentially accumulated in cardiac allografts and could prevent graft rejection. Furthermore, we found that allogeneic cardiac allografts express high levels of CXCL13 mRNA compared with either recipient native hearts or nontransplanted donor hearts. Ab neutralization of CXCL13 abrogated DN Treg cell migration in vitro and prevented in vivo homing of DN Treg clones into allografts. These data demonstrate that DN Treg cells preferentially express CXCR5, and interaction of this chemokine receptor with its ligand CXCL13 plays an important role in DN Treg cell migration both in vitro and in vivo.

Expression profiling of murine double-negative regulatory T cells suggest mechanisms for prolonged cardiac allograft survival.

Recent studies have demonstrated that both mouse and human alpha beta TCR(+)CD3(+)NK1.1(-)CD4(-)CD8- double-negative regulatory T (DN Treg) cells can suppress Ag-specific immune responses mediated by CD8+ and CD4+ T cells. To identify molecules involved in DN Treg cell function, we generated a panel of murine DN Treg clones, which specifically kill activated syngeneic CD8+ T cells. Through serial cultivation of DN Treg clones, mutant clones arose that lost regulatory capacity in vitro and in vivo. Although all allogeneic cardiac grafts in animals preinfused with tolerant CD4/CD8 negative 12 DN Treg clones survived over 100 days, allograft survival is unchanged following infusion of mutant clones (19.5 +/- 11.1 days) compared with untreated controls (22.8 +/- 10.5 days; p < 0.001). Global gene expression differences between functional DN Treg cells and nonfunctional mutants were compared. We found 1099 differentially expressed genes (q < 0.025%), suggesting increased cell proliferation and survival, immune regulation, and chemotaxis, together with decreased expression of genes for Ag presentation, apoptosis, and protein phosphatases involved in signal transduction. Expression of 33 overexpressed and 24 underexpressed genes were confirmed using quantitative real-time PCR. Protein expression of several genes, including Fc epsilon RI gamma subunit and CXCR5, which are >50-fold higher, was also confirmed using FACS. These findings shed light on the mechanisms by which DN Treg cells down-regulate immune responses and prolong cardiac allograft survival.