Tatyana Chernova

PD-L2 is a second ligand for PD-1 and inhibits T cell activation.

Programmed death 1 (PD-1)–deficient mice develop a variety of autoimmune-like diseases, which suggests that this immunoinhibitory receptor plays an important role in tolerance. We identify here PD-1 ligand 2 (PD-L2) as a second ligand for PD-1 and compare the function and expression of PD-L1 and PD-L2. Engagement of PD-1 by PD-L2 dramatically inhibits T cell receptor (TCR)-mediated proliferation and cytokine production by CD4+ T cells. At low antigen concentrations, PD-L2–PD-1 interactions inhibit strong B7-CD28 signals. In contrast, at high antigen concentrations, PD-L2–PD-1 interactions reduce cytokine production but do not inhibit T cell proliferation. PD-L–PD-1 interactions lead to cell cycle arrest in G0/G1 but do not increase cell death. In addition, ligation of PD-1 + TCR leads to rapid phosphorylation of SHP-2, as compared to TCR ligation alone. PD-L expression was up-regulated on antigen-presenting cells by interferon γ treatment and was also present on some normal tissues and tumor cell lines. Taken together, these studies show overlapping functions of PD-L1 and PD-L2 and indicate a key role for the PD-L–PD-1 pathway in regulating T cell responses.

ICOS is critical for CD40-mediated antibody class switching

The inducible co-stimulatory molecule (ICOS) is a CD28 homologue implicated in regulating T-cell differentiation. Because co-stimulatory signals are critical for regulating T-cell activation, an understanding of co-stimulatory signals may enable the design of rational therapies for immune-mediated diseases. According to the two-signal model for T-cell activation, T cells require an antigen-specific signal and a second, co-stimulatory, signal for optimal T-cell activation. The co-stimulatory signal promotes T-cell proliferation, lymphokine secretion and effector function. The B7–CD28 pathway provides essential signals for T-cell activation, but does not account for all co-stimulation. We have generated mice lacking ICOS (ICOS-/- ) to determine the essential functions of ICOS. Here we report that ICOS-/- mice exhibit profound deficits in immunoglobulin isotype class switching, accompanied by impaired germinal centre formation. Class switching was restored in ICOS-/- mice by CD40 stimulation, showing that ICOS promotes T-cell/B-cell collaboration through the CD40/CD40L pathway.

Mouse Inducible Costimulatory Molecule (ICOS) Expression Is Enhanced by CD28 Costimulation and Regulates Differentiation of CD4+ T Cells

The inducible costimulatory (ICOS) molecule is expressed by activated T cells and has homology to CD28 and CD152. ICOS binds B7h, a molecule expressed by APC with homology to CD80 and CD86. To investigate regulation of ICOS expression and its role in Th responses we developed anti-mouse ICOS mAbs and ICOS-Ig fusion protein. Little ICOS is expressed by freshly isolated mouse T cells, but ICOS is rapidly up-regulated on most CD4+ and CD8+ T cells following stimulation of the TCR. Strikingly, ICOS up-regulation is significantly reduced in the absence of CD80 and CD86 and can be restored by CD28 stimulation, suggesting that CD28-CD80/CD86 interactions may optimize ICOS expression. Interestingly, TCR-transgenic T cells differentiated into Th2 expressed significantly more ICOS than cells differentiated into Th1. We used two methods to investigate the role of ICOS in activation of CD4+ T cells. First, CD4+ cells were stimulated with beads coated with anti-CD3 and either B7h-Ig fusion protein or control Ig fusion protein. ICOS stimulation enhanced proliferation of CD4+ cells and production of IFN-γ, IL-4, and IL-10, but not IL-2. Second, TCR-transgenic CD4+ T cells were stimulated with peptide and APC in the presence of ICOS-Ig or control Ig. When the ICOS:B7h interaction was blocked by ICOS-Ig, CD4+ T cells produced more IFN-γ and less IL-4 and IL-10 than CD4+ cells differentiated with control Ig. These results demonstrate that ICOS stimulation is important in T cell activation and that ICOS may have a particularly important role in development of Th2 cells.

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.

Nitric Oxide Signaling in Brain Function, Dysfunction, and Dementia

Nitric oxide (NO) is an important signaling molecule that is widely used in the nervous system. With recognition of its roles in synaptic plasticity (long-term potentiation, LTP; long-term depression, LTD) and elucidation of calcium-dependent, NMDAR-mediated activation of neuronal nitric oxide synthase (nNOS), numerous molecular and pharmacological tools have been used to explore the physiology and pathological consequences for nitrergic signaling. In this review, the authors summarize the current understanding of this subtle signaling pathway, discuss the evidence for nitrergic modulation of ion channels and homeostatic modulation of intrinsic excitability, and speculate about the pathological consequences of spillover between different nitrergic compartments in contributing to aberrant signaling in neurodegenerative disorders. Accumulating evidence points to various ion channels and particularly voltage-gated potassium channels as signaling targets, whereby NO mediates activity-dependent control of intrinsic neuronal excitability; such changes could underlie broader mechanisms of synaptic plasticity across neuronal networks. In addition, the inability to constrain NO diffusion suggests that spillover from endothelium (eNOS) and/or immune compartments (iNOS) into the nervous system provides potential pathological sources of NO and where control failure in these other systems could have broader neurological implications. Abnormal NO signaling could therefore contribute to a variety of neurodegenerative pathologies such as stroke/excitotoxicity, Alzheimer’s disease, multiple sclerosis, and Parkinson’s disease.

Pulmonary toxicity of carbon nanotubes and asbestos - similarities and differences.

Carbon nanotubes are a valuable industrial product but there is potential for human pulmonary exposure during production and their fibrous shape raises the possibility that they may have effects like asbestos, which caused a worldwide pandemic of disease in the20th century that continues into present. CNT may exist as fibres or as more compact particles and the asbestos-type hazard only pertains to the fibrous forms of CNT. Exposure to asbestos causes asbestosis, bronchogenic carcinoma, mesothelioma, pleural fibrosis and pleural plaques indicating that both the lungs and the pleura are targets. The fibre pathogenicity paradigm was developed in the 1970s-80s and has a robust structure/toxicity relationship that enables the prediction of the pathogenicity of fibres depending on their length, thickness and biopersistence. Fibres that are sufficiently long and biopersistent and that deposit in the lungs can cause oxidative stress and inflammation. They may also translocate to the pleura where they can be retained depending on their length, and where they cause inflammation and oxidative stress in the pleural tissues. These pathobiological processes culminate in pathologic change - fibroplasia and neoplasia in the lungs and the pleura. There may also be direct genotoxic effects of fibres on epithelial cells and mesothelium, contributing to neoplasia. CNT show some of the properties of asbestos and other types of fibre in producing these types of effects and more research is needed. In terms of the molecular pathways involved in the interaction of long biopersistent fibres with target tissue the events leading to mesothelioma have been a particular area of interest. A variety of kinase pathways important in proliferation are activated by asbestos leading to pre-malignant states and investigations are under way to determine whether fibrous CNT also affects these molecular pathways. Current research suggests that fibrous CNT can elicit effects similar to asbestos but more research is needed to determine whether they, or other nanofibres, can cause fibrosis and cancer in the long term.

NMDAR‐mediated EPSCs are maintained and accelerate in time course during maturation of mouse and rat auditory brainstem in vitro

NMDA receptors (NMDARs) mediate a slow EPSC at excitatory glutamatergic synapses throughout the brain. In many areas the magnitude of the NMDAR‐mediated EPSC declines with development and is associated with changes in subunit composition, but the mature channel composition is often unknown. We have employed the calyx of Held terminal with its target, the principal neuron of the medial nucleus of the trapezoid body (MNTB), to examine the NMDAR‐mediated EPSC during synapse maturation from P10 to P40. Our data show that the calyx has reached a mature state by around P18. The NMDAR‐mediated EPSC amplitude (and dominant decay τ) fell from around 5 nA (τ: 40–50 ms) at P10/11 to 0.3–0.5 nA (τ: 10–15 ms) by P18. The mature NMDAR‐EPSC showed no sensitivity to ifenprodil, indicating lack of NR2B subunits, and no block by submicromolar concentrations of zinc, consistent with NR1‐1b subunit expression. Additionally, from P11 to P18 there was a reduction in voltage‐dependent block and the apparent dissociation constant for [Mg2+]o (Ko) changed from 7.5 to 14 mm. Quantitative PCR showed that the relative expression of NR2A and NR2C increased, while immunohistochemistry confirmed the presence of NR2A, NR2B and NR2C protein. Although the mature NMDAR‐EPSC is small, it is well coupled to NO signalling, as indicated by DAR‐4M imaging. We conclude that native mature NMDAR channels at the calyx of Held have a fast time course and reduced block by [Mg2+]o, consistent with dominance of NR2C subunits and functional exclusion of NR2B subunits. The pharmacology suggests a single channel type and we postulate that the mature NMDARs consist of heterotrimers of NR1‐1b–NR2A–NR2C.

Neurite Degeneration Induced by Heme Deficiency Mediated via Inhibition of NMDA Receptor-Dependent Extracellular Signal-Regulated Kinase 1/2 Activation

The early stages of many neurodegenerative diseases and age-related degeneration are characterized by neurite damage and compromised synaptic function that precede neuronal cell death. We investigated the signaling mechanisms underlying neurite degeneration using cortical neuron cultures. Inhibition of heme synthesis caused neurite damage, without neuronal death, and was mediated by reduced NMDA receptor (NMDAR) expression and phosphorylation. The signaling toward the degenerative phenotype involved suppression of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, and electrophysiological recording showed that the neurodegeneration is accompanied by reduced NMDAR current and Ca2+ influx, as well as reduced voltage-gated sodium currents, consistent with compromised neurite integrity. Rescue from the degenerative phenotype by heme replacement was dependent on restoration of NR2B subunit phosphorylation and expression of NMDAR currents with higher Ca2+ permeability, consistent with triggering prosurvival ERK1/2 signaling to maintain and extend neurites. This study demonstrated a new mechanism of neurodegeneration in which impaired heme synthesis led to NMDAR signaling dysfunction, suppression of the prosurvival ERK1/2 pathway, and progressive fragmentation of neuronal projections.

Hepatic Gene Expression in Protoporphyic Fech Mice Is Associated with Cholestatic Injury but Not a Marked Depletion of the Heme Regulatory Pool

BALB/c Fech(m1Pas) mice have a mutated ferrochelatase gene resulting in protoporphyria that models the hepatic injury occurring sporadically in human erythropoietic protoporphyria. We used this mouse model to study the development of the injury and to compare the dysfunction of heme synthesis with hepatic gene expression of liver metabolism, oxidative stress, and cellular injury/inflammation. From an early age expression of total cytochrome P450 and many of its isoforms was significantly lower than in wild-type mice. However, despite massive accumulation of protoporphyrin in the liver, expression of the main genes controlling heme synthesis and catabolism (Alas1 and Hmox1, respectively) were only modestly affected even in the presence of the cytochrome P450-inducing CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene. In contrast, in BALB/c mice exhibiting griseofulvin-induced hepatic protoporphyria with induction and destruction of cytochrome P450, both Alas1 and Hmox1 genes were markedly up-regulated. Other expression profiles in BALB/c Fech(m1Pas) mice identified roles for oxidative mechanisms in liver injury while modulated gene expression of hepatocyte transport proteins and cholesterol and bile acid synthesis illustrated the development of cholestasis. Subsequent inflammation and cirrhosis were also shown by the up-regulation of cytokine, cell cycling, and procollagen genes. Thus, gene expression profiles studied in Fech(m1Pas) mice may provide candidates for human polymorphisms that explain the sporadic hepatic consequences of erythropoietic protoporphyria.

Regulation of Kv channel expression and neuronal excitability in rat medial nucleus of the trapezoid body maintained in organotypic culture.

Principal neurons of the medial nucleus of the trapezoid body (MNTB) express a spectrum of voltage‐dependent K+ conductances mediated by Kv1–Kv4 channels, which shape action potential (AP) firing and regulate intrinsic excitability. Postsynaptic factors influencing expression of Kv channels were explored using organotypic cultures of brainstem prepared from P9–P12 rats and maintained in either low (5 mm, low‐K) or high (25 mm, high‐K) [K+]o medium. Whole cell patch‐clamp recordings were made after 7–28 days in vitro. MNTB neurons cultured in high‐K medium maintained a single AP firing phenotype, while low‐K cultures had smaller K+ currents, enhanced excitability and fired multiple APs. The calyx of Held inputs degenerated within 3 days in culture, having lost their major afferent input; this preparation of calyx‐free MNTB neurons allowed the effects of postsynaptic depolarisation to be studied with minimal synaptic activity. The depolarization caused by the high‐K aCSF only transiently increased spontaneous AP firing (<2 min) and did not measurably increase synaptic activity. Chronic depolarization in high‐K cultures raised basal levels of [Ca2+]i, increased Kv3 currents and shortened AP half‐widths. These events relied on raised [Ca2+]i, mediated by influx through voltage‐gated calcium channels (VGCCs) and release from intracellular stores, causing an increase in cAMP‐response element binding protein (CREB) phosphorylation. Block of VGCCs or of CREB function suppressed Kv3 currents, increased AP duration, and reduced Kv3.3 and c‐fos expression. Real‐time PCR revealed higher Kv3.3 and Kv1.1 mRNA in high‐K compared to low‐K cultures, although the increased Kv1.1 mRNA was mediated by a CREB‐independent mechanism. We conclude that Kv channel expression and hence the intrinsic membrane properties of MNTB neurons are homeostatically regulated by [Ca2+]i‐dependent mechanisms and influenced by sustained depolarization of the resting membrane potential.