T-cell Activation Responses Research Articles

Inhibitory action of gonadotropin-inhibitory hormone on the signaling pathways induced by kisspeptin and vasoactive intestinal polypeptide in GnRH neuronal cell line, GT1-7.

Gonadotropin-inhibitory hormone (GnIH) acts as a negative regulator of reproduction by acting on gonadotropes and gonadotropin-releasing hormone (GnRH) neurons. Despite its functional significance, the molecular mechanism of GnIH action in the target cells has not been fully elucidated. To expand our previous study on GnIH actions in gonadotropes, we investigated the potential signal transduction pathway that conveys the inhibitory action of GnIH in GnRH neurons by using the GnRH neuronal cell line, GT1-7. We examined whether GnIH inhibits the action of kisspeptin and vasoactive intestinal polypeptide (VIP), positive regulators of GnRH neurons. Although GnIH significantly suppressed the stimulatory effect of kisspeptin on GnRH release in hypothalamic culture, GnIH had no inhibitory effect on kisspeptin stimulation of serum response element and nuclear factor of activated T-cell response element activities and ERK phosphorylation, indicating that GnIH may not directly inhibit kisspeptin signaling in GnRH neurons. On the contrary, GnIH effectively eliminated the stimulatory effect of VIP on p38 and ERK phosphorylation, c-Fos mRNA expression, and GnRH release. The use of pharmacological modulators strongly demonstrated the specific inhibitory action of GnIH on the adenylate cyclase/cAMP/protein kinase A pathway, suggesting a common inhibitory mechanism of GnIH action in GnRH neurons and gonadotropes.-Son, Y. L., Ubuka, T., Soga, T., Yamamoto, K., Bentley, G. E., Tsutsui, K. Inhibitory action of gonadotropin-inhibitory hormone on the signaling pathways induced by kisspeptin and vasoactive intestinal polypeptide in GnRH neuronal cell line, GT1-7.

Abstract 18244: Macrophages Infiltrate the Heart With Activation of Adaptive Immunity During Compensated Pressure Overload Hypertrophy

As opposed to chronic ischemic heart failure (HF), spleen-derived macrophages (Mf) are not prominent disease mediators during the chronic stages of pressure-overload (PO) HF. Increases in Mf during the early stages of PO, however, may serve as important drivers of both remodeling and activation of T-cell responses during later stages. Additionally, recent evidence indicates distinct groups of resident and circulating monocyte-derived infiltrating Mf that have different reparative vis-à-vis pro-inflammatory capacities. We hypothesized that cardiac Mf are expanded during the early stages of PO, primarily due to an increase in infiltrating Mf, and contribute to long-term T-cell activation in the PO heart. Cardiac Mf were characterized by flow cytometry as MerTK + CD64 + F480 + MHCII + cells, with infiltrating Mf populations identified as expressing chemokine receptor 2 (CCR2). One week after transverse aortic constriction (TAC), C57Bl/6 mice exhibited significant (p < 0.05) hypertrophy (heart weight/body weight 7.7 ± 0.5 vs 5.7 ± 0.2 mg/g; LV wall thickness 0.84 ± 0.03 vs 0.74 ± 0.02), without change in LVEF as compared with sham. There were robust increases in cardiac macrophages (3.15 ± 0.64 vs 1.09 ± 0.24%), specifically in the infiltrating CCR2 + MHCII hi population (1.6 ± 0.5 vs 0.5 ± 0.1%). TAC mice also exhibited higher circulating Gr1 – CD11b + Ly6C hi monocytes (2.6 ± 0.5 vs 1.3 ± 0.2%) that are recruited to tissue via CCR2. To determine activation of the adaptive immune response, we analyzed the mediastinal heart draining lymph nodes (MLN) for T-cells and antigen presenting cells. As compared with sham mice, the MLN of TAC mice were larger with greater cellularity (10.8 x 10 5 vs 4.2 x 10 5 ), especially related to MerTK + CD169 + Mf and CD3 + CD8 + T cells. We conclude that there is both an organ-specific and systemic immune response during compensatory PO hypertrophy, despite the absence of ischemic tissue injury. As infiltrating Mf are known to be less reparative, potently pro-inflammatory, and robust activators of adaptive immune cells, specifically targeting Ly6C hi monocyte recruitment and CCR2 + infiltrating Mf during the early compensated stages of pressure-overload may prevent subsequent inflammatory activation, adverse remodeling, and the transition to HF.

Cytotoxic granule secretion by lymphocytes and its link to immune homeostasis.

The granule-dependent cytotoxic activity of T and natural killer lymphocytes has progressively emerged as an important effector pathway not only for host defence but also for immune regulation. The analysis of an early-onset, severe, primary immune dysregulatory syndrome known as hemophagocytic lymphohistiocytosis (HLH) has been decisive in highlighting this latter role and identifying key effectors on the basis of gene mutation analyses and mediators in the maturation and secretion of cytotoxic granules. Studies of cytotoxicity-deficient murine counterparts have helped to define primary HLH as a syndrome in which uncontrolled T-cell activation in response to lymphocytic choriomeningitis virus infection results in excessive macrophage activation and inflammation-associated cytopenia. Recent recognition of late-onset HLH, which occurs in a variety of settings, in association with hypomorphic, monoallelic mutations in genes encoding components of the granule-dependent cytotoxic pathway or even in the absence of such mutations has broadened our view about the mechanisms that underlie the perturbation of immune homeostasis. These findings have led to the development of a model in which disease occurs when a threshold is reached through the accumulation of genetic and environmental risk factors. Nevertheless, validation of this model will require further investigations.

Toll-like receptor signaling in hematopoietic homeostasis and the pathogenesis of hematologic diseases.

Toll-like receptors (TLRs), which are found in innate immune cells, are essential mediators of rapid inflammatory responses and appropriate T-cell activation in response to infection and tissue damage. Accumulating evidence suggests that TLR signaling is involved in normal hematopoiesis and specific hematologic pathologies. Particular TLRs and their downstream signaling mediators are expressed not only in terminally differentiated innate immune cells but also in early hematopoietic progenitors. Sterile activation of TLR signaling is required to generate early embryonic hematopoietic progenitor cells. In adult animals, TLR signaling directly or indirectly promotes differentiation of myeloid cells at the expense of that of lymphoid cells and the self-renewal of hematopoietic stem cells during infection and tissue damage. Activating mutations of the MyD88 gene, which codes for a key adaptor involved in TLR signaling, are commonly detected in B-cell lymphomas and other B-cell hematopathologies. Dysregulated TLR signaling contributes to the pathogenesis of many hematopoietic disorders, including bone marrow failure, myelodysplastic syndrome, and acute myeloid leukemia. Complete elucidation of the molecular mechanisms by which TLR signaling mediates the regulation of both normal and pathogenic hematopoiesis will prove valuable to the development of targeted therapies and strategies for improved treatment of hematopoietic disorders.

CytoSource: Current Issues for Cytopathology.

CytoSource: Current Issues for Cytopathology.

Elevated serum adenosine deaminase levels in secondary hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a severe hyperinflammatory condition caused by activated T cells and macrophages. Adenosine deaminase (ADA) plays an important role in immune regulation, especially in the proliferation, maturation, and differentiation of lymphocytes. Its role has been studied in inflammation and malignancy. Here, we present for the first time the alteration of ADA in secondary HLH (sHLH). Serum ADA levels were measured in 20 cases with lymphoma-associated hemophagocytic syndrome (LAHS), 15 with infection-associated hemophagocytic syndrome (IAHS), six with macrophage activation syndrome (MAS) as experimental group. Additionally, we enrolled 20 cases with lymphoma, 15 with infection, six with autoimmunity who were all not associated with HLH as conditional control group and 20 healthy subjects as blank group. We also demonstrated the ADA levels in 20 LAHS cases and 21 benign disease-associated HLH cases (IAHS and MAS). Serum ADA levels were significantly higher in patients with LAHS, IAHS, and MAS compared to the healthy subjects (P < 0.001) and conditional control group (P < 0.05). Serum ADA levels of patients with LAHS were significantly higher than benign disease-associated HLH cases (P < 0.05). The optimum ADA cutoff point for LAHS was 89.25 U/L, with a sensitivity and specificity of 85.0% and 76.2%, respectively. Serum ADA levels were increased in sHLH suggesting a partial role of activated T-cell response in the disease pathophysiology. Serum ADA levels were particularly higher in LAHS and probably be a potential indicator of underlying lymphoma in sHLH patients.

Clinical testing of a dendritic cell targeted therapeutic vaccine in patients with chronic hepatitis C virus infection

The lack of antiviral cellular immune responses in patients with chronic hepatitis C virus (HCV) infection suggests that T-cell vaccines may provide therapeutic benefit. Due to the central role that dendritic cells (DC) play in the activation of T-cell responses, our aim was to carry out a therapeutic vaccination clinical trial in HCV patients using DC. Five patients with chronic HCV infection were vaccinated with three doses of 5 × 106 or 107 autologous DC transduced with a recombinant adenovirus encoding NS3 using the adapter protein CFh40L, which facilitates DC transduction and maturation. No significant adverse effects were recorded after vaccination. Treatment caused no changes in serum liver enzymes nor in viral load. Vaccination induced weak but consistent expansion of T-cell responses against NS3 and adenoviral antigens. Patients’ DC, as opposed to murine DC or DC from healthy subjects, secreted high IL-10 levels after transduction, inducing the activation of IL-10–producing T cells. IL-10 blockade during vaccine preparation restored its ability to stimulate anti-NS3 Th1 responses. Thus, vaccination with adenovirus-transduced DC is safe and induces weak antiviral immune responses. IL-10 associated with vaccine preparation may be partly responsible for these effects, suggesting that future vaccines should consider concomitant inhibition of this cytokine.

Immunotherapy in prostate cancer: review of the current evidence

Prostate cancer is the most common male malignancy in the Western world. Once it metastasizes, it is incurable. The current gold standard for metastatic disease is the combined docetaxel/prednisone regimen. Prostate cancer shows several characteristics that make it a suitable candidate for immunotherapy, as recently exemplified by the approval of sipuleucel-T, the first vaccine to treat any malignancy. Here, we review different tumor-associated antigen immunotherapy strategies currently being investigated, from a humanized radiolabeled monoclonal antibody (J-591) that targets radiation into tumor cells, moving on to vaccines and through to immunomodulator agents such as anti-CPLA-4 and anti-PD-1 monoclonal antibodies that activate T-cell responses via immune checkpoint inhibition. We explore different opinions on the best approach to integrate immunotherapy into existing standard therapies, such as androgen-deprivation therapy, radiotherapy or chemotherapy, and review different combination sequences, patient types and time points during the course of the disease to achieve a lasting immune response. We present data from recent phase III clinical trials that call for a change in trial endpoint design with immunotherapy agents, from the traditional tumor progression to overall survival and how such trials should include immune response measurements as secondary or intermediate endpoints to help identify patient clinical benefit in the earlier phases of treatment. Finally, we join in the recent questioning on the validity of RECIST criteria to measure response to immunotherapeutic agents, as initial increases in the size of tumors/lymph nodes, which are part of a normal immune response, could be categorized as disease progression under RECIST.

CEACAM1 regulates TIM-3-mediated tolerance and exhaustion.

T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral infection and cancers. Under some conditions, TIM-3 expression has also been shown to be stimulatory. Considering that TIM-3, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), is being targeted for cancer immunotherapy, it is important to identify the circumstances under which TIM-3 can inhibit and activate T-cell responses. Here we show that TIM-3 is co-expressed and forms a heterodimer with carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition. Biochemical, biophysical and X-ray crystallography studies show that the membrane-distal immunoglobulin-variable (IgV)-like amino-terminal domain of each is crucial to these interactions. The presence of CEACAM1 endows TIM-3 with inhibitory function. CEACAM1 facilitates the maturation and cell surface expression of TIM-3 by forming a heterodimeric interaction in cis through the highly related membrane-distal N-terminal domains of each molecule. CEACAM1 and TIM-3 also bind in trans through their N-terminal domains. Both cis and trans interactions between CEACAM1 and TIM-3 determine the tolerance-inducing function of TIM-3. In a mouse adoptive transfer colitis model, CEACAM1-deficient T cells are hyper-inflammatory with reduced cell surface expression of TIM-3 and regulatory cytokines, and this is restored by T-cell-specific CEACAM1 expression. During chronic viral infection and in a tumour environment, CEACAM1 and TIM-3 mark exhausted T cells. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses with improved elimination of tumours in mouse colorectal cancer models. Thus, CEACAM1 serves as a heterophilic ligand for TIM-3 that is required for its ability to mediate T-cell inhibition, and this interaction has a crucial role in regulating autoimmunity and anti-tumour immunity.

Abstract 4613: Identification of tumor necrosis factor receptor II as a regulatory T cell target for cancer immunotherapy using designed ankyrin repeat protein phenotypic selections

Abstract Regulatory T cells (Treg cells) are observed within multiple tumor types and contribute to immune evasion. Effective and specific targeting of Treg cells by antibodies is hampered by the lack of a unique surface marker. With the aim of identifying such a marker, human Treg cells were isolated from peripheral blood, expanded in vitro and used as a source of antigen in phage display selections with a diverse library of designed ankyrin repeat proteins (DARPins). The resulting DARPins were screened for binding to multiple cell-types, and approximately thirty with preferential binding to Treg cells were identified. All of these DARPins were determined to bind to the third and/or fourth cysteine-rich extracellular domains of tumor necrosis factor receptor II (TNFRII, also known as TNFRSF1B or CD120b). Expression of TNFRII has previously been demonstrated on Treg cells, and TNFRII is shown here also to be expressed on CD8+ T cells following exposure to cognate antigen and on the surface of tumor-infiltrating T cells in the 4T1 mouse tumor model. Furthermore, TNFRII mRNA was detected at increased levels in tumor-infiltrating leukocytes isolated from cancer patients. Functional characterisation of the anti-TNFRII DARPins and mAbs revealed an ability to enhance T-cell activation in response to polyclonal and antigen specific stimuli, and in Treg cell suppression assays. These studies demonstrate the utility of DARPins for phenotypic selections to identify drug targets using primary human lymphocytes and highlight TNFRII as a potential therapeutic target enriched on Treg cells. Citation Format: Geoff Williams, Judith Anderton, Vahe Bedian, Jane Coates Ulrichsen, Andrea Gonzalez-Munoz, Sandrine Guillard, Olivia Harris, James Hair, Andrew Leishman, Arthur Lewis, Jacques Moissan, Ralph Minter, Bina Mistry, Julie Parmentier, Edmund Poon, Amy Popple, Steve Rust, Alan Sandercock, Ross Stewart, Viia Valge-Archer, Robert W. Wilkinson. Identification of tumor necrosis factor receptor II as a regulatory T cell target for cancer immunotherapy using designed ankyrin repeat protein phenotypic selections. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4613. doi:10.1158/1538-7445.AM2014-4613

Abstract LB-157: VISTA deficiency synergizes with a non-redundant immune checkpoint pathway and leads to enhanced immune activation

Abstract V-domain Ig suppressor of T cell activation (VISTA) is a novel negative checkpoint ligand that suppresses T-cell mediated immune responses. Previous studies using VISTA-neutralizing monoclonal antibody show that VISTA-blockade enhances T cell-activation in an inflammatory disease model EAE, as well as in murine tumor models. Current study describes a comprehensive characterization of VISTA knockout (KO) mice. We show that despite the apparent normal hematopoietic development in young ko mice, VISTA genetic deficiency leads to a pro-inflammatory phenotype in aged animals, as well as enhanced T-cell activation in response to acute antigen immunization. In addition, we show that VISTA deficiency significantly enhanced disease development in a spontaneous model of autoimmune disease, which is correlated with the spontaneous activation of auto-antigen specific CD4+ T cells. Lastly, when combined with the genetic deficiency of another checkpoint molecule, synergistic or additive immune activation was observed. The implication of such enhanced immunity in cancer development and treatment will be discussed. Citation Format: Li Wang, Isabelle LeMercier, Arief Suriawinata, Wenna Chen, Randolph J. Noelle, Jiannan Li. VISTA deficiency synergizes with a non-redundant immune checkpoint pathway and leads to enhanced immune activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-157. doi:10.1158/1538-7445.AM2014-LB-157

Characterization of the modes of action of deoxynivalenol (DON) in the human Jurkat T-cell line

Deoxynivalenol (DON) is one of the most abundant mycotoxins worldwide and mostly detected in cereals and grains. As such, DON poses a risk for many adverse health effects to human and animals. In particular, immune cells are very sensitive to DON, with the initiating step leading to toxicity being a binding to the eukaryotic 60S ribosomal subunit and induction of ribotoxic stress. The present study aimed to: (1) extend insight into the mechanism of action (MOA) of DON in immune cells; and (2) understand why immune cells are more sensitive to DON than most other cell types. Previously published microarray studies have described the effects of DON on immune cells. To build upon these findings, here, immunocytological and biochemical studies were performed using human T-lymphocyte Jurkat cells that were exposed for 3 h to 0.5 µM DON. Induction of ER stress by DON was confirmed by immunocytology demonstrating increased protein expression of two major ER stress markers ATF3 and DDIT3. T-cell activation was confirmed by induction of phosphorylation of protein kinases JNK and AKT, activation of NF-κB (p65), and increased expression of NFAT target gene NUR77; each of these are known inducers of the T-cell activation response. Induction of an oxidative stress response was also confirmed by monitoring the nuclear translocation of major oxidative stress markers NRF2 and KEAP1, as well as by changes (i.e. decreases) in cell levels of reduced glutathione. Lastly, this study showed that DON induced cleavage of caspase-3, an event known to mediate apoptosis. Taken together, these results allowed us to formulate a potential mechanism of action of DON in immune cells, i.e. binding to eukaryotic 60S ribosomal subunit → ribotoxic stress → ER stress → calcium release from the ER into cytoplasm → T-cell activation and oxidative stress → apoptosis. It is proposed that immune cells are more sensitive to DON than other cell types due to the induction of a T-cell activation response by increased intracellular calcium levels.

VISTA deficiency synergizes with a non-redundant immune checkpoint pathway and leads to enhanced immune activation (IRM10P.744)

Abstract V-domain Ig suppressor of T cell activation (VISTA) is a novel negative checkpoint ligand that suppresses T-cell mediated immune responses. Previous studies using VISTA-neutralizing monoclonal antibody show that VISTA-blockade enhances T cell-activation in an inflammatory disease model EAE, as well as in murine tumor models. Current study describes a comprehensive characterization of VISTA knockout (KO) mice. We show that despite the apparent normal hematopoietic development in young ko mice, VISTA genetic deficiency leads to a pro-inflammatory phenotype in aged animals, as well as enhanced T-cell activation in response to acute antigen immunization. In addition, we show that VISTA deficiency significantly enhanced disease development in a spontaneous model of autoimmune disease, which is correlated with the spontaneous activation of auto-antigen specific CD4+ T cells. Lastly, when combined with the genetic deficiency of another checkpoint molecule, synergistic or additive immune activation was observed.

In silico characterization of a putative ORF-MAP1138c of Mycobacterium avium subspecies paratuberculosis(MAP) with its implications in virulence

Background Johne’s disease is a chronic mycobacterial infection of the small intestine affecting ruminants worldwide. It is estimated that over 50% of the European Union (EU) dairy holdings is infected [1]. The causal agent is Mycobacterium avium subspecies paratuberculosis (MAP), a slow-growing, acid-fast bacterium. It is a part of the Mycobacterium avium complex (MAC), which also comprises of opportunistic pathogens of humans, as well as innocuous, environmental bacteria [2]. MAP generally interacts with macrophages via different types of receptors, including Toll-like receptors (TLRs) [3,4]. It has been demonstrated of late that H37Rv1411c (LprG) enhances the recognition of triacylated Mycobacterium tuberculosis glycolipids by TLR2 and thereby restraining the expression of MHC-II molecules and processing of antigen and presentation of MHC restricted antigens by macrophages in a TLR2dependent manner [5,6]. However, little is known about how M. paratuberculosis evades and resists this active CD4 T-cell response and survives and infects other macrophages, a hallmark of mycobacterial infections. In this context, the identification of antigenic proteins is useful in understanding the immune evasion mechanism of MAP within host macrophages.

Synthetic FXR Agonist GW4064 Is a Modulator of Multiple G Protein–Coupled Receptors

The synthetic nuclear bile acid receptor (farnesoid X receptor [FXR]) agonist GW4064 is extensively used as a specific pharmacological tool to illustrate FXR functions. We noticed that GW4064 activated empty luciferase reporters in FXR-deficient HEK-293T cells. We postulated that this activity of GW4064 might be routed through as yet unknown cellular targets and undertook an unbiased exploratory approach to identify these targets. Investigations revealed that GW4064 activated cAMP and nuclear factor for activated T-cell response elements (CRE and NFAT-RE, respectively) present on these empty reporters. Whereas GW4064-induced NFAT-RE activation involved rapid intracellular Ca(2+) accumulation and NFAT nuclear translocation, CRE activation involved soluble adenylyl cyclase-dependent cAMP accumulation and Ca(2+)-calcineurin-dependent nuclear translocation of transducers of regulated CRE-binding protein 2. Use of dominant negative heterotrimeric G-protein minigenes revealed that GW4064 caused activation of Gαi/o and Gq/11 G proteins. Sequential pharmacological inhibitor-based screening and radioligand-binding studies revealed that GW4064 interacted with multiple G protein-coupled receptors. Functional studies demonstrated that GW4064 robustly activated H1 and H4 and inhibited H2 histamine receptor signaling events. We also found that MCF-7 breast cancer cells, reported to undergo GW4064-induced apoptosis in an FXR-dependent manner, did not express FXR, and the GW4064-mediated apoptosis, also apparent in HEK-293T cells, could be blocked by selective histamine receptor regulators. Taken together, our results demonstrate identification of histamine receptors as alternate targets for GW4064, which not only necessitates cautious interpretation of the biological functions attributed to FXR using GW4064 as a pharmacological tool but also provides a basis for the rational designing of new pharmacophores for histamine receptor modulation.

Adenoviral Delivery of Recombinant Hepatitis B Virus Expressing Foreign Antigenic Epitopes for Immunotherapy of Persistent Viral Infection

We previously reported a proof-of-concept study for curing chronic hepatitis B virus (HBV) infection using a foreign-antigen recombinant HBV (rHBV) as a gene therapy vector. Targeted elimination of wild-type HBV (wtHBV)-infected cells could be achieved by functionally activating an in situ T-cell response against the foreign antigen. However, as chronic HBV infection spreads to all hepatocytes, specific targeting of virus-infected cells is thought to be less critical. It is also feared that rHBV may not induce active immunization in a setting resembling natural infection. For this immunotherapeutic approach to be practically viable, in the present study, we used a recombinant adenovirus (rAd) vector for rHBV delivery. The rAd vector allowed efficient transduction of wtHBV-producing HepG2 cells, with transferred rHBV undergoing dominant viral replication. Progeny rHBV virions proved to be infectious, as demonstrated in primary tupaia hepatocytes. These results greatly expanded the antiviral capacity of the replication-defective rAd/rHBV in wtHBV-infected liver tissue. With prior priming in the periphery, transduction with rAd/rHBV attracted a substantial influx of the foreign-antigen-specific T-effector cells into the liver. Despite the fully activated T-cell response, active expression of rHBV was observed for a prolonged time, which is essential for rHBV to achieve sustained expansion. In a mouse model of HBV persistence established by infection with a recombinant adeno-associated virus carrying the wtHBV genome, rAd/rHBV-based immunotherapy elicited a foreign-antigen-specific T-cell response that triggered effective viral clearance and subsequent seroconversion to HBV. It therefore represents an efficient strategy to overcome immune tolerance, thereby eliminating chronic HBV infection. Adenovirus-delivered rHBV activated a foreign-antigen-specific T-cell response that abrogated HBV persistence in a mouse model. Our study provides further evidence of the potential of foreign-antigen-based immunotherapy for the treatment of chronic HBV infection.

The interaction of influenza H5N1 viral hemagglutinin with sialic acid receptors leads to the activation of human γδ T cells

Highly pathogenic avian influenza H5N1 epidemics are a significant public health hazard. Genetically engineered H5N1 viruses with mammalian transmission activity highlight the potential risk of a human influenza H5N1 pandemic. Understanding the underlying principles of the innate immune system in response to influenza H5N1 viruses will lead to improved prevention and control of these potentially deadly viruses. γδ T cells act as the first line of defense against microbial infection and help initiate adaptive immune responses during the early stages of viral infection. In this study, we investigated the molecular mechanisms of γδ T cells in response to influenza H5N1 viral infection. We found that recombinant hemagglutinin (rHA) derived from three different strains of influenza H5N1 viruses elicited the activation of γδ T cells cultured in peripheral blood mononuclear cells (PBMCs). Both the cell surface expression of CD69, an early activation marker on γδ T cells, and the production of interferon-γ (IFN-γ) were significantly increased. Notably, the rHA protein-induced γδ T-cell activation was not mediated by TCRγδ, NKG2D or pattern recognition receptors (PRRs) or NKp46 receptors. The interaction of rHA proteins with sialic acid receptors may play a critical role in γδ T-cell activation. Our data may provide insight into the mechanisms underlying γδ T-cell activation in response to infection with H5N1 viruses.

An Influenza Virus M2 Protein Specific Chimeric Antigen Receptor Modulates Influenza A/WSN/33 H1N1 Infection In Vivo

A potential target for the development of universal vaccine strategies against Influenza A is the M2 protein – a membrane protein with a highly conserved extracellular domain. In this study we developed engineered T-cell receptors, by fusing M2-specific antibody sequences with T-cell receptor transmembrane and signaling domains to target influenza infected cells. When expressed on T-cells, these novel T-cell receptors (chimeric antigen receptors - CARs) are able to recognize specific antigens on the surface of target cells via an MHC-independent mechanism. Using an existing monoclonal antibody (14C2) specific for the M2 ectodomain (M2e), we generated an M2-specific CAR. We tested the specificity of this M2 CAR in vitro by measuring the activation of T-cells in response to M2-specific peptides or M2-expressing cell lines. Both Jurkat T-cells and peripheral blood mononuclear cells expressing the M2-specific CAR responded to specific antigen stimulation by upregulating NFAT and producing γ-interferon. To test whether the M2-specific CAR are effective at recognizing influenza infected cells in vivo we used an established BALB/c murine infection model. At day 4 post-infection, when M2 CAR expressing splenocytes could be detected in the lung, the Influenza A/WSN/33 virus titre was around 50% of that in control mice. Although the lung virus titre later increased in the treated group, virus was cleared in both groups of mice by day 8. The results provide support for the development of M2e as a target for cell mediated immunotherapy.

Serum Profile of T Helper 1 and T Helper 2 Cytokines in Hepatitis C Virus Infected Patients

BackgroundT-helper (Th) lymphocyte cytokine production may be important in the immune pathogenesis of hepatitis C virus (HCV) infections. Th1 cytokines such as; interleukin-2 (IL-2), and interferon gamma (IFN-gamma) are necessary for host antiviral immune responses, while Th2 cytokines (IL-4, IL-10) can inhibit the development of these effector mechanisms.ObjectivesThe aim of the present study was to assess the serum profile of Th1 and Th2 cytokines in treated and non-treated HCV infected individuals.Patients and MethodsThis study was carried out in 63 HCV infected patients (31 under treatment and 32 untreated) and 32 matched HCV-sero negative healthy subjects. Serum samples were checked with an enzyme-linked immune sorbent assay (ELISA) for IL-2, IL-4, IL-10 and IFN-gamma.ResultsLevels of circulating IL-2, IL-4, IL-10 and IFN-gamma were significantly elevated in HCV patients versus normal controls (2 822.6 ± 1 259.92 vs. 950.8 ± 286.9 pg/mL; 1 987 ± 900.69 vs. 895.91 ± 332.33 pg/mL; 1 688.5 ± 1 405.1 vs. 519.03 ± 177.64 pg/mL and 1 501.9 ± 1 298 vs. 264.66 ± 71.59 pg/mL, respectively; P < 0.001). The serum levels of all cytokines were significantly lower in the patients under treatment than those of the untreated patients (P < 0.001).ConclusionsOn the basis of our data, the simultaneous increase of Th1 and Th2 related cytokines may indicate that both Thl and Th2 cytokines are involved in the pathogenesis of HCV infections. Moreover, this activated T-cell response in HCV infected patients may be regulated by treatment.

Interferon Regulation Factor‐3 is a Critical Regulator of the Mature of Dendritic Cells from Mice

Interferon regulatory factor-3 (IRF-3) plays an important role in virus and double-stranded RNA-mediated induction of type I interferon and RANTES (regulated on activation normal T cell expressed and secreted), DNA damage signalling, tumour suppression and virus-induced apoptosis. IRF-3 had recently been shown to contribute to T-cell activation in response to pathogens, which implicated an extensive immunological role for IRF-3. Dendritic cells (DCs) played critical roles as professional APCs in the development of immune responses. However, it was unclear whether IRF-3 had any effect on phenotype or function of DCs. In this study, it was shown that IRF-3 acted as a promoter of DC maturation. The level of IRF-3 expression was transiently upregulated and accumulated in the nucleus in TNF-α-induced immune maturation of mice DC cells. Knockdown of IRF-3 by small interfering RNA in DC cells resulted in both phenotypic and functional immaturation, even without TNF-α treatment. Overall, our data demonstrated for the first time that IRF-3 was a critical regulator of mice DC maturation.