Early Signaling in Primary T Cells Activated by Antigen Presenting Cells Is Associated with a Deep and Transient Lamellal Actin Network

Cellular signaling transduction critically depends on molecular interactions that are in turn governed by dynamic subcellular distributions of the signaling system components. Comprehensive insight into signal transduction requires an understanding of such distributions and cellular structures driving them. To investigate the activation of primary murine T cells by antigen presenting cells (APC) we have imaged more than 60 signaling intermediates during T cell stimulation with microscopy across resolution limits. A substantial number of signaling intermediates associated with a transient, wide, and actin-associated lamellum extending from an interdigitated T cell:APC interface several micrometers into the T cell, as characterized in detail here. By mapping the more than 60 signaling intermediates onto the spatiotemporal features of cell biological structures, the lamellum and other ones previously described, we also define distinct spatial and temporal characteristics of T cell signal initiation, amplification, and core signaling in the activation of primary T cells by APCs. These characteristics differ substantially from ones seen when T cells are activated using common reductionist approaches.     

CD8+ T Cell Priming by Dendritic Cell Vaccines Requires Antigen Transfer to Endogenous Antigen Presenting Cells

Background

Immunotherapeutic strategies to stimulate anti-tumor immunity are promising approaches for cancer treatment. A major barrier to their success is the immunosuppressive microenvironment of tumors, which inhibits the functions of endogenous dendritic cells (DCs) that are necessary for the generation of anti-tumor CD8+ T cells. To overcome this problem, autologous DCs are generated ex vivo, loaded with tumor antigens, and activated in this non-suppressive environment before administration to patients. However, DC-based vaccines rarely induce tumor regression.

Methodology/Principal Findings

We examined the fate and function of these DCs following their injection using murine models, in order to better understand their interaction with the host immune system. Contrary to previous assumptions, we show that DC vaccines have an insignificant role in directly priming CD8+ T cells, but instead function primarily as vehicles for transferring antigens to endogenous antigen presenting cells, which are responsible for the subsequent activation of T cells.

Conclusions/Significance

This reliance on endogenous immune cells may explain the limited success of current DC vaccines to treat cancer and offers new insight into how these therapies can be improved. Future approaches should focus on creating DC vaccines that are more effective at directly priming T cells, or abrogating the tumor induced suppression of endogenous DCs.     

Antigen Presenting B Cells Facilitate CD4 T Cell Cooperation Resulting in Enhanced Generation of Effector and Memory CD4 T Cells

We show that the in vivo generation of cytokine-producing CD4 T cells specific for a given major histocompatibility class-II (MHCII)-binding peptide of hen egg lysozyme (HEL) is facilitated when mice are immunized with splenic antigen presenting cells (APC) pulsed with this HEL peptide and another peptide that binds a different MHCII molecule. This enhanced generation of peptide-specific effector CD4 T cells requires that the same splenic APC be pulsed with both peptides. Pulsed B cells, but not pulsed dendritic cells (DCs), can mediate CD4 T cell cooperation, which can be blocked by disrupting OX40-OX40L (CD134-CD252) interactions. In addition, the generation of HEL peptide-specific CD4 T cell memory is greater when mice are primed with B cells pulsed with the two peptides than with B cells pulsed with the HEL- peptide alone. Based on our findings, we suggest CD4 T cell cooperation is important for vaccine design, underlies the phenomenon of “epitope-spreading” seen in autoimmunity, and that the efficacy of B cell-depletion in the treatment of human cell-mediated autoimmune disease is due to the abrogation of the interactions between autoimmune CD4 T cells that facilitates their activation.     

Reduced SERCA Function Preferentially Affects Wnt Signaling by Retaining E-Cadherin in the Endoplasmic Reticulum

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Emergent Group Dynamics Governed by Regulatory Cells Produce a Robust Primary T Cell Response

The currently accepted paradigm for the primary T cell response is that effector T cells commit to autonomous developmental programs. This concept is based on several experiments that have demonstrated that the dynamics of a T cell response is largely determined shortly after antigen exposure and that T cell dynamics do not depend on the level and duration of antigen stimulation. Another experimental study has also shown that T cell responses are robust to variations in antigen-specific precursor frequency.     

Comparison of T Cell Receptor-Induced Proximal Signaling and Downstream Functions in Immortalized and Primary T Cells

Background

Human T cells play an important role in pathogen clearance, but their aberrant activation is also linked to numerous diseases. T cells are activated by the concurrent induction of the T cell receptor (TCR) and one or more costimulatory receptors. The characterization of signaling pathways induced by TCR and/or costimulatory receptor activation is critical, since these pathways are excellent targets for novel therapies for human disease. Although studies using human T cell lines have provided substantial insight into these signaling pathways, no comprehensive, direct comparison of these cell lines to activated peripheral blood T cells (APBTs) has been performed to validate their usefulness as a model of primary T cells.

Methodology/Principal Findings

We used quantitative biochemical techniques to compare the activation of two widely used human T cell lines, Jurkat E6.1 and HuT78 T cells, to APBTs. We found that HuT78 cells were similar to APBTs in proximal TCR-mediated signaling events. In contrast, Jurkat E6.1 cells had significantly increased site-specific phosphorylation of Pyk2, PLCγ1, Vav1, and Erk1/Erk2 and substantially more Ca²⁺ flux compared to HuT78 cells and APBTs. In part, these effects appear to be due to an overexpression of Itk in Jurkat E6.1 cells compared to HuT78 cells and APBTs. Both cell lines differ from APBTs in the expression and function of costimulatory receptors and in the range of cytokines and chemokines released upon TCR and costimulatory receptor activation.

Conclusions/Significance

Both Jurkat E6.1 and HuT78 T cells had distinct similarities and differences compared to APBTs. Both cell lines have advantages and disadvantages, which must be taken into account when choosing them as a model T cell line.     

Tunable Chemokine Production by Antigen Presenting Dendritic Cells in Response to Changes in Regulatory T Cell Frequency in Mouse Reactive Lymph Nodes

Background

Although evidence exists that regulatory T cells (Tregs) can suppress the effector phase of immune responses, it is clear that their major role is in suppressing T cell priming in secondary lymphoid organs. Recent experiments using two photon laser microscopy indicate that dendritic cells (DCs) are central to Treg cell function and that the in vivo mechanisms of T cell regulation are more complex than those described in vitro.

Principal Findings

Here we have sought to determine whether and how modulation of Treg numbers modifies the lymph node (LN) microenvironment. We found that pro-inflammatory chemokines—CCL2 (MCP-1) and CCL3 (MIP-la)—are secreted in the LN early (24 h) after T cell activation, that this secretion is dependent on antigen-specific DC–T cell interactions, and that it was inversely related to the frequency of Tregs specific for the same antigen. Furthermore, we demonstrate that Tregs modify the chemoattractant properties of antigen-presenting DCs, which, as the frequency of Tregs increases, fail to produce CCL2 and CCL3 and to attract antigen-specific T cells.

Conclusions

These results substantiate a major role of Tregs in LN patterning during antigen-specific immune responses.     

Interference of BCR-ABL1 Kinase Activity with Antigen Receptor Signaling in B cell Precursor Leukemia Cells

The chromosomal translocation t(9;22), resulting in the fusion of the BCR and ABL1 genes, represents a recurrent aberration in B cell precursor leukemia cells. Their normal counterparts, B cell precursor cells, are positively selected for survival signals through the antigen receptor, whose expression requires a functional immunoglobulin heavy chain (IGH) gene rearrangement. Unexpectedly, B cell precursor leukemia cells harboring a BCR-ABL1 gene rearrangement do not depend on antigen receptor mediated survival signals. Genes involved in the signaling cascade of the antigen receptor are silenced and in most cases, the dominant tumor clone does not carry a functional IGH gene rearrangement. However, upon inhibition of the BCR-ABL1 kinase activity by STI571, only leukemia cells expressing an antigen receptor are able to survive. Since resistance to STI571 is frequent in the therapy of BCR-ABL1+ B cell precursor leukemia is frequent, antigen receptor signaling may represent a mechanism through which these cells can temporarily evade STI571-induced apoptosis. This may open a time frame, during which leukemia cells may acquire secondary transforming events that confer definitive resistance to STI571.     

ERK Positive Feedback Regulates a Widespread Network of Tyrosine Phosphorylation Sites across Canonical T Cell Signaling and Actin Cytoskeletal Proteins in Jurkat T Cells

Competing positive and negative signaling feedback pathways play a critical role in tuning the sensitivity of T cell receptor activation by creating an ultrasensitive, bistable switch to selectively enhance responses to foreign ligands while suppressing signals from self peptides. In response to T cell receptor agonist engagement, ERK is activated to positively regulate T cell receptor signaling through phosphorylation of Ser⁵⁹ Lck. To obtain a wide-scale view of the role of ERK in propagating T cell receptor signaling, a quantitative phosphoproteomic analysis of 322 tyrosine phosphorylation sites by mass spectrometry was performed on the human Jurkat T cell line in the presence of U0126, an inhibitor of ERK activation. Relative to controls, U0126-treated cells showed constitutive decreases in phosphorylation through a T cell receptor stimulation time course on tyrosine residues found on upstream signaling proteins (CD3 chains, Lck, ZAP-70), as well as downstream signaling proteins (VAV1, PLCγ1, Itk, NCK1). Additional constitutive decreases in phosphorylation were found on the majority of identified proteins implicated in the regulation of actin cytoskeleton pathway. Although the majority of identified sites on T cell receptor signaling proteins showed decreases in phosphorylation, Tyr⁵⁹⁸ of ZAP-70 showed elevated phosphorylation in response to U0126 treatment, suggesting differential regulation of this site via ERK feedback. These findings shed new light on ERK’s role in positive feedback in T cell receptor signaling and reveal novel signaling events that are regulated by this kinase, which may fine tune T cell receptor activation.     

P2X7 Receptor Activation Impairs Exogenous MHC Class I Oligopeptides Presentation in Antigen Presenting Cells

Major histocompatibility complex class I (MHC I) on antigen presenting cells (APCs) is a potent molecule to activate CD8⁺ T cells and initiate immunity. P2X7 receptors (P2X7Rs) are present on the plasma membrane of APCs to sense the extracellular danger signal adenosine-5′-triphosphate (ATP). P2X7R activates the inflammasome and the release of IL-1β in macrophages and other immune cells to initiate the inflammatory response. Here we show that P2X7R stimulation by ATP in APCs decreased the amount of MHC I at the plasma membrane. Specific antagonism or genetic ablation of P2X7R inhibited the effects of ATP on levels of cellular MHC I. Furthermore, P2X7R stimulation was able to inhibit activation of CD8⁺ T cells via specific MHC I-oligopeptide complexes. Our study suggests that P2X7R activation on APCs is a novel inhibitor of adaptive CD8⁺ T cell immunity.     

The Us3 Protein of Herpes Simplex Virus 1 Inhibits T Cell Signaling by Confining Linker for Activation of T Cells (LAT) Activation via TRAF6 Protein

Herpes simplex virus 1 (HSV-1) is the most prevalent human virus and causes global morbidity because the virus is able to infect multiple cell types. Remarkably, HSV infection switches between lytic and latent cycles, where T cells play a critical role. However, the precise way of virus-host interactions is incompletely understood. Here we report that HSV-1 productively infected Jurkat T-cells and inhibited antigen-induced T cell receptor activation. We discovered that HSV-1-encoded Us3 protein interrupted TCR signaling and interleukin-2 production by inactivation of the linker for activation of T cells. This study unveils a mechanism by which HSV-1 intrudes into early events of TCR-mediated cell signaling and may provide novel insights into HSV infection, during which the virus escapes from host immune surveillance.     

NOD1 Cooperates with TLR2 to Enhance T Cell Receptor-Mediated Activation in CD8 T Cells

Pattern recognition receptors (PRR), like Toll-like receptors (TLR) and NOD-like receptors (NLR), are involved in the detection of microbial infections and tissue damage by cells of the innate immune system. Recently, we and others have demonstrated that TLR2 can additionally function as a costimulatory receptor on CD8 T cells. Here, we establish that the intracytosolic receptor NOD1 is expressed and functional in CD8 T cells. We show that C12-iEDAP, a synthetic ligand for NOD1, has a direct impact on both murine and human CD8 T cells, increasing proliferation and effector functions of cells activated via their T cell receptor (TCR). This effect is dependent on the adaptor molecule RIP2 and is associated with an increased activation of the NF-κB, JNK and p38 signaling pathways. Furthermore, we demonstrate that NOD1 stimulation can cooperate with TLR2 engagement on CD8 T cells to enhance TCR-mediated activation. Altogether our results indicate that NOD1 might function as an alternative costimulatory receptor in CD8 T cells. Our study provides new insights into the function of NLR in T cells and extends to NOD1 the recent concept that PRR stimulation can directly control T cell functions.     

Anti-Tumor Activity of Yuanhuacine by Regulating AMPK/mTOR Signaling Pathway and Actin Cytoskeleton Organization in Non-Small Cell Lung Cancer Cells

Yuanhuacine (YC), a daphnane diterpenoid from the flowers of Daphne genkwa, exhibited a potential growth inhibitory activity against human non-small cell lung cancer (NSCLC) cells. YC also suppressed the invasion and migration of lung cancer cells. However, the precise molecular mechanisms remain to be elucidated. In the present study, we report that YC significantly activated AMP-activated protein kinase (AMPK) signaling pathway and suppressed mTORC2-mediated downstream signaling pathway in H1993 human NSCLC cells. AMPK plays an important role in energy metabolism and cancer biology. Therefore, activators of AMPK signaling pathways can be applicable to the treatment of cancer. YC enhanced the expression of p-AMPKα. The co-treatment of YC and compound C (an AMPK inhibitor) or metformin (an AMPK activator) also confirmed that YC increases p-AMPKα. YC also suppressed the activation of the mammalian target of rapamycin (mTOR) expression, a downstream target of AMPK. Further study revealed that YC modulates mTORC2-associated downstream signaling pathways with a decreased expressions of p-Akt, p-protein kinase C alpha (PKCα), p-ras-related C3 botulinum toxin substrate 1 (Rac1) and filamentous actin (F-actin) that are known to activate cell growth and organize actin cytoskeleton. In addition, YC inhibited the tumor growth in H1993 cell-implanted xenograft nude mouse model. These data suggest the YC could be a potential candidate for cancer chemotherapeutic agents derived from natural products by regulating AMPK/mTORC2 signaling pathway and actin cytoskeleton organization.     

Lack ofl-Iduronic Acid in Heparan Sulfate Affects Interaction with Growth Factors and Cell Signaling

HSEPI (glucuronyl C5-epimerase) catalyzes the conversion of d-glucuronic acid to l-iduronic acid in heparan sulfate (HS) biosynthesis. Disruption of the Hsepi gene in mice yielded a lethal phenotype with selective organ defects but had remarkably little effect on other organ systems. We have approached the underlying mechanisms by examining the course and effects of FGF2 signaling in a mouse embryonic fibroblast (MEF) cell line derived from the Hsepi^−/− mouse. The HS produced by these cells is devoid of l-iduronic acid residues but shows up-regulated N- and 6-O-sulfation compared with wild type (WT) MEF HS. In medium fortified with 10% fetal calf serum, the Hsepi^−/− MEFs proliferated and migrated similarly to WT cells. Under starvation conditions, both cell types showed attenuated proliferation and migration that could be restored by the addition of FGF2 to WT cells, whereas Hsepi^−/− cells were resistant. Moreover, ERK phosphorylation following FGF2 stimulation was delayed in Hsepi^−/− compared with WT cells. Assessment of HS-growth factor interaction by nitrocellulose filter trapping revealed a strikingly aberrant binding property of FGF2 and glia-derived neurotropic factor to Hsepi^−/− but not to WT HS. glia-derived neurotropic factor has a key role in kidney development, defective in Hsepi^−/− mice. By contrast, Hsepi^−/− and WT HS interacted similarly and in conventional mode with FGF10. These findings correlate defective function of growth factors with their mode of HS interaction and may help explain the partly modest organ phenotypes observed after genetic ablation of selected enzymes in HS biosynthesis.Signaling activities of numerous growth factors and morphogens during development involve cell surface receptor systems consisting of a tyrosine kinase-type receptor along with a heparan sulfate proteoglycan (HSPG)2 co-receptor (1, 2). The complex and heterogeneous HSPG macromolecules occur in the extracellular matrix and on the surfaces of virtually all animal cells (3). HS side chains of HSPGs show great structural variability; hence, they interact with a multitude of proteins and influence a variety of biological processes, including growth factor signaling (4).The structural diversity of HS is best envisaged through an account of its biosynthesis (Fig. 1). The process is initiated by glycosylation reactions that generate saccharide sequences composed of alternating GlcA and GlcNAc units covalently bound to a core protein (through a specific “linker” tetrasaccharide sequence). The resulting polymer of (GlcAβ1,4-GlcNAcα1,4-)_n disaccharide repeats is modified through a series of reactions, including N-deacetylation/N-sulfation of GlcNAc residues, C5-epimerization of GlcA to l-iduronic acid (IdoA) units, and O-sulfation at various positions of the hexuronic acid and glucosamine residues (4, 5). Modulation of these modification reactions, through as yet poorly understood mechanisms, yields HS chains of strictly regulated saccharide composition, varying with tissue source and age (6, 7). Interactions between protein ligands and HS involve selective ionic binding of peptide sequences containing basic amino acid residues to saccharide domains with clustered sulfate groups and IdoA residues. The latter components are considered to promote ligand apposition through their conformational flexibility (8).Open in a separate windowFIGURE 1.Schematic display of HS biosynthesis. A precursor structure composed of alternating GlcA and GlcNAc residues linked to a core protein is modified through the series of enzymatic reactions indicated and outlined under“Results.” The modifications yield domains of consecutive N-acetylated or N-sulfated disaccharide units, along with mixed sequences. Wild type HS contains both GlcA and IdoA residues, whereas Hsepi^−/− HS lacks IdoA units but shows increased N- and 6-O-sulfation. In particular, the mutant HS features extended sequences of -GlcA-GlcNS6S- disaccharide units.Targeted disruption of genes encoding HS biosynthesis enzymes demonstrated critical roles for HSPGs in developmental processes (9–11). The observed phenotypes vary dramatically in severity, from gastrulation failure to subtle disturbance of organ development. This variability may reflect redundancy due to the occurrence of isoforms for some of the enzymes. Alternatively, developmental events might be critically dependant on HS involvement but not on the fine structure of the polysaccharide chain, such that even structurally deranged HS would fulfill a functional role (12). The glucuronyl C5-epimerase (HSEPI) that catalyzes the conversion of GlcA to IdoA in HS biosynthesis is encoded by a single gene (13). Targeted disruption of this gene in mice resulted in an abnormal HS structure, completely lacking IdoA residues and with severely distorted sulfation pattern. The Hsepi-deficient mice die shortly after birth with multiple developmental defects, such as skeletal malformations and kidney agenesis. Intriguingly, however, other major organ systems, such as the brain and vascular system, known to depend on HS-supported signaling processes, developed seemingly normally (14).The present study was undertaken to obtain insight into the mechanisms behind the developmental defects in the Hsepi mutant mice, using a model system based on murine embryonic fibroblast (MEF) cell lines. The mutant MEF cells responded poorly to FGF2 in proliferation and migration experiments and showed defective FGF2-dependent intracellular signaling. Moreover, binding studies revealed an aberrant mode of interaction between mutant HS chains and certain growth factors, of potential relevance to growth factor signaling.     

A Neoglycoconjugate Containing the Human Milk Sugar LNFPIII Drives Anti-Inflammatory Activation of Antigen Presenting Cells in a CD14 Dependent Pathway

The milk pentasaccharide LNFPIII has therapeutic action for metabolic and autoimmune diseases and prolongs transplant survival in mice when presented as a neoglycoconjugate. Within LNFPIII is the Lewis^x trisaccharide, expressed by many helminth parasites. In humans, LNFPIII is found in human milk and also known as stage-specific embryonic antigen-1. LNFPIII-NGC drives alternative activation of macrophages and dendritic cells via NFκB activation in a TLR4 dependent mechanism. However, the connection between LNFPIII-NGC activation of APCs, TLR4 signaling and subsequent MAP kinase signaling leading to anti-inflammatory activation of APCs remains unknown. In this study we determined that the innate receptor CD14 was essential for LNFPIII-NGC induction of both ERK and NFkB activation in APCs. Induction of ERK activation by LNFPIII-NGC was completely dependent on CD14/TLR4-Ras-Raf1/TPL2-MEK axis in bone marrow derived dendritic cells (BMDCs). In addition, LNFPIII-NGC preferentially induced the production of Th2 “favoring” chemokines CCL22 and matrix metalloprotease protein-9 in a CD14 dependent manner in BMDCs. In contrast, LNFPIII-NGC induces significantly lower levels of Th1 “favoring” chemokines, MIP1α, MIP1β and MIP-2 compared to levels in LPS stimulated cells. Interestingly, NGC of the identical human milk sugar LNnT, minus the alpha 1–3 linked fucose, failed to activate APCs via TLR4/MD2/CD14 receptor complex, suggesting that the alpha 1–3 linked fucose in LNFPIII and not on LNnT, is required for this process. Using specific chemical inhibitors of the MAPK pathway, we found that LNFPIII-NGC induction of CCL22, MMP9 and IL-10 production was dependent on ERK activation. Over all, this study suggests that LNFPIII-NGC utilizes CD14/TLR4-MAPK (ERK) axis in modulating APC activation to produce anti-inflammatory chemokines and cytokines in a manner distinct from that seen for the pro-inflammatory PAMP LPS. These pathways may explain the in vivo therapeutic effect of LNFPIII-NGC treatment for inflammation based diseases.     

Programmed Cell Death-1 Deficiency Exacerbates T Cell Activation and Atherogenesis despite Expansion of Regulatory T Cells in Atherosclerosis-Prone Mice

T cell activation represents a double-edged sword in atherogenesis, as it promotes both pro-inflammatory T cell activation and atheroprotective Foxp3⁺ regulatory T cell (Treg) responses. Here, we investigated the role of the co-inhibitory receptor programmed cell death-1 (PD-1) in T cell activation and CD4⁺ T cell polarization towards pro-atherogenic or atheroprotective responses in mice. Mice deficient for both low density lipoprotein receptor and PD-1 (Ldlr^−/−Pd1^−/−) displayed striking increases in systemic CD4⁺ and CD8⁺ T cell activation after 9 weeks of high fat diet feeding, associated with an expansion of both pro-atherogenic IFNγ-secreting T helper 1 cells and atheroprotective Foxp3⁺ Tregs. Importantly, PD-1 deficiency did not affect Treg suppressive function in vitro. Notably, PD-1 deficiency exacerbated atherosclerotic lesion growth and entailed a massive infiltration of T cells in atherosclerotic lesions. In addition, aggravated hypercholesterolemia was observed in Ldlr^−/−Pd1^−/− mice. In conclusion, we here demonstrate that although disruption of PD-1 signaling enhances both pro- and anti-atherogenic T cell responses in Ldlr^−/− mice, pro-inflammatory T cell activation prevails and enhances dyslipidemia, vascular inflammation and atherosclerosis.     

Activation of the B Cell Antigen Receptor Triggers Reactivation of Latent Kaposi's Sarcoma-Associated Herpesvirus in B Cells

Kaposi''s sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus and the cause of Kaposi''s sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman''s disease. Latently infected B cells are the main reservoir of this virus in vivo, but the nature of the stimuli that lead to its reactivation in B cells is only partially understood. We established stable BJAB cell lines harboring latent KSHV by cell-free infection with recombinant virus carrying a puromycin resistance marker. Our latently infected B cell lines, termed BrK.219, can be reactivated by triggering the B cell receptor (BCR) with antibodies to surface IgM, a stimulus imitating antigen recognition. Using this B cell model system we studied the mechanisms that mediate the reactivation of KSHV in B cells following the stimulation of the BCR and could identify phosphatidylinositol 3-kinase (PI3K) and X-box binding protein 1 (XBP-1) as proteins that play an important role in the BCR-mediated reactivation of latent KSHV.