bluuebunny/crossref_metadata_2025 huggingface dataset:
APC-Independent Activation of NK Cells by the Toll-Like Receptor 3 Agonist Double-Stranded RNA
Authors: Schmidt Kerstin N. , Leung Beatrice , Kwong Mandy , Zarember Kol A. , Satyal Sanjeev , Navas Tony A. , Wang Fay , Godowski Paul J.
Published: April 2014

Toll-like receptors (TLRs) play a fundamental role in the recognition of bacteria and viruses. TLR3 is activated by viral dsRNA and polyinosinic-polycytidylic acid (poly(I:C)), a synthetic mimetic of viral RNA. We show that NK cells, known for their capacity to eliminate virally infected cells, express TLR3 and up-regulate TLR3 mRNA upon poly(I:C) stimulation. Treatment of highly purified NK cells with poly(I:C) significantly augments NK cell-mediated cytotoxicity. Poly(I:C) stimulation also leads to up-regulation of activation marker CD69 on NK cells. Furthermore, NK cells respond to poly(I:C) by producing proinflammatory cytokines like IL-6 and IL-8, as well as the antiviral cytokine IFN-γ. The induction of cytokine production by NK cells was preceded by activation of NF-κB. We conclude that the ability of NK cells to directly recognize and respond to viral products is important in mounting effective antiviral responses.
Cognate B Cell Signaling via MHC Class II: Differential Regulation of B Cell Antigen Receptor and MHC Class II/Ig-αβ Signaling by CD22
Authors: Mills David M. , Stolpa John C. , Cambier John C.
Published: April 2014

Recent studies demonstrate that MHC class II molecules can signal via associated Ig-αβ dimers, signal transducers previously thought to function only in B cell Ag receptor (BCR) signaling. Surprisingly, the biologic outputs of MHC class II and BCR ligation (by thymus-dependent Ags) differ, e.g., MHC class II signaling leads to robust proliferation and extension of pseudopods. It seemed possible that these differences might be due, at least in part, to differential use of inhibitory coreceptors thought to modulate membrane Ig signals. In this study, we demonstrate that CD22, an inhibitory BCR coreceptor, neither associates with nor functions in MHC class II/Ig-αβ signaling. Interestingly, CD22 is actively excluded from cell surface MHC class II aggregates.
Cutting Edge: An Endogenous Pathway to Systemic Inflammatory Response Syndrome (SIRS)-Like Reactions through Toll-Like Receptor 4
Authors: Johnson Geoffrey B. , Brunn Gregory J. , Platt Jeffrey L.
Published: April 2014

Systemic inflammatory response syndrome (SIRS) is typically associated with trauma, surgery, or acute pancreatitis. SIRS resembles sepsis, triggered by exogenous macromolecules such as LPS acting on Toll-like receptors. What triggers SIRS in the absence of infection, however, is unknown. In this study, we report that a SIRS-like response can be induced in mice by administration of soluble heparan sulfate, a glycosaminoglycan associated with nucleated cells and extracellular matrices, and by elastase, which cleaves and releases heparan sulfate proteoglycans. The ability of heparan sulfate and elastase to induce SIRS depends on functional Toll-like receptor 4, because mutant mice lacking that receptor or its function do not respond. These results provide a molecular explanation for the initiation of SIRS.
Inducible Heat Shock Protein 70 Promotes Myelin Autoantigen Presentation by the HLA Class II
Authors: Mycko Marcin P. , Cwiklinska Hanna , Szymanski Jacek , Szymanska Bozena , Kudla Grzegorz , Kilianek Lukasz , Odyniec Artur , Brosnan Celia F. , Selmaj Krzysztof W.
Published: April 2014

In this study, we investigated the role of the inducible form of heat shock protein 70 (hsp70) in the presentation of the major putative autoantigen in multiple sclerosis, myelin basic protein (MBP), in the context of appropriate MHC class II. By coimmunoprecipitation, we found that MBP is associated with hsp70 in APC in an ATP/ADP-dependent manner. Additionally, using confocal microscopy, hsp70 was detected in the endocytic pathway of APC, where it colocalized with MBP and HLA-DR. The immunodominant epitopes of MBP 85–99 and 80–99 were shown to bind selectively and specifically to hsp70 by surface plasmon resonance. The functional significance of MBP interaction with hsp70 was demonstrated by the detection of enhanced responses of an MBP-specific T cell hybridoma to MBP and MBP 80–99 with increasing levels of hsp70 and reduced responses when hsp70 expression was diminished within APC-expressing DRA*0101, DRB1*1501 (DR1501). However, when MBP 85–99 was used as the stimulus, T cell hybridoma responses were not enhanced by hsp70 overexpression within APC, suggesting that hsp70 contributes to Ag processing rather than Ag presentation. The importance of a direct association between MBP and hsp70 in the presentation pathways was demonstrated by enhanced efficacy of MBP presentation by APC transfected with a plasmid vector encoding a fusion hsp70-MBP protein. This is the first report on the involvement of self-inducible hsp70 in MHC class II-dependent autoantigen processing by APC. It implicates that aberrant self hsp expression may lead to the enhancement/modulation of autoimmune responses.
Effects of T Cell Frequency and Graft Size on Transplant Outcome in Mice
Authors: He Chunshui , Schenk Soren , Zhang Qiwei , Valujskikh Anna , Bayer Jörg , Fairchild Robert L. , Heeger Peter S.
Published: April 2014

The features that determine whether graft-reactive T lymphocytes develop into effector cells capable of mediating organ destruction are not well understood. To investigate potential factors involved in this process, we first confirmed that female recipient mice acutely rejected minor Ag-disparate male skin, but not heart transplants. Despite this difference in outcome, heart and skin transplantation induced antidonor T cell responses of similar magnitude, specificity, and cytokine profile. The heart-graft-primed T cells transiently infiltrated the graft and ultimately induced the development of chronic transplant vasculopathy. Increasing the frequency of donor-reactive T cells by presensitization or by using TCR (CD8+ antimale)-transgenic recipients did not mediate acute rejection but accelerated the pace and severity of the vasculopathy. Surprisingly, decreasing the tissue mass of the donor heart by 50% resulted in acute rejection of these smaller grafts without increasing the frequency of antidonor effector T cells in the recipients. In complementary studies, placement of one or two male skin grafts on a single recipient did not affect the frequency or cytokine profile of the induced antimale T cell repertoire. Nonetheless, the recipients of single grafts acutely rejected the transplanted skin while the recipients of two skin grafts did not. These results provide new insight into the pathogenesis of transplant vasculopathy and provide an explanation for the difference in outcome between murine skin and heart transplants by highlighting the novel concept that the efficiency of transplant-reactive T cell immunity is heavily influenced by the tissue burden it encounters at the effector stage.
Colorectal Cancer Cells Induce Lymphocyte Apoptosis by an Endothelial Monocyte-Activating Polypeptide-II-Dependent Mechanism
Authors: Murray J. Clifford , Symonds Peter , Ward Wynne , Huggins Mary , Tiga Anna , Rice Katherine , Heng Yee M. , Todd Ian , Robins R. Adrian
Published: April 2014

Endothelial monocyte-activating polypeptide-II (EMAP-II) was first isolated from cell growth medium conditioned by tumor cells, and is closely related or identical with the p43 component of the mammalian multisynthase complex. In its secreted form, EMAP-II has multiple cytokine-like activities in vitro, inducing procoagulant activity on the surface of endothelial cells, increasing expression of E- and P-selectins and TNF-R1, and directing migration of monocytes and neutrophils. EMAP-II has also been shown to induce apoptosis in endothelial cells, leading to the suggestion that it is a proinflammatory polypeptide with antiangiogenic activity. The role of secreted EMAP-II in tumors remains poorly understood, and we hypothesized that EMAP-II may play a role in immune evasion by tumor cells. We investigated its effects on lymphocytes, using recombinant protein, or colorectal cancer cell lines, as a source of native EMAP-II. Recombinant EMAP-II inhibits DNA synthesis and cell division, and induces apoptosis in mitogen-activated lymphocytes in PBMC preparations, and in Jurkat T cells. Native EMAP-II, released by or expressed on the surface of colorectal carcinoma cells, also induces activation of caspase 8 and apoptosis of PBLs and Jurkat cells, which are partially blocked by addition of Abs against EMAP-II. Thus, activated lymphocytes, along with proliferating endothelial cells, are targets for the cytotoxic activity of EMAP-II. Membrane-bound and soluble EMAP-II appear to play multiple roles in the tumor microenvironment, one of which is to assist in immune evasion.
The α1β1 and αEβ7 Integrins Define a Subset of Dendritic Cells in Peripheral Lymph Nodes with Unique Adhesive and Antigen Uptake Properties
Authors: Pribila Jonathan T. , Itano Andrea A. , Mueller Kristen L. , Shimizu Yoji
Published: April 2014

Dendritic cells (DCs) are a heterogeneous population of APCs with critical roles in T cell activation and immune regulation. We report in this study the identification and characterization of a novel subset of DCs resident in skin-draining peripheral lymph nodes of normal mice. This subset of CD11chighCD40highCD8αintermediate (int) DCs expresses the collagen-binding integrin, α1β1, and the E-cadherin-binding integrin, αEβ7. Although α1β1 and αEβ7 are also expressed on CD11chighCD40intCD8αhigh lymphoid DCs, CD11chighCD40highCD8αint DCs demonstrate preferential integrin-mediated adhesion to collagen and fibronectin. This DC subset most likely acquires expression of these integrins in peripheral lymph node, as this subset is not found in the spleen or mesenteric lymph node, and recent DC migrants from the skin lack expression of α1β1 and αEβ7 integrins. Resident CD40high DCs express α1β1 integrin and colocalize with collagen in lymph nodes. When compared with CD11chighCD40highCD8αint DCs lacking expression of these integrins, the α1β1+αEβ7+ DC subset exhibits more efficient formation of Ag-independent conjugates with T cells, and a decreased ability to acquire soluble Ag. Thus, the α1β1 and αEβ7 integrins define a unique population of peripheral lymph node-derived DCs with altered functional properties and adhesive potential that localizes these cells to sites in lymph nodes where Ag presentation to T cells occurs.
Dermal Enhancement: Bacterial Products on Intact Skin Induce and Augment Organ-Specific Autoimmune Disease
Authors: Riminton D. Sean , Kandasamy Rama , Dravec Danijela , Basten Antony , Baxter Alan G.
Published: April 2014

The skin is both an essential barrier for host defense and an important organ of immunity. In this study, we show that the application of cholera toxin to intact mouse skin induces and enhances autoimmune diseases affecting organs at distant anatomic sites, whereas its administration by the mucosal route has been reported to have the opposite effect. First, the CNS autoantigen myelin oligodendrocyte glycoprotein 35–55, when applied repeatedly with cholera toxin to the intact skin of healthy C57BL/6 mice, induced relapsing paralysis with demyelinating immunopathologic features similar to multiple sclerosis. Second, the application of cholera toxin in the absence of autoantigen exacerbated the severity of conventional experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein in CFA. Third, the application of cholera toxin to the intact skin of NOD/Lt mice, with or without insulin B peptide 9–23, exacerbated insulitis and T lymphocyte-derived IFN-γ and IL-4 production in the islets of Langerhans, resulting in an increased incidence and rate of onset of autoimmune diabetes. The data presented in this study highlight the different outcomes of adjuvant administration by different routes. Because dermal application of cholera toxin, and other bacterial products with similar adjuvant activities, is being developed as a clinical vaccination strategy, these data raise the possibility that it could precipitate autoimmune disease in genetically susceptible humans.
Pulmonary Tumors Inefficiently Prime Tumor-Specific T Cells
Authors: O’Mara Leigh A. , Allen Paul M.
Published: April 2014

The lung is a common site of metastatic and primary tumor growth, and has been shown to be an immunosuppressive environment. We tested the impact of the lung environment on the development of tumor-specific T cell responses against the CMS5 fibrosarcoma, and found a deficit in the efficacy of naive tumor-specific DUC18 T cells against tumors established in the lung. One hundred-fold more naive tumor-specific T cells were required to protect against tumor development or reject established tumors in the lung than an identical tumor challenge delivered s.c. in the flank. Importantly, CMS5 growing in the flank facilitated the rejection of tumors present in the lungs. In the presence of flank tumors, transferred T cells were not phenotypically altered but were present in much greater numbers in the parabronchial lymph nodes, bronchoalveolar lavage, and lung parenchyma than in mice bearing lung tumors alone. We hypothesized that APC present in the lung and skin draining lymph nodes were differentially initiating T cell proliferation, leading to differences in the size of the final effector populations. A direct comparison of DUC18 T cell proliferation against APC from flank or lung draining lymph nodes showed profoundly greater proliferation to flank draining lymph node APC. The impaired stimulation of naive T cell proliferation by lung draining APC provides one mechanistic explanation for the lower overall immune response, and inability to effectively reject tumors, in the lung.
B7 Expression on T Cells Down-Regulates Immune Responses through CTLA-4 Ligation via R-T Interactions
Authors: Taylor Patricia A. , Lees Christopher J. , Fournier Sylvie , Allison James P. , Sharpe Arlene H. , Blazar Bruce R.
Published: April 2014

Although B7 on APCs has a well-recognized role in T cell costimulation, little is known about the functional significance of constitutive and activation-induced B7 expression that also occurs on T cells. To analyze the role of B7 on T cells, B7-1/B7-2-deficient mice (B7 double knockout) and mice overexpressing B7-2 exclusively on T cells (B7-2 transgenic) were used as T cell donors for allogeneic transplant recipients, and graft-vs-host disease (GVHD) was assessed. B7 double-knockout T cells resulted in significant GVHD acceleration compared with wild-type T cells. Conversely, B7-2 transgenic donor T cells mediated reduced GVHD mortality compared with wild-type T cells. Data indicated that B7 expression on T cells down-regulated alloresponses through CTLA-4 ligation. This study is the first to provide definitive in vivo data illustrating the importance of T cell-associated B7 as a negative regulator of immune responses in a clinically relevant murine model of GVHD. The up-regulation of B7 on T cells may be an important component of normal immune homeostasis.
Partitioning of Rearranged Ig Genes by Mutation Analysis Demonstrates D-D Fusion and V Gene Replacement in the Expressed Human Repertoire
Authors: Collins Andrew M. , Ikutani Masashi , Puiu Daniela , Buck Gregory A. , Nadkarni Aradhita , Gaeta Bruno
Published: April 2014

The accurate partitioning of Ig H chain VHDJH junctions and L chain VLJL junctions is problematic. We have developed a statistical approach for the partitioning of such sequences, by analyzing the distribution of point mutations between a determined V gene segment and putative Ig regions. The establishment of objective criteria for the partitioning of sequences between VH, D, and JH gene segments has allowed us to more carefully analyze intervening putative nontemplated (N) nucleotides. An analysis of 225 IgM H chain sequences, with five or fewer V mutations, led to the alignment of 199 sequences. Only 5.0% of sequences lacked N nucleotides at the VHD junction (N1), and 10.6% at the DJH junction (N2). Long N regions (>9 nt) were seen in 20.6% of N1 regions and 17.1% of N2 regions. Using a statistical analysis based upon known features of N addition, and mutation analysis, two of these N regions aligned with D gene segments, and a third aligned with an inverted D gene segment. Nine additional sequences included possible alignments with a second D segment. Four of the remaining 40 long N1 regions included 5′ sequences having six or more matches to V gene end motifs, which may be the result of V gene replacement. Such sequences were not seen in long N2 regions. The long N regions frequently seen in the expressed repertoire of human Ig gene rearrangements can therefore only partly be explained by V gene replacement and D-D fusion.
A Functional C5a Anaphylatoxin Receptor in a Teleost Species
Authors: Holland M. Claire H. , Lambris John D.
Published: April 2014

The anaphylatoxins are potent, complement-derived low m.w. proteins that bind to specific seven-transmembrane receptors to elicit and amplify a variety of inflammatory reactions. C5a is the most potent of these phlogistic peptides and is a strong chemoattractant for neutrophils and macrophages/monocytes. Although lower vertebrates possess complement systems that are believed to function similarly to those of mammals, anaphylatoxin receptors have not previously been characterized in any nonmammalian vertebrate. To study the functions of C5a in teleost fish, we generated recombinant C5a of the rainbow trout, Oncorhynchus mykiss (tC5a), and used fluoresceinated tC5a (tC5aF) and flow cytometry to identify the C5a receptor (C5aR) on trout leukocytes. Granulocytes/Macrophages present in cell suspensions of the head kidney (HKL), the main hemopoietic organ in teleosts, showed a univariate type of receptor expression, whereas those from the peripheral blood demonstrated either a low or high level of expression. The binding of tC5aF was inhibited by excess amounts of unlabeled tC5a or tC5adesArg, demonstrating that sites other than the C-terminal of tC5a interact with the C5aR. Both tC5a and tC5adesArg were able to induce chemotactic responses in granulocytes in a concentration-dependent manner, but the desArg derivative was at least 10-fold less active. Homologous desensitization occurred after HKL were exposed to continuous or high concentrations of tC5a, with a loss of tC5aF binding and an 80% reduction in chemotactic responses toward tC5a. Pertussis toxin reduced the migration of HKL toward tC5a by 40%, suggesting only a partial involvement of pertussis toxin-sensitive Gi proteins in tC5a-mediated chemotaxis.
The Tetraspanin CD81 Is Necessary for Partitioning of Coligated CD19/CD21-B Cell Antigen Receptor Complexes into Signaling-Active Lipid Rafts
Authors: Cherukuri Anu , Shoham Tsipi , Sohn Hae Won , Levy Shoshana , Brooks Stephen , Carter Robert , Pierce Susan K.
Published: April 2014

Tetraspanins have been hypothesized to facilitate the organization of functional multimolecular membrane complexes. In B cells the tetraspanin CD81 is a component of the CD19/CD21 complex. When coligated to the B cell Ag receptor (BCR), the CD19/CD21 complex significantly enhances BCR signaling in part by prolonging the association of the BCR with signaling-active lipid rafts. In this study CD81 is shown to associate with lipid rafts upon coligation of the BCR and the CD19/CD21 complex. Using B cells from CD81-deficient mice we demonstrate that in the absence of CD81, coligated BCR and CD19/CD21 complexes fail to partition into lipid rafts and enhance BCR signaling from rafts. Furthermore, a chimeric CD19 protein that associates only weakly if at all with CD81 fails to promote the association of coligated BCR with lipid rafts. The requirement for CD81 to promote lipid raft association may define a novel mechanism by which tetraspanins function as molecular facilitators of signaling receptors.
A Role for TCR Affinity in Regulating Naive T Cell Homeostasis
Authors: Kieper William C. , Burghardt J. Theodore , Surh Charles D.
Published: April 2014

Homeostatic signals that control the overall size and composition of the naive T cell pool have recently been identified to arise from contact with self-MHC/peptide ligands and a cytokine, IL-7. IL-7 presumably serves as a survival factor to keep a finite number of naive cells alive by preventing the onset of apoptosis, but how TCR signaling from contact with self-MHC/peptide ligands regulates homeostasis is unknown. To address this issue, murine polyclonal and TCR-transgenic CD8+ cells expressing TCR with different affinities for self-MHC/peptide ligands, as depicted by the CD5 expression level, were analyzed for their ability to respond to and compete for homeostatic factors under normal and lymphopenic conditions. The results suggest that the strength of the TCR affinity determines the relative “fitness” of naive T cells to compete for factors that support cell survival and homeostatic proliferation.
Organism-Specific Neutrophil-Endothelial Cell Interactions in Response to Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus
Authors: Moreland Jessica G. , Bailey Gail , Nauseef William M. , Weiss Jerrold P.
Published: April 2014

The recruitment of polymorphonuclear leukocytes (PMNs) from the vascular space into the lung interstitium and airspace is an early step in the host innate immune response to bacterial invasion of these sites. To determine the ability of intact bacteria to directly elicit PMN migration across an endothelial monolayer, we studied in vitro migration of PMNs across a monolayer of human pulmonary microvascular endothelial cells in response to Streptococcus pneumoniae, Staphylococcus aureus, and Escherichia coli, as well as to purified E. coli LPS. Bacterial induction of PMN migration was dose dependent and elicited by ≥104 bacteria/ml of each of the species tested. Pretreatment of PMNs with blocking Abs to CD18 significantly inhibited migration of PMN in response to all stimuli tested, but had the most profound effect on migration to S. pneumoniae and S. aureus. Intact E. coli were 10 times more potent in inducing transmigration of PMNs than a corresponding amount of purified LPS. Bacterial induction of PMN migration did not correlate with up-regulation of surface endothelial ICAM-1 expression (purified LPS ≫ intact E. coli > S. aureus and S. pneumoniae) nor up-regulation of VCAM-1 and E-selectin. Neutralizing Ab to ICAM-1 had no effect on PMN migration to any of the bacteria or to purified LPS. These findings demonstrate that diverse bacterial pathogens induce PMN migration across a pulmonary microvascular endothelial cell monolayer in a fashion that appears to be organism specific. In addition, intact bacteria elicit PMN-endothelial cell interactions distinct from those seen when purified bacterial products are used as agonists.
Dendritic Cells from Malaria-Infected Mice Are Fully Functional APC
Authors: Perry James A. , Rush Adam , Wilson Randy J. , Olver Christine S. , Avery Anne C.
Published: April 2014

Malaria infection has long been associated with diminished T cell responses in vitro and more recently in experimental studies in vivo. Suppression of T cell-proliferative responses during malaria has been attributed to macrophages in a variety of murine and human systems. More recently, however, attention has been directed at the role of dendritic cells in this phenomenon, with several studies suggesting that maturation of dendritic cells is inhibited in vitro by the presence of malaria-infected E. In the studies reported here, we have examined the function of dendritic cells taken directly from infected mice. We found that they express high levels of costimulatory proteins and class II MHC, can activate naive T cells to produce IL-2 as efficiently as dendritic cells from uninfected mice, and support high levels of IFN-γ production by naive T cells through an IL-12-dependent mechanism. Dendritic cells from infected mice also support higher levels of TNF-α production by naive T cells. These same dendritic cells present parasite Ag to a malaria-specific T cell hybridoma, a finding that demonstrates that dendritic cells participate in the generation of Ag-specific immunity during infection. Our findings challenge the contention that dendritic cell function is inhibited by malaria infection.
Conserved Hierarchy of Helper T Cell Responses in a Chimpanzee during Primary and Secondary Hepatitis C Virus Infections
Authors: Shoukry Naglaa H. , Sidney John , Sette Alessandro , Walker Christopher M.
Published: April 2014

Control of hepatitis C virus (HCV) infection could be influenced by the timing and magnitude of CD4+ T cell responses against individual epitopes. We characterized CD4+ T cells targeting seven Pan troglodytes (Patr) class II-restricted epitopes during primary and secondary HCV infections of a chimpanzee. All Patr-DR-restricted HCV epitopes bound multiple human HLA-DR molecules, indicating the potential for overlap in epitopes targeted by both species. Some human MHC class II molecules efficiently stimulated IL-2 production by chimpanzee virus-specific T cell clones. Moreover, one conserved epitope designated NS31248 (GYKVLVLNPSV) overlapped a helper epitope that is presented by multiple HLA-DR molecules in humans who spontaneously resolved HCV infection. Resolution of primary infection in the chimpanzee was associated with an initial wave of CD4+ T cells targeting a limited set of dominant epitopes including NS31248. A second wave of low-frequency CD4+ T cells targeting other subdominant epitopes appeared in blood several weeks later after virus replication was mostly contained. During a second infection 7 years later, CD4+ T cells against all epitopes appeared in blood sooner and at higher frequencies but the pattern of dominance was conserved. In summary, primary HCV infection in this individual was characterized by T cell populations targeting two groups of MHC class II-restricted epitopes that differed in frequency and kinetics of appearance in blood. The hierarchial nature of the CD4+ T cell response, if broadly applicable to other HCV-infected chimpanzees and humans, could be a factor governing the outcome of HCV infection.
Differential Regulation of IgG Anti-Capsular Polysaccharide and Antiprotein Responses to Intact Streptococcus pneumoniae in the Presence of Cognate CD4+ T Cell Help
Authors: Khan Abdul Q. , Lees Andrew , Snapper Clifford M.
Published: April 2014

The relative lack of memory for IgG antipolysaccharide responses is believed to be secondary to the inability of polysaccharides to associate with MHC class II molecules and thus a failure to recruit cognate CD4+ T cell help. However, little is known concerning the role of T cells and the generation of memory for antipolysaccharide Ig responses to intact extracellular bacteria. We used heat-killed, intact Streptococcus pneumoniae, capsular type 14 (Pn14), to evaluate the IgM and IgG responses specific for the capsular polysaccharide (PPS14), the phosphorylcholine determinant of the cell wall C-polysaccharide, and the cell wall protein, pneumococcal surface protein A (PspA). We demonstrate that the IgG (but not IgM), anti-PPS14, and anti-PspA responses to Pn14 are CD4+ T cell dependent and TCR specific. Nevertheless, in contrast to the anti-PspA response, the IgG anti-PPS14 response shows no apparent memory, an accelerated kinetics of primary Ig induction, and a more rapid delivery of CD4+ T cell help. In contrast, the IgG anti-phosphorylcholine response, although also dependent on CD4+ T cells, is TCR nonspecific. We make similar observations using soluble conjugates of PPS14-PspA and C-polysaccharide-PspA. These data lead us to suggest that the central issue concerning the mechanisms underlying different functional outcomes for anti-bacterial IgG responses to capsular polysaccharide vs protein Ags is not necessarily based on the ability to recruit cognate CD4+ T cell help, but perhaps on the nature of the B cell Ag receptor signaling that occurs and/or on the responding B cell subpopulations.
Membrane-Associated IL-1 Contributes to Chronic Synovitis and Cartilage Destruction in Human IL-1α Transgenic Mice
Authors: Niki Yasuo , Yamada Harumoto , Kikuchi Toshiyuki , Toyama Yoshiaki , Matsumoto Hideo , Fujikawa Kyosuke , Tada Norihiro
Published: April 2014

IL-1 molecules are encoded by two distinct genes, IL-1α and IL-1β. Both isoforms possess essentially identical activities and potencies, whereas IL-1α, in contrast to IL-1β, is known to act as a membrane-associated IL-1 (MA-IL-1) and plays an important role in a variety of inflammatory situations. The transgenic (Tg) mouse line (Tg1706), which was generated in our laboratory, overexpresses human IL-1α (hIL-1α) and exhibits a severe arthritic phenotype characterized by autonomous synovial proliferation with subsequent cartilage destruction. Because the transgene encoded Lys64 to Ala271 of the hIL-1α amino acid sequence, Tg mice may overproduce MA-IL-1 as well as soluble IL-1α. The present study investigated whether MA-IL-1 contributes to synovial proliferation and cartilage destruction in the development of arthritis. Flow cytometric analysis revealed that both macrophage-like and fibroblast-like synoviocytes constitutively produce MA-IL-1. D10 cell proliferation assay revealed MA-IL-1 bioactivity of paraformaldehyde-fixed synoviocytes and the further induction of endogenous mouse MA-IL-1 via autocrine mechanisms. MA-IL-1 expressed on synoviocytes triggered synoviocyte self-proliferation through cell-to-cell (i.e., juxtacrine) interactions and also promoted proteoglycan release from the cartilage matrix in chondrocyte monolayer culture. Interestingly, the severity of arthritis was significantly correlated with MA-IL-1 activity rather than with soluble IL-1α activity or concentration of serum hIL-1α. Moreover, when the Tg1706 line was compared with the Tg101 line, which selectively overexpresses the 17-kDa mature hIL-1α, the severity of arthritis was significantly higher in the Tg1706 line than in the Tg101 line. These results suggest that MA-IL-1 contributes to synoviocyte self-proliferation and subsequent cartilage destruction in inflammatory joint disease such as rheumatoid arthritis.
Soluble Fractalkine Prevents Monocyte Chemoattractant Protein-1-Induced Monocyte Migration via Inhibition of Stress-Activated Protein Kinase 2/p38 and Matrix Metalloproteinase Activities
Authors: Vitale Sébastien , Schmid-Alliana Annie , Breuil Véronique , Pomeranz Manuel , Millet Marie-Ange , Rossi Bernard , Schmid-Antomarchi Heidy
Published: April 2014

In this study, we address the question of the cross-talk between two chemokines that are cosecreted during inflammation, namely monocyte chemoattractant protein-1 (MCP-1) and soluble fractalkine (s-FKN), toward monocyte migration. We found that s-FKN fails to induce MonoMac6 cell migration per se. Interestingly, this chemokine antagonizes transendothelial migration and chemotaxis of MonoMac6 cells and freshly isolated human monocytes induced by MCP-1, indicating a direct effect of s-FKN on monocytic cells. In this study, we found that stress-activated protein kinase (SAPK)1/c-Jun N-terminal kinase 1 and SAPK2/p38 are involved in the control of MCP-1-induced MonoMac6 cell migration. We demonstrated that s-FKN abrogates the MCP-1-induced SAPK2/p38 activation as well as the upstream Pyk2 activity. Furthermore, we observed that s-FKN also inhibits the activity of a major matrix metalloproteinase (MMP), namely MMP-2. Taken collectively, our results indicate that the s-FKN antagonizes the chemoattractant effect of MCP-1 on monocytes, likely by inhibiting crucial signaling pathways, like SAPK2/p38 and MMP-2 activities.
Sustained IL-12 Signaling Is Required for Th1 Development
Authors: Athie-Morales Veronica , Smits Hermelijn H. , Cantrell Doreen A. , Hilkens Catharien M. U.
Published: April 2014

STAT4 is an essential transcription factor for Th1 cell development. IL-12 and IFN-α both activate STAT4, but with different kinetics. In this study we compared their capacities to drive differentiation of human naive Th cells toward the Th1 phenotype. The Th1-polarizing activity of IFN-α was much weaker than that of IL-12, correlating with a marked difference in the kinetics of STAT4 activation; the response to IL-12 was sustained (>48 h), whereas the response to IFN-α was transient (4 h). The continuous presence of IL-12 was required for sustained STAT4 activation. Similarly, optimal Th1 polarization was only achieved upon prolonged exposure to IL-12 and could not be induced by a transient IL-12 pulse. Furthermore, the cytokine IL-2 potentiated sustained IL-12/STAT4 responses through up-regulation of IL-12R expression and synergized with IL-12 in driving Th1 cell development. Transient IFN-α responses, on the other hand, were not prolonged by IL-2. IFN-α treatment induced down-regulation of IFN-αβ receptor subunit 1, rendering cells refractory to IFN-α, but did not trans-inhibit the IL-12/STAT4 response. These data indicate that sustained IL-12 signaling is essential for optimal Th1 cell development and that transient activation of STAT4 in response to IFN-α may explain the poor Th1-polarizing capacity of this cytokine. Collectively these data show that the duration of cytokine signaling is important for determining the biological response.
CD4+ T Cells Mediate Murine Syngeneic Graft-versus-Host Disease-Associated Colitis
Authors: Bryson J. Scott , Zhang Lining , Goes Sarah W. , Jennings C. Darrell , Caywood Betty E. , Carlson Sonia L. , Kaplan Alan M.
Published: April 2014

Syngeneic graft-vs-host disease (SGVHD) develops following lethal irradiation, reconstitution with syngeneic bone marrow, and treatment with a 21-day course of the immunosuppressive agent cyclosporin A (CsA). Following cessation of CsA, this inducible disease is characterized by weight loss, diarrhea, and development of inflammation in the colon and liver. Although nonspecific effector cells and Th1 cytokines have been shown to participate in disease induction, the role of T cells has not been fully elucidated. Initial studies demonstrated significant increases in CD4+ T cells, but not other T cell populations in the colons of diseased animals relative to transplant control animals. To demonstrate a functional linkage between increases in colonic CD4+ T cells and disease induction, in vivo T cell depletion studies were performed. Beginning on the day of bone marrow transplantation, groups of control and CsA-treated animals were treated with mAb against either CD4 or CD8 for 21 days. Treatment with anti-CD4, but not anti-CD8, eliminated clinical symptoms and colon pathology. Interestingly, neither anti-CD4 nor anti-CD8 therapy affected the development of liver pathology associated with SGVHD. These findings demonstrated that CD4+ T cells initiate development of the intestinal inflammation associated with murine SGVHD.
A CD91-Positive Subset of CD11c+ Blood Dendritic Cells: Characterization of the APC that Functions to Enhance Adaptive Immune Responses against CD91-Targeted Antigens
Authors: Hart Justin P. , Gunn Michael D. , Pizzo Salvatore V.
Published: April 2014

Dendritic cells (DC) and other APCs rely on a number of specialized receptors to facilitate the uptake and intracellular accumulation of Ags. In this capacity, APCs use receptor-mediated endocytosis to enhance Ag presentation and the stimulation of Ag-specific T cells. Studies have demonstrated that the targeted delivery of Ags in vivo to CD91/the low-density lipoprotein receptor-related protein (CD91/LRP) induces enhanced activation of the adaptive immune system. However, the APC that mediates these augmented, Ag-specific responses remains to be characterized. In this study, we show that a subset of CD11c+ lineage-negative (lin−) DC expresses the scavenger receptor CD91/LRP and that these rare APC are primarily responsible for the T cell activation that occurs following CD91/LRP-mediated Ag uptake in whole blood. The targeting of Ags to CD91/LRP results in enhanced receptor-mediated uptake within both lin− DCs and monocytes, and this uptake results in markedly increased T cell activation. Finally, purified cellular populations were used to demonstrate that CD11c+ lin− DC, but not monocytes, are capable of stimulating T cell activation following CD91/LRP-mediated Ag uptake. Therefore, CD11c+ lin− DC use CD91/LRP to facilitate the uptake and subsequent presentation of an array of Ags complexed within the CD91/LRP ligand, the activated form of α2-macroglobulin (α2M*).
The IL-12 Signature: NK Cell Terminal CD56+high Stage and Effector Functions
Authors: Loza Matthew J. , Perussia Bice
Published: April 2014

We report that human peripheral NK cells expressing high CD56 levels (CD56+high) are terminally differentiated cells indistinguishable from mature NK cells recently activated in the presence of IL-12, and not a functionally distinct NK-cell subset or progenitors to mature CD56+low NK cells. CD56+high NK cells coexpress all differentiation Ags constitutive or inducible in mature (CD56+) NK cells, except CD16, present at lower level than on most mature NK cells. Also, activation markers, activating receptors and adhesion molecules, and most inducible receptors are expressed exclusively and constitutively and are inducible at higher levels on CD56+high than on CD56+low NK cells. Consistent with their activated phenotype, many CD56+high NK cells are cycling and mediate heightened effector functions (proliferation, IFN-γ and IL-10 but not IL-13 production) in response to IL-12 and other NK cell-specific stimuli. Conversely, IL-12 induces on CD56+low NK cells all markers constitutively expressed on the CD56+high NK cells, concomitantly preventing the IL-2 (and IL-15)-inducible expression of NKp44 and CD16 re-expression after immune complex-induced down-modulation, and CD56−/+low NK cells acquire a CD56+high NK cell phenotype in short term in vitro culture with IL-12. The significance of these findings to the NK cell-mediated regulation of immune responses and NK cell development is discussed.
Cutting Edge: Extracellular Signal-Regulated Kinases 1/2 Function as Integrators of TCR Signal Strength
Authors: Schade Andrew E. , Levine Alan D.
Published: April 2014

Altered signaling through the TCR is currently showing promise for immunotherapy. However, the molecular mechanisms are not completely understood. Therefore, we investigated whether varying the strength of TCR engagement in various human T cells would yield different second messenger responses. The kinetics and duration of extracellular signal-regulated kinase (ERK) activation, central to multiple cellular responses, are distinctly dependent on the T cell activation state (naive vs effector), strength of TCR cross-linking, and input from the phosphatidylinositol-3 kinase pathway, which is regulated by cytokines and growth factors. Moreover, the duration of ERK activation affects c-Fos expression, a component of the AP-1 transcription complex. Thus, the character of ERK activation, transient or sustained, acts as a signal integrator to quantify the strength of TCR engagement and direct the cellular response.
Cutting Edge: TREM-Like Transcript-1, a Platelet Immunoreceptor Tyrosine-Based Inhibition Motif Encoding Costimulatory Immunoreceptor that Enhances, Rather than Inhibits, Calcium Signaling via SHP-2
Authors: Barrow Alexander D. , Astoul Emmanuelle , Floto Andres , Brooke Gary , Relou Ingrid A. M. , Jennings Nicola S. , Smith Kenneth G. C. , Ouwehand Willem , Farndale Richard W. , Alexander Denis R. , Trowsdale John
Published: April 2014

To date, immunoreceptor tyrosine-based inhibition motifs (ITIMs) have been shown to mediate inhibitory properties. We report a novel triggering receptor expressed on myeloid cells (TREM) family member, TREM-like transcript-1 (TLT1), which differs from the activating members because its cytoplasmic tail contains two ITIMs at Y245 and Y281. A TLT1 splice variant (TLT1sp) encodes a different cytoplasmic tail lacking ITIMs. Both isoforms are expressed in resting platelet α-granules, which are up-regulated to the cell surface following activation. TLT1 recruited Src homology 2 domain-containing tyrosine phosphatase (SHP)-2 to the “classical” ITIM (Y281) but not the “nonclassical” ITIM (Y245). In contrast to previously characterized ITIM receptors, TLT1 enhanced, rather than inhibited, FcεRI-mediated calcium signaling in rat basophilic leukemia cells, a property dependent on the SHP-2 recruiting classical Y281 ITIM. Therefore, TLT1 represents a new costimulatory ITIM immunoreceptor and is the second ITIM-bearing receptor to be identified in platelets after platelet endothelial cell adhesion molecule-1.
CpG Motifs as Proinflammatory Factors Render Autochthonous Tumors Permissive for Infiltration and Destruction
Authors: Garbi Natalio , Arnold Bernd , Gordon Siamon , Hämmerling Günter J. , Ganss Ruth
Published: April 2014

In a transgenic mouse model expressing SV40 T Ag (Tag) as a de novo tumor Ag, immune surveillance fails and islet cell carcinomas grow progressively. To develop an anticancer strategy that would be effective in eradicating solid, autochthonously growing tumors, we evaluated the effectiveness of immunostimulatory oligodeoxynucleotides (ODN) with cytosine-guanine-rich (CpG) motifs (CpG-ODN). In a classical vaccination protocol, Tag was administered with CpG-ODN as adjuvant. The antitumor vaccination, however, was only effective in a prophylactic setting, despite the successful activation of a Tag-specific CTL response in vivo. Histological examination demonstrated that even primed immune cells failed to infiltrate tumors once a malignant environment was established. To ensure that effector cells were not limiting, highly activated tumor Ag-specific T cells were transferred into tumor-bearing mice. However, this treatment also failed to result in tumor infiltration and rejection. Therefore, we further tested the efficacy of CpG-ODN as a proinflammatory agent in combination with the transfer of preactivated Tag-specific CD4+ and CD8+ T cells. Indeed, this combination therapy proved to be highly effective, because CpG-ODN rendered insulinomas permissive for massive infiltration and destruction. The opening of tumor tissue correlated with uptake of CpG-ODN by tissue-resident macrophages and a strong up-regulation of adhesion molecules such as ICAM and VCAM on blood vessel endothelia. These data demonstrate that systemic application of proinflammatory reagents drastically enhances extravasation of effector cells into tumor tissue, an observation that is of general importance for immunotherapy of solid tumors in a clinical setting.
CD4 Raft Association and Signaling Regulate Molecular Clustering at the Immunological Synapse Site
Authors: Balamuth Fran , Brogdon Jennifer L. , Bottomly Kim
Published: April 2014

T cell activation is associated with the partitioning of TCRs and other signaling proteins, forming an immunological synapse. This study demonstrates a novel function for the CD4 coreceptor in regulating molecular clustering at the immunological synapse site. We show using transgenic mouse and retroviral reconstitution studies that CD4 is required for TCR/protein kinase C (PKC) θ clustering. Specifically, we demonstrate that CD4 palmitoylation sequences are required for TCR/PKCθ raft association and subsequent clustering, indicating a particular role for raft-associated CD4 molecules in regulating immune synapse organization. Although raft association of CD4 is necessary, it is not sufficient to mediate clustering, as cytoplasmic tail deletion mutants are able to localize to rafts, but are unable to mediate TCR/PKCθ clustering, indicating an additional requirement for CD4 signaling. These studies suggest that CD4 coreceptor function is regulated not only through its known signaling function, but also by posttranslational lipid modifications which regulate localization of CD4 in lipid rafts.
The Inducible Costimulator Plays the Major Costimulatory Role in Humoral Immune Responses in the Absence of CD28
Authors: Suh Woong-Kyung , Tafuri Anna , Berg-Brown Nancy N. , Shahinian Arda , Plyte Suzanne , Duncan Gordon S. , Okada Hitoshi , Wakeham Andrew , Odermatt Bernhard , Ohashi Pamela S. , Mak Tak W.
Published: April 2014

CD28 plays crucial costimulatory roles in T cell proliferation, cytokine production, and germinal center response. Mice that are deficient in the inducible costimulator (ICOS) also have defects in cytokine production and germinal center response. Because the full induction of ICOS in activated T cells depends on CD28 signal, the T cell costimulatory capacity of ICOS in the absence of CD28 has remained unclear. We have clarified this issue by comparing humoral immune responses in wild-type, CD28 knockout (CD28 KO), and CD28-ICOS double-knockout (DKO) mice. DKO mice had profound defects in Ab responses against environmental Ags, T-dependent protein Ags, and vesicular stomatitis virus that extended far beyond those observed in CD28 KO mice. However, DKO mice mounted normal Ab responses against a T-independent Ag, indicating that B cell function itself was normal. Restimulated CD4+ DKO T cells that had been primed in vivo showed decreased proliferation and reduced IL-4 and IL-10 production compared with restimulated CD4+ T cells from CD28 KO mice. Thus, in the absence of CD28, ICOS assumes the major T cell costimulatory role for humoral immune responses. Importantly, CD28-mediated ICOS up-regulation is not essential for ICOS function in vivo.
An Inhibitory Ig Superfamily Protein Expressed by Lymphocytes and APCs Is Also an Early Marker of Thymocyte Positive Selection
Authors: Han Peggy , Goularte Olivia D. , Rufner Kevin , Wilkinson Beverley , Kaye Jonathan
Published: April 2014

Positive selection of developing thymocytes is associated with changes in cell function, at least in part caused by alterations in expression of cell surface proteins. Surprisingly, however, few such proteins have been identified. We have analyzed the pattern of gene expression during the early stages of murine thymocyte differentiation. These studies led to identification of a cell surface protein that is a useful marker of positive selection and is a likely regulator of mature lymphocyte and APC function. The protein is a member of the Ig superfamily and contains conserved tyrosine-based signaling motifs. The gene encoding this protein was independently isolated recently and termed B and T lymphocyte attenuator (Btla). We describe in this study anti-BTLA mAbs that demonstrate that the protein is expressed in the bone marrow and thymus on developing B and T cells, respectively. BTLA is also expressed by all mature lymphocytes, splenic macrophages, and mature, but not immature bone marrow-derived dendritic cells. Although mice deficient in BTLA do not show lymphocyte developmental defects, T cells from these animals are hyperresponsive to anti-CD3 Ab stimulation. Conversely, anti-BTLA Ab can inhibit T cell activation. These results implicate BTLA as a negative regulator of the activation and/or function of various hemopoietic cell types.