bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Granulocyte-Macrophage Colony-Stimulating Factor Signaling and Microglial Proliferation by Anti-CD45RO: Role of Hck Tyrosine Kinase and Phosphatidylinositol 3-Kinase/Akt
Authors: Suh Hyeon-Sook , Kim Mee-Ohk , Lee Sunhee C.
Published: April 2014

Increasing evidence suggests that CD45, a transmembrane protein tyrosine phosphatase, is an important modulator of macrophage activation. Microglia, resident brain macrophages, express CD45 and proliferate under pathologic conditions. In this study, we examined the role of CD45 in modulating GM-CSF-induced proliferation and signal transduction in primary human microglial cultures. Soluble, but not immobilized anti-CD45RO induced tyrosine phosphatase activity and inhibited GM-CSF-induced microglial proliferation. Microglial proliferation was also inhibited by PP2 (Src inhibitor), LY294002 (PI3K inhibitor), and U0126 (MEK inhibitor). GM-CSF induced phosphorylation of Jak2, Stat5, Hck (the myeloid-restricted Src kinase), Akt, Stat3, and Erk MAPKs in microglia. Of these, anti-CD45RO inhibited phosphorylation of Hck and Akt, and PP2 inhibited phosphorylation of Hck and Akt. In a macrophage cell line stably overexpressing wild-type or kinase-inactive Hck, GM-CSF increased proliferation of the control (empty vector) and wild-type but not kinase-inactive cells, and this was inhibited by anti-CD45RO. Together, these results demonstrate that, in macrophages, Hck tyrosine kinase is activated by GM-CSF, and that Hck plays a pivotal role in cell proliferation and survival by activating the PI3K/Akt pathway. Ab-mediated activation of macrophage and microglial CD45 tyrosine phosphatase may have therapeutic implications for CNS inflammatory diseases.
IL-6 Plays a Unique Role in Initiating c-Maf Expression during Early Stage of CD4 T Cell Activation
Authors: Yang Yu , Ochando Jordi , Yopp Adam , Bromberg Jonathan S. , Ding Yaozhong
Published: April 2014

The transcription factor c-Maf plays a critical and selective role in IL-4 gene transcription. Little is known about the mechanism that guides c-Maf regulation during early T cell activation. We report that IL-6 but not IL-4 or other cytokines, rapidly up-regulates c-Maf transcription, as early as 3 h after TCR activation in naive CD4+ T cells. c-Maf induction requires both IL-6- and TCR-initiated signals, and is independent of IL-4/Stat6 signals. Cyclosporin A and FK506, which target calcineurin and thereby inhibit TCR-mediated Ca2+ signal pathways, block IL-6-mediated c-Maf expression. We show that Stat3 binds the c-maf promoter in CD4 T cells after IL-6 stimulation, and also transactivates the c-maf promoter in reporter gene assays. IL-6 induces similar c-Maf expression in protein kinase Cθ-deficient CD4+ T cells. Furthermore, IL-6 enhances IL-4 gene expression very early after TCR activation in both wild-type and Stat6-deficient CD4+ T cells. Our findings suggest that IL-6 plays a unique role in initiating c-Maf expression after TCR engagement, and may subsequently regulate early IL-4 production and Th2 commitment.
In Vivo and in Absence of a Thymus, the Enforced Expression of the Notch Ligands Delta-1 or Delta-4 Promotes T Cell Development with Specific Unique Effects
Authors: de La Coste Alix , Six Emmanuelle , Fazilleau Nicolas , Mascarell Laurent , Legrand Nicolas , Mailhé Marie-Pierre , Cumano Ana , Laâbi Yacine , Freitas Antonio A.
Published: April 2014

The role of Notch signaling in T cell commitment during lymphoid development is well established. However, the identity of the ligand that triggers this critical signal in vivo is still unclear. By overexpressing Delta-1 and Delta-4 ligands in the hemopoietic cells of athymic nu/nu host mice, we demonstrate that, in vivo and in the absence of a thymus, Delta-1 or Delta-4 expression is sufficient to promote T cell development from the most immature progenitor stages to complete maturation of both CD8+ and CD4+ αβ T cells. The mature T cells developing in a Delta-1- or Delta-4-enriched environment express a diverse TCR repertoire, are able to proliferate upon in vitro TCR stimulation, but show different profiles of cytokine production after in vitro anti-CD3 stimulation.
Pro-IL-16 Regulation in Activated Murine CD4+ Lymphocytes
Authors: Ren Fucheng , Zhan Xin , Martens Gregory , Lee Jinhee , Center David , Hanson Sue Kim , Kornfeld Hardy
Published: April 2014

Prior DNA microarray studies suggested that IL-16 mRNA levels decrease following T cell activation, a property unique among cytokines. We examined pro-IL-16 mRNA and protein expression in resting and anti-CD3 mAb-activated primary murine CD4+ T cells. Consistent with the microarray reports, pro-IL-16 mRNA levels fell within 4 h of activation, and this response is inhibited by cyclosporin A. Total cellular pro-IL-16 protein also fell, reaching a nadir at 48 h. Pro-IL-16 comprises a C-terminal cytokine domain and an N-terminal prodomain that are cleaved by caspase-3. Pro-IL-16 expressed in transfected tumor cells was previously shown to translocate to the nucleus and to promote G0/G1 arrest by stabilizing the cyclin-dependent kinase inhibitor p27Kip1. In the present study, we observed increased S-phase kinase-associated protein 2 mRNA expression in IL-16 null mice, but basal expression and activation-dependent regulation of p27Kip1 were no different from wild-type mice. Stimulation with anti-CD3 mAb induced transiently greater thymidine incorporation in IL-16-deficient CD4+ T cells than wild-type controls, but there was no difference in cell survival or in the CFSE dilution profiles. Analysis of CD4+ T cell proliferation in vivo using BrdU labeling similarly failed to identify a hyperproliferative phenotype in T cells lacking IL-16. These data demonstrate that pro-IL-16 mRNA and protein expression are dynamically regulated during CD4+ T cell activation by a calcineurin-dependent mechanism, and that pro-IL-16 might influence T cell cycle regulation, although not in a dominant manner.
Different Diabetogenic Potential of Autoaggressive CD8+ Clones Associated with IFN-γ-Inducible Protein 10 (CXC Chemokine Ligand 10) Production but Not Cytokine Expression, Cytolytic Activity, or Homing Characteristics
Authors: Ejrnaes Mette , Videbaek Nicoline , Christen Urs , Cooke Anne , Michelsen Birgitte K. , von Herrath Matthias
Published: April 2014

Type 1 diabetes mellitus is an autoimmune disease characterized by T cell-mediated destruction of the insulin-producing β cells in the islets of Langerhans. From studies in animal models, CD8+ T cells recognizing autoantigens such as islet-specific glucose-6-phosphatase catalytic subunit-related protein, insulin, or glutamic acid decarboxylase (GAD) are believed to play important roles in both the early and late phases of β cell destruction. In this study, we investigated the factors governing the diabetogenic potential of autoreactive CD8+ clones isolated from spleens of NOD mice that had been immunized with GAD65515–524 or insulin B-chain15–23 peptides. Although these two clones were identical in most phenotypic and functional aspects, for example cytokine production and killing of autologous β cells, they differed in the expression of IFN-γ-inducible protein-10, which was only produced at high levels by the insulin-specific clone, but not by the GAD65-specific clone, and other autoantigen-specific nonpathogenic CD8 T cell clones. Interestingly, upon i.p. injection into neonatal mice, only the insulin B-chain15–23-reactive CD8+ T clone accelerated diabetes in all recipients after 4 wk, although both insulin- and GAD-reactive clones homed to pancreas and pancreatic lymph nodes with similar kinetics. Diabetes was associated with increased pancreatic T cell infiltration and, in particular, recruitment of macrophages. Thus, secretion of IFN-γ-inducible protein-10 by autoaggressive CD8+ lymphocytes might determine their diabetogenic capacity by affecting recruitment of cells to the insulitic lesion.
Inability of bm14 Mice to Respond to Theiler’s Murine Encephalomyelitis Virus Is Caused by Defective Antigen Presentation, Not Repertoire Selection
Authors: Block Matthew S. , Mendez-Fernandez Yanice V. , Van Keulen Virginia P. , Hansen Michael J. , Allen Kathleen S. , Taboas Anya L. , Rodriguez Moses , Pease Larry R.
Published: April 2014

Natural selection drives diversification of MHC class I proteins, but the mechanism by which selection for polymorphism occurs is not known. New variant class I alleles differ from parental alleles both in the nature of the CD8 T cell repertoire formed and the ability to present pathogen-derived peptides. In the current study, we examined whether T cell repertoire differences, Ag presentation differences, or both account for differential viral resistance between mice bearing variant and parental alleles. We demonstrate that nonresponsive mice have inadequate presentation of viral Ag, but have T cell repertoires capable of mounting Ag-specific responses. Although previous work suggests a correlation between the ability to present an Ag and the ability to generate a repertoire responsive to that Ag, we show that the two functions of MHC class I are independent.
The Impact of Misfolding versus Targeted Degradation on the Efficiency of the MHC Class I-Restricted Antigen Processing
Authors: Golovina Tatiana N. , Morrison Susan E. , Eisenlohr Laurence C.
Published: April 2014

Evidence suggests that most epitopes presented by MHC class I molecules are derived from those newly synthesized proteins that are defective due to errors during manufacture. We examined epitope production from model cytosolic and exocytic proteins modified in various ways. Substrates containing a degradation targeting sequence demonstrated very rapid turnover and enhanced epitope production, as was the case for substrate retargeted from endoplasmic reticulum to cytosol. For less radical alterations, including point mutation and deletion and elimination of glycosylation sites, despite detectable changes in folding, half-life was only moderately decreased and there were no significant increases in epitope production. Puromycin, which causes premature termination of protein synthesis, also had no impact upon epitope production. It appears that most defective proteins are not rapidly dispensed with and the targeting of most nascent proteins for Ag processing is not tied to quality control.
Constitutively Active Aryl Hydrocarbon Receptor Expressed Specifically in T-Lineage Cells Causes Thymus Involution and Suppresses the Immunization-Induced Increase in Splenocytes
Authors: Nohara Keiko , Pan Xiaoqing , Tsukumo Shin-ichi , Hida Azumi , Ito Tomohiro , Nagai Haruko , Inouye Kaoru , Motohashi Hozumi , Yamamoto Masayuki , Fujii-Kuriyama Yoshiaki , Tohyama Chiharu
Published: April 2014

The aryl hydrocarbon receptor (AhR) is a transcription factor belonging to the basic helix-loop-helix-PER-ARNT-SIM superfamily. Xenobiotics, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, bind the receptor and trigger diverse biological reactions. Thymocyte development and T cell-dependent immune reactions are sensitive targets of AhR-dependent 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity. However, the exact role of the AhR in T cells in animals exposed to exogenous ligands has not been clarified because indirect effects of activated AhR in other cell types cannot be excluded. In this study, we generated transgenic (Tg) mice expressing a constitutively active mutant of AhR under the regulation of a T cell-specific CD2 promoter to examine AhR function in T cells. The mRNAs of the constitutively active mutant of AhR and an AhR-induced gene, CYP1A1, were expressed in the thymus and spleen of the Tg mice. The transgene expression was clearly detected in the thymocytes, CD4, and CD8 T cells, but not in the B cells or thymus stromal cells. These Tg mice had a decreased number of thymocytes and an increased percentage of CD8 single-positive thymocytes, but their splenocytes were much less affected. By contrast, the increase in number of T cells and B cells taking place in the spleen after immunization was significantly suppressed in the Tg mice. These results clearly show that AhR activation in the T-lineage cells is directly involved in thymocyte loss and skewed differentiation. They also indicate that AhR activation in T cells and not in B cells suppresses the immunization-induced increase in both T cells and B cells.
Overlapping Signaling Pathways of Sphingosine 1-Phosphate and TGF-β in the Murine Langerhans Cell Line XS52
Authors: Radeke Heinfried H. , von Wenckstern Henrik , Stoidtner Kirsten , Sauer Bettina , Hammer Stefanie , Kleuser Burkhard
Published: April 2014

TGF-β has been defined as a key mediator for the induction and maintenance of immunological tolerance. Concomitantly, it is essential for homeostasis of specialized epithelial dendritic cells, namely, Langerhans cells (LC). Our data reveal that TGF-β induces migration of the immature LC, XS52, a cell line expressing the signaling components, TGF-β type I and II receptors and Smad2, 3, and 4 mRNA. TGF-β stimulation induced transient Smad3/4 oligomerization and Smad3/DNA binding. Antisense oligonucleotides (ASO) targeting Smad3 abrogated TGF-β-induced XS52 chemotaxis, proving the involvement of this Smad protein in the TGF-β-dependent migration. In contrast, the typical CCR6-dependent chemotaxis of immature LC induced by CCL20/MIP-3α was not affected by Smad3 ASO. Most notably, we also identified the lysophospholipid sphingosine 1-phosphate (S1P) as a potent chemoattractant for immature LC, which expressed mRNA transcripts of lysophospholipid receptors S1P1–4. Additional experiments with specific ASO showed that the Gαi-coupled receptors S1P1 and S1P3 were dominantly involved in the S1P-induced migration. In contrast, lysophosphatidic acid (LPA), also binding to members of the lysophospholipid receptor family, failed to induce XS52 migration. Intriguingly, we raised evidence that TGF-β and S1P signal transduction pathways are indeed overlapping, as S1P augmented Smad activation and targeted DNA binding with kinetics comparable to TGF-β. Finally, S1P failed to stimulate XS52 chemotaxis when Smad3 protein expression was abrogated. Thus, our data indicate a cross-communication between S1P and TGF-β signaling that might be relevant for more than only migratory activities of immature LC.
Generation of Antigen-Specific, Foxp3-Expressing CD4+ Regulatory T Cells by Inhibition of APC Proteosome Function
Authors: Cong Yingzi , Konrad Astrid , Iqbal Nuzhat , Hatton Robin D. , Weaver Casey T. , Elson Charles O.
Published: April 2014

We tested the hypothesis that immature APC, whose NF-κB-signaling pathway and thus maturation was blocked by the proteosome inhibitor benzyloxycarbonyl-isoleucyl-glutamyl(O-tert-butyl)-alanyl-leucinal (PSI), could be a source of Ag-specific regulatory T (Treg) cells. DO11.10 CD4+ T cells that were incubated with Ag- and PSI-pulsed APC proliferated poorly, produced less IL-2, IFN-γ, and IL-10 in secondary cultures, and inhibited the response of both naive and memory CD4+ T cells stimulated by Ag-pulsed APC. The generation of PSI-APC Treg cells required IL-10 production by APC. PSI-APC Treg cell inhibition required cell-cell contact but not IL-10 or TGF-β. Addition of IL-2 did not reverse, but Ab to CTLA-4 did reverse partially the inhibitory effect. Depletion of CD25+ T cells before initial culture with PSI-APC did not affect Treg generation. PSI-APC Treg cells expressed high levels of Foxp3, inhibited proliferation of naive DO11.10 T cells in vivo, and abrogated colitis driven by a memory Th1 response to bacterial-associated Ag. We conclude that NF-κB-blocked, immature APC are able to induce the differentiation of Treg cells that can function in vitro and in vivo in an Ag-specific manner.
Synergistic Effect of IL-2, IL-12, and IL-18 on Thymocyte Apoptosis and Th1/Th2 Cytokine Expression
Authors: Rodriguez-Galán Maria Cecilia , Bream Jay H. , Farr Andrew , Young Howard A.
Published: April 2014

In the periphery, IL-18 synergistically induces the expression of the Th1 cytokine IFN-γ in the presence of IL-12 and the Th2 cytokines IL-5 and IL-13 in the presence of IL-2. Although the expression of these cytokines has been described in the thymus, their role in thymic development and function remains uncertain. We report here that freshly isolated thymocytes from C57BL/6 and BALB/c mice stimulated in vitro with IL-2-plus-IL-18 or IL-12-plus-IL-18 produce large amounts of IFN-γ and IL-13. Analysis of the thymic subsets, CD4−CD8− (DN), CD4+CD8+, CD4+CD8−, and CD4−CD8+ revealed that IL-18 in combination with IL-2 or IL-12 induces IFN-γ and IL-13 preferentially from DN cells. Moreover, DN2 and DN3 thymocytes contained more IFN-γ+ cells than cells in the later stage of maturation. Additionally, IL-18 in combination with IL-2 induces CCR4 (Th2-associated) and CCR5 (Th1-associated) gene expression. In contrast, IL-18-plus-IL-12 specifically induced CCR5 expression. The IL-2-plus-IL-18 or IL-12-plus-IL-18 effect on IFN-γ and IL-13 expression is dependent on Stat4 and NF-κB but independent of Stat6, T-bet, or NFAT. Furthermore, IL-12-plus-IL-18 induces significant thymocyte apoptosis when expressed in vivo or in vitro, and this effect is exacerbated in the absence of IFN-γ. IL-12-plus-IL-18-stimulated thymocytes can also induce IA-IE expression on cortical and medullary thymic epithelial cells in an IFN-γ-dependent manner. Thus, the combination of IL-2, IL-12, and IL-18 can induce phenotypic and functional changes in thymocytes that may alter migration, differentiation, and cell death of immature T cells inside the thymus and potentially affect the Th1/Th2 bias in peripheral immune compartments.
IgH V-Region Sequence Does Not Predict the Survival Fate of Human Germinal Center B Cells
Authors: Jackson Stephen M. , Capra J. Donald
Published: April 2014

Germinal center (GC) B cell survival fate is governed in part by the outcome of successful/failed BCR-mediated interactions with accessory cells. However, the extent to which the BCR primary sequence influences such interactions is not fully understood. Over 1000 IgVH4 family cDNAs were sequenced from living (annexin V−) and apoptotic (annexin V+ or from within tingible body macrophages) GC B cell fractions from seven tonsils. Results surprisingly demonstrate that living and dying GC B cells do not significantly differ in IgVH, D, or JH gene segment use; HCDR3 length or positive charge; or mutation frequency. Additionally, equivalent IgH cDNA sequences were identified in both fractions, suggesting that BCR sequence alone is an unreliable predictor of GC B cell survival.
EBV-Induced Gene 3 Transcription Is Induced by TLR Signaling in Primary Dendritic Cells via NF-κB Activation
Authors: Wirtz Stefan , Becker Christoph , Fantini Massimo C. , Nieuwenhuis Edward E. , Tubbe Ingrid , Galle Peter R. , Schild Hans-Jörg , Birkenbach Mark , Blumberg Richard S. , Neurath Markus F.
Published: April 2014

The EBV-induced gene 3 (EBI3) is expressed in dendritic cells (DCs) and part of the cytokine IL-27 that controls Th cell development. However, its regulated expression in DCs is poorly understood. In the present study we demonstrate that EBI3 is expressed in splenic CD8−, CD8+, and plasmacytoid DC subsets and is induced upon TLR signaling. Cloning and functional analysis of the EBI3 promoter using in vivo footprinting and mutagenesis showed that stimulation via TLR2, TLR4, and TLR9 transactivated the promoter in primary DCs via NF-κB and Ets binding sites at −90 and −73 bp upstream of the transcriptional start site, respectively. Furthermore, we observed that NF-κB p50/p65 and PU.1 were sufficient to transactivate the EBI3 promoter in EBI3-deficient 293 cells. Finally, induced EBI3 gene expression in DCs was reduced or abrogated in TLR-2/TLR4, TLR9, and MyD88 knockout mice, whereas both basal and inducible EBI3 mRNA levels in DCs were strongly suppressed in NF-κB p50-deficient mice. In summary, these data suggest that EBI3 expression in DCs is transcriptionally regulated by TLR signaling via MyD88 and NF-κB. Thus, EBI3 gene transcription in DCs is induced rapidly by TLR signaling during innate immune responses preceding cytokine driven Th cell development.
Involvement of TNF and NF-κB in the Transcriptional Control of Cyclooxygenase-2 Expression by IFN-γ in Macrophages
Authors: Vila-del Sol Virginia , Fresno Manuel
Published: April 2014

IFN-γ induces cyclooxygenase (COX)-2 expression and PG production in mouse macrophage cells. IFN-γ activates COX-2 promoter-driven transcription. Deletion of the IFN sequence regulatory element (ISRE) I −1541/−1522 and ISRE II −1215/−1206 sites of the mouse COX-2 promoter minimally decrease this IFN-γ induction. In contrast, deletion of the −965/−150 region from the COX-2 promoter abrogated IFN-γ induction. In this region a NF-κB site has been described and mutation of this site impairs the induction of the full COX-2 promoter by IFN-γ. Moreover, IFN-γ induction of the COX-2 promoter was also strongly reduced by transfection of plasmid encoding the NF-κB inhibitor, IκBα. Interestingly, IFN-γ induction of the COX-2 and PGE2 synthesis was absent in macrophages from TNF−/− mice, and neutralizing anti-TNF Abs inhibited COX-2 promoter induction by IFN-γ in RAW 264.7 macrophages. Moreover, NF-κB activity was induced late after stimulation with IFN-γ correlating with the effect of autocrine TNF, and this NF-κB activation was absent in macrophages from TNF−/− mice. Taken together our results suggest a model in which IFN-γ-induced TNF activates NF-κB, which is required for full COX-2 expression.
The Igκ3′ Enhancer Is Activated by Gradients of Chromatin Accessibility and Protein Association
Authors: McDevit Daniel C. , Perkins Leslie , Atchison Michael L. , Nikolajczyk Barbara S.
Published: April 2014

The Igκ locus is recombined following initiation of a signaling cascade during the early pre-B stage of B cell development. The Ig κ3′ enhancer plays an important role in normal B cell development by regulating κ locus activation. Quantitative analyses of κ3′ enhancer chromatin structure by restriction endonuclease accessibility and protein association by chromatin immunoprecipitation in a developmental series of primary murine B cells and murine B cell lines demonstrate that the enhancer is activated progressively through multiple steps as cells mature. Moderate κ3′ chromatin accessibility and low levels of protein association in pro-B cells are increased substantially as the cells progress from pro- to pre-B, then eventually mature B cell stages. Chromatin immunoprecipitation assays suggest transcriptional regulators of the κ3′ enhancer, specifically PU.1 and IFN regulatory factor-4, exploit enhanced accessibility by increasing association as cells mature. Characterization of histone acetylation patterns at the κ3′ enhancer and experimental inhibition of histone deacetylation suggest changes therein may determine changes in enzyme and transcription factor accessibility. This analysis demonstrates κ activation is a multistep process initiated in early B cell precursors before Igμ recombination and finalized only after the pre-B cell stage.
Proteolytic Regulation of Activated STAT6 by Calpains
Authors: Zamorano Jose , Rivas Maria Dolores , Setien Fernando , Perez-G Moises
Published: April 2014

The transcription factor STAT6 plays an important role in cell responses to IL-4. Its activation is tightly regulated. STAT6 phosphorylation is associated with JAKs, whereas dephosphorylation is associated with specific phosphatases. Several studies indicate that proteases can also regulate STAT6. The aim of this study was to investigate the nature of these proteases in mouse T cell lines. We found that STAT6 was degraded in cell extracts by calcium-dependent proteases. This degradation was specifically prevented by calpain inhibitors, suggesting that STAT6 was a target for these proteases. This was supported by the cleavage of STAT6 by recombinant calpains. The proteolytic regulation of STAT6 was more complex in vivo. Calcium signaling was not sufficient to induce STAT6 degradation. However, treatment of IL-4-stimulated cells with calcium ionophores resulted in the absence of phosphorylated STAT6. This effect correlated with the loss of STAT6 protein and was prevented by calpain inhibitors. Cytoplasmic calpains seemed to be responsible for STAT6 degradation. Calpains can target signaling proteins; in this study we found that they can negatively regulate activated STAT6.
Localization of Src Homology 2 Domain-Containing Phosphatase 1 (SHP-1) to Lipid Rafts in T Lymphocytes: Functional Implications and a Role for the SHP-1 Carboxyl Terminus
Authors: Fawcett Vicki C. J. , Lorenz Ulrike
Published: April 2014

The protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1) has previously been shown to be a negative regulator of signaling mediated via the TCR. A growing body of evidence indicates that the regulated localization of proteins within certain membrane subdomains, referred to as lipid rafts, is important for the successful transduction of signaling events downstream of the TCR. However, considerably less is known about the localization of negative regulators during these lipid raft-dependent signaling events. In this study we have investigated the subcellular localization of SHP-1 and its role in regulation of TCR-mediated signaling. Our studies demonstrate that in a murine T cell hybridoma as well as in primary murine thymocytes, a fraction of SHP-1 localizes to the lipid rafts, both basally and after TCR stimulation. Interestingly, although SHP-1 localized in the nonraft fractions is tyrosine phosphorylated, the SHP-1 isolated from the lipid rafts lacks the TCR-induced tyrosine phosphorylation, suggesting physical and/or functional differences between these two subpopulations. We identify a requirement for the C-terminal residues of SHP-1 in optimal localization to the lipid rafts. Although expression of SHP-1 that localizes to lipid rafts potently inhibits TCR-mediated early signaling events and IL-2 production, the expression of lipid raft-excluded SHP-1 mutants fails to elicit any of the inhibitory effects. Taken together these studies reveal a key role for lipid raft localization of SHP-1 in mediating the inhibitory effects on T cell signaling events.
In Situ Diversification of the Antibody Repertoire in Chronic Lyme Arthritis Synovium
Authors: Ghosh Srimoyee , Steere Allen C. , Stollar B. David , Huber Brigitte T.
Published: April 2014

Lyme arthritis is initiated by the tick-borne spirochete, Borrelia burgdorferi. In a subset of patients, symptoms do not resolve in response to standard courses of antibiotics. Chronic joint inflammation may persist despite spirochetal killing, suggesting an autoimmune etiology. The pathogenic mechanisms that sustain chronic Lyme arthritis have not been fully elucidated, although T cells are believed to play a role. The synovial lesion contains elements of a peripheral lymph node, with lymphoid aggregates, plasma cells and follicular dendritic cells. An analysis of activated cells at the site of injury could yield clues regarding the nature of the response and the identity of potential autoantigens. Using laser-capture microdissection, we have isolated plasma cells from the joint tissue of chronic Lyme arthritis patients who underwent synovectomy. Expressed Ig V regions were amplified by RT-PCR. A majority of isolated cells expressed γ H chains, which is indicative of a class-switched response. There were a large number of nucleotide substitutions from germline, with a higher fraction of replacement mutations in the CDRs, suggesting a process of Ag-driven selection. We have recovered clonal clusters of cells containing identical junctions and V(D)J rearrangements. Sequence analysis reveals a hierarchy of shared somatic mutations between members of a given clone. Intraclonal diversity among plasma cells of close physical proximity points toward an ongoing process of diversification and affinity maturation, possibly driven by the chronic presence of an autoantigen.
The Two Major Oligomeric Forms of Human Mannan-Binding Lectin: Chemical Characterization, Carbohydrate-Binding Properties, and Interaction with MBL-Associated Serine Proteases
Authors: Teillet Florence , Dublet Bernard , Andrieu Jean-Pierre , Gaboriaud Christine , Arlaud Gérard J. , Thielens Nicole M.
Published: April 2014

Mannan-binding lectin (MBL) is an oligomeric C-type lectin assembled from homotrimeric structural units that binds to neutral carbohydrates on microbial surfaces. It forms individual complexes with MBL-associated serine proteases (MASP)-1, -2, -3 and a truncated form of MASP-2 (MAp19) and triggers the lectin pathway of complement through MASP-2 activation. To characterize the oligomerization state of the two major MBL forms present in human serum, both proteins were analyzed by mass spectrometry. Mass values of 228,098 ± 170 Da (MBL-I) and 304,899 ± 229 Da (MBL-II) were determined for the native proteins, whereas reduction of both species yielded a single chain with an average mass of 25,340 ± 18 Da. This demonstrates that MBL-I and -II contain 9 and 12 disulfide-linked chains, respectively, and therefore are trimers and tetramers of the structural unit. As shown by surface plasmon resonance spectroscopy, trimeric and tetrameric MBL bound to immobilized mannose-BSA and N-acetylglucosamine-BSA with comparable KD values (2.2 and 0.55 nM and 1.2 and 0.96 nM, respectively). However, tetrameric MBL exhibited significantly higher maximal binding capacity and lower dissociation rate constants for both carbohydrates. In contrast, no significant difference was detected for binding of the recombinant MASPs or MAp19 to immobilized trimeric or tetrameric MBL. As shown by gel filtration, both MBL species formed 1:2 complexes with MASP-3 or MAp19. These results provide the first precise analysis of the major human MBL oligomers. The oligomerization state of MBL has a direct effect on its carbohydrate-binding properties, but no influence on the interaction with the MASPs.
Interactions between NKG2x Immunoreceptors and HLA-E Ligands Display Overlapping Affinities and Thermodynamics
Authors: Kaiser Brett K. , Barahmand-pour Fariba , Paulsene Wendy , Medley Scott , Geraghty Daniel E. , Strong Roland K.
Published: April 2014

The NKG2x/CD94 family of C-type lectin-like immunoreceptors (x = A, B, C, E, and H) mediates surveillance of MHC class Ia cell surface expression, often dysregulated during infection or tumorigenesis, by recognizing the MHC class Ib protein HLA-E that specifically presents peptides derived from class Ia leader sequences. In this study, we determine the affinities and interaction thermodynamics between three NKG2x/CD94 receptors (NKG2A, NKG2C, and NKG2E) and complexes of HLA-E with four representative peptides. Inhibitory NKG2A/CD94 and activating NKG2E/CD94 receptors bind HLA-E with indistinguishable affinities, but with significantly higher affinities than the activating NKG2C/CD94 receptor. Despite minor sequence differences, the peptide presented by HLA-E significantly influenced the affinities; HLA-E allelic differences had no effect. These results reveal important constraints on the integration of opposing activating and inhibitory signals driving NK cell effector functions.
Hagfish Leukocytes Express a Paired Receptor Family with a Variable Domain Resembling Those of Antigen Receptors
Authors: Suzuki Takashi , Shin-I Tadasu , Fujiyama Asao , Kohara Yuji , Kasahara Masanori
Published: April 2014

Jawed vertebrates are equipped with TCR and BCR with the capacity to rearrange their V domains. By contrast, jawless vertebrates, represented by hagfish and lampreys, apparently lack such receptors. We describe in this study a family of hagfish genes carrying a single V-type domain resembling those of TCR/BCR. This multigene family, which we call agnathan paired receptors resembling Ag receptors (APAR), is expressed in leukocytes and predicted to encode a group of membrane glycoproteins with organizations characteristic of paired Ig-like receptors, consisting of activating and inhibitory forms. APAR has a J region in its V-type domain, and its V and J regions are encoded in a single exon. Thus, APAR is a member of the emerging families of diversified, innate immune-type receptors with TCR/BCR-like V-type domains and has many of the features expected for a primordial TCR/BCR-like receptor. The extracellular domain of APAR may be descended from a V-type domain postulated to have acquired recombination signal sequences in a jawed vertebrate lineage.
Intracellular Salmonella Inhibit Antigen Presentation by Dendritic Cells
Authors: Cheminay Cédric , Möhlenbrink Annette , Hensel Michael
Published: April 2014

Dendritic cells (DC) are important APCs linking innate and adaptive immunity. During analysis of the intracellular activities of Salmonella enterica in DC, we observed that viable bacteria suppress Ag-dependent T cell proliferation. This effect was dependent on the induction of inducible NO synthase by DC and on the function of virulence genes in Salmonella pathogenicity island 2 (SPI2). Intracellular activities of Salmonella did not affect the viability, Ag uptake, or maturation of DC, but resulted in reduced presentation of antigenic peptides by MHC class II molecules. Increased resistance to reinfection was observed after vaccination of mice with SPI2-deficient Salmonella compared with mice vaccinated with SPI2-proficient Salmonella, and this correlated with an increased amount of CD4+ as well as CD8+ T cells. Our study is the first example of interference of an intracellular bacterial pathogen with Ag presentation by DC. The subversion of DC functions is a novel strategy deployed by this pathogen to escape immune defense, colonize host organs, and persist in the infected host.
Loss of CD127 Expression Defines an Expansion of Effector CD8+ T Cells in HIV-Infected Individuals
Authors: Paiardini Mirko , Cervasi Barbara , Albrecht Helmut , Muthukumar Alagarraju , Dunham Richard , Gordon Shari , Radziewicz Henry , Piedimonte Giuseppe , Magnani Mauro , Montroni Maria , Kaech Susan M. , Weintrob Amy , Altman John D. , Sodora Donald L. , Feinberg Mark B. , Silvestri Guido
Published: April 2014

The immunodeficiency that follows HIV infection is related to the virus-mediated killing of infected CD4+ T cells, the chronic activation of the immune system, and the impairment of T cell production. In this study we show that in HIV-infected individuals the loss of IL-7R (CD127) expression defines the expansion of a subset of CD8+ T cells, specific for HIV as well as other Ags, that show phenotypic (i.e., loss of CCR7 and CD62 ligand expression with enrichment in activated and/or proliferating cells) as well as functional (i.e., production of IFN-γ, but not IL-2, decreased ex vivo proliferative potential and increased susceptibility to apoptosis) features of effector T cells. Importantly, in HIV-infected individuals the levels of CD8+CD127− T cells are directly correlated with the main markers of disease progression (i.e., plasma viremia and CD4+ T cell depletion) as well as with the indices of overall T cell activation. In all, these results identify the expansion of CD8+CD127− effector-like T cells as a novel feature of the HIV-associated immune perturbation. Further studies are thus warranted to determine whether measurements of CD127 expression on CD8+ T cells may be useful in the clinical management of HIV-infected individuals.
A Crucial Role for Tryptophan Catabolism at the Host/Candida albicansInterface
Authors: Bozza Silvia , Fallarino Francesca , Pitzurra Lucia , Zelante Teresa , Montagnoli Claudia , Bellocchio Silvia , Mosci Paolo , Vacca Carmine , Puccetti Paolo , Romani Luigina
Published: April 2014

By mediating tryptophan catabolism, the enzyme indoleamine 2,3-dioxygenase (IDO) has a complex role in immunoregulation in infection, pregnancy, autoimmunity, transplantation, and neoplasia. We hypothesized that IDO might affect the outcome of the infection in mice infected with Candida albicans by virtue of its potent regulatory effects on inflammatory and T cell responses. IDO expression was examined in mice challenged with the fungus along with the consequences of its blockade by in vivo treatment with an enzyme inhibitor. We found that IDO activity was induced at sites of infection as well as in dendritic cells and effector neutrophils via IFN-γ- and CTLA-4-dependent mechanisms. IDO inhibition greatly exacerbated infection and associated inflammatory pathology as a result of deregulated innate and adaptive/regulatory immune responses. However, a role for tryptophan catabolism was also demonstrated in a fungus-autonomous fashion; its blockade in vitro promoted yeast-to-hyphal transition. These results provide novel mechanistic insights into complex events that, occurring at the fungus/pathogen interface, relate to the dynamics of host adaptation to the fungus. The production of IFN-γ may be squarely placed at this interface, where IDO activation probably exerts a fine control over fungal morphology as well as inflammatory and adaptive antifungal responses.
Development and Migration of Protective CD8+ T Cells into the Nervous System following Ocular Herpes Simplex Virus-1 Infection
Authors: Lang Anna , Nikolich-Žugich Janko
Published: April 2014

After infection of epithelial surfaces, HSV-1 elicits a multifaceted antiviral response that controls the virus and limits it to latency in sensory ganglia. That response encompasses the CD8+ T cells, whose precise role(s) is still being defined; immune surveillance in the ganglia and control of viral spread to the brain were proposed as the key roles. We tracked the kinetics of the CD8+ T cell response across lymphoid and extralymphoid tissues after ocular infection. HSV-1-specific CD8+ T cells first appeared in the draining (submandibular) lymph node on day 5 and were detectable in both nondraining lymphoid and extralymphoid tissues starting on day 6. However, although lymphoid organs contained both resting (CD43lowCFSEhigh) and virus-specific cells at different stages of proliferation and activation, extralymphoid sites (eye, trigeminal ganglion, and brain) contained only activated cells that underwent more than eight proliferations (CD43highCFSEneg) and promptly secreted IFN-γ upon contact with viral Ags. Regardless of the state of activation, these cells appeared too late to prevent HSV-1 spread, which was seen in the eye (from day 1), trigeminal ganglia (from day 2), and brain (from day 3) well before the onset of a detectable CD8+ T cell response. However, CD8+ T cells were critical in reducing viral replication starting on day 6 and for its abrogation between days 8 and 10; CD8-deficient animals failed to control the virus, exhibited persisting high viral titers in the brain after day 6, and died of viral encephalitis between days 7 and 12. Thus, CD8+ T cells do not control HSV-1 spread from primary to tertiary tissues, but, rather, attack the virus in infected organs and control its replication in situ.
The Staphylococcal Superantigen-Like Protein 7 Binds IgA and Complement C5 and Inhibits IgA-FcαRI Binding and Serum Killing of Bacteria
Authors: Langley Ries , Wines Bruce , Willoughby Natasha , Basu Indira , Proft Thomas , Fraser John D.
Published: April 2014

The staphylococcal superantigen-like proteins (SSLs) are close relatives of the superantigens but are coded for by a separate gene cluster within a 19-kb region of the pathogenicity island SaPIn2. rSSL7 (formally known as SET1) bound with high affinity (KD, 1.1 nM) to the monomeric form of human IgA1 and IgA2 plus serum IgA from primate, pig, rat, and horse. SSL7 also bound the secretory form of IgA found in milk from human, cow, and sheep, and inhibited IgA binding to cell surface FcαRI (CD89) and to a soluble form of the FcαRI protein. In addition to IgA, SSL7 bound complement factor C5 from human (KD, 18 nM), primate, sheep, pig, and rabbit serum, and inhibited complement-mediated hemolysis and serum killing of a Gram-negative organism Escherichia coli. SSL7 is a superantigen-like protein secreted from Staphylococcus aureus that blocks IgA-FcR interactions and inhibits complement, leading to increased survival of a sensitive bacterium in blood.
Nonhealing Infection despite Th1 Polarization Produced by a Strain of Leishmania major in C57BL/6 Mice
Authors: Anderson Charles F. , Mendez Susana , Sacks David L.
Published: April 2014

Experimental Leishmania major infection in mice has been of immense interest because it was among the first models to demonstrate the importance of the Th1/Th2 balance to infection outcome in vivo. However, the Th2 polarization that promotes the development of nonhealing cutaneous lesions in BALB/c mice has failed to adequately explain the mechanisms underlying nonhealing forms of leishmaniasis in humans. We have studied a L. major strain from a patient with nonhealing lesions that also produces nonhealing lesions with ulcerations and high parasite burden in conventionally resistant C57BL/6 mice. Surprisingly, these mice develop a strong, polarized, and sustained Th1 response, as evidenced by high levels of IFN-γ produced by Leishmania-specific cells in the draining lymph node and in the ear lesion, and an absence of IL-4 or IL-13. The parasites fail to be effectively cleared despite high level induction of inducible NO synthase in the lesion, and despite their sensitivity to killing by IFN-γ-activated macrophages in vitro. Infection of IL-10−/− mice, blockade of the IL-10R, or depletion of CD25+ cells during the chronic phase promotes parasite killing, indicating that IL-10 and regulatory T cells play a role in rendering the Th1 responses ineffective at controlling infection in the skin. Mice with nonhealing primary lesions are nonetheless resistant to reinfection in the other ear. We suggest that nonhealing infections in animal models that are explained not by aberrant Th2 development, but by overactivation of homeostatic pathways designed to control inflammation, provide better models to understand nonhealing or reactivation forms of leishmaniasis in humans.
Human TLR10 Is a Functional Receptor, Expressed by B Cells and Plasmacytoid Dendritic Cells, Which Activates Gene Transcription through MyD88
Authors: Hasan Uzma , Chaffois Claire , Gaillard Claude , Saulnier Virginie , Merck Estelle , Tancredi Sandra , Guiet Chantal , Brière Francine , Vlach Jaromir , Lebecque Serge , Trinchieri Giorgio , Bates Elizabeth E. M.
Published: April 2014

Human TLR10 is an orphan member of the TLR family. Genomic studies indicate that TLR10 is in a locus that also contains TLR1 and TLR6, two receptors known to function as coreceptors for TLR2. We have shown that TLR10 was not only able to homodimerize but also heterodimerized with TLRs 1 and 2. In addition, unlike TLR1 and TLR6, TLR10 was expressed in a highly restricted fashion as a highly N-glycosylated protein, which we detected in B cell lines, B cells from peripheral blood, and plasmacytoid dendritic cells from tonsil. We were also able to detect TLR10 in a CD1a+ DC subset derived from CD34+ progenitor cells which resemble Langerhans cells in the epidermis. Although we were unable to identify a specific ligand for TLR10, by using a recombinant CD4TLR10 molecule we also demonstrated that TLR10 directly associates with MyD88, the common Toll IL-1 receptor domain adapter. Additionally, we have characterized regions in the Toll IL-1 receptor domain of TLR10 that are essential in the activation of promoters from certain inflammatory cytokines. Even though TLR10 expression has not been detected in mice, we have identified a partial genomic sequence of the TLR10 gene that was present but nonfunctional and disrupted by a retroviral insertion in all mouse strains tested. However, a complete TLR10 sequence could be detected in the rat genome, indicating that a functional copy may be preserved in this species.
Breast Milk-Derived Antigen-Specific CD8+ T Cells: An Extralymphoid Effector Memory Cell Population in Humans
Authors: Sabbaj Steffanie , Ghosh Mrinal K. , Edwards Bradley H. , Leeth Ruth , Decker W. Don , Goepfert Paul A. , Aldrovandi Grace M.
Published: April 2014

Although mouse studies have demonstrated the presence of an effector memory population in nonlymphoid tissues, the phenotype of human CD8+ T cells present in such compartments has not been characterized. Because of the relatively large number of CD8+ T cells present in breast milk, we were able to characterize the phenotype of this cell population in HIV-infected and uninfected lactating women. CMV, influenza virus, EBV, and HIV-specific CD8+ T cells as measured by the IFN-γ ELISPOT and MHC class I tetramer staining were all present at greater frequencies in breast milk as compared with blood. Furthermore, a greater percentage of the breast milk CD8+ T cells expressed the intestinal homing receptor, CD103, and the mucosal homing receptor CCR9. Breast milk T cells were predominantly CD45RO+HLADR+ and expressed low levels of CD45RA, CD62L, and CCR7 consistent with an effector memory population. Conversely, T cells derived from blood were mainly characterized as central memory cells (CCR7+CD62L+). These results demonstrate a population of extralymphoid CD8+ T cells with an effector memory phenotype in humans, which could contribute to enhanced local virologic control and the relative lack of HIV transmission via this route.
CD4+CD25+ Regulatory T Cells Control Innate Immune Reactivity after Injury
Authors: Murphy Thomas J. , Choileain Niamh Ni , Zang Yan , Mannick John A. , Lederer James A.
Published: April 2014

Major injury initiates a systemic inflammatory response that can be detrimental to the host. We have recently reported that burn injury primes innate immune cells for a progressive increase in TLR4 and TLR2 agonist-induced proinflammatory cytokine production and that this inflammatory phenotype is exaggerated in adaptive immune system-deficient (Rag1−/−) mice. The present study uses a series of adoptive transfer experiments to determine which adaptive immune cell type(s) has the capacity to control innate inflammatory responses after injury. We first compared the relative changes in TLR4- and TLR2-induced TNF-α, IL-1β, and IL-6 production by spleen cell populations prepared from wild-type (WT), Rag1−/−, CD4−/−, or CD8−/− mice 7 days after sham or burn injury. Our findings indicated that splenocytes prepared from burn-injured CD8−/− mice displayed TLR-induced cytokine production levels similar to those in WT mice. In contrast, spleen cells from burn-injured CD4−/− mice produced cytokines at significantly higher levels, equivalent to those in Rag1−/− mice. Moreover, reconstitution of Rag1−/− or CD4−/− mice with WT CD4+ T cells reduced postinjury cytokine production to WT levels. Additional separation of CD4+ T cells into CD4+CD25+ and CD4+CD25− subpopulations before their adoptive transfer into Rag1−/− mice showed that CD4+CD25+ T cells were capable of reducing TLR-stimulated cytokine production levels to WT levels, whereas CD4+CD25− T cells had no regulatory effect. These findings suggest a previously unsuspected role for CD4+CD25+ T regulatory cells in controlling host inflammatory responses after injury.