bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Positive Selection of B1 Cells and Reduced B Cell Tolerance to Intracellular Antigens in c1q-Deficient Mice
Authors: Ferry Helen , Potter Paul K. , Crockford Tanya L. , Nijnik Anastasia , Ehrenstein Michael R. , Walport Mark J. , Botto Marina , Cornall Richard J.
Published: April 2014

Inherited deficiency of early components of the classical complement pathway is strongly associated with the targeting of intracellular self Ags in systemic lupus erythematosus, but the reasons for this association are debated. In this study, we show that C1q deficiency increases the positive selection of B1b B cells and IgM autoantibodies by an intracellular self Ag, which is exposed on dying cells, and decreases the negative selection of autoreactive conventional B cells by the same Ag. These effects are specific to intracellular Ag because C1q deficiency does not affect negative selection by extracellular self Ag or increase the positive selection of naive B cells. The B1-derived IgM autoantibody binds to the intracellular Ag when it is expressed on dying cells, leading to fixation of C1q and clearance of cells by phagocytosis. These findings suggest that the positive selection of autoreactive B1 cells by self Ags may contribute to the IgM and C1q-dependent clearance of dying cells in a feedback loop that limits exposure of conventional B cells to immunogenic self Ags. We show that exposure of intracellular Ag leads to the activation of conventional B cells, when there is a source of T cell help in vivo.
CD4+ and CD8+ T Cell Survival Is Regulated Differentially by Protein Kinase Cθ, c-Rel, and Protein Kinase B
Authors: Saibil Samuel D. , Jones Russell G. , Deenick Elissa K. , Liadis Nicole , Elford Alisha R. , Vainberg Mitchell G. , Baerg Heather , Woodgett James R. , Gerondakis Steve , Ohashi Pamela S.
Published: April 2014

An effective immune response requires the expansion and survival of a large number of activated T cells. This study compared the role of protein kinase C (PKC)θ and associated signaling molecules in the survival of activated primary CD4+ vs CD8+ murine T cells. We demonstrate that the absence of PKCθ resulted in a moderate survival defect in CD4+ T cells and a striking survival defect of CD8+ T lymphocytes. CD8+ T cells lacking the c-Rel, but not the NF-κB1/p50, member of the NF-κB family of transcription factors displayed a similar impairment in cell survival as PKCθ−/− CD8+ T lymphocytes. This implicates c-Rel as a key target of PKCθ-mediated survival signals in CD8+ T cells. In addition, both c-Rel−/− and PKCθ−/− T cells also displayed impaired expression of the antiapoptotic Bcl-xL protein upon activation. Changes in Bcl-xL expression, however, did not correlate with the survival of CD4+ or CD8+ lymphocytes. The addition of protein kinase B-mediated survival signals could restore partially CD4+ T cell viability, but did not dramatically influence CD8+ survival. Active protein kinase B was also unable to restore proliferative responses in CD8+ PKCθ−/− T cells. The survival of CD4+ and CD8+ T cells deficient in either PKCθ or c-Rel, however, was promoted by the addition of IL-2. Collectively, these data demonstrate that CD4+ and CD8+ T cell survival signals are differentially programmed.
Regulatory T Cells Dynamically Control the Primary Immune Response to Foreign Antigen
Authors: Haribhai Dipica , Lin Wen , Relland Lance M. , Truong Nga , Williams Calvin B. , Chatila Talal A.
Published: April 2014

The population dynamics that enable a small number of regulatory T (TR) cells to control the immune responses to foreign Ags by the much larger conventional T cell subset were investigated. During the primary immune response, the expansion and contraction of conventional and TR cells occurred in synchrony. Importantly, the relative accumulation of TR cells at peak response significantly exceeded that of conventional T cells, reflecting extensive cell division within the TR cell pool. Transfer of a polyclonal TR cell population before immunization antagonized both polyclonal and TCR transgenic responses, whereas blocking TR cell function enhanced those responses. These results define an inverse quantitative relationship between TR and conventional T cells that controls the magnitude of the primary immune response. The high frequency of dividing TR cells suggests degenerate TCR specificity enabling activation by a broad spectrum of Ags.
Requirement for a Complex of Wiskott-Aldrich Syndrome Protein (WASP) with WASP Interacting Protein in Podosome Formation in Macrophages
Authors: Tsuboi Shigeru
Published: April 2014

Chemotactic migration of macrophages is critical for the recruitment of leukocytes to inflamed tissues. Macrophages use a specialized adhesive structure called a podosome to migrate. Podosome formation requires the Wiskott-Aldrich syndrome protein (WASP), which is a product of the gene defective in an X-linked inherited immunodeficiency disorder, the Wiskott-Aldrich syndrome. Macrophages from WASP-deficient Wiskott-Aldrich syndrome patients lack podosomes, resulting in defective chemotactic migration. However, the molecular basis for podosome formation is not fully understood. I have shown that the WASP interacting protein (WIP), a binding partner of WASP, plays an important role in podosome formation in macrophages. I showed that WASP bound WIP to form a complex at podosomes and that the knockdown of WIP impairs podosome formation. When WASP binding to WIP was blocked, podosome formation was also impaired. When WASP expression was reduced by small interfering RNA transfection, the amount of the complex of WASP with WIP decreased, resulting in reduced podosome formation. Podosomes were restored by reconstitution of the WASP-WIP complex in WASP knockdown cells. These results indicate that the WASP-WIP complex is required for podosome formation in macrophages. When podosome formation was reduced by blocking WASP binding to WIP, transendothelial migration of macrophages, the most crucial process in macrophage trafficking, was impaired. These results suggest that a complex of WASP with WIP plays a critical role in podosome formation, thereby mediating efficient transendothelial migration of macrophages.
Transmembrane Mutations to FcγRIIA Alter Its Association with Lipid Rafts: Implications for Receptor Signaling
Authors: García-García Erick , Brown Eric J. , Rosales Carlos
Published: April 2014

Many immunoreceptors have been reported to associate with lipid rafts upon ligand binding. The way in which this association is regulated is still obscure. We investigated the roles for various domains of the human immunoreceptor FcγRIIA in regulating its association with lipid rafts by determining the resistance of unligated, or ligated and cross-linked, receptors to solubilization by the nonionic detergent Triton X-100, when expressed in RBL-2H3 cells. Deletion of the cytoplasmic domain, or destruction of the cytoplasmic palmitoylation site, had no effect on the association of the receptor with lipid rafts. A transmembrane mutant, A224S, lost the ability to associate with lipid rafts upon receptor cross-linking, whereas transmembrane mutants VA231-2MM and VVAL234-7GISF showed constitutive lipid raft association. Wild-type (WT) FcγRIIA and all transmembrane mutants activated Syk, regardless of their association with lipid rafts. WT FcγRIIA and mutants that associated with lipid rafts efficiently activated NF-κB, in an ERK-dependent manner. In contrast, WT FcγRIIA and the A224S mutant both presented efficient phagocytosis, while VA231-2MM and VVAL234-7GISF mutants presented lower phagocytosis, suggesting that phagocytosis may proceed independently of lipid raft association. These data identify the transmembrane domain of FcγRIIA as responsible for regulating its inducible association with lipid rafts and suggest that FcγRIIA-mediated responses, like NF-κB activation or phagocytosis, can be modulated by lipid raft association of the ligated receptor.
Hidden Epitopes Emerge in Secondary Influenza Virus-Specific CD8+ T Cell Reponses
Authors: Thomas Paul G. , Brown Scott A. , Keating Rachael , Yue Wen , Morris Melissa Y. , So Jenny , Webby Richard J. , Doherty Peter C.
Published: April 2014

Influenza A virus-specific CD8+ T cell responses in H2b mice are characterized by reproducible hierarchies. Compensation by the DbPB1-F262 epitope is apparent following infection with a variant H3N2 virus engineered to disrupt the prominent DbNP366 and DbPA224 epitopes (a double knockout or DKO). Analysis with a “triple” knockout (TKO) virus, which also compromises DbPB1-F262, did not reveal further compensation to the known residual, minor, and predicted epitopes. However, infection with this deletion mutant apparently switched protective immunity to an alternative Ab-mediated pathway. As expected, TKO virus clearance was significantly delayed in Ab-deficient MHC class II−/− and Ig−/− mice, which were much more susceptible following primary, intranasal infection with the TKO, but not DKO, virus. CD8+ T cell compensation was detected in DKO, but not TKO, infection of Ig-deficient mice, suggestive of cooperation among CD8+ T cell responses. However, after priming with a TKO H1N1 mutant, MHC II−/− mice survived secondary intranasal exposure to the comparable H3N2 TKO virus. Such prime/challenge experiments with the DKO and TKO viruses allowed the emergence of two previously unknown epitopes. The contrast between the absence of compensatory effect following primary exposure and the substantial clonal expansion after secondary challenge suggests that the key factor limiting the visibility of these “hidden” epitopes may be very low naive T cell precursor frequencies. Overall, these findings suggest that vaccine approaches using virus vectors to deliver an Ag may be optimized by disrupting key peptides in the normal CD8+ T cell response associated with common HLA types.
Human Peptidoglycan Recognition Proteins Require Zinc to Kill Both Gram-Positive and Gram-Negative Bacteria and Are Synergistic with Antibacterial Peptides
Authors: Wang Minhui , Liu Li-Hui , Wang Shiyong , Li Xinna , Lu Xiaofeng , Gupta Dipika , Dziarski Roman
Published: April 2014

Mammals have four peptidoglycan recognition proteins (PGRPs or PGLYRPs), which are secreted innate immunity pattern recognition molecules with effector functions. In this study, we demonstrate that human PGLYRP-1, PGLYRP-3, PGLYRP-4, and PGLYRP-3:4 have Zn2+-dependent bactericidal activity against both Gram-positive and Gram-negative bacteria at physiologic Zn2+ concentrations found in serum, sweat, saliva, and other body fluids. The requirement for Zn2+ can only be partially replaced by Ca2+ for killing of Gram-positive bacteria but not for killing of Gram-negative bacteria. The bactericidal activity of PGLYRPs is salt insensitive and requires N-glycosylation of PGLYRPs. The LD99 of PGLYRPs for Gram-positive and Gram-negative bacteria is 0.3–1.7 μM, and killing of bacteria by PGLYRPs, in contrast to killing by antibacterial peptides, does not involve permeabilization of cytoplasmic membrane. PGLYRPs and antibacterial peptides (phospholipase A2, α- and β-defensins, and bactericidal permeability-increasing protein), at subbactericidal concentrations, synergistically kill Gram-positive and Gram-negative bacteria. These results demonstrate that PGLYRPs are a novel class of recognition and effector molecules with broad Zn2+-dependent bactericidal activity against both Gram-positive and Gram-negative bacteria that are synergistic with antibacterial peptides.
Differential Recognition of TLR-Dependent Microbial Ligands in Human Bronchial Epithelial Cells
Authors: Mayer Anja K. , Muehmer Mario , Mages Jörg , Gueinzius Katja , Hess Christian , Heeg Klaus , Bals Robert , Lang Roland , Dalpke Alexander H.
Published: April 2014

Bronchial epithelial cells represent the first line of defense against invading airborne pathogens. They are important contributors to innate mucosal immunity and provide a variety of antimicrobial effectors. However, mucosal surfaces are prone to contact with pathogenic, as well as nonpathogenic microbes, and therefore, immune recognition principles have to be tightly controlled to avoid uncontrolled permanent activation. TLRs have been shown to recognize conserved microbial patterns and to mediate inducible activation of innate immunity. Our experiments demonstrate that bronchial epithelial cells express functional TLR1–6 and TLR9 and thus make use of a common principle of professional innate immune cells. Although it was observed that TLR2 ligands dependent on heterodimeric signaling either with TLR1 or TLR6 were functional, other ligands like lipoteichoic acid were not. Additionally, it was found that bronchial epithelial cells could be stimulated only marginally by Gram-positive bacteria bearing known TLR2 ligands while Gram-negative bacteria were easily recognized. This correlated with low expression of TLR2 and the missing expression of the coreceptor CD36. Transgenic expression of both receptors restored responsiveness to the complete set of TLR2 ligands and Staphylococcus aureus. Additional gene-array experiments confirmed hyporesponsiveness to this bacterium while Pseudomonas aeruginosa and respiratory syncytial virus induced common, as well as pathogen-specific, sets of genes. The findings indicate that bronchial epithelium regulates its sensitivity to recognize microbes by managing receptor expression levels. This could serve the special needs of controlled microbial recognition in mucosal compartments.
The Type I IFN Response to Infection withMycobacterium tuberculosisRequires ESX-1-Mediated Secretion and Contributes to Pathogenesis
Authors: Stanley Sarah A. , Johndrow James E. , Manzanillo Paolo , Cox Jeffery S.
Published: April 2014

The ESX-1 secretion system is a major determinant of Mycobacterium tuberculosis virulence, although the pathogenic mechanisms resulting from ESX-1-mediated transport remain unclear. By global transcriptional profiling of tissues from mice infected with either wild-type or ESX-1 mutant bacilli, we found that host genes controlled by ESX-1 in vivo are predominantly IFN regulated. ESX-1-mediated secretion is required for the production of host type I IFNs during infection in vivo and in macrophages in vitro. The macrophage signaling pathway leading to the production of type I IFN required the host kinase TANK-binding kinase 1 and occurs independently of TLR signaling. Importantly, the induction of type I IFNs during M. tuberculosis infection is a pathogenic mechanism as mice lacking the type I IFNR were more restrictive for bacterial growth in the spleen than wild-type mice, although growth in the lung was unaffected. We propose that the ESX-1 secretion system secretes effectors into the cytosol of infected macrophages, thereby triggering the type I IFN response for the manipulation of host immunity.
Mycobacterial Lipomannan Induces Granuloma Macrophage Fusion via a TLR2-Dependent, ADAM9- and β1 Integrin-Mediated Pathway
Authors: Puissegur Marie-Pierre , Lay Guillaume , Gilleron Martine , Botella Laure , Nigou Jérôme , Marrakchi Hedia , Mari Bernard , Duteyrat Jean-Luc , Guerardel Yann , Kremer Laurent , Barbry Pascal , Puzo Germain , Altare Frédéric
Published: April 2014

Tuberculous granulomas are the sites of interaction between the host response and the tubercle bacilli within infected individuals. They mainly consist of organized aggregations of lymphocytes and macrophages (Mf). A predominant role of mycobacterial envelope glycolipids in granulomas formation has been recently emphasized, yet the signaling events interfering with granuloma cell differentiation remain elusive. To decipher this molecular machinery, we have recently developed an in vitro human model of mycobacterial granulomas. In this study, we provide evidence that the mycobacterial proinflammatory phosphatidyl-myo-inositol mannosides and lipomannans (LM), as well as the anti-inflammatory lipoarabinomannan induce granuloma formation, yet only the proinflammatory glycolipids induce the fusion of granuloma Mf into multinucleated giant cells (MGC). We also demonstrate that LM induces large MGC resembling those found in vivo within the granulomas of tuberculosis patients, and that this process is mediated by TLR2 and is dependent on the β1 integrin/ADAM9 cell fusion machinery. Our results demonstrate for the first time that the Mf differentiation stage specifically occurring within granulomatous structures (i.e., MGC formation) is triggered by mycobacterial envelope glycolipids, which are capable of inducing the cell fusion machinery. This provides the first characterization of the ontogeny of human granuloma MGC, thus resulting in a direct modulation by a particular mycobacterial envelope glycolipid of the differentiation process of granuloma Mf.
Efficient Capture of Antibody Neutralized HIV-1 by Cells Expressing DC-SIGN and Transfer to CD4+ T Lymphocytes
Authors: van Montfort Thijs , Nabatov Alexey A. , Geijtenbeek Teunis B. H. , Pollakis Georgios , Paxton William A.
Published: April 2014

Infection of CD4+ T lymphocytes is enhanced by the capture and subsequent transfer of HIV-1 by dendritic cells (DCs) via the interaction with C-type lectins such as the DC-specific ICAM-grabbing nonintegrin (DC-SIGN). Numerous HIV-1 envelope-directed neutralizing Abs have been shown to successfully block the infection of CD4+ T lymphocytes. In this study, we find that HIV-1-neutralized with the mAb 2F5 is more efficiently captured by immature monocyte-derived DCs (iMDDCs) and DC-SIGN-expressing Raji cells (Raji-DC-SIGN). Furthermore, a 2F5-neutralized virus captured by these cells was able to subsequently infect CD4+ T lymphocytes upon the release of HIV-1 from iMDDCs, thereby enhancing infection. We show that upon transfer via DC-SIGN-expressing cells, HIV-1 is released from immune-complexes with the Abs 2F5 and 4E10 (gp41-directed) and 2G12, 4.8D, and 1.7b (gp120-directed). The nonneutralizing V3-21 (V3 region of the gp120-directed) Ab enhanced HIV-1 infection upon capture and transfer via Raji-DC-SIGN cells, whereas no infection was observed with the neutralizing b12 Ab (gp120-directed), indicating that different Abs have variant effects on inhibiting HIV-1 transfer to CD4+ T lymphocytes. The increased capture of the 2F5-neutralized virus by iMDDCs was negated upon blocking the Fc receptors. Blocking DC-SIGN on iMDDCs resulted in a 70–75% inhibition of HIV-1 capture at 37°C, whereas at 4°C a full block was observed, showing that the observed transfer is mediated via DC-SIGN. Taken together, we propose that DC-SIGN-mediated capture of neutralized HIV-1 by iMDDCs has the potential to induce immune evasion from the neutralization effects of HIV-1 Abs, with implications for HIV-1 pathogenesis and vaccine development.
The Contact Allergen Nickel Triggers a Unique Inflammatory and Proangiogenic Gene Expression Pattern via Activation of NF-κB and Hypoxia-Inducible Factor-1α
Authors: Viemann Dorothee , Schmidt Marc , Tenbrock Klaus , Schmid Sybille , Müller Verena , Klimmek Kerstin , Ludwig Stephan , Roth Johannes , Goebeler Matthias
Published: April 2014

Nickel compounds are prime inducers of contact allergy reactions in humans. To identify the signal transduction pathways mediating the cellular responses to nickel and to elucidate their hierarchy, we performed Affymetrix gene profiling using human primary endothelial cells, which strongly respond to nickel stimulation. Overall, we found 258 significantly modulated transcripts, comprising 140 up-regulated and 118 down-regulated genes. The bulk of those genes were identified as targets of two distinct signaling cascades, the IKK2/NF-κB pathway and a proangiogenic pathway mediated by HIF-1α, which accumulates upon exposure to nickel. Using dominant-interfering mutants and retroviral RNA interference technology, we demonstrate that both pathways act independently to regulate expression of nonoverlapping gene pools. NF-κB activation mediates most of the proinflammatory responses to nickel. Nickel-dependent HIF-1α activation primarily modulates expression of genes involved in proliferation, survival, metabolism, and signaling, albeit the induction of some proinflammatory nickel-response genes, most prominently IL-6, which we identified as novel bona fide HIF-1α target in this study, is also critically dependent on this pathway. Furthermore, we provide evidence that transactivation of both transcription factors partially depends on p38 MAPK activation that contributes to the intensity of at least some target genes. Taken together, our data provide mechanistic insight into the complex network of nickel-induced cellular events and identify IKK2/NF-κB and HIF-1α as important pathways involved in processes such as delivery of “second signals” in contact hypersensitivity reactions to nickel.
Retinoid-Related Orphan Receptor γ Controls Immunoglobulin Production and Th1/Th2 Cytokine Balance in the Adaptive Immune Response to Allergen
Authors: Tilley Stephen L. , Jaradat Maisa , Stapleton Cliona , Dixon Darlene , Hua Xiaoyang , Erikson Christopher J. , McCaskill Joshua G. , Chason Kelly D. , Liao Grace , Jania Leigh , Koller Beverly H. , Jetten Anton M.
Published: April 2014

The retinoid-related orphan receptors (ROR) comprise a distinct subfamily of nuclear receptors with the capacity to act as both repressors and activators of transcription. RORγ, the most recently identified member of the ROR family, has been shown to be important for the development of normal lymphocyte compartments as well as organogenesis of some lymphoid organs. In this report, we examine the capacity of RORγ-deficient mice to develop an adaptive immune response to Ag using OVA-induced inflammation in mice as a model for allergic airway disease. In sham-treated mice lacking RORγ, low-grade pulmonary inflammation was observed and characterized by the perivascular accumulation of B and T lymphocytes, increased numbers of inflammatory cells in the lung lavage fluid, and polyclonal Ig activation. Following sensitization and challenge, the capacity of these animals to develop the allergic phenotype was severely impaired as evidenced by attenuated eosinophilic pulmonary inflammation, reduced numbers of CD4+ lymphocytes, and lower Th2 cytokines/chemokine protein and mRNA expression in the lungs. IFN-γ and IL-10 production was markedly greater in splenocytes from RORγ-deficient mice following in vitro restimulation with OVA compared with wild-type splenocytes, and a shift toward a Th1 immune response was observed in sensitized/challenged RORγ-deficient animals in vivo. These data reveal a critical role for RORγ in the regulation of Ig production and Th1/Th2 balance in adaptive immunity.
TLR4 Hyperresponsiveness via Cell Surface Expression of Heat Shock Protein gp96 Potentiates Suppressive Function of Regulatory T Cells
Authors: Dai Jie , Liu Bei , Ngoi Soo Mun , Sun Shaoli , Vella Anthony T. , Li Zihai
Published: April 2014

As one of the main mediators of the endoplasmic reticulum unfolded protein response, heat shock protein gp96 is also an obligate chaperone for multiple TLRs including TLR4. We demonstrated recently that enforced cell surface expression of gp96 in a transgenic (Tg) mouse (96tm-Tg) conferred hyperresponsiveness to LPS and induced TLR4-dependent lupus-like autoimmune diseases. In this study, we investigated the function of CD4+CD25+ Foxp3+ regulatory T cells (Treg) in these mice in light of the important roles of Treg in the maintenance of peripheral tolerance against self-Ag as well as the increasing appreciation of TLR signaling on the regulation of Treg. We found that the development of Treg was not impaired in 96tm-Tg mice. Contrary to the prediction of dampened Treg activity, we discovered that the suppressive functions of Treg were increased in 96tm-Tg mice. Inactivation of Treg during the neonatal stage of life exacerbated not only organ-specific diseases but also systemic autoimmune diseases. By crossing 96tm-Tg mice into the TLR4 null background, we demonstrated the critical roles of TLR4 in the amplification of Treg suppressive function. These findings illustrate that gp96 plays dual roles in regulating immune responses by augmenting proinflammatory responses and inducing Treg function, both of which are dependent on its ability to chaperone TLR4. Our study provides strong support to the notion of compensatory Treg activation by TLR ligation to dampen inflammation and autoimmune diseases.
TCR Signaling Antagonizes Rapid IP-10-Mediated Transendothelial Migration of Effector Memory CD4+ T Cells
Authors: Manes Thomas D. , Shiao Stephen L. , Dengler Thomas J. , Pober Jordan S.
Published: April 2014

Human microvascular endothelial cells (ECs) constitutively express MHC class II in peripheral tissues, the function of which remains unknown. In vitro assays have established that the recognition of EC MHC class II can affect cytokine expression, proliferation, and delayed transendothelial migration of allogeneic memory, but not naive, CD4+ T cells. Previously, we have shown that effector memory CD4+ T cells will rapidly transmigrate in response to the inflammatory chemokine IFN-γ-inducible protein-10 (IP-10) in a process contingent upon the application of venular levels of shear stress. Using two models that provide polyclonal TCR signaling by ECs in this flow system, we show that TCR engagement antagonizes the rapid chemokine-dependent transmigration of memory CD4+ T cells. Inhibitor studies suggest that TCR signaling downstream of Src family tyrosine kinase(s) but upstream of calcineurin activation causes memory CD4+ T cell arrest on the EC surface, preventing the transendothelial migration response to IP-10.
Rate of Increase in Circulating IL-7 and Loss of IL-7Rα Expression Differ in HIV-1 and HIV-2 Infections: Two Lymphopenic Diseases with Similar Hyperimmune Activation but Distinct Outcomes
Authors: Albuquerque Adriana S. , Cortesão Catarina S. , Foxall Russell B. , Soares Rui S. , Victorino Rui M. M. , Sousa Ana E.
Published: April 2014

IL-7 is a nonredundant cytokine for T cell homeostasis. Circulating IL-7 levels increase in lymphopenic clinical settings, including HIV-1 infection. HIV-2 infection is considered a “natural” model of attenuated HIV disease given its much slower rate of CD4 decline than HIV-1 and limited impact on the survival of the majority of infected adults. We compared untreated HIV-1- and HIV-2-infected patients and found that the HIV-2 cohort demonstrated a delayed increase in IL-7 levels during the progressive depletion of circulating CD4 T cells as well as a dissociation between the acquisition of markers of T cell effector differentiation and the loss of IL-7Rα expression. This comparison of two persistent infections associated with progressive CD4 depletion and immune activation demonstrates that a better prognosis is not necessarily associated with higher levels of IL-7. Moreover, the delayed increase in IL-7 coupled with sustained expression of IL-7Rα suggests a maximization of available resources in HIV-2. The observation that increased IL-7 levels early in HIV-1 infection were unable to reduce the rate of CD4 loss and the impaired expression of the IL-7Rα irrespective of the state of cell differentiation raises concerns regarding the use of IL-7 therapy in HIV-1 infection.
Involvement of C-C Chemokine Ligand 2-CCR2 Interaction in Monocyte-Lineage Cell Recruitment of Normal Human Corneal Stroma
Authors: Ebihara Nobuyuki , Yamagami Satoru , Yokoo Seiichi , Amano Shiro , Murakami Akira
Published: April 2014

Bone marrow-derived cells (BMCs) reside in the anterior stroma of the central and paracentral cornea, as well as all stromal layers of the peripheral cornea, in normal human eyes. We investigated the factors regulating the constitutive distribution of BMCs in normal human corneal stroma. Cultured human corneal keratocytes expressed several chemokines (growth-related oncogene/CXCL1–3, IL-8/CXCL8, and MCP-1/CCL2) in the Ab array study. CCR2 and CCR7 mRNAs were detected in BMCs by multiplex RT-PCR. Keratocytes/corneal epithelial cells and BMCs selected from normal human donor corneas by using magnetic beads expressed MCP-1/CCL2 and CCR2 protein, respectively. BMCs isolated from human corneal stroma showed a chemotactic response to MCP-1/CCL2 in the Boyden chamber assay. The chemotactic effect of keratocyte supernatant was inhibited by blockade of MCP-1/CCL2. This is the first work on constitutive expression of CCR2 by BMCs from the corneal stroma and MCP-1/CCL2 by keratocytes/epithelial cells. Our findings suggest that the interaction between MCP-1/CCL2 and CCR2 determines the distribution of constitutive BMCs in normal human corneal stroma.
Cutting Edge: Influenza A Virus Activates TLR3-Dependent Inflammatory and RIG-I-Dependent Antiviral Responses in Human Lung Epithelial Cells
Authors: Le Goffic Ronan , Pothlichet Julien , Vitour Damien , Fujita Takashi , Meurs Eliane , Chignard Michel , Si-Tahar Mustapha
Published: April 2014

Influenza A virus (IAV) triggers a contagious acute respiratory disease that causes considerable mortality annually. Recently, we established a role for the pattern-recognition TLR3 in the response of lung epithelial cells to IAV-derived dsRNA. However, additional nucleic acid-recognition proteins have lately been implicated as key viral sensors, including the RNA helicases retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene (MDA)-5. In this study, we investigated the respective role of TLR3 vs RIG-I/MDA-5 signaling in human respiratory epithelial cells infected by IAV using BEAS-2B cells transfected with vectors encoding either a dominant-negative form of TLR3 or of mitochondrial antiviral signaling protein (MAVS; a signaling intermediate of RIG-I and MDA-5), or with plasmids overexpressing functional RIG-I or MDA-5. We demonstrate that the sensing of IAV by TLR3 primarily regulates a proinflammatory response, whereas RIG-I (but not MDA-5) mediates both a type I IFN-dependent antiviral signaling and a proinflammatory response.
Regulatory T Cells Are Resistant to Apoptosis via TCR but Not P2X7
Authors: Taylor Simon R. J. , Alexander Denis R. , Cooper Joanne C. , Higgins Christopher F. , Elliott James I.
Published: April 2014

Regulatory T cells (Tregs) are relatively autoreactive yet, paradoxically, have been found to display normal sensitivity to thymic deletion. The relationship between self-avidity, apoptosis, and the selection of Tregs therefore remains unclear. We show that thymic Tregs develop efficiently, even at low self-avidity, and are moderately resistant to apoptosis in comparison to conventional thymocytes. Consistent with this, although conventional self-reactive T cell populations undergo chronic peripheral deletion, self-reactive Tregs are largely spared removal. Similarly, the distribution of Tregs among peripheral CD4+ cells exhibits a linear inverse relationship with CD45RB expression, indicating relative apoptosis resistance of Tregs in chronic responses to environmental Ags. We also show that appropriate controls for CD45RB levels are important for comparisons of Treg and conventional T cell activity. When thus controlled, and contrary to previous reports, Tregs exhibit normal sensitivity to cell death through TCR-independent stimuli, such as the purinergic receptor, P2X7. Finally, although absence of CD45 in gene-targeted mice results in profound T cell hyporesponsiveness, there is little or no effect on thymic Treg frequency. In summary, the data support a model in which signal strength plays little part in Treg lineage specification, though moderate resistance of self-reactive Tregs to apoptosis may result in progressive biasing of peripheral Treg TCRs toward autoreactivity in comparison to those of conventional T cells.
Type I IFN Signaling on Both B and CD4 T Cells Is Required for Protective Antibody Response to Adenovirus
Authors: Zhu Jiangao , Huang Xiaopei , Yang Yiping
Published: April 2014

Recombinant adenoviruses have been used as vehicles for gene therapy as well as vaccination against infectious diseases and cancer. Efficient activation of host B cell response to adenoviral vectors that leads to the generation of protective, neutralizing Ab, represents a major barrier for gene therapy, but an attractive feature for vaccine development. What regulate(s) potent B cell response to adenoviral vectors remains incompletely defined. In this study, we showed that type I IFNs induced upon adenoviral infection are critical for multiple stages of adaptive B cell response to adenovirus including early B cell activation, germinal center formation, Ig isotype switching as well as plasma cell differentiation. We further demonstrated that although type I IFN signaling on dendritic cells was important for the production of virus-specific IgM, the generation of protective neutralizing Ab critically depended on type I IFN signaling on both CD4 T and B cells. The results may suggest potential strategies for improving adenovirus-mediated gene therapy in vivo and/or the design of effective vaccines for cancer and infectious diseases.
Increased Expression of Leukocyte Ig-Like Receptor-1 and Activating Role of UL18 in the Response to Cytomegalovirus Infection
Authors: Wagner Claudia S. , Riise Gerdt C. , Bergström Tomas , Kärre Klas , Carbone Ennio , Berg Louise
Published: April 2014

NK and T cells are important for combating CMV infection. Some NK and T cells express leukocyte Ig-like receptor-1 (LIR-1), an inhibitory receptor recognizing MHC class I and the CMV-encoded homolog UL18. We previously demonstrated an early increase in LIR-1-expressing blood lymphocytes in lung-transplanted patients later developing CMV disease. We now show that NK and T cells account for the observed LIR-1 augmentation. Coincubation of PBMC from CMV-seropositive donors with virus-infected lung fibroblasts led to a T cell-dependent secretion of IFN-γ, produced mainly by LIR-1+ T cells and by NK cells. Cytokine production during coculture with fibroblasts infected with virus containing the UL18 gene was augmented compared with the UL18 deletion virus, suggesting a stimulatory role for UL18. However, purified UL18Fc proteins inhibited IFN-γ production of LIR-1+ T cells. We propose that cytokine production in the transplant induces NK and T cells to express LIR-1, which may predispose to CMV disease by MHC/LIR-1-mediated suppression. Although the UL18/LIR-1 interaction could inhibit T cell responses, this unlikely plays a role in response to infected cells. Instead, our data point to an activating role for viral UL18 during infection, where indirect intracellular effects cannot be excluded.
Natural Regulation of Immunity to Minor Histocompatibility Antigens
Authors: Robertson Nathan J. , Chai Jian-Guo , Millrain Maggie , Scott Diane , Hashim Fazila , Manktelow Emily , Lemonnier François , Simpson Elizabeth , Dyson Julian
Published: April 2014

MHC-matched hemopoietic stem cell transplantation is commonly used for the treatment of some forms of leukemia. Conditioning regimens before transplant act to reduce the burden of leukemic cells and the graft-vs-leukemia (GvL) effect can eliminate residual disease. The GvL effect results largely from the recognition of minor histocompatibility Ags by donor T cells on recipient tissues. These Ags are generally widely expressed and also provoke graft-vs-host (GvH) disease. Manipulation of immunity to promote GvL while curtailing GvH would greatly improve clinical outcome. To develop strategies that may achieve this, the parameters which control immunity to minor histocompatibility Ags need to be defined. In this study, we have analyzed responses to the mouse HY minor histocompatibility Ag using hemopoietic cell and skin grafts as surrogate GvL and GvH targets, respectively. We show that natural regulation of CD8 T cell responses to HY operates at multiple levels. First, CD4 T cell help is required for primary CD8 responses directed at hemopoietic cells. However, although CD4 T cells of H2k mouse strains recognize HY, they provide ineffective help associated with a proportion of recipients developing tolerance. This was further investigated using TCR-transgenic mice which revealed H2k-restricted HY-specific CD4 T cells are highly susceptible to regulation by CD25+ regulatory T cells which expand in tolerant recipients. A second level of regulation, operating in the context of skin grafts, involves direct inhibition of CD8 T cell responses by CD94/NKG2 engagement of the nonclassical MHC class I molecule Qa1.
Normal TCR Signal Transduction in Mice That Lack Catalytically Active PTPN3 Protein Tyrosine Phosphatase
Authors: Bauler Timothy J. , Hughes Elizabeth D. , Arimura Yutaka , Mustelin Tomas , Saunders Thomas L. , King Philip D.
Published: April 2014

PTPN3 (PTPH1) is a cytoskeletal protein tyrosine phosphatase that has been implicated as a negative regulator of early TCR signal transduction and T cell activation. To determine whether PTPN3 functions as a physiological negative regulator of TCR signaling in primary T cells, we generated gene-trapped and gene-targeted mouse strains that lack expression of catalytically active PTPN3. PTPN3 phosphatase-negative mice were born in expected Mendelian ratios and exhibited normal growth and development. Furthermore, numbers and ratios of T cells in primary and secondary lymphoid organs were unaffected by the PTPN3 mutations and there were no signs of spontaneous T cell activation in the mutant mice with increasing age. TCR-induced signal transduction, cytokine production, and proliferation was normal in PTPN3 phosphatase-negative mice. This was observed using both quiescent T cells and recently stimulated T cells where expression of PTPN3 is substantially up-regulated. We conclude, therefore, that the phosphatase activity of PTPN3 is dispensable for negative regulation of TCR signal transduction and T cell activation.
Monocyte-Dependent Fibroblast CXCL8 Secretion Occurs in Tuberculosis and Limits Survival of Mycobacteria within Macrophages
Authors: O’Kane Cecilia M. , Boyle Joseph J. , Horncastle Donna E. , Elkington Paul T. , Friedland Jon S.
Published: April 2014

CXCL8 is a chemokine that is implicated in the formation of tuberculous (TB) granulomas and in immunity to Mycobacterium tuberculosis (Mtb). Fibroblast chemokine secretion is important for modulating inflammatory responses in chronic lung disease and inflammatory arthritis but has not been investigated in the pathophysiology of TB. In this study, we used a cellular model to examine monocyte/macrophage-dependent stimulation of fibroblasts by Mtb in the regulation of chemokine secretion, particularly that of CXCL8. Human lung fibroblasts grown in collagen were stimulated with conditioned medium from Mtb-infected monocytes (CoMTb). CoMTb-induced prolonged dose-dependent, p38-mediated expression of stable CXCL8 mRNA by fibroblasts accompanied by a >10-fold increase in CXCL8 secretion (487 ± 88 ng/ml vs 48.6 ± 34 ng/ml in controls) at 120 h. Fibroblasts strongly expressed CXCL8 in vivo in human TB granulomas. Inhibition of TNF-α or IL-1 in CoMTb abrogated the induction of CXCL8 at a pretranscriptional level. CXCL8 secretion was NF-κB, C/EBP, and JNK dependent. Sustained NF-κB activation was demonstrated beyond 24 h in response to CoMTb. Exogenous CXCL8 reduced the survival of Mtb within macrophages, and inhibition of CXCL8 was associated with intracellular mycobacterial proliferation. These data show that fibroblasts have a previously unrecognized role in modulating inflammation in TB by their CXCL8-dependent contribution to cell recruitment and mycobacterial killing within the granuloma.
Pyrin-Only Protein 2 Modulates NF-κB and Disrupts ASC:CLR Interactions
Authors: Bedoya Felipe , Sandler Laurel L. , Harton Jonathan A.
Published: April 2014

NF-κB is pivotal for transactivation of cell-cycle regulatory, cytokine, and adhesion molecule genes and is dysregulated in many cancers, neurodegenerative disorders, and inflammatory diseases. Proteins with pyrin and/or caspase recruitment domains have roles in apoptosis, innate immunity, and inflammation. Many pyrin domain (PYD) proteins modulate NF-κB activity as well as participate in assembling both the perinuclear “apoptotic speck” and the pro-IL1β/IL-18-converting inflammasome complex. “Pyrin-only” proteins (POP) are attractive as negative regulators of PYD-mediated functions and one such protein, POP1, has been reported. We report the identification and initial characterization of a second POP. POP2 is a 294 nt single exon gene located on human chromosome 3 encoding a 97-aa protein with sequence and predicted structural similarity to other PYDs. Highly similar to PYDs in CATERPILLER (CLR, NLR, NALP) family proteins, POP2 is less like the prototypic pyrin and ASC PYDs. POP2 is expressed principally in peripheral blood leukocytes and displays both cytoplasmic and nuclear expression patterns in transfected cells. TNF-α-stimulated and p65 (RelA)-induced NF-κB-dependent gene transcription is inhibited by POP2 in vitro by a mechanism involving changes in NF-κB nuclear import or distribution. While colocalizing with ASC in perinuclear specks, POP2 also inhibits the formation of specks by the CLR protein CIAS1/NALP3. Together, these observations demonstrate that POP2 is a negative regulator of NF-κB activity that may influence the assembly of PYD-dependent complexes.
FcαRI (CD89) Alleles Determine the Proinflammatory Potential of Serum IgA
Authors: Wu Jianming , Ji Chuanyi , Xie Fenglong , Langefeld Carl D. , Qian Kun , Gibson Andrew W. , Edberg Jeffrey C. , Kimberly Robert P.
Published: April 2014

The human IgA FcR (FcαRI; CD89) mediates a variety of immune system functions including degranulation, endocytosis, phagocytosis, cytokine synthesis, and cytokine release. We have identified a common, nonsynonymous, single nucleotide polymorphism (SNP) in the coding region of CD89 (844A→G) (rs16986050), which changes codon 248 from AGC (Ser248) to GGC (Gly248) in the cytoplasmic domain of the receptor. The two different alleles demonstrate significantly different FcαRI-mediated intracellular calcium mobilization and degranulation in rat basophilic leukemia cells and cytokine production (IL-6 and TNF-α) in murine macrophage P388D1 cells. In the absence of FcR γ-chain association in P388D1 cells, the Ser248-FcαRI allele does not mediate cytokine production, but the Gly248-FcαRI allele retains the capacity to mediate a robust production of proinflammatory cytokine. This allele-dependent difference is also seen with FcαRI-mediated IL-6 cytokine release by human neutrophils ex vivo. These findings and the enrichment of the proinflammatory Gly248-FcαRI allele in systemic lupus erythematosus populations in two ethnic groups compared with their respective non-systemic lupus erythematosus controls suggest that FcαRI (CD89) α-chain alleles may affect receptor-mediated signaling and play an important role in the modulation of immune responses in inflammatory diseases.
Human CD4+CD25low Adaptive T Regulatory Cells Suppress Delayed-Type Hypersensitivity during Transplant Tolerance
Authors: Xu Qingyong , Lee Junglim , Jankowska-Gan Ewa , Schultz Jackie , Roennburg Drew A. , Haynes Lynn D. , Kusaka Satoshi , Sollinger Hans W. , Knechtle Stuart J. , VanBuskirk Anne M. , Torrealba Jose R. , Burlingham William J.
Published: April 2014

Adaptive T regulatory (TR) cells mediate the suppression of donor-specific, delayed-type hypersensitivity (DTH) in tolerant organ transplant recipients. We hypothesized that cells belonging to the CD4+CD25+ T cell subset but distinct from natural TR cells may fulfill this role. To test this hypothesis, PBMC and biopsy samples from two tolerant kidney transplant recipients (K1 and K2) were analyzed. When transferred with recipient APC into a SCID mouse footpad, CD4+ T cells were hyporesponsive in DTH to donor type HLA-B Ags and derivative allopeptides. However, anti-human TGF–β1 Ab revealed a response to immunodominant allopeptides in both patients, suggesting that CD4+ T effector (TE) cells coexisted with suppressive, TGF–β1-producing CD4+ TR cells. During in vitro culture, allopeptide stimulation induced both IFN-γ-producing and surface TGF–β1+ T cells. The relative strength of the latter response in patient K1 was inversely correlated with the level of systemic anti-donor DTH, which varied over a 6-year interval. Allopeptide-induced surface TGF–β1 expression was found primarily in Forkhead box P3 (FoxP3)–negative CD4+CD25low T cells, which could adoptively transfer suppression of donor-specific DTH. Biopsy samples contained numerous surface TGF-β1+ mononuclear cells that costained for CD4 and, less frequently CD25, but were negative for FoxP3. The CD4+TGF-β1+ T cells were localized primarily to the tubulointerstitium, whereas TGF-β1−FoxP3+CD25+ cells were found mainly in lymphoid aggregates. Thus, adaptive TR cells suppressing TE cell responses to donor allopeptides in two tolerant patients appear to be functionally and phenotypically distinct from CD4+CD25highFoxP3+ T cells.
Function of the IL-2R for Thymic and Peripheral CD4+CD25+ Foxp3+ T Regulatory Cells
Authors: Bayer Allison L. , Yu Aixin , Malek Thomas R.
Published: April 2014

IL-2 contributes to the production, function, and homeostasis of CD4+CD25+ Treg cells. However, it remains uncertain whether IL-2 is essential for the development of Treg cells in the thymus, their homeostasis in the periphery, or both. The present study was undertaken to investigate the contribution of IL-2 during thymic Treg cell development and its maintenance in peripheral immune tissue. Relying on genetic mouse models where IL-2R signaling was either completely blocked or selectively inhibited in peripheral CD4+CD25+ Treg cells, we show that the IL-2/IL-2R interaction is active in the thymus at the earliest stage of the development of Treg cells to promote their expansion and to up-regulate Foxp3 and CD25 to normal levels. Furthermore, CD4+CD25+Foxp3+ Treg cells with impaired IL-2-induced signaling persist in the periphery and control autoimmunity without constant thymic output. These peripheral Treg cells with poor responsiveness to IL-2 exhibited slower growth and extended survival in vivo, somewhat lower suppressive activity, and poor IL-2-dependent survival in vitro. Mixed thymic and bone marrow chimeric mice showed that wild-type-derived Treg cells were substantially more effective in populating peripheral immune tissue than Treg cells with impaired IL-2 signaling. Collectively, these data support the notion that normally IL-2 is a dominant mechanism controlling the number of thymic and peripheral Treg cells.
The Blood Contains Multiple Distinct Progenitor Populations with Clonogenic B and T Lineage Potential
Authors: Umland Oliver , Mwangi William N. , Anderson Bradley M. , Walker Juliette C. , Petrie Howard T.
Published: April 2014

The thymus is seeded by bone marrow-derived progenitors that circulate in the blood. Multiple cell types can be found in the thymus early after i.v. administration or in steady state, but most fail to satisfy the known characteristics of true T progenitors. Cells that do conform to classical definitions retain multilineage potential, but surprisingly, cannot make B cells. Because acquisition of the T lineage fate among noncommitted progenitors is a lengthy process, the absence of B cell potential in early thymocytes suggests that B and T lineages diverge prethymically. To test this suggestion, we screened numerous presumptive progenitor populations for T cell growth and differentiation potential, as well as for clonogenic T or B cell development. We find that blood and marrow each contain multiple distinct subsets that display growth and differentiation potential consistent with being canonical T progenitors. Assessment of clonogenic potential further shows that although all blood and marrow populations have high T cell cloning potential, no T/non-B cells are apparent. These data suggest that either true thymic reconstitution potential derives from a small T/non-B cell subset of one of these populations, or that most of the cells defined as canonical progenitors within the thymus do not, in fact, reside in the mainstream of T progenitor differentiation.