bluuebunny/crossref_metadata_2025 huggingface dataset:
Uncoupled Anti-HIV and Immune-Enhancing Effects when Combining IFN-α and IL-7
Authors: Audigé Annette , Schlaepfer Erika , Joller Helen , Speck Roberto F.
Published: April 2014

Cytokine-based therapies have been examined for purging viral reservoirs and immunomodulation in HIV infection. However, single cytokines did not result in either HIV eradication or an efficient HIV-specific immune response. We hypothesize that cytokines with distinct biologic effects need to be combined for immunotherapy of HIV infection. In this study, we investigated the anti-HIV activity and immune-enhancing effects of the combination of IFN-α and IL-7. In human lymphocyte aggregate cultures infected ex vivo with the X4 HIV strain NL4-3, IFN-α/IL-7 potently inhibited HIV replication and preserved CD4+ T cells, probably by up-regulating Bcl-2. IFN-α/IL-7 also strongly inhibited R5 HIV replication. Furthermore, in allogeneic MLRs, IFN-α/IL-7 increased T cell proliferation and IFN-γ production. IFN-α alone also had strong anti-HIV activity, but neither preserved CD4+ T cells nor increased T cell responses in MLRs. IL-7 alone maintained T cells and enhanced T cell activation in MLRs, but only moderately inhibited or increased HIV replication. Thus, coadministration of IFN-α/IL-7 combines the potent anti-HIV activity of IFN-α with the beneficial effects of IL-7 on T cell survival and function. We speculate that IFN-α will block viral replication, activate APCs, and up-regulate MHC molecules, thus allowing IL-7 to display its effects for generating an efficient immune response. In this scenario, the known reactivation of latent HIV by IL-7 may be advantageous.
Are Extensive T Cell Epitope Polymorphisms in the Plasmodium falciparum Circumsporozoite Antigen, a Leading Sporozoite Vaccine Candidate, Selected by Immune Pressure?
Authors: Kumkhaek Chutima , Phra-ek Kooruethai , Rénia Laurent , Singhasivanon Pratap , Looareesuwan Sornchai , Hirunpetcharat Chakrit , White Nicholas J. , Brockman Alan , Grüner Anne Charlotte , Lebrun Nicolas , Alloueche Ali , Nosten François , Khusmith Srisin , Snounou Georges
Published: April 2014

Protective cellular immune responses depend on MHC presentation of pathogen-derived Ag fragments. MHC diversity renders this process sensitive to point mutations coding for altered amino acid sequence of the short target Ag-derived peptides epitopes. Thus, in a given host, a pathogen with an altered epitope sequence will be more likely to escape detection and elimination by the immune system. At a population level, selection by immune pressure will increase the likelihood of polymorphism in important pathogen antigenic epitopes. This mechanism of immune evasion is found in viruses and other pathogens. The detection of polymorphic hot spots in an Ag is often taken as a strong indication of its role in protective immunity. We provide evidence that polymorphisms in the T cell epitopes of a malaria vaccine candidate are unlikely to have been selected by immune pressure in the human host.
Influence of CD14 on Ligand Interactions between Lipopolysaccharide and Its Receptor Complex
Authors: Gangloff Sophie C. , Zähringer Ulrich , Blondin Catherine , Guenounou Moncef , Silver Jack , Goyert Sanna M.
Published: April 2014

The interaction of LPS (endotoxin) with the CD14-TLR4 receptor complex modulates the host innate immune response. Several studies using partial structures of LPS have suggested that TLR4 determines the ligand specificity of this complex, and that CD14 indiscriminately serves to deliver the ligand to TLR4. This conclusion has been made despite observations that the response of TLR4+/+,CD14−/− macrophages to LPS is very weak. To determine whether CD14 itself plays a role in specific ligand recognition, the influences of various partial structures of LPS on induction of the proinflammatory cytokine, TNF, by CD14+/+ and CD14−/− macrophages were compared. These studies show that the ligand specificities of CD14+/+ and CD14−/− macrophages are very different. When CD14 is present, the receptor complex shows exquisite specificity for smooth LPS, the major form expressed by Gram-negative bacteria; however, as increasing amounts of carbohydrate are removed from smooth LPS, the sensitivity of CD14+/+ macrophages decreases as much as 500-fold. In contrast, CD14−/− macrophages are unable to distinguish between smooth LPS and its various partial structures. Furthermore, CD14−/− macrophages are 150,000-fold less sensitive than CD14+/+ macrophages to smooth LPS. A similar ability to distinguish the differing LPS structures of various bacteria such as Bacteroides fragilis and Salmonella abortus are observed for CD14+/+, but not CD14−/−, macrophages. Thus, CD14+/+, but not CD14−/−, macrophages are highly sensitive to stimulation by natural forms of LPS and show the ability to distinguish between various LPS ligands, consistent with CD14 being a highly specific receptor.
Treatment of Passive Experimental Autoimmune Encephalomyelitis in SJL Mice with a Recombinant TCR Ligand Induces IL-13 and Prevents Axonal Injury
Authors: Offner Halina , Subramanian Sandhya , Wang Chunhe , Afentoulis Michael , Vandenbark Arthur A. , Huan Jianya , Burrows Gregory G.
Published: April 2014

The major goal of this study was to evaluate the efficacy and mechanism of a rTCR ligand (RTL) construct (I-As/proteolipid protein (PLP)-139–151 peptide = RTL401) for treatment of SJL/J mice developing passive experimental autoimmune encephalomyelitis (EAE) that did not involve coimmunization with the highly inflammatory CFA. Our results demonstrated clearly that RTL401 was highly effective in treating passive EAE, with kinetics of recovery from disease very similar to treatment of actively induced EAE. The potent RTL401 treatment effect was reflected by a partial reduction of infiltrating mononuclear cells into CNS, minimal inflammatory lesions in spinal cord, and preservation of axons injured in vehicle-treated mice during the progression of EAE. Interestingly, in the absence of CFA, RTL401 treatment strongly enhanced production of the Th2 cytokine, IL-13, in spleen, blood, and spinal cord tissue, with variable effects on other Th1 and Th2 cytokines, and no significant effect on the Th3 cytokine, TGF-β1, or on FoxP3 that is expressed by regulatory T cells. Moreover, pretreatment of PLP-139–151-specific T cells with RTL401 in vitro induced high levels of secreted IL-13, with lesser induction of other pro- and anti-inflammatory cytokines. Given the importance of IL-13 for protection against EAE, these data strongly implicate IL-13 as a dominant regulatory cytokine induced by RTL therapy. Pronounced IL-13 levels coupled with marked reduction in IL-6 levels secreted by PLP-specific T cells from blood after treatment of mice with RTL401 indicate that IL-13 and IL-6 may be useful markers for following effects of RTL therapy in future clinical trials in multiple sclerosis.
Cutting Edge: TLR2-Mediated Proinflammatory Cytokine and Chemokine Production by Microglial Cells in Response to Herpes Simplex Virus
Authors: Aravalli Rajagopal N. , Hu Shuxian , Rowen Timothy N. , Palmquist Joseph M. , Lokensgard James R.
Published: April 2014

Recent studies indicate that TLRs are critical in generating innate immune responses during infection with HSV-1. In this study, we investigated the role of TLR2 signaling in regulating the production of neuroimmune mediators by examining cytokine and chemokine expression using primary microglial cells obtained from TLR2−/− as well as wild-type mice. Data presented here demonstrate that TLR2 signaling is required for the production of proinflammatory cytokines and chemokines: TNF-α, IL-1β, IL-6, IL-12, CCL7, CCL8, CCL9, CXCL1, CXCL2, CXCL4, and CXCL5. CXCL9 and CXCL10 were also induced by HSV, but their production was not dependent upon TLR2 signaling. Because TLR2−/− mice display significantly reduced mortality and diminished neuroinflammation in response to brain infection with HSV, the TLR2-dependent cytokines identified here might function as key players influencing viral neuropathogenesis.
CIN85 Regulates the Ligand-Dependent Endocytosis of the IgE Receptor: A New Molecular Mechanism to Dampen Mast Cell Function
Authors: Molfetta Rosa , Belleudi Francesca , Peruzzi Giovanna , Morrone Stefania , Leone Laura , Dikic Ivan , Piccoli Mario , Frati Luigi , Torrisi Maria Rosaria , Santoni Angela , Paolini Rossella
Published: April 2014

Ligation of the high-affinity receptor for IgE (FcεRI), constitutively expressed on mast cells and basophils, promotes cell activation and immediate release of allergic mediators. Furthermore, FcεRI up-regulation on APC from atopic donors is involved in the pathophysiology of allergic diseases. In consideration of the clinical relevance of the IgE receptor, the down-modulation of FcεRI expression in mast cells may represent a potential target for handling atopic diseases. In an effort to identify new molecular mechanisms involved in attenuating FcεRI expression and signaling, we focused our attention on CIN85, a scaffold molecule that regulates, in concert with the ubiquitin ligase Cbl, the clathrin-mediated endocytosis of several receptor tyrosine kinases. In the present study, we show that endogenous CIN85 is recruited in Cbl-containing complexes after engagement of the FcεRI on a mast cell line and drives ligand-induced receptor internalization. By confocal microscopic analysis, we provide evidence that CIN85 directs a more rapid receptor sorting in early endosomes and delivery to a lysosomal compartment. Furthermore, biochemical studies indicate that CIN85 plays a role in reducing the expression of receptor complex. Finally, we demonstrate that CIN85-overexpressing mast cells are dramatically impaired in their ability to degranulate following Ag stimulation, suggesting that the accelerated internalization of activated receptors by perturbing the propagation of FcεRI signaling may contribute to dampen the functional response. This role of CIN85 could be extended to include other multimeric immune receptors, such as the T and B cell receptors, providing a more general molecular mechanism for attenuating immune responses.
Absence of Caprin-1 Results in Defects in Cellular Proliferation
Authors: Wang Bin , David Muriel D. , Schrader John W.
Published: April 2014

Cytoplasmic activation/proliferation-associated protein-1 (Caprin-1) is a cytoplasmic phosphoprotein that is the prototype of a novel family of highly conserved proteins. Its levels, except in the brain, are tightly correlated with cellular proliferation. We disrupted caprin-1 alleles in the chicken B lymphocyte line DT40 using homologous recombination. We readily obtained clones with one disrupted allele (31% of transfectants), but upon transfection of heterozygous cells we obtained a 10-fold lower frequency of clones with disruption of the remaining allele. Clones of caprin-1-null DT40 cells exhibited marked reductions in their proliferation rate. To obviate the problem that we had selected for caprin-1-null clones with characteristics that partially compensated for the lack of Caprin-1, we generated clones of DT40 cells heterozygous for the caprin-1 gene in which, during disruption of the remaining wild-type allele of the chicken caprin-1 gene, the absence of endogenous Caprin-1 would be complemented by conditional expression of human Caprin-1. Suppression of expression of human Caprin-1 resulted in slowing of the proliferation rate, due to prolongation of the G1 phase of the cell cycle, formally demonstrating that Caprin-1 was essential for normal cellular proliferation.
Diabetes Is Predicted by the β Cell Level of Autoantigen
Authors: Byersdorfer Craig A. , Schweitzer George G. , Unanue Emil R.
Published: April 2014

Two novel transgenic (Tg) strains were created expressing hen egg-white lysozyme (HEL) in a pancreas-specific fashion. RmHP.111 mice had levels of HEL per cell similar to that of the established ILK-3 strain, while RmHP.117 mice had 10-fold lower levels (50,000 molecules per cell). When bred to 3A9 TCR Tg mice, negative selection occurred equally in all three double-Tg combinations, yet only ILK-3 × 3A9 and RmHP.111 × 3A9 mice became diabetic. Additionally, activated 3A9 cells readily transferred diabetes into ILK-3 or RmHP.111 mice, but only marginally into the RmHP.117 strain. In the peripancreatic lymph node, division of naive 3A9 cells was similar between RmHP.111 and RmHP.117 strains, but pancreatic APCs from RmHP.111 × 3A9 mice stimulated HEL-reactive cells to a much greater degree than those from RmHP.117 × 3A9 mice. In this model, diabetes was dependent upon both initial priming in the peripancreatic lymph node and subsequent presentation in the pancreas, with disease incidence predicted by the β cell level of autoantigen.
Signal 3 Tolerant CD8 T Cells Degranulate in Response to Antigen but Lack Granzyme B to Mediate Cytolysis
Authors: Curtsinger Julie M. , Lins Debra C. , Johnson Christopher M. , Mescher Matthew F.
Published: April 2014

Naive CD8 T cells that respond in vivo to Ag and costimulation in the absence of a third signal, such as IL-12, fail to develop cytolytic function and become tolerized. We show in this study that CD8 T cells purified from TCR transgenic mice and stimulated in vitro in the presence or absence of IL-12 form conjugates with specific target cells, increase intracellular Ca2+, and undergo degranulation to comparable extents. Perforin is also expressed at comparable levels in the absence or presence of a third signal, but expression of granzyme B depends upon IL-12. Levels of granzyme B also correlate strongly with the cytolytic activity of cells responding in vivo. In contrast, an increase in CD107a (lysosomal-associated membrane protein 1) expression resulting from degranulation cannot distinguish in vivo generated lytic effector cells from tolerized, noncytolytic cells. Thus, it appears that cells rendered tolerant as a result of stimulation in the absence of a third signal fail to lyse target cells because they are “shooting blanks.”
Lipopolysaccharide-Induced Heme Oxygenase-1 Expression in Human Monocytic Cells Is Mediated via Nrf2 and Protein Kinase C
Authors: Rushworth Stuart A. , Chen Xi-Lin , Mackman Nigel , Ogborne Richard M. , O’Connell Maria A.
Published: April 2014

Monocytes play a key role in mobilization of the immune response during sepsis. In response to LPS, monocytes produce both proinflammatory mediators and regulatory proteins that counteract the inflammation and oxidative stress. In murine macrophages, LPS stimulates expression of heme oxygenase 1 (HO-1), a cytoprotective enzyme that catalyzes the degradation of heme. The HO-1 5′-untranslated region, similarly to other cytoprotective genes, contains antioxidant-response elements (AREs) that can bind the transcription factor NF-E2-related factor 2 (Nrf2). At present, the role of Nrf2 in LPS-induced HO-1 expression in monocytic cells has not been investigated. In this study, LPS induced HO-1 mRNA and protein expression in human monocytes and THP-1 cells. Nrf2 translocated from the cytosol to the nucleus in response to LPS and bound to the ARE site in the human HO-1 promoter. In addition, a dominant negative Nrf2 mutant inhibited LPS-induced HO-1 mRNA expression but not TNF-α mRNA expression in THP-1 cells. Ro-31-8220, a pan-protein kinase C (PKC) inhibitor, and Go6976, a classical PKC inhibitor, blunted LPS-induced HO-1 mRNA expression in monocytes and THP-1 cells. Both PKC inhibitors also blocked LPS-induced Nrf2 binding to the ARE. These results indicate that LPS-induced HO-1 expression in human monocytic cells requires Nrf2 and PKC.
Analyzing Expression of Perforin, Runx3, and Thpok Genes during Positive Selection Reveals Activation of CD8-Differentiation Programs by MHC II-Signaled Thymocytes
Authors: Liu Xiaolong , Taylor Barbara J. , Sun Guangping , Bosselut Rémy
Published: April 2014

Intrathymic positive selection matches CD4-CD8 lineage differentiation to MHC specificity. However, it is unclear whether MHC signals induce lineage choice or simply select thymocytes of the appropriate lineage. To investigate this issue, we assessed thymocytes undergoing positive selection for expression of the CD8 lineage markers perforin and Runx3. Using both population-based and single-cell RT-PCR analyses, we found large subsets of MHC class II (MHC-II)-signaled thymocytes expressing these genes within the CD4+8+ and CD4+8int, but not the CD4+8− populations of signaling competent mice. This indicates that MHC-II signals normally fail to impose CD4 differentiation and further implies that the number of mature CD8 single-positive (SP) thymocytes greatly underestimates CD8 lineage choice. We next examined whether MHC-II-restricted CD4+8− thymocytes remain competent to initiate CD8 lineage gene expression. In mice in which expression of the tyrosine kinase Zap70 and thereby TCR signaling were impaired selectively in SP thymocytes, MHC-II-signaled CD4+8− thymocytes expressed perforin and Runx3 and failed to up-regulate the CD4 marker Thpok. This indicated that impairing TCR signals at the CD4 SP stage switched gene expression patterns from CD4- to CD8-lineage specific. We conclude from these findings that MHC-II-signaled thymocytes remain competent to initiate CD8-specific gene expression even after CD8 down-regulation and that CD4 lineage differentiation is not fixed before the CD4 SP stage.
Id3 Induces Growth Arrest and Caspase-2-Dependent Apoptosis in B Lymphocyte Progenitors
Authors: Kee Barbara L.
Published: April 2014

The E-protein transcription factors E2A, HEB, and E2-2 play an essential role in the differentiation, proliferation, and survival of B lymphocyte progenitors (BLPs). In this study, we show that the E-protein antagonist Id3 induces apoptosis of both primary and transformed BLPs through a caspase-2-dependent mechanism that does not require p53 and is not inhibited by bcl-2. Id3 expressing B lineage cells show reduced expression of known E-protein target genes as well as multiple genes involved in cell proliferation. We hypothesize that Id3 induces activation of caspase-2 as a consequence of severe or “catastrophic” growth arrest. In support of this hypothesis, we show that chemical-induced growth arrest is sufficient to activate caspase-2 and induce apoptosis in BLPs. Our data suggest that E-proteins function in the control of differentiation and proliferation and that diminished E-protein activity results in apoptosis as a consequence of growth arrest.
IL-16 Is Critical for Tropheryma whipplei Replication in Whipple’s Disease
Authors: Desnues Benoît , Raoult Didier , Mege Jean-Louis
Published: April 2014

Whipple’s disease (WD) is a rare systemic disease caused by Tropheryma whipplei. We showed that T. whipplei was eliminated by human monocytes but replicated in monocyte-derived macrophages (Mφ) by inducing an original activation program. Two different host molecules were found to be key elements for this specific pattern. Thioredoxin, through its overexpression in infected monocytes, was involved in bacterial killing because adding thioredoxin to infected Mφ inhibited bacterial replication. IL-16, which was up-regulated in Mφ, enabled T. whipplei to replicate in monocytes and increased bacterial replication in Mφ. In addition, anti-IL-16 Abs abolished T. whipplei replication in Mφ. IL-16 down-modulated the expression of thioredoxin and up-regulated that of IL-16 and proapoptotic genes. In patients with WD, T. whipplei replication was higher than in healthy subjects and was related to high levels of circulating IL-16. Both events were corrected in patients who successfully responded to antibiotics treatment. This role of IL-16 was not reported previously and gives an insight into the understanding of WD pathophysiology.
Increased Susceptibility of Mice Lacking T-bet to Infection withMycobacterium tuberculosisCorrelates with Increased IL-10 and Decreased IFN-γ Production
Authors: Sullivan Brandon M. , Jobe Ousman , Lazarevic Vanja , Vasquez Kristine , Bronson Roderick , Glimcher Laurie H. , Kramnik Igor
Published: April 2014

A sustained CD4+ Th1-dominated type 1 immune response is required to successfully control Mycobacterium tuberculosis infection. Considerable work has demonstrated that the transcription factor, T-bet, is required for IFN-γ expression and fundamental to the generation of type 1 immunity in multiple cell types. Mice lacking T-bet are susceptible to virulent M. tuberculosis infection. Susceptibility of T-bet-deficient mice is associated with increased systemic bacterial burden, diminished IFN-γ production, and the striking accumulation of eosinophilic macrophages and multinucleated giant cells in the lung. Interestingly, T-bet−/− mice did not develop a fully polarized Th2 response toward M. tuberculosis, but exhibited selective elevation of IL-10 production. These results indicate that T-bet plays a central role in controlling M. tuberculosis disease progression, in part through the regulation of both IFN-γ and IL-10.
A Novel Role of CD30/CD30 Ligand Signaling in the Generation of Long-Lived Memory CD8+ T Cells
Authors: Nishimura Hitoshi , Yajima Toshiki , Muta Hiromi , Podack Eckhard R. , Tani Kenzaburo , Yoshikai Yasunobu
Published: April 2014

Memory CD8+ T cells can be divided into two subsets, central memory (TCM) and effector memory (TEM) CD8+ T cells. We found that CD30, a member of the TNFR-associated factor (TRAF)-linked TNFR superfamily, signaling is involved in differentiation of long-lived CD8+ TCM cells following Listeria monocytogenes infection. Although CD8+ TEM cells transiently accumulated in the nonlymphoid tissues of CD30 ligand (CD153−/−) mice after infection, long-lived memory CD8+ TCM cells were poorly generated in these mice. CCR7 mRNA expression was down-regulated in CD8+ T cells of the spleen of CD153−/− mice in vivo and the expression was up-regulated in CD8+ TEM cells by anti-CD30 mAb cross-linking in vitro. These results suggest that CD30/CD30 ligand signaling plays an important role in the generation of long-lived memory CD8+ T cells at least partly by triggering homing receptors for TCM cells.
A Dipalmitoylated Lipoprotein fromMycoplasma pneumoniaeActivates NF-κB through TLR1, TLR2, and TLR6
Authors: Shimizu Takashi , Kida Yutaka , Kuwano Koichi
Published: April 2014

The pathogenesis of Mycoplasma pneumoniae infection is considered to be in part attributed to excessive immune responses. Recently, lipoproteins from mycoplasmas have been reported to induce NF-κB activation. In this study, we examined the ability of lipoproteins from M. pneumoniae to activate NF-κB, and the active component responsible for the NF-κB activation was identified. Lipid-associated membrane proteins from M. pneumoniae were found to induce NF-κB through TLR 2 in a human monocytic cell line, THP-1. The active component of the Lipid-associated membrane proteins was a subunit b of F0F1-type ATPase (F0F1-ATPase). The F0F1-ATPase is assumed to contain two palmitic acids. The activation of NF-κB by the F0F1-ATPase was inhibited by a dominant negative construct of TLR1 and TLR6. These results indicate that the activation of NF-κB by F0F1-ATPase is dependent on TLR1, TLR2, and TLR6. The activity of the F0F1-ATPase was decreased with pretreatment of lipoprotein lipase but not protease, indicating that the lipid moiety of the F0F1-ATPase was important for the NF-κB activation. Thus, a dipalmitoylated lipoprotein from M. pneumoniae was found to activate NF-κB through TLR1, TLR2, and TLR6.
Molecular Basis of Reduced Potency of Underacylated Endotoxins
Authors: Teghanemt Athmane , Zhang DeSheng , Levis Erika N. , Weiss Jerrold P. , Gioannini Theresa L.
Published: April 2014

Potent TLR4-dependent cell activation by Gram-negative bacterial endotoxins depends on sequential endotoxin-protein and protein-protein interactions with LPS-binding protein, CD14, myeloid differentiation protein 2 (MD-2), and TLR4. Previous studies have suggested that reduced agonist potency of underacylated endotoxins (i.e., tetra- or penta- vs hexa-acylated) is determined by post-CD14 interactions. To better define the molecular basis of the differences in agonist potency of endotoxins differing in fatty acid acylation, we compared endotoxins (lipooligosaccharides (LOS)) from hexa-acylated wild-type (wt), penta-acylated mutant msbB meningococcal strains as well as tetra-acylated LOS generated by treatment of wt LOS with the deacylating enzyme, acyloxyacylhydrolase. To facilitate assay of endotoxin:protein and endotoxin:cell interactions, the endotoxins were purified after metabolic labeling with [3H]- or [14C]acetate. All LOS species tested formed monomeric complexes with MD-2 in an LPS-binding protein- and CD14-dependent manner with similar efficiency. However, msbB LOS:MD-2 and acyloxyacylhydrolase-treated LOS:MD-2 were at least 10-fold less potent in inducing TLR4-dependent cell activation than wt LOS:MD-2 and partially antagonized the action of wt LOS:MD-2. These findings suggest that underacylated endotoxins produce decreased TLR4-dependent cell activation by altering the interaction of the endotoxin:MD-2 complex with TLR4 in a way that reduces receptor activation. Differences in potency among these endotoxin species is determined not by different aggregate properties, but by different properties of monomeric endotoxin:MD-2 complexes.
Inflammatory Responses to Pneumovirus Infection in IFN-αβR Gene-Deleted Mice
Authors: Garvey Tara L. , Dyer Kimberly D. , Ellis John A. , Bonville Cynthia A. , Foster Barbara , Prussin Calman , Easton Andrew J. , Domachowske Joseph B. , Rosenberg Helene F.
Published: April 2014

Pneumonia virus of mice (PVM; family Paramyxoviridae) is a natural pathogen of rodents that reproduces important clinical features of severe respiratory syncytial virus infection in humans. As anticipated, PVM infection induces transcription of IFN antiviral response genes preferentially in wild-type over IFN-αβR gene-deleted (IFN-αβR−/−) mice. However, we demonstrate that PVM infection results in enhanced expression of eotaxin-2 (CCL24), thymus and activation-regulated chemokine (CCL17), and the proinflammatory RNase mouse eosinophil-associated RNase (mEar) 11, and decreased expression of monocyte chemotactic protein-5, IFN-γ-inducible protein-10, and TLR-3 in lung tissue of IFN-αβR−/− mice when compared with wild type. No differential expression of chemokines MIP-1α or MIP-2 or Th2 cytokines IL-4 or IL-5 was observed. Differential expression of proinflammatory mediators was associated with distinct patterns of lung pathology. The widespread granulocytic infiltration and intra-alveolar edema observed in PVM-infected, wild-type mice are replaced with patchy, dense inflammatory foci localized to the periphery of the larger blood vessels. Bronchoalveolar lavage fluid from IFN-αβR−/− mice yielded 7- to 8-fold fewer leukocytes overall, with increased percentages of eosinophils, monocytes, and CD4+ T cells, and decreased percentage of CD8+ T cells. Differential pathology is associated with prolonged survival of the IFN-αβR−/− mice (50% survival at 10.8 ± 0.6 days vs the wild type at 9.0 ± 0.3 days; p < 0.02) despite increased virus titers. Overall, our findings serve to identify novel transcripts that are differentially expressed in the presence or absence of IFN-αβR-mediated signaling, further elucidating interactions between the IFN and antiviral inflammatory responses in vivo.
HIV-Specific CD8+ Lymphocytes in Semen Are Not Associated with Reduced HIV Shedding
Authors: Sheth Prameet M. , Danesh Ali , Shahabi Kamnoosh , Rebbapragada Anuradha , Kovacs Colin , Dimayuga Rowena , Halpenny Roberta , MacDonald Kelly S. , Mazzulli Tony , Kelvin David , Ostrowski Mario , Kaul Rupert
Published: April 2014

Sexual contact with HIV-infected semen is a major driving force behind the global HIV pandemic. Little is known regarding the immune correlates of virus shedding in this compartment, although HIV-1-specific CD8+ T cells are present in semen. We collected blood and semen from 27 chronically HIV-infected, therapy-naive men without common sexually transmitted infections or urethral inflammation and measured HIV-1 RNA viral load and cytokine/chemokine levels in both compartments. HIV-1 RNA levels were 10-fold higher in blood than semen, but discordantly high semen shedding was associated with higher semen levels of the proinflammatory cytokines IL-6, IL-8, IL-12, and IFN-γ. Virus-specific CD8+ T cell epitopes were mapped in blood by IFN-γ ELISPOT, using an overlapping HIV-1 clade B peptide matrix, and blood and semen CD8+ T cell responses were then assayed ex vivo using intracellular IFN-γ staining. HIV-specific CD8+ responses were detected in 70% of semen samples, and their frequency was similar to or higher than blood. There was no correlation between the presence of virus-specific CD8+ T cells in semen and levels of HIV-1 RNA shedding. Among participants with detectable CD8+ IFN-γ semen responses, their relative frequency was not associated with reduced HIV-1 RNA shedding, and their absolute number was correlated with higher levels of HIV-1 RNA semen shedding (r = 0.6; p = 0.03) and of several proinflammatory cytokines. Neither the presence nor the frequency of semen HIV-specific CD8+ T cell IFN-γ responses in semen correlated with reduced levels of HIV RNA in semen.
Role of CXC Chemokine Ligand 13, CC Chemokine Ligand (CCL) 19, and CCL21 in the Organization and Function of Nasal-Associated Lymphoid Tissue
Authors: Rangel-Moreno Javier , Moyron-Quiroz Juan , Kusser Kim , Hartson Louise , Nakano Hideki , Randall Troy D.
Published: April 2014

Nasal-associated lymphoid tissue (NALT) orchestrates immune responses to Ags in the upper respiratory tract. Unlike other lymphoid organs, NALT develops independently of lymphotoxin-α (LTα). However, the structure and function of NALT are impaired in Ltα−/− mice, suggesting a link between LTα and chemokine expression. In this study we show that the expression of CXCL13, CCL19, CCL21, and CCL20 is impaired in the NALT of Ltα−/− mice. We also show that the NALT of Cxcl13−/− and plt/plt mice exhibits some, but not all, of the structural and functional defects observed in the NALT of Ltα−/− mice. Like the NALT of Ltα−/− mice, the NALT in Cxcl13−/− mice lacks follicular dendritic cells, BP3+ stromal cells, and ERTR7+ lymphoreticular cells. However, unlike the NALT of Ltα−/− mice, the NALT of Cxcl13−/− mice has peripheral node addressin+ high endothelial venules (HEVs). In contrast, the NALT of plt/plt mice is nearly normal, with follicular dendritic cells, BP3+ stromal cells, ERTR7+ lymphoreticular cells, and peripheral node addressin+ HEVs. Functionally, germinal center formation and switching to IgA are defective in the NALT of Ltα−/− and Cxcl13−/− mice. In contrast, CD8 T cell responses to influenza are impaired in Ltα−/− mice and plt/plt mice. Finally, the B and T cell defects in the NALT of Ltα−/− mice lead to delayed clearance of influenza from the nasal mucosa. Thus, the B and T cell defects in the NALT of Ltα−/− mice can be attributed to the impaired expression of CXCL13 and CCL19/CCL21, respectively, whereas impaired HEV development is directly due to the loss of LTα.
Killer Cell Ig-Like Receptor-Dependent Signaling by Ig-Like Transcript 2 (ILT2/CD85j/LILRB1/LIR-1)
Authors: Kirwan Sheryl E. , Burshtyn Deborah N.
Published: April 2014

Inhibitory killer cell Ig-like receptors (KIR) signal by recruitment of the tyrosine phosphatase Src homology region 2 domain-containing phosphatase-1 to ITIM. In the present study, we show that, surprisingly, KIR lacking ITIM are able to signal and inhibit in the human NK cell line NK92, but not in mouse NK cells. Signaling by mutant KIR is weaker than the wild-type receptor, does not require the transmembrane or cytoplasmic tail of KIR, and is blocked by overexpression of a catalytically inactive Src homology region 2 domain-containing phosphatase-1 molecule. We also demonstrate that mutant KIR signaling is blocked by Abs, which disrupt the interaction between KIR and human leukocyte Ag-C or Abs, which block the interaction between Ig-like transcript 2 (ILT2) and the α3 domain of HLA class I molecules. Thus, although ILT2 expressed in NK92 is insufficient to signal in response to human leukocyte Ag-C alone, ILT2 can signal in a KIR-dependent manner revealing functional cooperation between receptors encoded by two distinct inhibitory receptor families.
Heterogeneity of Flt3-Expressing Multipotent Progenitors in Mouse Bone Marrow
Authors: Lai Anne Y. , Lin Simon M. , Kondo Motonari
Published: April 2014

Mechanisms of lymphoid and myeloid lineage choice by hemopoietic stem cells remain unclear. In this study we show that the multipotent progenitor (MPP) population, which is immediately downstream of hemopoietic stem cells, is heterogeneous and can be subdivided in terms of VCAM-1 expression. VCAM-1+ MPPs were fully capable of differentiating into both lymphoid and myeloid lineages. In contrast, VCAM-1− MPPs gave rise to lymphocytes predominately in vivo. T and B cell development from VCAM-1− MPPs was 1 wk faster than that from VCAM-1+ MPPs. Furthermore, VCAM-1+ MPPs gave rise to common myeloid progenitors and VCAM-1− MPPs in vivo, indicating that VCAM-1− MPPs are progenies of VCAM-1+ MPPs. VCAM-1− MPPs, in turn, developed into lymphoid lineage-restricted common lymphoid progenitors. These results establish a hierarchy of developmental relationship between MPP subsets and lymphoid and myeloid progenitors. In addition, VCAM-1+ MPPs may represent the branching point between the lymphoid and myeloid lineages.
Rapid Production of TNF-α following TCR Engagement of Naive CD8 T Cells
Authors: Brehm Michael A. , Daniels Keith A. , Welsh Raymond M.
Published: April 2014

The acquisition of effector functions by naive CD8 T cells following TCR engagement is thought to occur sequentially with full functionality being gained only after the initiation of division. We show that naive CD8 T cells are capable of immediate effector function following TCR engagement, which stimulates the rapid production of TNF-α. Stimulation of splenocytes from naive mice of differing genetic backgrounds with anti-CD3ε mAb resulted in significant production of TNF-α by naive CD8 T cells within 5 h. Moreover, naive lymphocytic choriomeningitis virus-specific TCR-transgenic CD8 T cells stimulated with either their cognate peptide ligand or virus-infected cells produced TNF-α as early as 2 h poststimulation, with production peaking by 4 h. Naive CD8 T cells produced both membrane-bound and soluble TNF-α. Interfering with TNF-α activity during the initial encounter between naive CD8 T cells and Ag loaded dendritic cells altered the maturation profile of the APC and diminished the overall viability of the APC population. These findings suggest that production of TNF-α by naive CD8 T cells immediately after TCR engagement may have an unappreciated impact within the local environment where Ag presentation is occurring and potentially influence the development of immune responses.
Both Regulatory T Cells and Antitumor Effector T Cells Are Primed in the Same Draining Lymph Nodes during Tumor Progression
Authors: Hiura Toru , Kagamu Hiroshi , Miura Satoru , Ishida Akira , Tanaka Hiroshi , Tanaka Junta , Gejyo Fumitake , Yoshizawa Hirohisa
Published: April 2014

The peripheral tolerance mechanism prevents effective antitumor immunity, even though tumor cells possess recognizable tumor-associated Ags. Recently, it has been elucidated that regulatory T cells (Treg) play a critical role in maintaining not only self-tolerance, but also tolerance of tumor cells. However, because the Treg that maintain self-tolerance arise naturally in the thymus and are thought to be anergic in peripheral, it is still unclear where and when Treg for tumor cells are generated. In this study we analyze tumor-draining lymph nodes (LNs) and demonstrate that both antitumor effector T cells and Treg capable of abrogating the antitumor reactivity of the effector T cells are primed in the same LNs during tumor progression. The regulatory activity generated in tumor-draining LNs exclusively belonged to the CD4+ T cell subpopulation that expresses both CD25 and a high level of CD62L. Forkhead/winged helix transcription factor gene expression was detected only in the CD62LhighCD4+CD25+ T cells. CD62LhighCD4+CD25+ Treg and CD62LlowCD4+CD25+ T cells, which possess effector T cell functions, had comparable expression of LFA-1, VLA-4, CTLA-4, lymphocyte activation gene-3, and glucocorticoid-induced TNFR. Thus, only CD62L expression could distinguish regulatory CD4+CD25+ cells from effector CD4+CD25+ cells in draining LNs as a surface marker. The Treg generated in tumor-draining LNs possess the same functional properties as the Treg that arise naturally in the thymus but recognize tumor-associated Ag. CD62LhighCD4+CD25+ Treg contained a subpopulation that expressed CD86. Blocking experiments revealed that ligation of CTLA-4 on effector T cells by CD86 on Treg plays a pivotal role in regulating CD4+ effector T cells.
Treatment of Mice with the Suppressor of Cytokine Signaling-1 Mimetic Peptide, Tyrosine Kinase Inhibitor Peptide, Prevents Development of the Acute Form of Experimental Allergic Encephalomyelitis and Induces Stable Remission in the Chronic Relapsing/Remitting Form
Authors: Mujtaba Mustafa G. , Flowers Lawrence O. , Patel Chintak B. , Patel Ravi A. , Haider Mohammad I. , Johnson Howard M.
Published: April 2014

We have previously characterized a novel tyrosine kinase inhibitor peptide (Tkip) that is a mimetic of suppressor of cytokine signaling 1 (SOCS-1) and inhibits JAK2 phosphorylation of the transcription factor STAT1α. We show in this study that Tkip protects mice against experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis. Mice are immunized with myelin basic protein (MBP) for induction of disease. Tkip (63 μg) administered every other day suppressed the development of acute EAE in 75% of New Zealand White (NZW) mice. Furthermore, Tkip completely protected SJL/J mice, which where induced to get the relapsing/remitting form of EAE, against relapses compared with control groups in which >70% of the mice relapsed after primary incidence of disease. Protection of mice by Tkip was similar to that seen with the type I IFN, IFN-τ. Protection of mice correlated with lower MBP Ab titers in Tkip-treated groups as well as suppression of MBP-induced proliferation of splenocytes taken from EAE-afflicted mice. Cessation of Tkip and IFN-τ administration resulted in SJL/J mice relapsing back into disease. Prolonged treatment of mice with Tkip produced no evidence of cellular toxicity or weight loss. Consistent with its JAK2 inhibitory function, Tkip also inhibited the activity of the inflammatory cytokine TNF-α, which uses the STAT1α transcription factor. The data presented in this study show that Tkip, like the type I IFN, IFN-τ, inhibits both the autoreactive cellular and humoral responses in EAE and ameliorates both the acute and chronic relapsing/remitting forms of EAE.
BMP Signaling Is Required for Normal Thymus Development
Authors: Bleul Conrad C. , Boehm Thomas
Published: April 2014

The microenvironment of the thymus fosters the generation of a diverse and self-tolerant T cell repertoire from a pool of essentially random specificities. Epithelial as well as mesenchymal cells contribute to the thymic stroma, but little is known about the factors that allow for communication between the two cells types that shape the thymic microenvironment. In this study, we investigated the role of bone morphogenetic protein (BMP) signaling in thymus development. Transgenic expression of the BMP antagonist Noggin in thymic epithelial cells under the control of a Foxn1 promoter in the mouse leads to dysplastic thymic lobes of drastically reduced size that are ectopically located in the neck at the level of the hyoid bone. Interestingly, the small number of thymocytes in these thymic lobes develops with normal kinetics and shows a wild-type phenotype. Organ initiation of the embryonic thymic anlage in these Noggin transgenic mice occurs as in wild-type mice, but the tight temporal and spatial regulation of BMP4 expression is abrogated in subsequent differentiation stages. We show that transgenic Noggin blocks BMP signaling in epithelial as well as mesenchymal cells of the thymic anlage. Our data demonstrate that BMP signaling is crucial for thymus development and that it is the thymic stroma rather than developing thymocytes that depends on BMP signals.
Discovery and Pharmacological Characterization of a Novel Rodent-Active CCR2 Antagonist, INCB3344
Authors: Brodmerkel Carrie M. , Huber Reid , Covington Maryanne , Diamond Sharon , Hall Leslie , Collins Robert , Leffet Lynn , Gallagher Karen , Feldman Patricia , Collier Paul , Stow Mark , Gu Xiaomei , Baribaud Frederic , Shin Niu , Thomas Beth , Burn Tim , Hollis Greg , Yeleswaram Swamy , Solomon Kim , Friedman Steve , Wang Anlai , Xue Chu Biao , Newton Robert C. , Scherle Peggy , Vaddi Kris
Published: April 2014

This report describes the characterization of INCB3344, a novel, potent and selective small molecule antagonist of the mouse CCR2 receptor. The lack of rodent cross-reactivity inherent in the small molecule CCR2 antagonists discovered to date has precluded pharmacological studies of antagonists of this receptor and its therapeutic relevance. In vitro, INCB3344 inhibits the binding of CCL2 to mouse monocytes with nanomolar potency (IC50 = 10 nM) and displays dose-dependent inhibition of CCL2-mediated functional responses such as ERK phosphorylation and chemotaxis with similar potency. Against a panel of G protein-coupled receptors that includes other CC chemokine receptors, INCB3344 is at least 100-fold selective for CCR2. INCB3344 possesses good oral bioavailability and systemic exposure in rodents that allows in vivo pharmacological studies. INCB3344 treatment results in a dose-dependent inhibition of macrophage influx in a mouse model of delayed-type hypersensitivity. The histopathological analysis of tissues from the delayed-type hypersensitivity model demonstrates that inhibition of CCR2 leads to a substantial reduction in tissue inflammation, suggesting that macrophages play an orchestrating role in immune-based inflammatory reactions. These results led to the investigation of INCB3344 in inflammatory disease models. We demonstrate that therapeutic dosing of INCB3344 significantly reduces disease in mice subjected to experimental autoimmune encephalomyelitis, a model of multiple sclerosis, as well as a rat model of inflammatory arthritis. In summary, we present the first report on the pharmacological characterization of a selective, potent and rodent-active small molecule CCR2 antagonist. These data support targeting this receptor for the treatment of chronic inflammatory diseases.
A Genetic Basis for IFN-γ Production and T-bet Expression in Humans
Authors: Höhler Thomas , Reuss Esther , Adams Petra , Bartsch Brigitte , Weigmann Benno , Wörns Markus , Galle Peter R. , Victor Anja , Neurath Markus F.
Published: April 2014

Th1 and Th2 cytokines secreted by polarized effector T cells play a pivotal role in the development of autoimmune and allergic diseases. However, the genetic basis of cytokine production by T lymphocytes in humans is poorly understood. In this study, we investigated the genetic contribution to cytokine production and regulation of T cell-specific transcription factors in a prospective twin study. We found a substantial genetic contribution to the production of Th1 cytokines such as IFN-γ and TNF-α with heritabilities of 0.85 (95% confidence intervals, 0.74–0.95) and 0.72 (0.50–0.93), respectively, whereas no genetic influence on production of the Th2 signature cytokine IL-4 was observed. Furthermore, the intrapair variability in IFN-γ production by isolated T cells was lower in monozygotic than in dizygotic twins. In contrast to GATA-3, NFAT, and NF-κB, intrapair variability of T-bet, the master transcription factor of Th1 cells, was very low among monozygotic and high among dizygotic twins, indicative of a strong genetic influence on T-bet (heritability 0.93, 95% confidence interval, 0.84–1.0). Our data provide novel insights into the genetic regulation of human Th cell polarization. These data suggest that signature cytokines and cytokine signaling events of Th1 rather than Th2 cells are genetically determined and implicate that Th2-associated diseases in humans might be due to genetic variations in Th1 cytokine regulation via T-bet. This concept is highlighted by the recent finding that inactivation of the T-bet gene in mice results in development of clinical hallmark features of asthma.
Cutting Edge: MyD88 Controls Phagocyte NADPH Oxidase Function and Killing of Gram-Negative Bacteria
Authors: Laroux F. Stephen , Romero Xavier , Wetzler Lee , Engel Pablo , Terhorst Cox
Published: April 2014

MyD88 is an adaptor protein for the TLR family of proteins that has been implicated as a critical mediator of innate immune responses to pathogen detection. In this study, we report that MyD88 plays a crucial role in killing Gram-negative bacteria by primary macrophages via influencing NADPH oxidase function. Peritoneal macrophages from MyD88−/− mice exhibited a marked inability to kill Escherichia coli (F18) or an attenuated strain of Salmonella typhimurium (sseB) in vitro. This defect in killing was due to diminished NADPH oxidase-mediated production of superoxide anion in response to bacteria by MyD88−/− phagocytes as a consequence of defective NADPH oxidase assembly. Defective oxidase assembly in MyD88-deficient macrophages resulted from impaired p38 MAPK activation and subsequent phosphorylation of p47phox. Together these data demonstrate a pivotal role for MyD88 in killing Gram-negative bacteria via modulation of NADPH oxidase activity in phagocytic cells.
Cutting Edge: CpG Oligonucleotides Induce Splenic CD19+ Dendritic Cells to Acquire Potent Indoleamine 2,3-Dioxygenase-Dependent T Cell Regulatory Functions via IFN Type 1 Signaling
Authors: Mellor Andrew L. , Baban Babak , Chandler Phillip R. , Manlapat Anna , Kahler David J. , Munn David H.
Published: April 2014

CpG oligodeoxynucleotides (CpG-ODNs) stimulate innate and adaptive immunity by binding to TLR9 molecules. Paradoxically, expression of the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) is induced following i.v. CpG-ODN administration to mice. CpG-ODNs induced selective IDO expression by a minor population of splenic CD19+ dendritic cells (DCs) that did not express the plasmacytoid DC marker 120G8. Following CpG-ODN treatment, CD19+ DCs acquired potent IDO-dependent T cell suppressive functions. Signaling through IFN type I receptors was essential for IDO up-regulation, and CpG-ODNs induced selective activation of STAT-1 in CD19+ DCs. Thus, CpG-ODNs delivered systemically at relatively high doses elicited potent T cell regulatory responses by acting on a discrete, minor population of splenic DCs. The ability of CpG-ODNs to induce both stimulatory and regulatory responses offers novel opportunities for using them as immunomodulatory reagents but may complicate therapeutic use of CpG-ODNs to stimulate antitumor immunity in cancer patients.