bluuebunny/crossref_metadata_2025 huggingface dataset:
Neonatal Immunity Develops in a Transgenic TCR Transfer Model and Reveals a Requirement for Elevated Cell Input to Achieve Organ-Specific Responses
Authors: Li Lequn , Legge Kevin L. , Min Booki , Bell J. Jeremiah , Gregg Randal , Caprio Jacque , Zaghouani Habib
Published: April 2014

In recent years, it has become clear that neonatal exposure to Ag induces rather than ablates T cell immunity. Moreover, rechallenge with the Ag at adult age can trigger secondary responses that are distinct in the lymph node vs the spleen. The question addressed in this report is whether organ-specific secondary responses occur as a result of the diversity of the T cell repertoire or could they arise with homogeneous TCR-transgenic T cells. To test this premise, we used the OVA-specific DO11.10 TCR-transgenic T cells and established a neonatal T cell transfer system suitable for these investigations. In this system, neonatal T cells transferred from 1-day-old DO11.10/SCID mice into newborn (1-day-old) BALB/c mice migrate to the host’s spleen and maintain stable frequency. The newborn BALB/c hosts were then given Ig-OVA, an Ig molecule carrying the OVA peptide, and challenged with the OVA peptide in CFA at the age of 7 wk; then their secondary responses were analyzed. The findings show that the lymph node T cells were deviated and produced IL-4 instead of IFN-γ and the splenic T cells, although unable to proliferate or produce IFN-γ, secreted a significant level of IL-2. Supply of exogenous IL-12 during Ag stimulation restores both proliferation and IFN-γ production by the splenic T cells. This restorable form of splenic unresponsiveness referred to as IFN-γ-dependent anergy required a transfer of a high number of neonatal DO11.10/SCID T cells to develop. Thus, the frequency of neonatal T cell precursors rather than repertoire diversity exerts control on the development of organ-specific neonatal immunity.
Inhibition of Activated/Memory (CD45RO+) T Cells by Oxidative Stress Associated with Block of NF-κB Activation
Authors: Malmberg Karl-Johan , Arulampalam Velmurugesan , Ichihara Fumiko , Petersson Max , Seki Kazutake , Andersson Tove , Lenkei Rodica , Masucci Giuseppe , Pettersson Sven , Kiessling Rolf
Published: April 2014

Impaired immune responses in cancer patients have been associated with oxidative stress. Increased levels of reactive oxygen species released from activated, tumor-infiltrating macrophages or granulocytes may therefore constitute a hurdle for effective immunotherapy against cancer. In this study, we investigated functional consequences and molecular events in T cells exposed to low levels of oxidative stress. We observed that cytokine production of human PBMC, upon stimulation with an HLA-A*0201-restricted influenza peptide and nonspecific receptor cross-linking, was reduced after exposure to micromolar levels of H2O2. Functional impairment as measured by IFN-γ release occurred earlier and at lower doses of exogenously added H2O2 than required to induce apoptosis. This suggests that there is a dose window of oxidative stress leading to T cell unresponsiveness in the absence of apoptosis. The reduction of Th1 cytokines, induced by H2O2, was predominantly observed in memory/effector (CD45RO+) T cells and correlated with a block in NF-κB activation. IL-10 production was more profoundly influenced by low doses of H2O2 than IFN-γ, TNF-α, and IL-2. The influence of H2O2 on production of IL-10 was not significantly different between memory/activated and naive T cells. These observations suggest that Th1 and Th2 cytokines are differently regulated under conditions of oxidative stress. Taken together, these findings may explain why Ag-experienced, CD45RO+, T cells found in the tumor milieu are functionally suppressed.
Genomic DNA Released by Dying Cells Induces the Maturation of APCs
Authors: Ishii Ken J. , Suzuki Koichi , Coban Cevayir , Takeshita Fumihiko , Itoh Yasushi , Matoba Hana , Kohn Leonard D. , Klinman Dennis M.
Published: April 2014

Mature APCs play a key role in the induction of Ag-specific immunity. This work examines whether genomic DNA released by dying cells provides a stimulus for APC maturation. Double-stranded but not single-stranded genomic DNA triggered APC to up-regulate expression of MHC class I/II and various costimulatory molecules. Functionally, dsDNA enhanced APC function in vitro and improved primary cellular and humoral immune responses in vivo. These effects were dependent on the length and concentration of the dsDNA but were independent of nucleotide sequence. The maturation of APC induced by dsDNA may promote host survival by improving immune surveillance at sites of tissue injury/infection.
Regulation of IL-12 p40 Promoter Activity in Primary Human Monocytes: Roles of NF-κB, CCAAT/Enhancer-Binding Protein β, and PU.1 and Identification of a Novel Repressor Element (GA-12) That Responds to IL-4 and Prostaglandin E2
Authors: Becker Christoph , Wirtz Stefan , Ma Xiaojing , Blessing Manfred , Galle Peter R. , Neurath Markus F.
Published: April 2014

Appropriate regulation of IL-12 expression is critical for cell-mediated immune responses. In the present study, we have analyzed the regulation of IL-12 p40 promoter activity in primary human monocytes in vivo. Accordingly, we analyzed the p40 promoter by in vivo footprinting in resting and activated primary human blood CD14+ monocytes. Interestingly, footprints at binding sites for trans-activating proteins such as C/EBP, NF-κB, and ETS were only found upon stimulation with LPS and IFN-γ. In contrast, a footprint over a purine-rich sequence at −155, termed GA-12 (GATA sequence in the IL-12 promoter), was observed in resting, but not activated, cells. Further characterization of this site revealed specific complex formation at a protected GATA core motif in unstimulated primary monocytes and RAW264.7 macrophages. Mutagenesis within the GA-12 sequence caused a significant up-regulation of inducible IL-12 p40 promoter activity in both transient and stable transfection systems, suggesting a repressor function of this site. Furthermore, binding activity of the GA-12 binding protein GAP-12 was increased by treatment with two potent inhibitors of IL-12 expression, IL-4 and PGE2. Finally, we observed that IL-4-mediated repression of IL-12 p40 promoter activity is critically dependent on an intact GA-12 sequence. In summary, our data underline the complex regulation of the human IL-12 p40 promoter and identify GA-12 as a potent, novel repressor element that mediates IL-4-dependent suppression of inducible promoter activity in monocytes. Regulation of GAP-12 binding may thus modulate IL-12 p40 gene expression.
The Recognition of HLA-B27 by Human CD4+ T Lymphocytes
Authors: Boyle Louise H. , Goodall Jane C. , Opat Stephen S. , Gaston J. S. Hill
Published: April 2014

HLA-B27 transgenic animal models suggest a role for CD4+ T lymphocytes in the pathogenesis of the spondyloarthropathies, and murine studies have raised the possibility that unusual forms of B27 may be involved in disease. We demonstrate that CD4+ T cells capable of recognizing B27 can be isolated from humans by coculture with the MHC class II-negative cell line T2 transfected with B27. These CD4+ T cells recognize a panel of B27-transfected cell lines that are defective in Ag-processing pathways, but not the nontransfected parental cell lines, in a CD4-dependent fashion. Inhibition of responses by the MHC class I-specific mAb w6/32 and the B27 binding mAb ME1 implicates the recognition of a form of B27 recognized by both of these Abs. We suggest that B27-reactive CD4+ T cells may be pathogenic in spondyloarthropathies, particularly if factors such as infection influence expression of abnormal forms of B27.
Connection Between B Lymphocyte and Osteoclast Differentiation Pathways
Authors: Manabe Noriyo , Kawaguchi Hiroshi , Chikuda Hirotaka , Miyaura Chisato , Inada Masaki , Nagai Ryozo , Nabeshima Yo-ichi , Nakamura Kozo , Sinclair Angus M. , Scheuermann Richard H. , Kuro-o Makoto
Published: April 2014

Osteoclasts differentiate from the hemopoietic monocyte/macrophage cell lineage in bone marrow through cell-cell interactions between osteoclast progenitors and stromal/osteoblastic cells. Here we show another osteoclast differentiation pathway closely connected with B lymphocyte differentiation. Recently the TNF family molecule osteoclast differentiation factor/receptor activator of NF-κB ligand (ODF/RANKL) was identified as a key membrane-associated factor regulating osteoclast differentiation. We demonstrate that B-lymphoid lineage cells are a major source of endogenous ODF/RANKL in bone marrow and support osteoclast differentiation in vitro. In addition, B-lymphoid lineage cells in earlier developmental stages may hold a potential to differentiate into osteoclasts when stimulated with M-CSF and soluble ODF/RANKL in vitro. B-lymphoid lineage cells may participate in osteoclastogenesis in two ways: they 1) express ODF/RANKL to support osteoclast differentiation, and 2) serve themselves as osteoclast progenitors. Consistent with these observations in vitro, a decrease in osteoclasts is associated with a decrease in B-lymphoid cells in klotho mutant mice (KL−/−), a mouse model for human aging that exhibits reduced turnover during bone metabolism, rather than a decrease in the differentiation potential of osteoclast progenitors. Taken together, B-lymphoid lineage cells may affect the pathophysiology of bone disorders through regulating osteoclastogenesis.
Efficient Presentation of Both Cytosolic and Endogenous Transmembrane Protein Antigens on MHC Class II Is Dependent on Cytoplasmic Proteolysis
Authors: Mukherjee Paushali , Dani Aadish , Bhatia Sumeena , Singh Nagendra , Rudensky Alexander Y. , George Anna , Bal Vineeta , Mayor Satyajit , Rath Satyajit
Published: April 2014

Peptides from extracellular proteins presented on MHC class II are mostly generated and loaded in endolysosomal compartments, but the major pathways responsible for loading peptides from APC-endogenous sources on MHC class II are as yet unclear. In this study, we show that MHC class II molecules present peptides from proteins such as OVA or conalbumin introduced into the cytoplasm by hyperosmotic pinosome lysis, with efficiencies comparable to their presentation via extracellular fluid-phase endocytosis. This cytosolic presentation pathway is sensitive to proteasomal inhibitors, whereas the presentation of exogenous Ags taken up by endocytosis is not. Inhibitors of nonproteasomal cytosolic proteases can also inhibit MHC class II-restricted presentation of cytosolically delivered protein, without inhibiting MHC class I-restricted presentation from the same protein. Cytosolic processing of a soluble fusion protein containing the peptide epitope I-Eα52–68 yields an epitope that is similar to the one generated during constitutive presentation of I-Eα as an endogenous transmembrane protein, but is subtly different from the one generated in the exogenous pathway. Constitutive MHC class II-mediated presentation of the endogenous transmembrane protein I-Eα is also specifically inhibited over time by inhibitors of cytosolic proteolysis. Thus, Ag processing in the cytoplasm appears to be essential for the efficient presentation of endogenous proteins, even transmembrane ones, on MHC class II, and the proteolytic pathways involved may differ from those used for MHC class I-mediated presentation.
Cellular Localization and Functional Role of Phosphatidylcholine-Specific Phospholipase C in NK Cells
Authors: Ramoni Carlo , Spadaro Francesca , Menegon Michela , Podo Franca
Published: April 2014

Although several classes of phospholipases have been implicated in NK cell-mediated cytotoxicity, no evidence has been reported to date on involvement of phosphatidylcholine-specific phospholipase C (PC-PLC) in NK activation by lymphokines and/or in lytic granule exocytosis. This study demonstrated the expression of two PC-PLC isoforms (Mr 40 and 66 kDa) and their IL-2-dependent distribution between cytoplasm and ectoplasmic membrane surface in human NK cells. Following cell activation by IL-2, cytoplasmic PC-PLC translocated from the microtubule-organizing center toward cell periphery, essentially by kinesin-supported transport along microtubules, while PC-PLC exposed on the outer cell surface increased 2-fold. Preincubation of NK cells with a PC-PLC inhibitor, tricyclodecan-9-yl-xanthogenate, strongly reduced NK-mediated cytotoxicity. In IL-2-activated cells, this loss of cytotoxicity was associated with a decrease of PC-PLC exposed on the cell surface, and accumulation of cytoplasmic PC-PLC in the Golgi region. Massive colocalization of PC-PLC-rich particles with perforin-containing granules was found in the cytoplasm of NK-activated (but not NK-resting) cells; both organelles clustered at the intercellular contact region of effector-target cell conjugates. These newly detected mechanisms of PC-PLC translocation and function support an essential role of this enzyme in regulated granule exocytosis and NK-mediated cytotoxicity.
Intestinal Macrophages Lack CD14 and CD89 and Consequently Are Down-Regulated for LPS- and IgA-Mediated Activities
Authors: Smith Phillip D. , Smythies Lesley E. , Mosteller-Barnum Meg , Sibley Don A. , Russell Michael W. , Merger Michael , Sellers Marty T. , Orenstein Jan M. , Shimada Toshihide , Graham Martin F. , Kubagawa Hiromi
Published: April 2014

The intestinal mucosa normally displays minimal inflammation despite the close proximity between mucosal macrophages and lumenal bacteria. Macrophages interact with bacteria and their products through CD14, a surface receptor involved in the response to LPS, and CD89, the receptor for IgA (FcαR). Here we show that resident macrophages isolated from normal human intestine lack CD14 and CD89. The absence of CD14 and CD89 was not due to the isolation procedure or mucosal cell products, but was evident at the transcriptional level, as the macrophages expressed neither CD14- nor CD89-specific mRNAs, but did express Toll-like receptor 2 and 4 transcripts. Consistent with their CD14− phenotype, lamina propria macrophages displayed markedly reduced LPS-induced cytokine production and LPS-enhanced phagocytosis. In addition, IgA-enhanced phagocytosis was sharply reduced in lamina propria macrophages. Thus, the absence of CD14 and CD89 on resident intestinal macrophages, due to down-regulated gene transcription, causes down-modulated LPS- and IgA-mediated functions and probably contributes to the low level of inflammation in normal human intestinal mucosa.
Abnormal T Cell Receptor Signal Transduction of CD4 Th Cells in X-Linked Lymphoproliferative Syndrome
Authors: Nakamura Hiroyuki , Zarycki Jodi , Sullivan John L. , Jung Jae U.
Published: April 2014

The molecular basis of X-linked lymphoproliferative (XLP) disease has been attributed to mutations in the signaling lymphocytic activation molecule-associated protein (SAP), an src homology 2 domain-containing intracellular signaling molecule known to interact with the lymphocyte-activating surface receptors signaling lymphocytic activation molecule and 2B4. To investigate the effect of SAP defects on TCR signal transduction, herpesvirus saimiri-immortalized CD4 Th cells from XLP patients and normal healthy individuals were examined for their response to TCR stimulation. CD4 T cells of XLP patients displayed elevated levels of tyrosine phosphorylation compared with CD4 T cells from healthy individuals. In addition, downstream serine/threonine kinases are constitutively active in CD4 T cells of XLP patients. In contrast, TCR-mediated activation of Akt, c-Jun-NH2-terminal kinases, and extracellular signal-regulated kinases in XLP CD4 T cells was transient and rapidly diminished when compared with that in control CD4 T cells. Consequently, XLP CD4 T cells exhibited severe defects in up-regulation of IL-2 and IFN-γ cytokine production upon TCR stimulation and in MLRs. Finally, SAP specifically interacted with a 75-kDa tyrosine-phosphorylated protein upon TCR stimulation. These results demonstrate that CD4 T cells from XLP patients exhibit aberrant TCR signal transduction and that the defect in SAP function is likely responsible for this phenotype.
Regulatory Potential of Fever-Range Whole Body Hyperthermia on Langerhans Cells and Lymphocytes in an Antigen-Dependent Cellular Immune Response
Authors: Ostberg Julie R. , Gellin Caren , Patel Rahul , Repasky Elizabeth A.
Published: April 2014

The febrile response is one of the most common features of infection and inflammation. However, temperature is rarely a variable in experimental immunological investigations. To determine whether the thermal microenvironment has any immunoregulatory potential in an Ag-dependent response, we applied a mild fever-range whole body hyperthermia (FR-WBH) protocol to BALB/c mice experiencing the contact hypersensitivity (CHS) reaction. We observed that the timing of this FR-WBH treatment relative to the different phases of the CHS response was crucial to the outcome. FR-WBH treatment before sensitization with a 0.5% FITC solution resulted in a depressed CHS response. This appears to be due to direct effects of FR-WBH on epidermal Langerhans cell trafficking to the draining lymph nodes. In contrast, application of FR-WBH directly after application of the elicitation dose of FITC solution resulted in an enhanced reaction. This result correlates with increased homing of lymphocytes to the site of elicitation. Overall, these data have important implications regarding the role of thermal changes experienced during infection and the clinical use of FR-WBH relative to immunotherapeutic strategies.
Progressive Depletion of Peripheral B Lymphocytes in 4-1BB (CD137) Ligand/I-Eα-Transgenic Mice
Authors: Zhu Gefeng , Flies Dallas B. , Tamada Koji , Sun Yonglian , Rodriguez Moses , Fu Yang-Xin , Chen Lieping
Published: April 2014

Interaction of 4-1BB (CD137) and its ligand (4-1BBL) is thought to positively regulate cell-mediated and humoral immune responses. We have prepared transgenic mouse strains that express 4-1BBL cDNA under the control of MHC class II I-Eα promoter. The 4-1BBL-transgenic mice show progressive splenomegaly and selective depletion of B220+ B cells accompanied with low levels of circulating IgG and defective humoral responses to Ag challenge. In addition, splenocytes from the transgenic mice fail to provide stimulation for allogeneic T cells in both lymphoproliferative and CTL responses in vitro, whereas their T cells remain functionally normal. Our results reveal unexpected functions of 4-1BBL in the regulation of humoral immune responses and Ag presentation.
Inhibition of NFATx Activation by an Oligopeptide: Disrupting the Interaction of NFATx with Calcineurin
Authors: Liu Jie , Arai Ken-ichi , Arai Naoko
Published: April 2014

Calcium-dependent phosphatase calcineurin (CN) regulates the activation and nuclear translocation of NFAT. We identify here a novel CN-binding motif in one member of the NFAT family, NFATx, and a peptide based on this motif, Pep3. Pep3 binds CN and competes with wild-type NFATx for CN interaction. Amino acid mutations within Pep3 show that multiple amino acid residues are required for the effective functions of Pep3. Ectopic expression of Pep3 in a Th clone via a retrovirus-mediated gene transfer could selectively block the nuclear translocation of endogenous NFATx, whereas it had little effect on the nuclear translocation of another member of the NFAT family, NFATp. Furthermore, in transfection experiments, Pep3 also blocked the nuclear translocation of transfected NFATx, but not NFATp, in the B cell line M12, demonstrating specific inhibition of Pep3 for NFATx. Importantly, several cytokines produced by the T cell clone were severely repressed by ectopic Pep3, and indeed, the production of these cytokines was enhanced by the expression of wild-type NFATx. Our results show selective inhibition of NFATx activation and cytokine expression by Pep3 and suggest a new approach for studying the biology of each NFAT family member. This approach may provide an opportunity for pharmacological targeting of Ca2+-dependent signaling events.
Immunomodulation of Experimental Autoimmune Encephalomyelitis with Ordered Peptides Based on MHC-TCR Binding Motifs
Authors: Ruiz Pedro J. , DeVoss Jason J. , Nguyen Louis-Vu T. , Fontoura Paulo P. , Hirschberg David L. , Mitchell Dennis J. , Garcia K. Christopher , Steinman Lawrence
Published: April 2014

T cell-mediated destruction of the myelin sheath causes inflammatory damage of the CNS in multiple sclerosis (MS). The major T and B cell responses in MS patients who are HLA-DR2 (about two-thirds of MS patients) react to a region between residues 84 and 103 of myelin basic protein (1 ). The crystal structure of HLA-DR2 complexed with myelin basic protein84–102 confirmed that Lys91 is the major TCR contact site, whereas Phe90 is a major anchor to MHC and binds the hydrophobic P4 pocket (2 ). We have tested peptides containing repetitive 4-aa sequences designed to bind critical MHC pockets and to interfere with T cell activation. One such sequence, EYYKEYYKEYYK, ameliorates experimental autoimmune encephalomyelitis in Lewis rats, an animal model of MS.
Identification, Cloning, and Recombinant Expression of Procalin, a Major Triatomine Allergen
Authors: Paddock Christopher D. , McKerrow James H. , Hansell Elizabeth , Foreman K. W. , Hsieh Ivy , Marshall Neal
Published: April 2014

Among the most frequent anaphylactic reactions to insects are those attributed to reduviid bugs. We report the purification and identification of the major salivary allergen of these insects. This 20-kDa protein (procalin) is a member of the lipocalin family, which includes salivary allergens from other invertebrates and mammals. An expression system capable of producing reagent quantities of recombinant allergen was developed in Saccharomyces cerevisiae. Antisera produced against recombinant protein cross-reacts with ELISA with salivary allergen. Recombinant Ag is also shown to react with sera from an allergic patient but not with control sera. By immunolocalization, the source of the salivary Ag is the salivary gland epithelium and its secretions.
Negative Regulation of the NFAT1 Factor by CD45: Implication in HIV-1 Long Terminal Repeat Activation
Authors: Barbeau Benoit , Robichaud Gilles A. , Fortin Jean-François , Tremblay Michel J.
Published: April 2014

HIV-1 gene regulation is greatly dependent on the presence of the −104/−81 enhancer region which is regulated by both NF-κB and NFAT transcription factors. We have found that a greater induction in HIV-1 long terminal repeat-driven gene expression was observed upon PMA/ionomycin (Iono) stimulation of a CD45-deficient cell line (J45.01) in comparison to the parental Jurkat cells. Unlike NF-κB which was not affected by the absence of CD45, NFAT showed a much greater augmentation in nuclear translocation and transcriptional activity in J45.01 cells upon PMA/Iono stimulation. PMA/Iono-induced NFAT activation, NFAT translocation and calcium influx peaked at similar time points for both Jurkat and J45.01 cell lines. The NFAT-dependent promoters from the IL-2 and TNF-α genes were also more potently activated by PMA/Iono in J45.01 cells. Interestingly, higher levels of intracellular calcium were consistently demonstrated in PMA/Iono-induced CD45-deficient cell lines (J45.01 and HPB45.0). Furthermore, PMA/Iono induction of calcium mobilization in both Jurkat and J45.01 cell lines was observed to be EGTA-sensitive. Mechanistic studies revealed that CD3ζ and ZAP-70 were more heavily tyrosine phosphorylated in J45.01 cells than Jurkat cells. Analysis of the HIV-1 enhancer by EMSAs demonstrated that the bound NFAT complex was present at higher levels in J45.01 nuclear extracts and that the NFAT1 member was predominant. In conclusion, our results indicate that NFAT activation by stimuli acting in a more distal fashion from the TCR-mediated signaling pathway can be down-regulated by CD45 and that this CD45-dependent regulation in turn affects HIV-1 long terminal repeat activation.
Phosphatidylinositol 3-Kinase Potentiates, but Does Not Trigger, T Cell Proliferation Mediated by the IL-2 Receptor
Authors: Moon James J. , Nelson Brad H.
Published: April 2014

Proliferative signaling by the IL-2R can occur through two distinct pathways, one mediated by Stat5 and one by the adaptor protein Shc. Although Stat5 induces T cell proliferation by serving as a transcription factor, the mechanism of proliferative signaling by Shc is poorly defined. We examined the roles of two major signaling pathways downstream of Shc, the p44/p42 mitogen-activated protein kinase (extracellular signal-related kinase (Erk)) and phosphatidylinositol 3-kinase (PI3K) pathways, in promitogenic gene induction and proliferation in the IL-2-dependent T cell line CTLL-2. Using IL-2R mutants and specific pharmacologic inhibitors, we found that the PI3K, but not Erk, pathway is required for maximal induction of c-myc, cyclin D2, cyclin D3, cyclin E, and bcl-xL by Shc. To test whether the PI3K pathway is sufficient for proliferative signaling, a tamoxifen-regulated form of PI3K (mp110*ER) was expressed in CTLL-2 cells. Activation of the PI3K pathway through mp110*ER failed to up-regulate expression of the c-myc, cyclin D2, cyclin D3, cyclin E, bcl-2, or bcl-xL genes or down-regulate expression of p27Kip1, even when coactivated with the Janus kinases (Jak) or the Raf/Erk pathway. Moreover, mp110*ER induced modest levels of thymidine incorporation without subsequent cell division. Although insufficient for mitogenesis, mp110*ER enhanced Stat5-mediated proliferative signaling through a mechanism independent of Stat5 transcriptional activity. Thus, in addition to serving a necessary, but insufficient role in Shc-mediated promitogenic gene expression, the PI3K pathway contributes to T cell proliferation by potentiating mitogenic signaling by Stat5.
Monomorphic Molecules Function as Additional Recognition Structures on Haptenated Target Cells for HLA-A1-Restricted, Hapten-Specific CTL
Authors: Stöckl Johannes , Majdic Otto , Fischer Gottfried , Maurer Dieter , Knapp Walter
Published: April 2014

Hapten-specific T cells have been shown to recognize haptenated peptides with high avidity and, in some instances, with promiscuous MHC restriction. In this study, the impact of Ag density on MHC restriction of a CTL response specific to the trinitrophenyl (TNP) hapten was investigated. In this study, we demonstrate a novel recognition mechanism used by TNP-specific CD8+ CTL in the presence of high Ag doses. Although low levels of TNP epitopes on target cells allowed for HLA-A1-restricted CTL activity only, entirely MHC-independent target cell recognition became operative at high TNP loading. In both cases, recognition was mediated by the TCR. This MHC-independent recognition is target cell type restricted and critically involves in our model direct recognition of the ectonucleotidase family surface molecule CD39 by the CTL.
CD4+ and CD8+ T Cells Kill IntracellularMycobacterium tuberculosisby a Perforin and Fas/Fas Ligand-Independent Mechanism
Authors: Canaday David H. , Wilkinson Robert J. , Li Qing , Harding Clifford V. , Silver Richard F. , Boom W. Henry
Published: April 2014

Cytotoxic effector phenotype and function of MHC-restricted Mycobacterium tuberculosis (MTB)-reactive CD4+ and CD8+ T lymphocytes were analyzed from healthy tuberculin skin test-positive persons. After stimulation in vitro with MTB, both CD4+ and CD8+ T cells up-regulated mRNA expression for granzyme A and B, granulysin, perforin, and CD95L (Fas ligand). mRNA levels for these molecules were greater for resting CD8+ than CD4+ T cells. After MTB stimulation, mRNA levels were similar for both T cell subsets. Increased perforin and granulysin protein expression was confirmed in both in CD4+ and CD8+ T cells by flow cytometry. Both T cell subsets lysed MTB-infected monocytes. Biochemical inhibition of the granule exocytosis pathway in CD4+ and CD8+ T cells decreased cytolytic function by >90% in both T cell subsets. Ab blockade of the CD95-CD95L interaction decreased cytolytic function for both T cell populations by 25%. CD4+ and CD8+ T cells inhibited growth of intracellular MTB in autologous monocytes by 74% and 84%, respectively. However, inhibition of perforin activity, the CD95-CD95L interaction, or both CTL mechanisms did not affect CD4+ and CD8+ T cell mediated restriction of MTB growth. Thus, perforin and CD95-CD95L were not involved in CD4+ and CD8+ T cell mediated restriction of MTB growth.
Vpr Is Preferentially Targeted by CTL During HIV-1 Infection
Authors: Altfeld Marcus , Addo Marylyn M. , Eldridge Robert L. , Yu Xu G. , Thomas Seddon , Khatri Ashok , Strick Daryld , Phillips Mary N. , Cohen George B. , Islam Sabina A. , Kalams Spyros A. , Brander Christian , Goulder Philip J. R. , Rosenberg Eric S. , Walker Bruce D. ,   the HIV Study Collaboration
Published: April 2014

The HIV-1 accessory proteins Vpr, Vpu, and Vif are essential for viral replication, and their cytoplasmic production suggests that they should be processed for recognition by CTLs. However, the extent to which these proteins are targeted in natural infection, as well as precise CTL epitopes within them, remains to be defined. In this study, CTL responses against HIV-1 Vpr, Vpu, and Vif were analyzed in 60 HIV-1-infected individuals and 10 HIV-1-negative controls using overlapping peptides spanning the entire proteins. Peptide-specific IFN-γ production was measured by ELISPOT assay and flow-based intracellular cytokine quantification. HLA class I restriction and cytotoxic activity were confirmed after isolation of peptide-specific CD8+ T cell lines. CD8+ T cell responses against Vpr, Vpu, and Vif were found in 45%, 2%, and 33% of HIV-1-infected individuals, respectively. Multiple CTL epitopes were identified in functionally important regions of HIV-1 Vpr and Vif. Moreover, in infected individuals in whom the breadth of HIV-1-specific responses was assessed comprehensively, Vpr and p17 were the most preferentially targeted proteins per unit length by CD8+ T cells. These data indicate that despite the small size of these proteins Vif and Vpr are frequently targeted by CTL in natural HIV-1 infection and contribute importantly to the total HIV-1-specific CD8+ T cell responses. These findings will be important in evaluating the specificity and breadth of immune responses during acute and chronic infection, and in the design and testing of candidate HIV vaccines.
Concurrent Naive and Memory CD8+ T Cell Responses to an Influenza A Virus
Authors: Turner Stephen J. , Cross Richard , Xie Weidong , Doherty Peter C.
Published: April 2014

Memory Thy-1+CD8+ T cells specific for the influenza A virus nucleoprotein (NP366–374) peptide were sorted after staining with the DbNP366 tetramer, labeled with CFSE, and transferred into normal Thy-1.2+ recipients. The donor DbNP366+ T cells recovered 2 days later from the spleens of the Thy-1.2+ hosts showed the CD62LlowCD44highCD69low phenotype, characteristic of the population analyzed before transfer, and were present at frequencies equivalent to those detected previously in mice primed once by a single exposure to an influenza A virus. Analysis of CFSE-staining profiles established that resting tetramer+ T cells divided slowly over the next 30 days, while the numbers in the spleen decreased about 3-fold. Intranasal infection shortly after cell transfer with a noncross-reactive influenza B virus induced some of the donor DbNP366+ T cells to cycle, but there was no increase in the total number of transferred cells. By contrast, comparable challenge with an influenza A virus caused substantial clonal expansion, and loss of the CFSE label. Unexpectedly, the recruitment of naive Thy-1.2+CD8+DbNP366+ host DbNP366+ T cells following influenza A challenge was not obviously diminished by the presence of the memory Thy-1.1+CD8+DbNP366+ donor DbNP366+ set. Furthermore, the splenic response to an epitope (DbPA224) derived from the influenza acid polymerase (PA224–233) was significantly enhanced in the mice given the donor DbNP366+ memory population. These experiments indicate that an apparent recall response may be comprised of both naive and memory CD8+ T cells.
Critical Role of Lipopolysaccharide-Binding Protein and CD14 in Immune Responses against Gram-Negative Bacteria
Authors: Le Roy Didier , Di Padova Franco , Adachi Yoshiyuki , Glauser Michel Pierre , Calandra Thierry , Heumann Didier
Published: April 2014

LPS-binding protein (LBP) and CD14 potentiate cell activation by LPS, contributing to lethal endotoxemia. We analyzed the contribution of LBP/CD14 in models of bacterial infection. Mice pretreated with mAbs neutralizing CD14 or LBP showed a delay in TNF-α production and died of overwhelming infection within 24 h, after a challenge with 250 CFU of virulent Klebsiella pneumoniae. Blockade of TNF-α also increased lethality, whereas pretreatment with TNF-α protected mice, even in the presence of LBP and CD14 blockade. Anti-LBP or anti-CD14 mAbs did not improve or decrease lethality with a higher inoculum (105 K. pneumoniae) and did not affect outcome following injections of low or high inocula of Escherichia coli O111. These results point to the essential role of LBP/CD14 in innate immunity against virulent bacteria.
HIV-1 Tat Induces Microvascular Endothelial Apoptosis Through Caspase Activation
Authors: Park In-Woo , Ullrich Christina K. , Schoenberger Elena , Ganju Ramesh K. , Groopman Jerome E.
Published: April 2014

HIV-1 Tat, in addition to its critical role in viral transcription, is secreted from infected cells and can act as a proto-cytokine. We studied the effects of HIV-1 Tat in primary human microvascular endothelial cells of lung origin and found that it caused apoptosis. This apoptosis occurred without induction of either Fas or TNF, known mediators of programmed cell death. Tat, like Fas ligand, induced cleavage of chromatin structure, as evidenced by changes in DNA laddering, incorporation of fluorescein into the nicked chromosomal DNA (TUNEL assay), and mono- or oligonucleosomes. Furthermore, Tat treatment caused cleavage of poly(A/DP)-ribose polymerase, a substrate of caspases. Caspase-3, but not caspase-9, was activated following treatment of primary human microvascular endothelial cells of lung origin with either Tat or anti-Fas agonist Ab (anti-Fas). Inhibition of caspase-3 activity markedly reduced apoptosis. Although Fas-mediated apoptosis involved changes in Bcl-2, Bax, and Bad regulatory proteins, such alterations were not observed with Tat. Taken together, these data demonstrate that HIV-1 Tat is able to activate apoptosis in microvascular endothelium by a mechanism distinct from TNF secretion or the Fas pathway.
Lipoxin A4 and Aspirin-Triggered 15-Epi-Lipoxin A4 Antagonize TNF-α-Stimulated Neutrophil-Enterocyte Interactions In Vitro and Attenuate TNF-α-Induced Chemokine Release and Colonocyte Apoptosis in Human Intestinal Mucosa Ex Vivo
Authors: Goh Jason , Baird Alan W. , O’Keane Conor , Watson R. William G. , Cottell David , Bernasconi Giovanni , Petasis Nicos A. , Godson Catherine , Brady Hugh R. , MacMathuna Padraic
Published: April 2014

Lipoxins (LXs) are lipoxygenase-derived eicosanoids and putative endogenous braking signals for inflammation in the gastrointestinal tract and other organs. Aspirin triggers the production of 15-epimers during cell-cell interaction in a cytokine-primed milieu, and aspirin-triggered 15-epi-5(S),6(R),15(S)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid (15-epi-LXA4) may contribute to the bioactivity profile of this prototype nonsteroidal anti-inflammatory drug in vivo. We determined the effect of LXA4, 15-(R/S)-methyl-11,12-dehydro-LXA4 methyl ester (15-(R/S)-methyl-LXA4), and stable analogs of LXA4 on TNF-α-stimulated neutrophil-enterocyte interaction in vitro and TNF-α-stimulated chemokine release, changes in mucosal architecture, and enterocyte apoptosis in cytokine-activated intact human colonic mucosa ex vivo. LXA4, 15-(R/S)-epi-LXA4, and 16-phenoxy-11,12-dehydro-17,18,19,20-tetranor-LXA4 methyl ester (16-phenoxy-LXA4) inhibited TNF-α-stimulated neutrophil adherence to epithelial monolayers at nanomolar concentrations. In parallel experiments involving human colonic mucosa ex vivo, LXA4potently attenuated TNF-α-stimulated release of the C-X-C chemokine IL-8, and the C-C chemokines monocyte-chemoattractant protein-1 (MCP-1) and RANTES. Exposure of strips of normal human colonic mucosa to TNF-α induced disruption of mucosa architecture and enhanced colonocyte apoptosis via a caspase-3-independent mechanism. Prior exposure of the mucosa strips to 15-(R/S)-methyl-LXA4 attenuated TNF-α-stimulated colonocyte apoptosis and protected the mucosa against TNF-α-induced mucosal damage. In aggregate, our data demonstrate that lipoxins and aspirin-triggered 15-epi-LXA4 are potent antagonists of TNF-α-mediated neutrophil-enterocyte interactions in vitro, attenuate TNF-α-triggered chemokine release and colonocyte apoptosis, and are protective against TNF-α-induced morphological disruption in human colonic strips ex vivo. Our observations further expand the anti-inflammatory profile of these lipoxygenase-derived eicosanoids and suggest new therapeutic approaches for the treatment of inflammatory bowel disease.
Elimination of Colonic Patches with Lymphotoxin β Receptor-Ig Prevents Th2 Cell-Type Colitis
Authors: Dohi Taeko , Rennert Paul D. , Fujihashi Kohtaro , Kiyono Hiroshi , Shirai Yuko , Kawamura Yuki I. , Browning Jeffrey L. , McGhee Jerry R.
Published: April 2014

Past studies have shown that colonic patches, which are the gut-associated lymphoreticular tissues (GALT) in the colon, become much more pronounced in hapten-induced murine colitis, and this was associated with Th2-type T cell responses. To address the role of GALT in colonic inflammation, experimental colitis was induced in mice either lacking organized GALT or with altered GALT structures. Trinitrobenzene sulfonic acid was used to induce colitis in mice given lymphotoxin-β receptor-Ig fusion protein (LTβR-Ig) in utero, a treatment that blocked the formation of both Peyer’s and colonic patches. Mice deficient in colonic patches developed focal acute ulcers with Th1-type responses, whereas lesions in normal mice were of a diffuse mucosal type with both Th1- and Th2-type cytokine production. We next determined whether LTβR-Ig could be used to treat colitis in normal or Th2-dominant, IFN-γ gene knockout (IFN-γ−/−) mice. Four weekly treatments with LTβR-Ig resulted in deletion of Peyer’s and colonic patches with significant decreases in numbers of dendritic cells. This pretreatment protected IFN-γ−/− mice from trinitrobenzene sulfonic acid-induced colitis; however, in normal mice this weekly treatment was less protective. In these mice hypertrophy of colonic patches was seen after induction of colitis. We conclude that Th2-type colitis is dependent upon the presence of colonic patches. The effect of LTβR-Ig was mediated through prevention of colonic patch hypertrophy in the absence of IFN-γ. Thus, LTβR-Ig may offer a possible treatment for the Th2-dominant form of colitis.
Increased Susceptibility of Decay-Accelerating Factor Deficient Mice to Anti-Glomerular Basement Membrane Glomerulonephritis
Authors: Sogabe Hajime , Nangaku Masaomi , Ishibashi Yoshitaka , Wada Takehiko , Fujita Toshiro , Sun Xiujun , Miwa Takashi , Madaio Michael P. , Song Wen-Chao
Published: April 2014

To prevent complement-mediated autologous tissue damage, host cells express a number of membrane-bound complement inhibitors. Decay-accelerating factor (DAF, CD55) is a GPI-linked membrane complement regulator that is widely expressed in mammalian tissues including the kidney. DAF inhibits the C3 convertase of both the classical and alternative pathways. Although DAF deficiency contributes to the human hematological syndrome paroxysmal nocturnal hemoglobinuria, the relevance of DAF in autoimmune tissue damage such as immune glomerulonephritis remains to be determined. In this study, we have investigated the susceptibility of knockout mice that are deficient in GPI-anchored DAF to nephrotoxic serum nephritis. Injection of a subnephritogenic dose of rabbit anti-mouse glomerular basement membrane serum induced glomerular disease in DAF knockout mice but not in wild-type controls. When examined at 8 days after anti-glomerular basement membrane treatment, DAF knockout mice had a much higher percentage of diseased glomeruli than wild-type mice (68.8 ± 25.0 vs 10.0 ± 3.5%; p < 0.01). Morphologically, DAF knockout mice displayed increased glomerular volume (516 ± 68 vs 325 ± 18 × 103 μm3 per glomerulus; p < 0.0001) and cellularity (47.1 ± 8.9 vs 32.0 ± 3.1 cells per glomerulus; p < 0.01). Although the blood urea nitrogen level showed no difference between the two groups, proteinuria was observed in the knockout mice but not in the wild-type mice (1.4 ± 0.7 vs 0.02 ± 0.01 mg/24 h albumin excretion). The morphological and functional abnormalities in the knockout mouse kidney were associated with evidence of increased complement activation in the glomeruli. These results support the conclusion that membrane C3 convertase inhibitors like DAF play a protective role in complement-mediated immune glomerular damage in vivo.
Murine Cytomegalovirus Infection Alters Th1/Th2 Cytokine Expression, Decreases Airway Eosinophilia, and Enhances Mucus Production in Allergic Airway Disease
Authors: Wu Carol A. , Puddington Lynn , Whiteley Herbert E. , Yiamouyiannis Carmen A. , Schramm Craig M. , Mohammadu Fusaini , Thrall Roger S.
Published: April 2014

Concomitant infection of murine CMV (MCMV), an opportunistic respiratory pathogen, altered Th1/Th2 cytokine expression, decreased bronchoalveolar lavage (BAL) fluid eosinophilia, and increased mucus production in a murine model of OVA-induced allergic airway disease. Although no change in the total number of leukocytes infiltrating the lung was observed between challenged and MCMV/challenged mice, the cellular profile differed dramatically. After 10 days of OVA-aerosol challenge, eosinophils comprised 64% of the total leukocyte population in BAL fluid from challenged mice compared with 11% in MCMV/challenged mice. Lymphocytes increased from 11% in challenged mice to 30% in MCMV/challenged mice, and this increase corresponded with an increase in the ratio of CD8+ to CD4+TCRαβ lymphocytes. The decline in BAL fluid eosinophilia was associated with a change in local Th1/Th2 cytokine profiles. Enhanced levels of IL-4, IL-5, IL-10, and IL-13 were detected in lung tissue from challenged mice by RNase protection assays. In contrast, MCMV/challenged mice transiently expressed elevated levels of IFN-γ and IL-10 mRNAs, as well as decreased levels of IL-4, IL-5, and IL-13 mRNAs. Elevated levels of IFN-γ and reduced levels of IL-5 were also demonstrated in BAL fluid from MCMV/challenged mice. Histological evaluation of lung sections revealed extensive mucus plugging and epithelial cell hypertrophy/hyperplasia only in MCMV/challenged mice. Interestingly, the development of airway hyperresponsiveness was observed in challenged mice, not MCMV/challenged mice. Thus, MCMV infection can modulate allergic airway inflammation, and these findings suggest that enhanced mucus production may occur independently of BAL fluid eosinophilia.
Eotaxin Represents the Principal Eosinophil Chemoattractant in a Novel Murine Asthma Model Induced by House Dust Containing Cockroach Allergens
Authors: Kim Jiyoun , Merry Andrew C. , Nemzek Jean A. , Bolgos Gerry L. , Siddiqui Javed , Remick Daniel G.
Published: April 2014

Asthma represents a serious health problem particularly for inner city children, and recent studies have identified that cockroach allergens trigger many of these asthmatic attacks. This study tested the concept that asthma-like pulmonary inflammation may be induced by house dust containing cockroach allergens. An aqueous extract was prepared from a house dust sample containing endotoxin and high levels of cockroach allergens. BALB/c mice were immunized with the house dust extract (HDE) and received two additional pulmonary challenges. Bronchoalveolar lavage (BAL) eosinophil counts and eotaxin levels were significantly increased in immunized mice exposed to the HDE, whereas neutrophils were the predominant BAL inflammatory cell in the unimmunized mice. Kinetics studies in immunized mice demonstrated a peak pulmonary inflammatory response 48 h after the last challenge. The allergic response in this model was further confirmed by histological and physiological studies demonstrating a significant influx of eosinophils and lymphocytes in the peribronchial area, and severe airway hyperreactivity through whole-body plethysmography. The specificity of the response was established by immunizing with HDE and challenging with purified cockroach allergen, which induced pulmonary eosinophilia and airway hyperreactivity. Ab inhibition of eotaxin significantly inhibited the number of BAL eosinophils. These data describe a novel murine model of asthma-like pulmonary inflammation induced by house dust containing endotoxin and cockroach allergens and further demonstrate that eotaxin represents the principal chemoattractant for the recruitment of the pulmonary eosinophils.
IL-8 Released Constitutively by Primary Bronchial Epithelial Cells in Culture Forms an Inactive Complex with Secretory Component
Authors: Marshall Lindsay J. , Perks Beatrice , Ferkol Thomas , Shute Janis K.
Published: April 2014

The bronchial epithelium is a source of both α and β chemokines and, uniquely, of secretory component (SC), the extracellular ligand-binding domain of the polymeric IgA receptor. Ig superfamily relatives of SC, such as IgG and α2-macroglobulin, bind IL-8. Therefore, we tested the hypothesis that SC binds IL-8, modifying its activity as a neutrophil chemoattractant. Primary bronchial epithelial cells were cultured under conditions to optimize SC synthesis. The chemokines IL-8, epithelial neutrophil-activating peptide-78, growth-related oncogene α, and RANTES were released constitutively by epithelial cells from both normal and asthmatic donors and detected in high m.w. complexes with SC. There were no qualitative differences in the production of SC-chemokine complexes by epithelial cells from normal or asthmatic donors, and in all cases this was the only form of chemokine detected. SC contains 15% N-linked carbohydrate, and complete deglycosylation with peptide N-glycosidase F abolished IL-8 binding. In micro-Boyden chamber assays, no IL-8-dependent neutrophil chemotactic responses to epithelial culture supernatants could be demonstrated. SC dose-dependently (IC50 ∼0.3 nM) inhibited the neutrophil chemotactic response to rIL-8 (10 nM) in micro-Boyden chamber assays and also inhibited IL-8-mediated neutrophil transendothelial migration. SC inhibited the binding of IL-8 to nonspecific binding sites on polycarbonate filters and endothelial cell monolayers, and therefore the formation of haptotactic gradients, without effects on IL-8 binding to specific receptors on neutrophils. The data indicate that in the airways IL-8 may be solubilized and inactivated by binding to SC.
The Affinity of Integrin α4β1 Governs Lymphocyte Migration
Authors: Rose David M. , Grabovsky Valentin , Alon Ronen , Ginsberg Mark H.
Published: April 2014

The interaction of integrin α4β1 with endothelial VCAM-1 controls the trafficking of lymphocytes from blood into peripheral tissues. Cells actively regulate the affinity of α4β1 for VCAM-1 (activation). To investigate the biological function of α4β1 activation, we isolated Jurkat T cell lines with defective α4β1 activation. Using these cells, we found that α4β1-stimulated αLβ2-dependent cell migration was dramatically reduced in cells with defects in α4β1 activation. These cells required 20 times more VCAM-1 to promote αLβ2-dependent cell migration. This defect was at the level of α4β1 affinity as an activating α4β1 Ab rescued α4β1-stimulated αLβ2-dependent migration. In contrast, migration of α4β1 activation-defective cells on VCAM-1 alone was enhanced at higher VCAM-1 densities. Thus, α4β1 activation determines a set point or threshold at which VCAM-1 can regulate αLβ2-dependent as well as α4β1-dependent cell migration. Changes in this set point may specify preferred anatomical sites of integrin-dependent leukocyte emigration from the bloodstream.