bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of an Antigen-Specific CTL Response by a Conformationally Biased Agonist of Human C5a Anaphylatoxin as a Molecular Adjuvant
Authors: Ulrich J. Terry , Cieplak Witold , Paczkowski Natalii J. , Taylor Stephen M. , Sanderson Sam D.
Published: April 2014

A conformationally biased decapeptide agonist of human C5a anaphylatoxin (YSFKPMPLaR) was used as a molecular adjuvant in stimulating an Ag-specific CTL response against murine P815S target cells expressing an Ld-restricted CTL epitope of the hepatitis B surface Ag (HBsAg). Groups of BALB/c mice (H-2d) were immunized with aqueous solutions of the HBsAg CTL epitopes (IPQSLDSWWTSL and IPQSLDSWWTSLRR); the C5a agonist (YSFKPMPLaR); the C5a agonist and HBsAg CTL epitopes admixed (IPQSLDSWWTSL and IPQSLDSWWTSLRR + YSFKPMPLaR); the C5a-active, HBsAg CTL epitope-C5a agonist constructs (IPQSLDSWWTSLYSFKPMPLaR, IPQSLDSWWTSLRRYSFKPMPLaR, and IPQSLDSWWTSLRVRRYSFPMPLaR); a C5a-inactive, reverse-moiety construct (YSFKPMPLaRRRIPQSLDSWWTSL); and a C5a-attenuated, carboxyl-terminal-blocked construct (IPQSLDSWWTSLRRYSFKPMPLaRG). Ag-specific CD8+ CTL responses were observed after the secondary boost in the absence of any added adjuvant only in mice that were immunized with C5a-active contructs, IPQSLDSWWTSLRRYSFKPMPLaR and IPQSLDSWWTSLRVRRYSFKPMPLaR. These two C5a-active immunogens contained potential subtilisin-sensitive linker sequences between the HBsAg CTL epitope and the C5a agonist; i.e., a double-Arg (RR) and a furin protease sensitive sequence (RVRR). The introduction of these potentially cleavable sequences may be a method of increasing the likelihood of liberating the CTL epitope from the C5a agonist by intracellular proteases, thereby facilitating entry of the epitope into Ag-processing pathways via an exogenous route.
Human Dendritic Cells Transfected with RNA Encoding Prostate-Specific Antigen Stimulate Prostate-Specific CTL Responses In Vitro
Authors: Heiser Axel , Dahm Philipp , R. Yancey Donna , Maurice Margaret A. , Boczkowski David , Nair Smita K. , Gilboa Eli , Vieweg Johannes
Published: April 2014

Although immunological tolerance to self Ags represents an important mechanism to prevent normal tissue injury, there is growing evidence that tolerance to tumor Ags, which often represent normal peripherally expressed proteins, is not absolute and can be effectively reverted. Prostate-specific Ag (PSA) is a self Ag expressed by both normal and malignant prostatic epithelium, and therefore offers a unique opportunity to examine the ability of self Ags to serve as specific CTL targets. In this study, we investigated the efficacy of autologous dendritic cells (DC) transfected with mRNA encoding PSA to stimulate CTL against PSA Ags in vitro. Ag in form of RNA carries the advantage to encode multiple epitopes for many HLA alleles, thus permitting induction of CTL responses among many cancer patients independent of their HLA repertoire. In this study, we show that PSA mRNA-transfected DC were capable of stimulating primary CTL responses against PSA Ags in vitro. The PSA-specific CTL did not cross-react with kallikrein Ags, a protein, which shares significant homology with PSA, suggesting that harmful autoimmune toxicity may not represent a significant problem with this approach. PSA RNA-transfected DC generated from male or female healthy volunteers or from cancer patients were equally effective in stimulating PSA-specific CTL in vitro, implying that neither natural tolerance to PSA Ags nor tumor-mediated T cell anergy may represent major barriers for CTL generation against the self Ag PSA. This study provides a preclinical rationale for using PSA RNA-transfected DC in active or adoptive immunization protocols.
Cutting Edge: B Cells Promote CD8+ T Cell Activation in MRL-FaslprMice Independently of MHC Class I Antigen Presentation
Authors: Chan Owen T. M. , Shlomchik Mark J.
Published: April 2014

Spontaneous CD8+ T cell activation in MRL-Faslpr mice is B cell dependent. It is unclear whether this B-dependent activation is mediated by direct Ag presentation via MHC class I proteins (i.e., cross-presentation) or whether activation occurs by an indirect mechanism, e.g., via effects on CD4+ cells. To determine how CD8+ T cell activation is promoted by B cells, we created mixed bone marrow chimeras where direct MHC class I Ag presentation by B cells was abrogated while other leukocyte compartments could express MHC class I. Surprisingly, despite the absence of B cell class I-restricted Ag presentation, CD8+ T cell activation was intact in the chimeric mice. Therefore, the spontaneous B cell-dependent CD8+ T cell activation that occurs in systemic autoimmunity is not due to direct presentation by B cells to CD8+ T cells.
Cutting Edge: Lipoxins Rapidly Stimulate Nonphlogistic Phagocytosis of Apoptotic Neutrophils by Monocyte-Derived Macrophages
Authors: Godson Catherine , Mitchell Siobhan , Harvey Killeen , Petasis Nicos A. , Hogg Nancy , Brady Hugh R.
Published: April 2014

Lipoxins (LX) are lipoxygenase-derived eicosanoids generated during inflammation. LX inhibit polymorphonuclear neutrophil (PMN) chemotaxis and adhesion and are putative braking signals for PMN-mediated tissue injury. In this study, we report that LXA4 promotes another important step in the resolution phase of inflammation, namely, phagocytosis of apoptotic PMN by monocyte-derived macrophages (Mφ). LXA4 triggered rapid, concentration-dependent uptake of apoptotic PMN. This bioactivity was shared by stable synthetic LXA4 analogues (picomolar concentrations) but not by other eicosanoids tested. LXA4-triggered phagocytosis did not provoke IL-8 or monocyte chemoattractant protein-1 release. LXA4-induced phagocytosis was attenuated by anti-CD36, αvβ3, and CD18 mAbs. LXA4-triggered PMN uptake was inhibited by pertussis toxin and by 8-bromo-cAMP and was mimicked by Rp-cAMP, a protein kinase A inhibitor. LXA4 attenuated PGE2-stimulated protein kinase A activation in Mφ. These results suggest that LXA4 is an endogenous stimulus for PMN clearance during inflammation and provide a novel rationale for using stable synthetic analogues as anti-inflammatory compounds in vivo.
Adenoviral-Mediated Overexpression of Monocyte Chemoattractant Protein-1 Differentially Alters the Development of Th1 and Th2 Type Responses In Vivo
Authors: Matsukawa Akihiro , Lukacs Nicholas W. , Standiford Theodore J. , Chensue Stephen W. , Kunkel Steven L.
Published: April 2014

The expression of chemokines during an immune response may participate in determining the intensity and type of the developing immune response. In the present study, we have examined the effect of overexpressing monocyte chemoattractant protein (MCP)-1 at the site of immunization during different stages of Th1- and Th2-type granulomatous responses. The overexpression of MCP-1 by MCP-1 adenovirus during the sensitization phase of the purified protein derivative Th1-type model significantly reduced the elicitation of the granulomatous response. In contrast, the overexpression of MCP-1 during the sensitization phase of the schistosome egg Ag Th2 response led to an enhanced granulomatous reaction. When cytokines were examined upon restimulation of splenocytes ex vivo, an altered cytokine profile was observed, as compared with control mice. IFN-γ and IL-12 were significantly reduced in the purified protein derivative Th1-type response, whereas IL-10 and IL-13 were up-regulated in the schistosome egg Ag Th2-type response. The regulation of the immune response was further examined by using the MCP-1 adenovirus at later time points during the elicitation phase. When MCP-1 was overexpressed during the elicitation phase of the responses, neither the Th1-type nor the Th2-type granuloma was altered. Likewise, the cytokine profiles after restimulation of splenocytes ex vivo were unchanged. Thus, the function of MCP-1 may depend on the stage and type of immune response.
Functional Maturation of Adult Mouse Resting Microglia into an APC Is Promoted by Granulocyte-Macrophage Colony-Stimulating Factor and Interaction with Th1 Cells
Authors: Aloisi Francesca , De Simone Roberta , Columba-Cabezas Sandra , Penna Giuseppe , Adorini Luciano
Published: April 2014

A precise knowledge of the early events inducing maturation of resting microglia into a competent APC may help to understand the involvement of this cell type in the development of CNS immunopathology. To elucidate whether signals from preactivated T cells are sufficient to induce APC features in resting microglia, microglia from the adult BALB/c mouse CNS were cocultured with Th1 and Th2 lines from DO11.10 TCR transgenic mice to examine modulation of APC-related molecules and Ag-presenting capacity. Upon Ag-specific interaction with Th1, but not Th2, cells, microglia strongly up-regulated the surface expression of MHC class II, CD40, and CD54 molecules. Induction of CD86 on mouse microglia did not require T cell-derived signals. Acutely isolated adult microglia stimulated Th1 cells to secrete IFN-γ and, to a lesser extent, IL-2, but were inefficient stimulators of IL-4 secretion by Th2 cells. Microglia exposed in vitro to IFN-γ showed enhanced expression of MHC class II, CD40, and CD54 molecules and became able to restimulate Th2 cells. In addition to IFN-γ, GM-CSF increased the ability of microglia to activate Th1, but not Th2, cells without up-regulating MHC class II, CD40, or CD54 molecules. These results suggest that interaction with Th1 cells and/or Th1-secreted soluble factors induces the functional maturation of adult mouse microglia into an APC able to sustain CD4+ T cell activation. Moreover, GM-CSF, a cytokine secreted by T cells as well as reactive astrocytes, could prime microglia for Th1-stimulating capacity, possibly by enhancing their responsiveness to Th1-derived signals.
Clonal Characterization of a Bipotent T Cell and NK Cell Progenitor in the Mouse Fetal Thymus
Authors: Michie Alison M. , Carlyle James R. , Schmitt Thomas M. , Ljutic Belma , Cho Sarah K. , Fong Quyen , Zúñiga-Pflücker Juan Carlos
Published: April 2014

We recently described a population of fetal thymocytes with a CD117+NK1.1+CD90lowCD25− phenotype, which were shown to contain committed T cell and NK cell progenitors. However, the characterization of a single cell with a restricted T and NK cell precursor potential was lacking. Here, using an in vitro model for T and NK cell differentiation, we provide conclusive evidence demonstrating the existence of a clonal lineage-restricted T and NK cell progenitor. These results establish that fetal thymocytes with a CD117+NK1.1+CD90lowCD25− phenotype represent bipotent T and NK cell progenitors.
CD27-Mediated Activation of Murine NK Cells
Authors: Takeda Kazuyoshi , Oshima Hideo , Hayakawa Yoshihiro , Akiba Hisaya , Atsuta Machiko , Kobata Tetsuji , Kobayashi Kimio , Ito Mamoru , Yagita Hideo , Okumura Ko
Published: April 2014

CD27, a member of the TNF receptor superfamily, has been implicated in T cell activation, T cell development, and T cell-dependent Ab production by B cells. In the present study we examined the expression and function of CD27 on murine NK cells. Murine NK cells constitutively expressed CD27 on their surface. Stimulation with immobilized anti-CD27 mAb or murine CD27 ligand (CD70) transfectans solely could induce proliferation and IFN-γ production of freshly isolated NK cells and enhanced the proliferation and IFN-γ production of anti-NK1.1-sutimulated NK cells. Although NK cell cytotoxicity was not triggered by anti-CD27 mAb or against CD70 transfectants, prestimulation via CD27 enhanced the cytotoxic activity of NK cells in an IFN-γ-dependent manner. These results suggest that CD27-mediated activation may be involved in the NK cell-mediated innate immunity against virus-infected or transformed cells expressing CD70.
Measles Virus Induces Abnormal Differentiation of CD40 Ligand-Activated Human Dendritic Cells
Authors: Servet-Delprat Christine , Vidalain Pierre-Olivier , Bausinger Huguette , Manié Serge , Le Deist Françoise , Azocar Olga , Hanau Daniel , Fischer Alain , Rabourdin-Combe Chantal
Published: April 2014

Measles virus (MV) infection induces a profound immunosuppression responsible for a high rate of mortality in malnourished children. MV can encounter human dendritic cells (DCs) in the respiratory mucosa or in the secondary lymphoid organs. The purpose of this study was to investigate the consequences of DC infection by MV, particularly concerning their maturation and their ability to generate CD8+ T cell proliferation. We first show that MV-infected Langerhans cells or monocyte-derived DCs undergo a maturation process similarly to the one induced by TNF-α or LPS, respectively. CD40 ligand (CD40L) expressed on activated T cells is shown to induce terminal differentiation of DCs into mature effector DCs. In contrast, the CD40L-dependent maturation of DCs is inhibited by MV infection, as demonstrated by CD25, CD69, CD71, CD40, CD80, CD86, and CD83 expression down-regulation. Moreover, the CD40L-induced cytokine pattern in DCs is modified by MV infection with inhibition of IL-12 and IL-1α/β and induction of IL-10 mRNAs synthesis. Using peripheral blood lymphocytes from CD40L-deficient patients, we demonstrate that MV infection of DCs prevents the CD40L-dependent CD8+ T cell proliferation. In such DC-PBL cocultures, inhibition of CD80 and CD86 expression on DCs was shown to require both MV replication and CD40 triggering. Finally, for the first time, MV was shown to inhibit tyrosine-phosphorylation level induced by CD40 activation in DCs. Our data demonstrate that MV replication modifies CD40 signaling in DCs, thus leading to impaired maturation. This phenomenon could play a pivotal role in MV-induced immunosuppression.
Processing and Reactivity of T Cell Epitopes Containing Two Cysteine Residues from Hen Egg-White Lysozyme (HEL74–90)
Authors: Kang Hee-Kap , Mikszta John A. , Deng Hongkui , Sercarz Eli E. , Jensen Peter E. , Kim Byung S.
Published: April 2014

The Ag processing and structural requirements involved in the generation of a major T cell epitope from the hen egg-white lysozyme protein (HEL74–88), containing two cysteine residues at positions 76 and 80, were investigated. Several T cell hybridomas derived from both low responder (I-Ab) and high responder (I-Ak) mice recognize this region. These hybridomas are strongly responsive to native HEL, but unresponsive to the reduced and carboxymethylated protein. Air-oxidized HEL74–88 peptide was unable to bind I-Ak molecules and failed to stimulate T cells in the absence of intracellular Ag processing. Further functional competition assays showed that alkylation of cysteine residues with bulky methyl groups interferes with the contacts for the MHC class II molecules (I-Ak) of high responder mice and the I-Ab-restricted TCR of low responder mice. Serine substitutions of the cysteine residues of HEL74–88 either enhanced or abrogated T cell stimulation by the peptides without significant alterations in the class II binding. These results suggest that the cysteine residues of peptides must be free from disulfide bonding for efficient stimulation of T cells and yet frequently used modifications of cysteine residues may not be suitable for peptide-based vaccine development.
Analysis of the Virus-Specific and Nonspecific B Cell Response to a Persistent B-Lymphotropic Gammaherpesvirus
Authors: Sangster Mark Y. , Topham David J. , D’Costa Sybil , Cardin Rhonda D. , Marion Tony N. , Myers Linda K. , Doherty Peter C.
Published: April 2014

Respiratory challenge of mice with murine gammaherpesvirus 68 (γHV68) results in acute replication in respiratory epithelial cells and persistent, latent infection of B cells and macrophages. γHV68 elicits virus-specific Ab, and also nonspecifically activates B cells to Ab production through a CD4+ T cell-dependent process. The current analysis characterizes virus-specific and nonspecific Ab production at the single cell level and investigates the requirements and nature of the nonspecific response. Virus-specific Ab-forming cell (AFC) numbers were dwarfed by the increase in total AFC in all sites examined, indicating substantial nonspecific Ab production. Clear increases and decreases in specific and total AFC numbers occurred in the lymph nodes draining the respiratory tract and the spleen, but AFC numbers in the bone marrow (BM) increased to a plateau and remained constant. The longevity of the BM response was reflected in a sustained increase in virus-specific and total serum Ab levels. Generally, the IgG2a and IgG2b isotypes predominated. Analysis of cytokine-deficient mice, CD40 ligand-deficient mice, and radiation BM chimeras lacking MHC class II expression specifically on B cells indicated that nonspecific Ab production is independent of IL-6 or IFN-γ, and dependent on cognate CD4+ T cell help. Several observations were consistent with polyclonal B cell activation by γHV68, including the induction of durable serum levels of IgG reactive with mammalian dsDNA and murine type II collagen. Our findings indicate new directions for studies of this valuable model of γ-herpesvirus pathogenesis.
Human Interdigitating Dendritic Cells Induce Isotype Switching and IL-13-Dependent IgM Production in CD40-Activated Naive B Cells
Authors: Johansson Bengt , Ingvarsson Sigurdur , Björck Pia , Borrebaeck Carl A. K.
Published: April 2014

Interdigitating dendritic cells (IDC) represent a mature progeny of dendritic cells (DC) in vivo and are exhibiting a strong lymphocyte stimulatory potential. Because of the restricted localization to secondary lymphoid organs where decisive cellular interactions take place in the initial events of immunity, IDC regulatory function was addressed in relation to naive B cells. In this study, we demonstrate that human tonsillar IDC induce a dual response from CD40-activated IgD+/CD38− naive B lymphocytes. IDC direct naive B cells toward either isotype switching or an IL-13-dependent IgM secretion. IDC-dependent proliferation, isotype switching, and Ig production are all strictly mediated by soluble factors, suggesting that such skewing in B cell activation is the result of differential cytokine expression. Moreover, IDC-expressed IL-13 represents a novel source of a cytokine with recently established effects in Th2 induction as well as in immunological disorders resulting in allergic reactions.
Cancer Immunotherapy Targeting Wilms’ Tumor Gene WT1 Product
Authors: Oka Yoshihiro , Udaka Keiko , Tsuboi Akihiro , Elisseeva Olga A. , Ogawa Hiroyasu , Aozasa Katsuyuki , Kishimoto Tadamitsu , Sugiyama Haruo
Published: April 2014

The Wilms’ tumor gene WT1 is expressed at high levels not only in acute myelocytic and lymphocytic leukemia and in chronic myelocytic leukemia but also in various types of solid tumors including lung cancers. To determine whether the WT1 protein can serve as a target Ag for tumor-specific immunity, three 9-mer WT1 peptides (Db126, Db221, and Db235), which contain H-2Db-binding anchor motifs and have a comparatively higher binding affinity for H-2Db molecules, were tested in mice (C57BL/6, H-2Db) for in vivo induction of CTLs directed against these WT1 peptides. Only one peptide, Db126, with the highest binding affinity for H-2Db molecules induced vigorous CTL responses. The CTLs specifically lysed not only Db126-pulsed target cells dependently upon Db126 concentrations but also WT1-expressing tumor cells in an H-2Db-restricted manner. The sensitizing activity to the Db126-specific CTLs was recovered from the cell extract of WT1-expressing tumor cells targeted by the CTLs in the same retention time as that needed for the synthetic Db126 peptide in RP-HPLC, indicating that the Db126-specific CTLs recognize the Db126 peptide to kill WT1-expressing target cells. Furthermore, mice immunized with the Db126 peptide rejected challenges by WT1-expressing tumor cells and survived for a long time with no signs of autoaggression by the CTLs. Thus, the WT1 protein was identified as a novel tumor Ag. Immunotherapy targeting the WT1 protein should find clinical application for various types of human cancers.
The Murine Liver-Specific Nonclassical MHC Class I Molecule Q10 Binds a Classical Peptide Repertoire
Authors: Zappacosta Francesca , Tabaczewski Piotr , Parker Kenneth C. , Coligan John E. , Stroynowski Iwona
Published: April 2014

The biological properties of the nonclassical class I MHC molecules secreted into blood and tissue fluids are not currently understood. To address this issue, we studied the murine Q10 molecule, one of the most abundant, soluble class Ib molecules. Mass spectrometry analyses of hybrid Q10 polypeptides revealed that α1α2 domains of Q10 associate with 8–9 long peptides similar to the classical class I MHC ligands. Several of the sequenced peptides matched intracellularly synthesized murine proteins. This finding and the observation that the Q10 hybrid assembly is TAP2-dependent supports the notion that Q10 groove is loaded by the classical class I Ag presentation pathway. Peptides eluted from Q10 displayed a binding motif typical of H-2K, D, and L ligands. They carried conserved residues at P2 (Gly), P6 (Leu), and Pω (Phe/Leu). The role of these residues as anchors/auxiliary anchors was confirmed by Ala substitution experiments. The Q10 peptide repertoire was heterogeneous, with 75% of the groove occupied by a multitude of diverse peptides; however, 25% of the molecules bound a single peptide identical to a region of a TCR V β-chain. Since this peptide did not display enhanced binding affinity for Q10 nor does its origin and sequence suggest that it is functionally significant, we propose that the nonclassical class I groove of Q10 resembles H-2K, D, and L grooves more than the highly specialized clefts of nonclassical class I Ags such as Qa-1, HLA-E, and M3.
Elimination of Fc Receptor-Dependent Effector Functions of a Modified IgG4 Monoclonal Antibody to Human CD4
Authors: Reddy Manjula P. , Kinney Cheryl Ann S. , Chaikin Margery A. , Payne Angela , Fishman-Lobell Jacqueline , Tsui Ping , Dal Monte Paul R. , Doyle Michael L. , Brigham-Burke Michael R. , Anderson Darrell , Reff Mitchell , Newman Roland , Hanna Nabil , Sweet Raymond W. , Truneh Alemseged
Published: April 2014

Several CD4 mAbs have entered the clinic for the treatment of autoimmune diseases or transplant rejection. Most of these mAbs caused CD4 cell depletion, and some were murine mAbs which were further hampered by human anti-mouse Ab responses. To obviate these concerns, a primatized CD4 mAb, clenoliximab, was generated by fusing the V domains of a cynomolgus macaque mAb to human constant regions. The heavy chain constant region is a modified IgG4 containing two single residue substitutions designed to ablate residual Fc receptor binding activity and to stabilize heavy chain dimer formation. This study compares and contrasts the in vitro properties of clenoliximab with its matched IgG1 derivative, keliximab, which shares the same variable regions. Both mAbs show potent inhibition of in vitro T cell responses, lack of binding to complement component C1q, and inability to mediate complement-dependent cytotoxicity. However, clenoliximab shows markedly reduced binding to Fc receptors and therefore does not mediate Ab-dependent cell-mediated cytotoxicity or modulation/loss of CD4 from the surface of T cells, except in the presence of rheumatoid factor or activated monocytes. Thus, clenoliximab retains the key immunomodulatory attributes of keliximab without the liability of strong Fcγ receptor binding. In initial clinical trials, these properties have translated to a reduced incidence of CD4+ T cell depletion.
Polymeric IgA Is Superior to Monomeric IgA and IgG Carrying the Same Variable Domain in Preventing Clostridium difficile Toxin A Damaging of T84 Monolayers
Authors: Stubbe Hiltrud , Berdoz José , Kraehenbuhl Jean-Pierre , Corthésy Blaise
Published: April 2014

The two exotoxins A and B produced by Clostridium difficile are responsible for antibiotic-associated enterocolitis in human and animals. When added apically to human colonic carcinoma-derived T84 cell monolayers, toxin A, but not toxin B, abolished the transepithelial electrical resistance and altered the morphological integrity. Apical addition of suboptimal concentration of toxin A made the cell monolayer sensitive to toxin B. Both toxins induced drastic and rapid epithelial alterations when applied basolaterally with a complete disorganization of tight junctions and vacuolization of the cells. Toxin A-specific IgG2a from hybridoma PCG-4 added apically with toxin A alone or in combination with toxin B abolished the toxin-induced epithelial alterations for up to 8 h. The Ab neutralized basolateral toxin A for 4 h, but not the mixture of the two toxins. Using an identical Ab:Ag ratio, we found that recombinant polymeric IgA (IgAd/p) with the same Fv fragments extended protection against toxin A for at least 24 h in both compartments. In contrast, the recombinant monomeric IgA counterpart behaved as the PCG-4 IgG2a Ab. The direct comparison between different Ig isotype and molecular forms, but of unique specificity, demonstrates that IgAd/p Ab is more efficient in neutralizing toxin A than monomeric IgG and IgA. We conclude that immune protection against C. difficile toxins requires toxin A-specific secretory Abs in the intestinal lumen and IgAd/p specific for both toxins in the lamina propria.
Murine Pro-B Cells Require IL-7 and Its Receptor Complex to Up-Regulate IL-7Rα, Terminal Deoxynucleotidyltransferase, and cμ Expression
Authors: Wei Chiju , Zeff Richard , Goldschneider Irving
Published: April 2014

Phenotypic analysis of bone marrow cells from IL-7 knockout (KO) mice revealed that B cell development is blocked precisely at the transition between pro-B cells and pre-B cells. In contrast, the generation of pre-pro-B cells and pro-B cells appeared to be normal, as judged by total cell numbers, proliferative indexes, D-JH and V-DJH gene rearrangements, and mRNA for recombinase-activating gene-1 (RAG-1), RAG-2, TdT, Igμ, λ5, and VpreB. However, upon closer inspection, several abnormalities in pro-B cell development were identified that could be corrected by injection of rIL-7 in vivo. These included the absence of the subset of late pro-B cells that initiates cμ expression for pre-B cell Ag receptor (BCR) formation, and the failure of pro-B cells to up-regulate TdT and the IL-7Rα (but not the common γ-chain) chain. Similar defects were present in common γ-chain and Jak3 KO mice, but not in λ5 or (excluding cytoplasmic Ig μ heavy chain (cμ)) RAG-1 KO mice, all of which also arrest at the late pro-B cell stage. Consequently, up-regulation of TdT and IL-7Rα expression requires signaling through the high affinity IL-7R, but does not require cμ expression or a functional pre-BCR. Taken together, these results suggest that IL-7 and its receptor complex are essential for 1) up-regulating the expression of TdT and IL-7Rα, 2) initiating the production of cμ, and 3) promoting the formation of a functional pre-BCR in/on pro-B cells. These key events, in turn, appear to be prerequisite both for differentiation of pro-B cells to pre-B cells and for proliferation of these cell subsets upon continued stimulation with IL-7.
Transfer of Immune Complexes from Erythrocyte CR1 to Mouse Macrophages
Authors: Reinagel Michele L. , Taylor Ronald P.
Published: April 2014

We are developing a potential therapeutic approach for removing pathogens from the circulation of primates in which the pathogen is bound to the complement receptor (CR1) on E using a bispecific mAb complex, a heteropolymer (HP). We have used mAb this approach to demonstrate that cleared prototype pathogens are localized to, phagocytosed in, and destroyed in the liver. Extension of this work to a clinical setting will require a detailed understanding of the mechanism by which the E-bound immune complex substrates are transferred to fixed tissue macrophages in the liver, the transfer reaction. Therefore, we examined an in vitro system to study this process using bacteriophage φX174 as a model pathogen. E containing φX174 (bound via an anti-CR1/anti-φX174 HP) were incubated with P388D1 murine macrophages, and the two cell types were separated by centrifugation through Ficoll. Both E and macrophages were then probed and analyzed by RIA or flow cytometry. The results indicate that all three components of the E-bound IC (φX174, HP, and CR1) were removed from the E and internalized by the macrophages. We found that transfer requires the Fc portion of IgG, because little transfer of φX174 occurs when it is bound to E CR1 using a HP containing only Fab fragments. These findings, taken in the context of other studies, suggest a general mechanism for the transfer reaction in which Fc receptors facilitate close juxtaposition of the macrophage to the E-bound IC which then allows a macrophage-associated protease to cleave CR1. The released IC are then internalized and processed by the macrophages.
NK Markers Are Expressed on a High Percentage of Virus-Specific CD8+ and CD4+ T Cells
Authors: Slifka Mark K. , Pagarigan Robb R. , Whitton J. Lindsay
Published: April 2014

NK cells have been phenotypically defined by the expression of specific markers such as NK1.1, DX5, and asialo-GM1 (ASGM1). In addition to NK cells, a small population of CD3+ T cells has been shown to express these markers, and a unique subpopulation of NK1.1+CD3+ T cells that expresses an invariant TCR has been named “NKT cells.” Here, we describe NK marker expression on a broad spectrum of MHC class I- and MHC class II-restricted T cells that are induced after acute viral infection. From 5 to >500 days post lymphocytic choriomeningitis virus (LCMV) infection, more than 90% of virus-specific CD8+ and CD4+ T cells coexpress one or more of these three prototypical NK markers. Furthermore, in vivo depletion of NK cells with anti-ASGM1 Ab resulted in the removal of 90% of virus-specific CD8+ T cells and 50–80% of virus-specific CD4+ T cells. This indicates that studies using in vivo depletion to determine the role of NK cells in immune defense could potentially be misinterpreted because of the unintended depletion of Ag-specific T cells. These results demonstrate that NK Ags are widely expressed on the majority of virus-specific T cells and indicate that the NK and T cell lineages may not be as distinct as previously believed. Moreover, the current nomenclature defining NKT cells will require comprehensive modification to include Ag-specific CD8+ and CD4+ T cells that express prototypical NK Ags.
CCR2 Expression Determines T1 Versus T2 Polarization During Pulmonary Cryptococcus neoformans Infection
Authors: Traynor Tim R. , Kuziel William A. , Toews Galen B. , Huffnagle Gary B.
Published: April 2014

Pulmonary clearance of the encapsulated yeast Cryptococcus neoformans requires the development of T1-type immunity. The objective of this study was to determine the role of CCR2 in leukocyte recruitment and development of T1-type cell-mediated immunity during pulmonary C. neoformans infection. Intratracheal inoculation of C. neoformans into CCR2 knockout (CCR2−/−) mice produced a prolonged pulmonary infection (5000-fold CFU at 6 wk compared with CCR2+/+ mice) and significant dissemination to the spleen and brain (160- and 800-fold greater). In addition, CCR2 deficiency resulted in significantly reduced recruitment of macrophages (weeks 1–3) and CD8+ T cells (weeks 1–2) into the lungs. The immune response in CCR2−/− mice was characterized by chronic pulmonary eosinophilia, crystal deposition in the lungs, pulmonary leukocyte production of IL-4 and IL-5 but not IFN-γ, lack of anticryptococcal delayed-type hypersensitivity, and high levels of serum IgE. These results demonstrate that expression of CCR2 is required for the development of a T1-type response to C. neoformans infection and lack of CCR2 results in a switch to a T2-type response. Thus, CCR2 plays a critical role in promoting the development of T1- over T2-type immune responses in the lung following cryptococcus infection.
Induction of E-Selectin Expression by Double-Stranded RNA and TNF-α Is Attenuated in Murine Aortic Endothelial Cells Derived from Double-Stranded RNA-Activated Kinase (PKR)-Null Mice
Authors: Bandyopadhyay Sudip K. , de la Motte Carol A. , Williams Bryan R. G.
Published: April 2014

The adherence of leukocytes on the endothelium is mediated in part by the transient expression of the E-selectin adhesion molecule. Because we have previously shown that the dsRNA-activated kinase PKR mediates dsRNA induction of NF-κB, we used murine aortic endothelial (MuAE) cells isolated from wild-type and PKR-null mice to investigate the role of PKR in the induction of E-selectin expression by dsRNA (pIC) and TNF-α. E-selectin mRNA and protein expression was inducible by both pIC and TNF-α in wild-type MuAE cells, whereas induction of E-selectin expression by these agents was defective in PKR-null MuAE cells. Induction of E-selectin promoter activity and NF-κB DNA binding activity were substantially reduced in pIC- or TNF-α-treated PKR-null cells, indicating a role for PKR in both pIC and TNF-α induction of E-selectin via an NF-κB-dependent pathway. In PKR-null cells, pIC-mediated degradation of IκBβ is deficient. Activation of this pathway requires the PKR-dependent degradation of the IκBβ protein. Moreover, both phosphorylated and unphosphorylated activating transcription factor 2 DNA-binding activities were reduced in PKR-null aortic endothelial cells. These results indicate that the PKR is required for full activation of E-selectin expression by pIC and TNF-α in primary mouse aortic endothelial cells identifying activating transcription factor 2 as a new target for PKR-dependent regulation and suggest a role for PKR in leukocyte adhesion.
Involvement of Cytosolic Phospholipase A2 and Secretory Phospholipase A2 in Arachidonic Acid Release from Human Neutrophils
Authors: Marshall John , Krump Eric , Lindsay Thomas , Downey Gregory , Ford David A. , Zhu Peihong , Walker Paul , Rubin Barry
Published: April 2014

The purpose of this study was to define the role of secretory phospholipase A2 (sPLA2), calcium-independent PLA2, and cytosolic PLA2 (cPLA2) in arachidonic acid (AA) release from fMLP-stimulated human neutrophils. While fMLP induced the release of extracellular sPLA2 activity and AA, 70% of sPLA2 activity remained associated with the cell. Treatment with the cell-impermeable sPLA2 inhibitors DTT or LY311-727, or the anti-sPLA2 Ab 3F10 all inactivated extracellular sPLA2 activity, but had minimal effect on neutrophil AA mass release. In contrast, coincubation of streptolysin-O toxin-permeabilized neutrophils with DTT, LY311-727, or 3F10 all decreased [3H8]AA release from [3H8]AA-labeled, fMLP-stimulated cells. Exposure to fMLP resulted in a decrease in the electrophoretic mobility of cPLA2, a finding consistent with cPLA2 phosphorylation, and stimulated the translocation of cPLA2 from cytosolic to microsomal and nuclear compartments. The role of cPLA2 was further evaluated with the cPLA2 inhibitor methyl arachidonyl fluorophosphonate, which attenuated cPLA2 activity in vitro and decreased fMLP-stimulated AA mass release by intact neutrophils, but had no effect on neutrophil sPLA2 activity. Inhibition of calcium-independent PLA2 with haloenol lactone suicide substrate had no effect on neutrophil cPLA2 activity or AA mass release. These results indicate a role for cPLA2 and an intracellular or cell-associated sPLA2 in the release of AA from fMLP-stimulated human neutrophils.
Production of IFN-γ and IL-10 to Shigella Invasins by Mononuclear Cells from Volunteers Orally Inoculated with a Shiga Toxin-Deleted Shigella dysenteriae Type 1 Strain
Authors: Samandari Taraz , Kotloff Karen L. , Losonsky Genevieve A. , Picking William D. , Sansonetti Philippe J. , Levine Myron M. , Sztein Marcelo B.
Published: April 2014

Volunteers were orally administered invasive, non-Shiga toxin-producing Shigella dysenteriae 1 to establish a challenge model to assess vaccine efficacy. In stepwise fashion, four separate groups were given 3 × 102, 7 × 103, 5 × 104, or 7 × 105 CFU. Using PBMC, proliferative responses and cytokine production were measured to S. dysenteriae whole-cell preparations and to purified recombinant invasion plasmid Ags (Ipa) C and IpaD. Anti-LPS and anti-Ipa Abs and Ab-secreting cells were also evaluated. Preinoculation PBMC produced considerable quantities of IL-10 and IFN-γ, probably secreted by monocytes and NK cells, respectively, of the innate immune system. Following inoculation, PBMC from 95 and 87% of volunteers exhibited an increased production of IFN-γ and IL-10, respectively, in response to Shigella Ags. These increases included responses to IpaC and IpaD among those volunteers receiving the lowest inoculum. No IL-4 or IL-5 responses were detected. Whereas there were no Ab or Ab-secreting cell responses in volunteers receiving the lowest inoculum, other dose groups had moderate to strong anti-LPS and anti-Ipa responses. These results suggest that in humans, type 1 responses play an important role in mucosal and systemic immunity to S. dysentariae 1.
IL-1β Protects Human Chondrocytes from CD95-Induced Apoptosis
Authors: Kühn Klaus , Hashimoto Sanshiro , Lotz Martin
Published: April 2014

This study addresses the effects of IL-1β on apoptosis induced by agonistic anti-CD95 (Fas) Ab. IL-1β inhibited anti-CD95 Ab-induced apoptosis in all preparations of normal human articular chondrocytes tested. Inhibitors of nitric oxide synthase or cyclooxygenase did not influence the protective effect of IL-1β, indicating that nitric oxide and PGs were not involved in the modulation of CD95-induced apoptosis. However, when the IL-1β-dependent induction of NF-κB was inhibited, the antiapoptotic effect of IL-1β was partially reversed, suggesting that NF-κB-mediated gene activation is part of the protective mechanism. In addition, IL-1β significantly increased the expression of Bcl-2. The protein tyrosine kinase inhibitor herbimycin A completely eliminated the protective effect of IL-1β on CD95-induced apoptosis. These findings suggest that IL-1β modulates the CD95 death cascade in chondrocytes by mechanisms that involve tyrosine phosphorylation events and NF-κB-dependent gene activation.
A Transcriptional Defect Underlies B Lymphocyte Dysfunction in a Patient Diagnosed with Non-X-Linked Hyper-IgM Syndrome
Authors: Bhushan Ameesha , Barnhart Bryan , Shone Scott , Song Charles , Covey Lori R.
Published: April 2014

To establish the underlying cause of hyper-IgM syndrome in one female patient, B cell function was examined in response to CD40- and IL-4-mediated pathways. When CD40-induced functional responses were measured in unfractionated B cells, CD80 up-regulation, de novo Cμ-Cγ recombination, and Iγ transcription were all found to be relatively unaffected. However, CD40- and IL-4-mediated CD23 up-regulation and VDJ-Cγ transcription were clearly diminished compared to control cells. IL-4-induced CD23 expression was measurably reduced in the CD20− population as well. These results suggested that the patient’s defect is positioned downstream of CD40 contact and affects both CD40− and IL-4 signal transduction pathways. Further analysis of B cell function in CD19+ B cells revealed a clear B cell defect with respect to Iγ and mature VDJ-Cγ transcription and IgG expression. However, under the same conditions Iε transcription was relatively normal. Partial restoration of B cell function occurred if PBMC or CD19+ B cells were cultured in vitro in the presence of CD154 plus IL-4. Because addition of IL-4 to cocultures containing activated T cells failed to induce B cells to undergo differentiation, the ability of the patient’s B cells to acquire a responsive phenotype correlated with receiving a sustained signal through CD40. These findings support a model in which the patient expresses an intrinsic defect that is manifested in the failure of specific genes to become transcriptionally active in response to either CD154 or IL-4 and results in a functionally unresponsive B cell phenotype.
A Bone Marrow-Derived APC in the Gut-Associated Lymphoid Tissue Captures Oral Antigens and Presents Them to Both CD4+ and CD8+ T Cells
Authors: Blanas Effrossini , Davey Gayle M. , Carbone Francis R. , Heath William R.
Published: April 2014

We have previously reported that feeding OVA to C57BL/6 mice can lead to a weak CTL response that is dependent on CD4+ T cell help and is capable of causing autoimmunity. In this study, we investigated the basis of the class I and class II-restricted Ag presentation required for such CTL induction. Two days after feeding OVA, Ag-specific CD4+ and CD8+ T cells were seen to proliferate in the Peyer’s patches and mesenteric lymph nodes. Little proliferation was evident in other lymphoid tissues, except at high Ags doses, in which case some dividing CD4+ T cells were observed in the spleen and peripheral lymph nodes. Using chimeric mice, the APC responsible for presenting orally derived Ags was shown to be derived from the bone marrow. Examination of the Ag dose required to activate either CD4+ or CD8+ T cells indicated that a single dose of 6 mg OVA was the minimum dose that consistently stimulated either T cell subset. These data indicate that oral Ags can be transported from the gut into the gut-associated lymphoid tissue, where they are captured by a bone marrow-derived APC and presented to both CD4+ and CD8+ T cells.
Normal Lymphoid Homeostasis and Lack of Lethal Autoimmunity in Mice Containing Mature T Cells with Severely Impaired IL-2 Receptors
Authors: Malek Thomas R. , Porter Brian O. , Codias Elaine K. , Scibelli Paul , Yu Aixin
Published: April 2014

The importance of IL-2Rβ function for immune regulation is highlighted by the severe impairment in lymphoid cell function in IL-2Rβ-deficient mice. It has been speculated that failed IL-2/IL-2R signaling in peripheral T cells causes the associated autoimmunity, imbalanced peripheral lymphoid homeostasis, and defective T cell function. This study explored the requirement for IL-2Rβ function in mature T lymphocytes. We show that transgenic thymic expression of the IL-2R β-chain in IL-2Rβ-deficient mice prevents lethal autoimmunity, restores normal production of B lymphocytes, and results in a peripheral T cell compartment that is responsive to triggering through the TCR, but not the IL-2R. The dysfunction of the IL-2R is illustrated by the near complete failure of mature T cells to proliferate to IL-2 in vitro and in vivo, to differentiate into CTL, and to up-regulate IL-2Rα expression. These data indicate that lymphoid homeostasis is largely maintained despite a nonfunctional IL-2R in mature T lymphocytes and suggest that IL-2Rβ provides an essential signal during thymic development to regulate self-reactivity.
Effects of Geldanamycin, a Heat-Shock Protein 90-Binding Agent, on T Cell Function and T Cell Nonreceptor Protein Tyrosine Kinases
Authors: Yorgin Peter D. , Hartson Steven D. , Fellah Abdul M. , Scroggins Bradley T. , Huang Wenjun , Katsanis Emmanuel , Couchman Jeff M. , Matts Robert L. , Whitesell Luke
Published: April 2014

The benzoquinoid ansamycins geldanamycin (GA), herbimycin, and their derivatives are emerging as novel therapeutic agents that act by inhibiting the 90-kDa heat-shock protein hsp90. We report that GA inhibits the proliferation of mitogen-activated T cells. GA is actively toxic to both resting and activated T cells; activated T cells appear to be especially vulnerable. The mechanism by which GA acts is reflected by its effects on an essential hsp90-dependent protein, the T cell-specific nonreceptor tyrosine kinase lck. GA treatment depletes lck levels in cultured T cells by a kinetically slow dose-dependent process. Pulse-chase analyses indicate that GA induces the very rapid degradation of newly synthesized lck molecules. GA also induces a slower degradation of mature lck populations. These results correlate with global losses in protein tyrosine kinase activity and an inability to respond to TCR stimuli, but the activity of mature lck is not immediately compromised. Although the specific proteasome inhibitor lactacystin provides marginal protection against GA-induced lck depletion, proteasome inhibition also induces changes in lck detergent solubility independent of GA application. There is no other evidence for the involvement of the proteosome. Lysosome inhibition provides quantitatively superior protection against degradation. These results indicate that pharmacologic inhibition of hsp90 chaperone function may represent a novel immunosuppressant strategy, and elaborate on the appropriate context in which to interpret losses of lck as a reporter for the pharmacology of GA in whole organisms.
Importance of Histamine in the Cytokine Network in the Lung Through H2 and H3 Receptors: Stimulation of IL-10 Production
Authors: Sirois Jocelyne , Ménard Geneviève , Moses Audric S. , Bissonnette Elyse Y.
Published: April 2014

Histamine, a well-known inflammatory mediator, has been implicated in various immunoregulatory effects that are poorly understood. Thus, we tested the hypothesis that histamine inhibits the release of a proinflammatory cytokine, namely TNF, by stimulating the release of an anti-inflammatory cytokine, IL-10. Alveolar macrophages (AMs) from humans, Sprague Dawley rats, and the AM cell line, NR8383, were treated with different concentrations of histamine (10−5-10−7 M) for 2 h prior to their stimulation with suboptimal concentration of LPS (1 ng/ml) for 4 h. Histamine inhibited TNF release in a dose-dependent manner. This inhibition was mimicked by H2 and H3 receptor agonists, but not by H1 receptor agonist. Furthermore, we demonstrated the expression of H3 receptor mRNA in human AMs. Interestingly, treatment of AMs with anti-IL-10, anti-PGE2, or a NO synthase inhibitor (Nω-nitro-l-arginine methyl ester) before the addition of histamine abrogated the inhibitory effect of the latter on TNF release. Histamine treatment (10−5 M) increased the release of IL-10 from unstimulated (2.2-fold) and LPS-stimulated (1.7-fold) AMs. Unstimulated AMs, NR8383, express few copies of IL-10 mRNA, as tested by quantitative PCR, but expression of IL-10 was increased by 1.5-fold with histamine treatment. Moreover, the stimulation of IL-10 release by histamine was abrogated by pretreatment with anti-PGE2 or the NO synthase inhibitor, Nω-nitro-l-arginine methyl ester. Thus, histamine increases the synthesis and release of IL-10 from AMs through PGE2 and NO production. These results suggest that histamine may play an important role in the modulation of the cytokine network.
Development and Analysis of Various Clonal Alloantigen- Dependent Cytotoxic Cell Lines from Channel Catfish
Authors: Stuge Tor B. , Wilson Melanie R. , Zhou He , Barker Katherine S. , Bengtén Eva , Chinchar Greg , Miller Norman W. , Clem L. William
Published: April 2014

To determine the phenotypes of cytotoxic cells in channel catfish, clonal alloantigen-dependent leukocyte lines were established from mixed leukocyte cultures. Each clone was analyzed for expression of TCR α and β genes by RT-PCR and for target cell specificity by 51Cr-release assay. Based on the above criteria, the following five different cell types were identified among the 19 clones analyzed: 1) TCR αβ+ allospecific cytotoxic cells, 2) TCR αβ+ nonspecific cytotoxic cells, 3) allospecific TCR αβ+ noncytotoxic cells, 4) TCR αβ− nonspecific cytotoxic cells, and 5) TCR αβ− allospecific cytotoxic cells. The demonstration of cloned, TCR αβ+, allospecific cytotoxic effectors provides the strongest evidence to date for the existence of cytotoxic T cells in fish.
Role of Activator Protein-1 in TCR-Mediated Regulation of the Murine fasl Promoter
Authors: Matsui Ken , Xiao Sheng , Fine Alan , Ju Shyr-Te
Published: April 2014

The present study demonstrates that transcription factor interactions are important in regulating the murine fasl promoter following TCR-mediated activation. We used DNase I-footprinting, EMSAs, and transient transfection assays to identify the minimal TCR signal-responsive region within the fasl promoter. This region contains the previously identified binding sites for NF-κB and Egr and the AP-1 site identified in this study. We found that TCR signaling induces AP-1 binding to this site and regulates the fasl promoter function in a fashion dependent on NF-κB binding. However, mutation in the AP-1 site alone did not show a significant effect on the promoter function. The data suggest that the minimal promoter required at least two transcription factors to function.