bluuebunny/crossref_metadata_2025 huggingface dataset:
Activating Ly-49 NK Receptors: Central Role in Cytokine and Chemokine Production
Authors: Ortaldo John R. , Bere Earl W. , Hodge Deborah , Young Howard A.
Published: April 2014

In an attempt to understand potential novel functions of receptors in vivo, we evaluated gene expression after cross-linking the activating Ly-49D mouse NK receptor. Gene expression was evaluated using a mouse GEM 2 microarray chip (Incyte Genomics, St. Louis, MO). Each chip displays a total of 8734 elements. The strongly induced genes fell into two categories: 1) soluble factors and 2) apoptotic genes. The majority of the strongly induced mRNAs as analyzed by microarray hybridization were chemokine genes. RNase protection assays and chemokine protein production analysis validated the microarray results, as cross-linking the Ly-49D mouse NK receptor induced high levels of IFN-γ, lymphotactin, macrophage-inflammatory protein (MIP)1α, and MIP1β. This gene expression was specific because other chemokines were not induced by anti-Ly-49D receptors. In addition, a series of pharmacological inhibitors were used to identify the key signaling pathways involved in the cellular response. The primary Ly-49D signaling for IFN-γ production is predominately mediated through Src kinase pathways involving membrane proximal events, whereas MIP1α and MIP1β gene induction is more complex and may involve multiple biochemical pathways. Thus, we conclude that a primary role for the activating NK receptors in vivo may be to trigger soluble factor production and regulation of the immune response. This would place NK cells and their activating Ly-49 receptors as important initiators of microbial immunity and key elements of the innate immune system.
Identification of Novel Functional Regions Important for the Activity of HOXB7 in Mammalian Cells
Authors: Yaron Yifah , McAdara Jeanne K. , Lynch Maureen , Hughes Elizabeth , Gasson Judith C.
Published: April 2014

Members of the HOX family of homeobox transcription factors play a role in pattern formation in diverse developmental systems. The clearly documented role of HOX genes in the proliferation and differentiation of primary hematopoietic cells and cell lines provides a convenient system to pursue a biochemical analysis of HOX gene function in mammalian cells. To explore the role of HOXB7 in myeloid hematopoiesis, a number of mutations and deletions in the gene were constructed that targeted sequences with known functions or in regions that had not been examined previously. The wild-type and mutant B7 constructs were introduced into the murine myelomonocytic cell line, 32D, and assayed for their effects on G-CSF-induced myeloid differentiation. Wild-type HOXB7 inhibited the differentiation of 32D cells, whereas mutations in the Pbx-binding pentapeptide motif or the DNA-binding homeodomain, as well as internal deletions of the N-terminal unique region, blocked this effect. Interestingly, mutations eliminating two target sites for casein kinase II, the glutamate-rich C terminus, or the first 14 amino acids of HOXB7, led to enhanced 32D differentiation. A model proposing a role for these regions of HOXB7 is presented.
Interaction Properties of Human Mannan-Binding Lectin (MBL)-Associated Serine Proteases-1 and -2, MBL-Associated Protein 19, and MBL
Authors: Thielens Nicole M. , Cseh Sándor , Thiel Steffen , Vorup-Jensen Thomas , Rossi Véronique , Jensenius Jens C. , Arlaud Gérard J.
Published: April 2014

The mannan-binding lectin (MBL) activation pathway of complement plays an important role in the innate immune defense against pathogenic microorganisms. In human serum, two MBL-associated serine proteases (MASP-1, MASP-2) and MBL-associated protein 19 (MAp19) were found to be associated with MBL. With a view to investigate the interaction properties of these proteins, human MASP-1, MASP-2, MAp19, as well as the N-terminal complement subcomponents C1r/C1s, Uegf, and bone morphogenetic protein-1-epidermal growth factor (CUB-EGF) segments of MASP-1 and MASP-2, were expressed in insect or human kidney cells, and MBL was isolated from human serum. Sedimentation velocity analysis indicated that the MASP-1 and MASP-2 CUB-EGF segments and the homologous protein MAp19 all behaved as homodimers (2.8–3.2 S) in the presence of Ca2+. Although the latter two dimers were not dissociated by EDTA, their physical properties were affected. In contrast, the MASP-1 CUB-EGF homodimer was not sensitive to EDTA. The three proteins and full-length MASP-1 and MASP-2 showed no interaction with each other as judged by gel filtration and surface plasmon resonance spectroscopy. Using the latter technique, MASP-1, MASP-2, their CUB-EGF segments, and MAp19 were each shown to bind to immobilized MBL, with KD values of 0.8 nM (MASP-2), 1.4 nM (MASP-1), 13.0 nM (MAp19 and MASP-2 CUB-EGF), and 25.7 nM (MASP-1 CUB-EGF). The binding was Ca2+-dependent and fully sensitive to EDTA in all cases. These data indicate that MASP-1, MASP-2, and MAp19 each associate as homodimers, and individually form Ca2+-dependent complexes with MBL through the CUB-EGF pair of each protein. This suggests that distinct MBL/MASP complexes may be involved in the activation or regulation of the MBL pathway.
Clustering of Class I HLA Oligomers with CD8 and TCR: Three-Dimensional Models Based on Fluorescence Resonance Energy Transfer and Crystallographic Data
Authors: Gáspár Rezső , Bagossi Péter , Bene László , Matkó János , Szöllősi János , Tőzsér József , Fésüs László , Waldmann Thomas A. , Damjanovich Sándor
Published: April 2014

Fluorescence resonance energy transfer (FRET) data, in accordance with lateral mobility measurements, suggested the existence of class I HLA dimers and oligomers at the surface of live human cells, including the B lymphoblast cell line (JY) used in the present study. Intra- and intermolecular class I HLA epitope distances were measured on JY B cells by FRET using fluorophore-conjugated Ag-binding fragments of mAbs W6/32 and L368 directed against structurally well-characterized heavy and light chain epitopes, respectively. Out-of-plane location of these epitopes relative to the membrane-bound BODIPY-PC (2-(4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine) was also determined by FRET. Computer-simulated docking of crystallographic structures of class I HLA and epitope-specific Ag-binding fragments, with experimentally determined interepitope and epitope to cell surface distances as constraints, revealed several sterically allowed and FRET-compatible class I HLA dimeric and tetrameric arrangements. Extension of the tetrameric class I HLA model with interacting TCR and CD8 resulted in a model of a supramolecular cluster that may exist physiologically and serve as a functionally significant unit for a network of CD8-HLA-I complexes providing enhanced signaling efficiency even at low MHC-peptide concentrations at the interface of effector and APCs.
N-Acetylglucosamine Prevents IL-1β-Mediated Activation of Human Chondrocytes
Authors: Shikhman Alexander R. , Kuhn Klaus , Alaaeddine Nada , Lotz Martin
Published: April 2014

Glucosamine represents one of the most commonly used drugs to treat osteoarthritis. However, mechanisms of its antiarthritic activities are still poorly understood. The present study identifies a novel mechanism of glucosamine-mediated anti-inflammatory activity. It is shown that both glucosamine and N-acetylglucosamine inhibit IL-1β- and TNF-α-induced NO production in normal human articular chondrocytes. The effect of the sugars on NO production is specific, since several other monosaccharides, including glucose, glucuronic acid, and N-acetylmannosamine, do not express this activity. Furthermore, N-acetylglucosamine polymers, including the dimer and the trimer, also do not affect NO production. The observed suppression of IL-1β-induced NO production is associated with inhibition of inducible NO synthase mRNA and protein expression. In addition, N-acetylglucosamine also suppresses the production of IL-1β-induced cyclooxygenase-2 and IL-6. The constitutively expressed cyclooxygenase-1, however, was not affected by the sugar. N-acetylglucosamine-mediated inhibition of the IL-1β response of human chondrocytes was not associated with the decreased inhibition of the mitogen-activated protein kinases c-Jun N-terminal kinase, extracellular signal-related kinase, and p38, nor with activation of the transcription factor NF-κB. In conclusion, these results demonstrate that N-acetylglucosamine expresses a unique range of activities and identifies a novel mechanism for the inhibition of inflammatory processes.
Enhanced Airway Th2 Response After Allergen Challenge in Mice Deficient in CC Chemokine Receptor-2 (CCR2)
Authors: Kim YongBok , Sung Sung-sang J. , Kuziel William A. , Feldman Sanford , Fu Shu Man , Rose C. Edward
Published: April 2014

To evaluate the role of CCR2 in allergic asthma, mutant mice deficient in CCR2 (CCR2−/−) and intact mice were sensitized with i.p. OVA with alum on days 0 and 7, and challenged by inhalation with nebulization of either OVA or saline. Airway hyperreactivity, measured by the methacholine-provoked increase in enhanced pause, was significantly increased (p < 0.05) in OVA-challenged CCR2−/− mutant mice, compared with comparably challenged CCR2+/+ mice. OVA-challenged CCR2−/− mutants also were also found to have enhanced bronchoalveolar lavage fluid eosinophilia, peribronchiolar cellular cuffing, and Ig subclass switching, with increase in OVA-specific IgG1 and IgE. In addition, RNase protection assay revealed increased whole lung expression of IL-13 in OVA-challenged CCR2−/− mutants. Unexpectedly, serum monocyte chemotactic protein-1 levels were 8-fold higher in CCR2−/− mutants than in CCR2+/+ mice sensitized to OVA, but OVA challenge had no additional effect on circulating monocyte chemotactic protein-1 in either genotype. Ag stimulation of lymphocytes isolated from OVA-sensitized CCR2 mutants revealed a significant increase (p < 0.05) in IL-5 production, which differed from OVA-stimulated lymphocytes from sensitized CCR2+/+ mice. These experiments demonstrate an enhanced response in airway reactivity and in lung inflammation in CCR2−/− mutant mice compared with comparably sensitized and challenged CCR2+/+ mice. These observations suggest that CC chemokines and their receptors are involved in immunomodulation of atopic asthma.
FcγRIII-Mediated Production of TNF-α Induces Immune Complex Alveolitis Independently of CXC Chemokine Generation
Authors: Chouchakova Nelli , Skokowa Julia , Baumann Ulrich , Tschernig Thomas , Philippens Karel M. H. , Nieswandt Bernhard , Schmidt Reinhold E. , Gessner J. Engelbert
Published: April 2014

We recently demonstrated a codominant role of C5aR and FcγRIII in the initiation of IgG immune complex-mediated inflammation in mice. In this study, we investigated the relative contribution of FcγRIII in the generation of several cytokines during experimental hypersensitivity pneumonitis/alveolitis in vivo. Induction of immune complex-alveolitis in C57BL/6 mice resulted in strong accumulation of neutrophils into the lung and enhanced chemotactic activity within bronchoalveolar lavage fluid accompanied by an increased production of the proinflammatory cytokines TNF-α and IL-1β as well as the ELR-CXC chemokines macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (KC). FcγRIII-deficient C57BL/6 mice (FcγRIII−/−) showed a marked reduction of the inflammatory response due to decreased production of TNF-α, IL-1β, and MIP-2. Results obtained in C57BL/6 mice either lacking the TNF-α class I receptor (TNF-αRI−/−) or treated with neutralizing anti-TNF-α mAb demonstrated an essential contribution of TNF-α for mediating IL-1β release, neutrophil influx, and hemorrhage. Surprisingly, MIP-2 and KC chemokine levels remained largely unaffected in TNF-αRI−/− mice or after functional inhibition of TNF-α. These data suggest that in immune complex alveolitis, the activation of FcγRIII may induce divergent downstream effector pathways with TNF-α acting independently of CXC chemokines to trigger the inflammatory response in C57BL/6 mice.
Spontaneous In Vivo Reversion of an Inherited Mutation in the Wiskott-Aldrich Syndrome
Authors: Ariga Tadashi , Kondoh Tatsuro , Yamaguchi Koji , Yamada Masafumi , Sasaki Satoshi , Nelson David L. , Ikeda Hisami , Kobayashi Kunihiko , Moriuchi Hiroyuki , Sakiyama Yukio
Published: April 2014

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency disease, arising from mutations of the WAS-protein (WASP) gene. Previously, we have reported that mononuclear cells from WAS patients showed lack/reduced of the intracellular WASP (WASPdim) by flow cytometric analysis, and analysis of WASP by flow cytometry (FCM-WASP) was useful for WAS diagnosis. In this study, we report a WAS patient who showed the unique pattern of FCM-WASP. The patient had the small population of normal expression of WASP (WASPbright) mononuclear cells together with the major WASPdim population. The WASPbright cells were detected in T cells, not in B cells or in monocytes. Surprisingly, the molecular studies of the WASPbright cells revealed that the inherited mutation of WASP gene was reversed to normal. His mother was proved as a WAS carrier, and HLA studies and microsatellite polymorphic studies proved that the WASPbright cells were derived from the patient himself. Therefore, we concluded that the WASPbright cells were resulted from spontaneous in vivo reversion of the inherited mutation. Furthermore, the scanning electron microscopic studies indicated that WASP-positive cells from the patient restored the dense microvillus surface projections that were hardly observed in the WASPdim cells. This case might have significant implications regarding the prospects of the future gene therapy for WAS patients.
Targeting Antigen in Mature Dendritic Cells for Simultaneous Stimulation of CD4+ and CD8+ T Cells
Authors: Bonini Chiara , Lee Steven P. , Riddell Stan R. , Greenberg Philip D.
Published: April 2014

Due to their potent immunostimulatory capacity, dendritic cells (DC) have become the centerpiece of many vaccine regimens. Immature DC (DCimm) capture, process, and present Ags to CD4+ lymphocytes, which reciprocally activate DCimm through CD40, and the resulting mature DC (DCmat) loose phagocytic capacity, but acquire the ability to efficiently stimulate CD8+ lymphocytes. Recombinant vaccinia viruses (rVV) provide a rapid, easy, and efficient method to introduce Ags into DC, but we observed that rVV infection of DCimm results in blockade of DC maturation in response to all activation signals, including CD40L, monocyte-conditioned medium, LPS, TNF-α, and poly(I:C), and failure to induce a CD8+ response. By contrast, DCmat can be infected with rVV and induce a CD8+ response, but, having lost phagocytic activity, fail to process the Ag via the exogenous class II pathway. To overcome these limitations, we used the CMV protein pp65 as a model Ag and designed a gene containing the lysosomal-associated membrane protein 1 targeting sequence (Sig-pp65-LAMP1) to target pp65 to the class II compartment. DCmat infected with rVV-Sig-pp65-LAMP1 induced proliferation of pp65-specific CD4+ clones and efficiently induced a pp65-specific CD4+ response, suggesting that after DC maturation the intracellular processing machinery for class II remains intact for at least 16 h. Moreover, infection of DCmat with rVV-Sig-pp65-LAMP1 resulted in at least equivalent presentation to CD8+ cells as infection with rVV-pp65. These results demonstrate that despite rVV interference with DCimm maturation, a single targeting vector can deliver Ags to DCmat for the effective simultaneous stimulation of both CD4+ and CD8+ cells.
T Cell Responses to HLA-A*0201-Restricted Peptides Derived from Human α Fetoprotein
Authors: Butterfield Lisa H. , Meng Wilson S. , Koh Andrew , Vollmer Charles M. , Ribas Antoni , Dissette Vivian B. , Faull Kym , Glaspy John A. , McBride William H. , Economou James S.
Published: April 2014

α fetoprotein (AFP)-derived peptide epitopes can be recognized by human T cells in the context of MHC class I. We determined the identity of AFP-derived peptides, presented in the context of HLA-A*0201, that could be recognized by the human (h) T cell repertoire. We screened 74 peptides and identified 3 new AFP epitopes, hAFP137–145, hAFP158–166, and hAFP325–334, in addition to the previously reported hAFP542–550. Each possesses two anchor residues and stabilized HLA-A*0201 on T2 cells in a concentration-dependent class I binding assay. The peptides were stable for 2–4 h in an off-kinetics assay. Each peptide induced peptide-specific T cells in vitro from several normal HLA-A*0201 donors. Importantly, these hAFP peptide-specific T cells also were capable of recognizing HLA-A*0201+/AFP+ tumor cells in both cytotoxicity assays and IFN-γ enzyme-linked immunospot assays. The immunogenicity of each peptide was tested in vivo with HLA-A*0201/Kb-transgenic mice. After immunization with each peptide emulsified in CFA, draining lymph node cells produced IFN-γ on recognition of cells stably transfected with hAFP. Furthermore, AFP peptide-specific T cells could be identified in the spleens of mice immunized with dendritic cells transduced with an AFP-expressing adenovirus (AdVhAFP). Three of four AFP peptides could be identified by mass spectrometric analysis of surface peptides from an HLA-A*0201 human hepatocellular carcinoma (HCC) cell line. Thus, compelling immunological and physiochemical evidence is presented that at least four hAFP-derived epitopes are naturally processed and presented in the context of class I, are immunogenic, and represent potential targets for hepatocellular carcinoma immunotherapy.
Enhancement of CD8+ T Cell Responses by ICOS/B7h Costimulation
Authors: Wallin Jeffrey J. , Liang Linda , Bakardjiev Anastasia , Sha William C.
Published: April 2014

Although the recently identified ICOS/B7h costimulatory counterreceptors are critical regulators of CD4+ T cell responses, their ability to regulate CD8+ responses is unclear. Here we report using a tumor-rejection model that ectopic B7h expression can costimulate rejection by CD8+ T cells in the absence of CD4+ T cells. Although responses of naive T cells were significantly augmented by priming with B7h, B7h was surprisingly effective in mobilizing recall responses of adoptively transferred T cells. To explore why secondary responses of CD8+ T cells were particularly enhanced by B7h, kinetics of ICOS up-regulation, proliferative responses, and cytokine production were compared from both naive and rechallenged 2C-transgenic T cells costimulated in vitro. Although B7h costimulated proliferative responses from both CD8+ populations, rechallenged cells were preferentially costimulated for IL-2 and IFN-γ production. These results indicate that ICOS/B7h counterreceptors likely function in vivo to enhance secondary responses by CD8+ T cells.
Molecular Characterization of B Cell Clonal Expansions in the Liver of Chronically Hepatitis C Virus-Infected Patients
Authors: Racanelli Vito , Sansonno Domenico , Piccoli Claudia , D’Amore Francesca Paola , Tucci Felicia Anna , Dammacco Franco
Published: April 2014

PCR DNA amplification of IgH genes was performed on liver biopsy samples of 42 unselected hepatitis C virus (HCV)-positive patients. Genotypic analysis and signal amplification by branched DNA were used to characterize and quantitate HCV RNA genomic sequences. Intraportal lymphoid follicle-like structures were isolated from surrounding hepatocytes by microdissection technique. IgH VDJ PCR products were cloned and sequenced. IgH VDJ gene rearrangements were detected in the liver of 26 (62%) patients. Unequivocal monoclonal or oligoclonal patterns of B cell expansions were found in 14 (33.3%) and 12 (28.6%) patients, respectively. Patients with intrahepatic B cell monoclonal expansions showed liver HCV RNA levels higher than those with oligoclonal or polyclonal features (1106.4 ± 593.5 vs 677.3 ± 424.3 vs 406.2 ± 354.3 pg HCV RNA/g tissue; p = 0.048 and p = 0.001, respectively). Although a single dominant band was obtained with total DNA, characterization of DNA recovered from intraportal inflammatory aggregates resulted in the detection of multiple IgH VDJ gene rearrangements, pointing to an oligoclonal pattern of lymphoproliferation. Cloning and sequence analyses showed that B cell clonalities were differently distributed in adjacent portal tracts of the same liver area. In addition, HCV RNA genomic sequences could be consistently amplified from each of the portal inflammatory aggregates examined. These data support the concept that in chronic HCV infection the intrahepatic B cell repertoire is frequently clonally restricted and that HCV may have a direct role in sustaining in situ B cell proliferation.
Positive Regulatory Role of IL-12 in Macrophages and Modulation by IFN-γ
Authors: Grohmann Ursula , Belladonna Maria L. , Vacca Carmine , Bianchi Roberta , Fallarino Francesca , Orabona Ciriana , Fioretti Maria C. , Puccetti Paolo
Published: April 2014

Similar to myeloid dendritic cells, murine macrophages and macrophage cell lines were found to express a surface receptor for IL-12. As a result, peritoneal macrophages could be primed by IL-12 to present an otherwise poorly immunogenic tumor peptide in vivo. Using binding analysis and RNase protection assay, we detected a single class of high affinity IL-12 binding sites (Kd of ∼35 pM) whose number per cell was increased by IFN-γ via up-regulation of receptor subunit expression. Autocrine production of IL-12 was suggested to be a major effect of IL-12 on macrophages when the cytokine was tested alone or after priming with IFN-γ in vitro. In vivo, combined treatment of macrophages with IFN-γ and IL-12 resulted in synergistic effects on tumor peptide presentation. Therefore, our findings suggest a general and critical role of IL-12 in potentiating the accessory function of myeloid APC.
Regulation of Dendritic Cell Recruitment into Resting and Inflamed Airway Epithelium: Use of Alternative Chemokine Receptors as a Function of Inducing Stimulus
Authors: Stumbles Philip A. , Strickland Deborah H. , Pimm Carolyn L. , Proksch Stephen F. , Marsh Amanda M. , McWilliam Andrew S. , Bosco Anthony , Tobagus Iriani , Thomas Jennifer A. , Napoli Sylvia , Proudfoot Amanda E. I. , Wells Timothy N. C. , Holt Patrick G.
Published: April 2014

Dendritic cells (DC) were purified by flow cytometry from rat tracheal mucosa; they exhibited the phenotypic characteristics of immature DC including high endocytic activity, low CD80/86 expression, and in vitro responsiveness to a broad range of CC chemokines. Daily treatment of adult rats with the selective CCR1 and CCR5 antagonist Met-RANTES reduced baseline numbers of tracheal intraepithelial DC by 50–60%, and pretreatment of animals with Met-RANTES before inhalation of aerosol containing heat-killed bacteria abolished the rapid DC influx into the epithelium that occurred in untreated controls, implicating CCR1 and CCR5 and their ligands in recruitment of immature DC precursors into resting airway tissues and during acute bacterial-induced inflammation. Comparable levels of DC recruitment were observed during airway mucosal Sendai virus infection and after aerosol challenge of sensitized animals with the soluble recall Ag OVA. However, Met-RANTES did not affect these latter responses, indicating the use of alternative chemokine receptors/ligands for DC recruitment, or possibly attraction of different DC subsets, depending on the nature of the eliciting stimulus.
B-Myb Overexpression Results in Activation and Increased Fas/Fas Ligand-Mediated Cytotoxicity of T and NK Cells
Authors: Powzaniuk Mark A. , Trotta Rossana , Loza Matthew J. , Harth Amy , Iozzo Renato V. , Eisenlohr Lawrence C. , Perussia Bice , Calabretta Bruno
Published: April 2014

The human B-myb gene encodes a transcriptional regulator that plays an important role in cell cycle progression, differentiation, and survival. To assess the in vivo role of B-myb, we investigated the phenotype of mouse transgenic lines in which B-Myb expression in lymphoid tissues was driven by the LCK proximal promoter. Overexpression of B-Myb had no measurable effect on the subsets of splenic and thymic lymphocytes, but was associated with increased expression of Fas ligand in NK and T cells. B-Myb-overexpressing splenocytes expressed higher IFN-γ levels and contained higher percentages of cytokine-producing cells than wild-type (wt) splenocytes, as detected by Western blot analysis and ELISPOT assays, respectively. Ex vivo-cultured transgenic thymocytes and splenocytes had decreased survival compared with the corresponding cells from wt mice, possibly dependent on increased expression of Fas ligand. In addition, Fas ligand-dependent cytotoxicity of transgenic T and NK cells was significantly higher than that mediated by their wt counterparts. Together, these results indicate that B-Myb overexpression results in T and NK cell activation and increased cytotoxicity. Therefore, in addition to its well-established role in proliferation and differentiation, B-myb also appears to be involved in activation of NK and T cells and in their regulation of Fas/Fas ligand-mediated cytotoxicity
Unequal VH Gene Rearrangement Frequency Within the Large VH7183 Gene Family Is Not Due to Recombination Signal Sequence Variation, and Mapping of the Genes Shows a Bias of Rearrangement Based on Chromosomal Location
Authors: Williams G. Stuart , Martinez Alan , Montalbano Alina , Tang Alan , Mauhar America , Ogwaro Kisani M. , Merz Denise , Chevillard Christophe , Riblet Roy , Feeney Ann J.
Published: April 2014

Much of the nonrandom usage of V, D, and J genes in the Ab repertoire is due to different frequencies with which gene segments undergo V(D)J rearrangement. The recombination signal sequences flanking each segment are seldom identical with consensus sequences, and this natural variation in recombination signal sequence (RSS) accounts for some differences in rearrangement frequencies in vivo. Here, we have sequenced the RSS of 19 individual VH7183 genes, revealing that the majority have one of two closely related RSS. One group has a consensus heptamer, and the other has a nonconsensus heptamer. In vitro recombination substrate studies show that the RSS with the nonconsensus heptamer, which include the frequently rearranging 81X, rearrange less well than the RSS with the consensus heptamer. Although 81X differs from the other 7183-I genes at three positions in the spacer, this does not significantly increase its recombination potency in vitro. The rearrangement frequency of all members of the family was determined in μMT mice, and there was no correlation between the in vitro recombination potential and VH gene rearrangement frequency in vivo. Furthermore, genes with identical RSS rearrange at different frequencies in vivo. This demonstrates that other factors can override differences in RSS potency in vivo. We have also determined the gene order of all VH7183 genes in a bacterial artificial chromosome contig and show that most of the frequently rearranging genes are in the 3′ half of the region. This suggests that chromosomal location plays an important role in nonrandom rearrangement of the VH7183 genes.
11β-Hydroxysteroid Dehydrogenase Type 1 Is Induced in Human Monocytes upon Differentiation to Macrophages
Authors: Thieringer Rolf , Le Grand Cheryl B. , Carbin Linda , Cai Tian-Quan , Wong Birming , Wright Samuel D. , Hermanowski-Vosatka Anne
Published: April 2014

11β-hydroxysteroid dehydrogenases (11β-HSD) perform prereceptor metabolism of glucocorticoids through interconversion of the active glucocorticoid, cortisol, with inactive cortisone. Although the immunosuppressive and anti-inflammatory activities of glucocorticoids are well documented, the expression of 11β-HSD enzymes in immune cells is not well understood. Here we demonstrate that 11β-HSD1, which converts cortisone to cortisol, is expressed only upon differentiation of human monocytes to macrophages. 11β-HSD1 expression is concomitant with the emergence of peroxisome proliferator activating receptor γ, which was used as a surrogate marker of monocyte differentiation. The type 2 enzyme, 11β-HSD2, which converts cortisol to cortisone, was not detectable in either monocytes or cultured macrophages. Incubation of monocytes with IL-4 or IL-13 induced 11β-HSD1 activity by up to 10-fold. IFN-γ, a known functional antagonist of IL-4 and IL-13, suppressed the induction of 11β-HSD1 by these cytokines. THP-1 cells, a human macrophage-like cell line, expressed 11β-HSD1 and low levels of 11β-HSD2. The expression of 11β-HSD1 in these cells is up-regulated 4-fold by LPS. In summary, we have shown strong expression of 11β-HSD1 in cultured human macrophages and THP-1 cells. The presence of the enzyme in these cells suggests that it may play a role in regulating the immune function of these cells.
Dendritic Cells Recruitment and In Vivo Priming of CD8+ CTL Induced by a Single Topical or Transepithelial Immunization Via the Buccal Mucosa with Measles Virus Nucleoprotein
Authors: Etchart Nathalie , Desmoulins Pierre-Olivier , Chemin Karine , Maliszewski Charles , Dubois Bertrand , Wild Fabian , Kaiserlian Dominique
Published: April 2014

The buccal mucosa, a prototype of pluristratified mucosal epithelia, contains a network of directly accessible class II+ epithelial dendritic cells (DC), similar to skin Langerhans cells. We showed that a single buccal immunization with measles virus nucleoprotein (NP), by either topical application onto or intradermal injection in the buccal mucosa, induced in vivo priming of protective class I-restricted specific CD8+ CTL. Both routes of immunization with NP induced a rapid recruitment of DC into the mucosa, which peaked at 2 h and decreased by 24 h. Treatment of mice with Flt3 ligand resulted in an increased number of DC in the buccal mucosa and enhanced the frequency of IFN-γ-producing NP-specific effectors and the NP-specific CTL response generated after buccal immunization with NP. Finally, NP-pulsed bone marrow-derived DC induced NP-specific IFN-γ-producing cells upon adoptive transfer to naive mice. These data demonstrate that a viral protein delivered to DC of the buccal mucosa induces in vivo priming of protective anti-viral CD8+ CTL.
The Distinct Roles of T Cell-Derived Cytokines and a Novel Follicular Dendritic Cell-Signaling Molecule 8D6 in Germinal Center-B Cell Differentiation
Authors: Zhang Xin , Li Li , Jung Jaeho , Xiang Shulin , Hollmann Christiane , Choi Yong Sung
Published: April 2014

Germinal center-B (GC-B) cells differentiate into memory B cells and plasma cells (PC) through interaction with T cells and follicular dendritic cells (FDC). Activated T cell and FDC play distinct roles in this process. The detailed kinetic experiments revealed that cytokines secreted by activated T cells determined the pathway of GC-B cell differentiation. IL-4 directs GC-B cells to differentiate into memory B cells, whereas IL-10 steers them into PC. FDC/HK cells do not direct either pathway, but provide signals for proliferation of GC-B cells. A novel FDC-signaling molecule 8D6 (FDC-SM-8D6) produced by FDC augments PC generation in the GC. FDC-SM-8D6-specific mAb blocked PC generation and IgG secretion but not memory B cell proliferation. COS cells expressing FDC-SM-8D6 enhanced GC-B cell proliferation and Ab secretion, which was blocked by mAb 8D6. In the cultures with B cell subsets, PC generation was inhibited by mAb 8D6 in the cultures with CD27+ B cells but not in the culture with CD27− B cells, suggesting that CD27+ PC precursor is the specific target of FDC-SM-8D6 stimulation.
Critical Role for IL-4 in the Development of Transplant Arteriosclerosis in the Absence of CD40-CD154 Costimulation
Authors: Ensminger Stephan M. , Spriewald Bernd M. , Sorensen Henrik V. , Witzke Oliver , Flashman Emily G. , Bushell Andrew , Morris Peter J. , Rose Marlene L. , Rahemtulla Amin , Wood Kathryn J.
Published: April 2014

Blockade of the CD40-CD154 pathway can inhibit CD4+ T cell activation but is unable to prevent immune responses mediated by CD8+ T cells. However, even in the absence of CD8+ T cells, inhibition of the CD40-CD154 pathway is insufficient to prevent the development of transplant arteriosclerosis. This study investigated the mechanisms of transplant arteriosclerosis in the absence of the CD40 pathway. C57BL/6 CD40−/− (H2b) recipients were transplanted with MHC-mismatched BALB/c (H2d) aortas. Transplant arteriosclerosis was evident in both CD40−/− and CD40+/− mice (intimal proliferation was 59 ± 5% for CD40−/− mice vs 58 ± 4% for CD40+/− mice) in the presence or absence of CD8+ T cells (intimal proliferation was 46 ± 7% for CD40−/− anti-CD8-treated mice vs 50 ± 10% for CD40+/− anti-CD8-treated mice), confirming that CD8+ T cells are not essential effector cells for the development of this disease. In CD40−/− recipients depleted of CD8+ T cells, the number of eosinophils infiltrating the graft was markedly increased (109 ± 24 eosinophils/grid for CD40−/− anti-CD8-treated mice vs 28 ± 7 for CD40+/− anti-CD8-treated mice). The increased presence of eosinophils correlated with augmented intragraft production of IL-4. To test the hypothesis that IL-4 was responsible for the intimal proliferation, CD8 T cell-depleted CD40−/− recipients were treated with anti-IL-4 mAb. This resulted in significantly reduced eosinophil infiltration into the graft (12 ± 5 eosinophils/grid for CD40−/− anti-CD8+, anti-IL-4-treated mice vs 109 ± 24 for CD40−/− anti-CD8-treated mice), intragraft eotaxin, CCR3 mRNA production, and the level of intimal proliferation (18 ± 5% for CD40−/− anti-CD8+-, anti-IL-4-treated mice vs 46 ± 7% for CD40−/− anti-CD8-treated mice). In conclusion, elevated intragraft IL-4 production results in an eosinophil infiltrate and is an important mechanism for CD8+ T cell-independent transplant arteriosclerosis in the absence of CD40-CD154 costimulation.
T Cell Epitope Mapping of the Smith Antigen Reveals That Highly Conserved Smith Antigen Motifs Are the Dominant Target of T Cell Immunity in Systemic Lupus Erythematosus
Authors: Talken Beth L. , Schäfermeyer Kim R. , Bailey Craig W. , Lee David R. , Hoffman Robert W.
Published: April 2014

B cell and T cell immunity to the Smith Ag (Sm) is a characteristic feature of systemic lupus erythematosus (SLE). We have shown that T cell immunity against Sm can be detected in SLE patients, and that T and B cell immunity against Sm are linked in vivo. TCR usage by Sm-reactive T cells is highly restricted and characteristic of an Ag-driven immune response. Sm is a well-characterized complex Ag consisting of proteins B1, B2, D1, D2, D3, E, F, and G. A unique feature of all Sm proteins is the presence of homologous motifs, Sm motif 1 and Sm motif 2. We used limiting dilution cloning and synthetic peptide Ags to characterize the human T cell immune response against Sm in seven SLE patients. We sought to determine the precise antigenic peptides recognized, the common features of antigenic structure recognized, and the evolution of the T cell response against Sm. We found there was a highly restricted set of Sm self-peptides recognized by T cells, with three epitopes on Sm-B and two epitopes on Sm-D. We found that T cell immunity against Sm-B and Sm-D was encoded within the highly conserved Sm motif 1 and Sm motif 2, and that immunity against these epitopes appeared stable. The present study supports the concept that T cell immunity to Sm is an Ag-driven immune response directed against a highly restricted set of self-peptides, encoded within Sm motif 1 and Sm motif 2, that is shared among all Sm proteins.
Therapeutic Alteration of Insulin-Dependent Diabetes Mellitus Progression by T Cell Tolerance to Glutamic Acid Decarboxylase 65 Peptides In Vitro and In Vivo
Authors: Wilson Stephen S. , White Todd C. , DeLuca Dominick
Published: April 2014

We have reported previously that nonobese diabetic (NOD) fetal pancreas organ cultures lose the ability to produce insulin when maintained in contact with NOD fetal thymus organ cultures (FTOC). Initial studies indicated that exposure to glutamic acid decarboxylase (GAD65) peptides in utero resulted in delay or transient protection from insulin-dependent diabetes mellitus (IDDM) in NOD mice. We also found that exposure of young adult NOD mice to the same peptides could result in acceleration of the disease. To more closely examine the effects of early and late exposure to diabetogenic Ags on T cells, we applied peptides derived from GAD65 (GAD AA 246–266, 509–528, and 524–543), to our “in vitro IDDM” (ivIDDM) model. T cells derived from NOD FTOC primed during the latter stages of organ culture, when mature T cell phenotypes are present, had the ability to proliferate to GAD peptides. ivIDDM was exacerbated under these conditions, suggesting that GAD responsiveness correlates with the ivIDDM phenotype, and parallels the acceleration of IDDM we had seen in young adult NOD mice. When GAD peptides were present during the initiation of FTOC, GAD proliferative responses were inhibited, and ivIDDM was reduced. This result suggests that tolerance to GAD peptides may reduce the production of diabetogenic T cells or their capacity to respond, as suggested by the in utero therapies studied in NOD mice.
Lymphotactin Expression by Engineered Myeloma Cells Drives Tumor Regression: Mediation by CD4+ and CD8+ T Cells and Neutrophils Expressing XCR1 Receptor
Authors: Cairns Chantelle M. , Gordon John R. , Li Fang , Baca-Estrada Maria E. , Moyana Terence , Xiang Jim
Published: April 2014

The C chemokine lymphotactin has been characterized as a T cell chemoattractant both in vitro and in vivo. To determine whether lymphotactin expression within tumors could influence tumor growth, we transfected an expression vector for lymphotactin into SP2/0 myeloma cells and tested their ability to form tumors in BALB/c and nude mice. Transfection did not alter cell growth in vitro. Whereas SP2/0 cells gave rise to a 100% tumor incidence, lymphotactin-expressing SP2/0-Lptn tumors invariably regressed in BALB/c mice and became infiltrated with CD4+ and CD8+ T cells and neutrophils. Regression of the SP2/0-Lptn tumors was associated with a type 1 cytokine response and dependent on both CD4+ and CD8+ T cells, but not NK cells. Both SP2/0 and SP2/0-Lptn tumors grew in nude mice, but growth of the latter tumors was retarded and associated with heavy neutrophil responses; this retardation of SP2/0-Lptn tumor growth was reversed by neutrophil depletion of the mice. Our data also indicate that mouse neutrophils express the lymphotactin receptor XCR1 and that lymphotactin specifically chemoattracts these cells in vitro. Thus, lymphotactin has natural adjuvant activities that may augment antitumor responses via effects on both T cells and neutrophils and thereby could be important in gene transfer immunotherapies for some cancers.
Nitric Oxide Inhibits the Transcription Repressor Yin-Yang 1 Binding Activity at the Silencer Region of the Fas Promoter: A Pivotal Role for Nitric Oxide in the Up-Regulation of Fas Gene Expression in Human Tumor Cells
Authors: Garbán Hermes J. , Bonavida Benjamin
Published: April 2014

NO has been increasingly implicated in control of the transcriptional machinery and serves as an intracellular second messenger to modify gene expression. We have demonstrated that NO up-regulated Fas receptor expression in ovarian carcinoma cell lines, albeit the mechanism involved is not known. Thus, we hypothesized that NO, directly or indirectly, may modify the transcriptional machinery that is responsible for the increased expression of the Fas gene. We examined the effect of NO on Fas gene expression using a Fas promoter-driven luciferase reporter system. Transient transfection of AD10 cells with pGL-3-FasP demonstrated that the IFN-γ-dependent NO generation increases the trans-activation of the Fas promoter, and this increase was blocked by the NOS inhibitor (NG-monomethyl-l-arginine), but could be restored by the addition of the NO donor S-nitroso-N-acetylpenicillamine. Systematic deletion of the Fas promoter revealed that the functional region responsible for the NO-mediated effect was located at the silencer region, suggesting that NO may be responsible for the disruption of a repressor mechanism. We demonstrate that NO up-regulates the expression of the Fas receptor on AD10 cells via the specific inactivation of the transcription repressor yin-yang 1 DNA binding activity to the silencer region of the Fas promoter. These findings reveal a new mechanism of NO-mediated gene regulation by interfering with a repressor transcription factor at the silencer region of the Fas promoter.
Role of CD28-B7 Interactions in Generation and Maintenance of CD8 T Cell Memory
Authors: Suresh M. , Whitmire Jason K. , Harrington Laurie E. , Larsen Christian P. , Pearson Thomas C. , Altman John D. , Ahmed Rafi
Published: April 2014

Although the role of CD28-B7 interaction in the activation of naive T cells is well established, its importance in the generation and maintenance of T cell memory is not well understood. In this study, we examined the requirement for CD28-B7 interactions in primary T cell activation and immune memory. Ag-specific CD8 T cell responses were compared between wild-type (+/+) and CD28-deficient (CD28−/−) mice following an acute infection with lymphocytic choriomeningitis virus (LCMV). During the primary response, there was a substantial activation and expansion of LCMV-specific CD8 T cells in both +/+ and CD28−/− mice. However, the magnitude of the primary CD8 T cell response to both dominant and subdominant LCMV CTL epitopes was ∼2- to 3-fold lower in CD28−/− mice compared with +/+ mice; the lack of CD28-mediated costimulation did not lead to preferential suppression of CD8 T cell responses to the weaker subdominant epitopes. As seen in CD28−/− mice, blockade of B7-mediated costimulation by CTLA4-Ig treatment of +/+ mice also resulted in a 2-fold reduction in the anti-LCMV CD8 T cell responses. Loss of CD28/B7 interactions did not significantly affect the generation and maintenance of CD8 T cell memory; the magnitude of CD8 T cell memory was ∼2-fold lower in CD28−/− mice as compared with +/+ mice. Further, in CD28−/− mice, LCMV-specific memory CD8 T cells showed normal homeostatic proliferation in vivo and also conferred protective immunity. Therefore, CD28 signaling is not necessary for the proliferative renewal and maintenance of memory CD8 T cells.
Regulation of IFN-γ Signaling Is Essential for the Cytotoxic Activity of CD8+ T Cells
Authors: Tau Gregory Z. , Cowan Simone N. , Weisburg Jeffrey , Braunstein Ned S. , Rothman Paul B.
Published: April 2014

Previous studies have demonstrated that, as naive murine CD4+ cells differentiate into Th1 cells, they lose expression of the second chain of IFN-γR (IFN-γR2). Hence, the IFN-γ-producing subset of Th cells is unresponsive to IFN-γ. Analysis of IFN-γ-producing CD8+ T cells demonstrates that, like Th1 cells, these cells do not express IFN-γR2. To define the importance of IFN-γ signaling for the development of functional CD8+ T cells, mice either lacking IFN-γR2 or overexpressing this protein were examined. While CD8+ T cell development and function appear normal in IFN-γR2−/− mice, CD8+ T cell function in IFN-γR2 transgenic is altered. IFN-γR2 transgenic CD8+ T cells are unable to lyse target cells in vitro. However, these cells produce Fas ligand, perforin, and granzyme B, the effector molecules required for killing. Interestingly, TG CD8+ T cells proliferate normally and produce cytokines, such as IFN-γ in response to antigenic stimulation. Therefore, although IFN-γ signaling is not required for the generation of normal cytotoxic T cells, constitutive IFN-γ signaling can selectively impair the cytotoxic function of CD8+ T cells.
Listeria monocytogenesInfection Overcomes the Requirement for CD40 Ligand in Exogenous Antigen Presentation to CD8+ T Cells
Authors: Hamilton Sara E. , Tvinnereim Amy R. , Harty John T.
Published: April 2014

In vivo priming of CD8+ T lymphocytes against exogenously processed model Ags requires CD4+ T cell help, specifically interactions between CD40 ligand (CD40L) expressed by activated CD4+ T cells and CD40, which is present on professional APC such as dendritic cells (DCs). To address this issue in the context of bacterial infection, we examined CD40L-CD40 interactions in CD8+ T cell priming against an exogenously processed, nonsecreted bacterial Ag. CD40L interactions were blocked by in vivo treatment with anti-CD40L mAb MR-1, which inhibited germinal center formation and CD8+ T cell cross-priming against an exogenous model Ag, OVA. In contrast, MR-1 treatment did not interfere with CD8+ T cell priming against a nonsecreted or secreted recombinant Ag expressed by Listeria monocytogenes. Memory and secondary responses of CD8+ T cells against nonsecreted and secreted bacterial Ags were also largely unimpaired by transient MR-1 treatment. When MR-1-treated mice were concurrently immunized with L. monocytogenes and OVA-loaded splenocytes, cross-priming of OVA-specific naive CD8+ T cells occurred. No significant decline in cross-priming against OVA was measured when either TNF or IFN-γ was neutralized in L. monocytogenes-infected animals, demonstrating that multiple signals exist to overcome CD40L blockade of CD8+ T cell cross-priming during bacterial infection. These data support a model in which DCs can be stimulated in vivo through signals other than CD40, becoming APC that can effectively stimulate CD8+ T cell responses against exogenous Ags during infection.
Antagonistic Roles for CTLA-4 and the Mammalian Target of Rapamycin in the Regulation of Clonal Anergy: Enhanced Cell Cycle Progression Promotes Recall Antigen Responsiveness
Authors: Vanasek Tracy L. , Khoruts Alexander , Zell Traci , Mueller Daniel L.
Published: April 2014

CD4+ T cells that undergo multiple rounds of cell division during primary Ag challenge in vivo produce IL-2 on secondary Ag rechallenge, whereas cells that fail to progress through the cell cycle are anergic to restimulation. Anti-CTLA-4 mAb treatment during primary Ag exposure increases cell cycle progression and enhances recall Ag responsiveness; however, simultaneous treatment with rapamycin, an inhibitor of the mammalian target of rapamycin and potent antiproliferative agent, prevents both effects. The data suggest that cell cycle progression plays a primary role in the regulation of recall Ag responsiveness in CD4+ T cells in vivo. CTLA-4 molecules promote clonal anergy development only indirectly by limiting cell cycle progression during the primary response.