bluuebunny/crossref_metadata_2025 huggingface dataset:
Vaccination with a Melan-A Peptide Selects an Oligoclonal T Cell Population with Increased Functional Avidity and Tumor Reactivity
Authors: Valmori Danila , Dutoit Valerie , Schnuriger Valerie , Quiquerez Anne-Lise , Pittet Mikaël J. , Guillaume Philippe , Rubio-Godoy Verena , Walker Paul R. , Rimoldi Donata , Liénard Danielle , Cerottini Jean-Charles , Romero Pedro , Dietrich Pierre-Yves
Published: April 2014

Both the underlying molecular mechanisms and the kinetics of TCR repertoire selection following vaccination against tumor Ags in humans have remained largely unexplored. To gain insight into these questions, we performed a functional and structural longitudinal analysis of the TCR of circulating CD8+ T cells specific for the HLA-A2-restricted immunodominant epitope from the melanocyte differentiation Ag Melan-A in a melanoma patient who developed a vigorous and sustained Ag-specific T cell response following vaccination with the corresponding synthetic peptide. We observed an increase in functional avidity of Ag recognition and in tumor reactivity in the postimmune Melan-A-specific populations as compared with the preimmune blood sample. Improved Ag recognition correlated with an increase in the t1/2 of peptide/MHC interaction with the TCR as assessed by kinetic analysis of A2/Melan-A peptide multimer staining decay. Ex vivo analysis of the clonal composition of Melan-A-specific CD8+ T cells at different time points during vaccination revealed that the response was the result of asynchronous expansion of several distinct T cell clones. Some of these T cell clones were also identified at a metastatic tumor site. Collectively, these data show that tumor peptide-driven immune stimulation leads to the selection of high-avidity T cell clones of increased tumor reactivity that independently evolve within oligoclonal populations.
Cutting Edge: Molecular Mechanisms of Synergy Between CD40 and the B Cell Antigen Receptor: Role for TNF Receptor-Associated Factor 2 in Receptor Interaction
Authors: Haxhinasto Sokol A. , Hostager Bruce S. , Bishop Gail A.
Published: April 2014

Optimal Ag-specific B lymphocyte activation requires both recognition of Ag by the B cell Ag receptor (BCR) and contact-mediated interactions with Ag-specific Th lymphocytes. One of these interactions involves ligation of B cell CD40 by T cell-expressed CD154. CD40 signaling is crucial for Ab production, isotype switching, up-regulation of surface molecules, development of germinal centers, and the humoral memory response. The signaling pathways emanating from the BCR and CD40 are able to cooperate, but the molecular mechanisms responsible for this interaction are incompletely understood. The present study explored the roles of signaling motifs in the CD40 cytoplasmic tail in this synergy. We find that threonine in the PXQXT motif in the TNFR-associated factor-2 binding site is critical for synergistic effects of CD40 and BCR signals, independent of its phosphorylation. Furthermore, data suggest an indirect role for TNFR-associated factor-2 in the cooperative signaling.
Direct Ex Vivo Analysis of Human CD4+ Memory T Cell Activation Requirements at the Single Clonotype Level
Authors: Bitmansour Arlene D. , Douek Daniel C. , Maino Vernon C. , Picker Louis J.
Published: April 2014

CD4+ memory T cells continuously integrate signals transmitted through the TCR and costimulatory molecules, only responding when the intensity of such signals exceeds an intrinsic activation threshold. Recent data suggest that these activation thresholds can be regulated independently of TCR specificity, and that threshold tuning may constitute a major mechanism for controlling T cell effector activity. In this work we take advantage of the profound clonotypic hierarchies of the large human CD4+ T cell response to CMV to study activation thresholds of fresh (unexpanded) memory T cells at the clonotypic level. We identified dominant responses to CMV matrix determinants mediated by single TCRB sequences within particular TCR-Vβ families. The specific response characteristics of these single, Ag-specific, TCRB-defined clonotypes could be unequivocally determined in fresh PBMC preparations by cytokine flow cytometry with gating on the appropriate Vβ family. These analyses revealed 1) optimal peptides capable of eliciting specific responses by themselves at doses as low as 2 pg/ml, with each log increase in dose eliciting ever-increasing frequencies of responding cells over a 4- to 5-log range; 2) significant augmentation of response frequencies at all submaximal peptide doses by CD28- and CD49d-mediated costimulation; 3) differential dose response and costimulatory characteristics for IFN-γ and IL-2 responses; and 4) no association of activation requirements with the CD27-defined CD4+ T cell memory differentiation pathway. Taken together these data confirm that triggering heterogeneity exists within individual CD4+ memory T cell clonotypes in vivo and demonstrate that such single clonotypes can manifest qualitatively different functional responses depending on epitope dose and relative levels of costimulation.
Priming Biologically Active Antibody Responses Against an Isolated, Conformational Viral Epitope by DNA Vaccination
Authors: Riedl Petra , El Kholy Shereen , Reimann Jörg , Schirmbeck Reinhold
Published: April 2014

The immunodominant, conformational “a” determinant of hepatitis B surface Ag (HBsAg) elicits Ab responses. We selectively expressed the Ab-binding, glycosylated, native a determinant (residue 120–147) of HBsAg in a fusion protein containing C-terminally the HBsAg fragment SII (residue 80–180) fused to a SV40 T-Ag-derived hsp73-binding 77 aa (T77) or non-hsp-binding 60 aa (T60) N terminus. A DNA vaccine encoding non-hsp-binding secreted T60-SII fusion protein-stimulated murine Ab responses with a similar efficacy as a DNA vaccine encoding the secreted, native, small HBsAg. A DNA vaccine encoding hsp73-binding, intracellular T77-SII fusion protein-stimulated murine Ab responses less efficiently but comparable to a DNA vaccine encoding the intracellular, native, large HBsAg. HBsAg-specific Abs elicited by either the T60-SII-expressing or the T77-SII-expressing DNA vaccine suppressed HBsAg antigenemia in transgenic mice that produce HBsAg from a transgene in the liver; hence, a biologically active B cell response cross-reacting with the native, viral envelope epitope was primed by both DNA vaccine constructs. HBsAg-specific Ab and CTL responses were coprimed when an S20–50 fragment (containing the immunodominant, Ld-binding epitope S28–39) of HBsAg was fused C-terminally to the pCI/T77-SII sequence (pCI/T77-SII-Ld DNA vaccine). Chimeric, polyepitope DNA vaccines encoding conformational, Ab-binding epitopes and MHC class I-binding epitopes can thus efficiently deliver antigenic information to different compartments of the immune system in an immunogenic way.
IFN Consensus Sequence Binding Protein/IFN Regulatory Factor-8 Guides Bone Marrow Progenitor Cells Toward the Macrophage Lineage
Authors: Tsujimura Hideki , Nagamura-Inoue Tokiko , Tamura Tomohiko , Ozato Keiko
Published: April 2014

IFN consensus sequence binding protein (ICSBP; IFN regulatory factor-8) is a transcription factor of the IFN regulatory factor family. Disruption of this gene results in a leukemia-like disease in mice. To investigate the role of ICSBP in myeloid cell development, lineage marker-negative (Lin−) bone marrow progenitor cells were purified from ICSBP+/+ and ICSBP−/− mice and tested for gene expression and colony-forming ability. ICSBP was expressed in Lin− progenitor cells, and its levels were markedly increased by IFN-γ. The colony-forming potential of ICSBP−/− progenitor cells was grossly abnormal, as they gave rise to a disproportionately high number of granulocyte colonies and many fewer macrophage colonies. IFN-γ inhibited colony formation, while promoting macrophage maturation in ICSBP+/+ cells. In contrast, the effects of IFN-γ were completely absent in ICSBP−/− progenitors. By retrovirus transduction we tested whether reintroduction of ICSBP restores a normal colony-forming potential in −/− progenitor cells. The wild-type ICSBP, but not transcriptionally defective mutants, corrected abnormal colony formation by increasing macrophage colonies and decreasing granulocyte colonies. Taken together, ICSBP plays a critical role in myeloid cell development by controlling lineage selection and is indispensable for IFN-γ-dependent modulation of progenitor cell maturation.
Two Distinct Domains Within the N-Terminal Region of Janus Kinase 1 Interact with Cytokine Receptors
Authors: Usacheva Anna , Kotenko Sergei , Witte Michael M. , Colamonici Oscar R.
Published: April 2014

The interaction between receptors and kinases of the Janus kinase (Jak) family is critical for signaling by growth factors, cytokines, and IFNs. Therefore, the characterization of the domains involved in these interactions is pivotal not only in understanding kinase activation but also in the development of drugs that mimic or inhibit signaling. In this report, we have characterized the domains of Jak1 required to associate with distinct cytokine receptor subunits: IFN-αRβL, IFN-γRα, IL-10Rα, IL-2Rβ, and IL-4Rα. We demonstrate that two regions of Jak1 are necessary for the interaction with cytokine receptors. First, a common N-terminal region that includes Jak homology (JH) domain 7 and the first 19 aa of JH6, and, second, a C-terminal region (JH6–3) that was different for distinct receptors. The contribution of the two different regions of Jak1 to cytokine receptor binding was also variable. Deletion of JH7–6 impaired the association of IL-2Rβ and IL-4Rα chains with Jak1 but did not have a major impact on the binding of Jak1 to IFN-αRβL or IL-10Rα. Interestingly, regardless of the effect on receptor binding, removal of JH7–6 completely abrogated kinase activation, indicating that this domain is required for ligand-driven kinase activation and, thus, for proper signaling through cytokine receptors.
Isolation and Detection of Human IgA Using a Streptococcal IgA-Binding Peptide
Authors: Sandin Charlotta , Linse Sara , Areschoug Thomas , Woof Jenny M. , Reinholdt Jesper , Lindahl Gunnar
Published: April 2014

Bacterial proteins that bind to the Fc part of IgG have found widespread use in immunology. A similar protein suitable for the isolation and detection of human IgA has not been described. Here, we show that a 50-residue synthetic peptide, designated streptococcal IgA-binding peptide (Sap) and derived from a streptococcal M protein, can be used for single-step affinity purification of human IgA. High affinity binding of IgA required the presence in Sap of a C-terminal cysteine residue, not present in the intact M protein. Passage of human serum through a Sap column caused depletion of >99% of the IgA, and elution of the column allowed quantitative recovery of highly purified IgA, for which the proportions of the IgA1 and IgA2 subclasses were the same as in whole serum. Moreover, immobilized Sap could be used for single-step purification of secretory IgA of both subclasses from human saliva, with a recovery of ∼45%. The Sap peptide could also be used to specifically detect IgA bound to Ag. Together, these data indicate that Sap is a versatile Fc-binding reagent that may open new possibilities for the characterization of human IgA.
The Rap GTPases Regulate B Cell Migration Toward the Chemokine Stromal Cell-Derived Factor-1 (CXCL12): Potential Role for Rap2 in Promoting B Cell Migration
Authors: McLeod Sarah J. , Li Anson H. Y. , Lee Rosaline L. , Burgess Anita E. , Gold Michael R.
Published: April 2014

Stromal cell-derived factor-1 (SDF-1) is a potent chemoattractant for B cells and B cell progenitors. Although the binding of SDF-1 to its receptor, CXCR4, activates multiple signaling pathways, the mechanism by which SDF-1 regulates cell migration is not completely understood. In this report we show that activation of the Rap GTPases is important for B cells to migrate toward SDF-1. We found that treating B cells with SDF-1 resulted in the rapid activation of both Rap1 and Rap2. Moreover, blocking the activation of Rap1 and Rap2 via the expression of a Rap-specific GTPase-activating protein significantly reduced the ability of B cells to migrate toward SDF-1. Conversely, expressing a constitutively active form of Rap2 increased SDF-1-induced B cell migration. Thus, the Rap GTPases control cellular processes that are important for B cells to migrate toward SDF-1.
Mitogen-Activated Protein Kinases and NF-κB Are Involved in TNF-α Responses to Group B Streptococci
Authors: Mancuso Giuseppe , Midiri Angelina , Beninati Concetta , Piraino Giovanna , Valenti Andrea , Nicocia Giacomo , Teti Diana , Cook James , Teti Giuseppe
Published: April 2014

TNF-α is a mediator of lethality in experimental infections by group B streptococcus (GBS), an important human pathogen. Little is known of signal transduction pathways involved in GBS-induced TNF-α production. Here we investigate the role of mitogen-activated protein kinases (MAPKs) and NF-κB in TNF-α production by human monocytes stimulated with GBS or LPS, used as a positive control. Western blot analysis of cell lysates indicates that extracellular signal-regulated kinase 1/2 (ERK 1/2), p38, and c-Jun N-terminal kinase MAPKs, as well as IκBα, became phosphorylated, and hence activated, in both LPS- and GBS-stimulated monocytes. The kinetics of these phosphorylation events, as well as those of TNF-α production, were delayed by 30–60 min in GBS-stimulated, relative to LPS-stimulated, monocytes. Selective inhibitors of ERK 1/2 (PD98059 or U0126), p38 (SB203580), or NF-κB (caffeic acid phenetyl ester (CAPE)) could all significantly reduce TNF-α production, although none of the inhibitors used alone was able to completely prevent TNF-α release. However, this was completely blocked by combinations of the inhibitors, including PD98059-SB203580, PD98059-CAPE, or SB203580-CAPE combinations, in both LPS- and GBS-stimulated monocytes. In conclusion, our data indicate that the simultaneous activation of multiple pathways, including NF-κB, ERK 1/2, and p38 MAPKs, is required to induce maximal TNF-α production. Accordingly, in septic shock caused by either GBS or Gram-negative bacteria, complete inhibition of TNF-α release may require treatment with drugs or drug combinations capable of inhibiting multiple activation pathways.
Antibodies Highly Effective in SCID Mice During Infection by the Intracellular BacteriumEhrlichia chaffeensisAre of Picomolar Affinity and Exhibit Preferential Epitope and Isotype Utilization
Authors: Li Julia Shu-yi , Chu Frederick , Reilly Andrew , Winslow Gary M.
Published: April 2014

Although often considered to be ineffective against intracellular bacteria, Abs, in the absence of lymphocytes, have been shown previously to protect SCID mice from lethal infection by the obligate intracellular bacterium Ehrlichia chaffeensis, even when administered well after infection has been established. To identify characteristics of Abs that are critical for host defense during this intracellular infection, a panel of Ehrlichia-specific mAbs was generated and analyzed. Among 100 Abs recovered, 39 recognized an amino-terminal hypervariable region of an outer membrane protein (OMP), demonstrating that the OMPs are both antigenically variable and immunodominant. A subset of 16 representative OMP-specific Abs was further examined to identify characteristics that were essential for in vivo efficacy. The highly effective Abs recognized a linear epitope within the first hypervariable region of OMP-1g. Only IgG were found to be effective, and among the effective IgG, the following hierarchy was observed: IgG2a > IgG3 = IgG2b. The most striking characteristics of the highly effective Abs were their picomolar binding affinities and long binding t1/2. Thus, although epitope recognition and isotype use may contribute to efficacy, high affinity may be a critical characteristic of Abs that can act effectively during this intracellular bacterial infection.
Viral and Bacterial Infections Induce Expression of Multiple NK Cell Receptors in Responding CD8+ T Cells
Authors: McMahon Christopher W. , Zajac Allan J. , Jamieson Amanda M. , Corral Laura , Hammer Gianna E. , Ahmed Rafi , Raulet David H.
Published: April 2014

NK cells express several families of receptors that play central roles in target cell recognition. These NK cell receptors are also expressed by certain memory phenotype CD8+ T cells, and in some cases are up-regulated in T cells responding to viral infection. To determine how the profile of NK receptor expression changes in murine CD8+ T cells as they respond to intracellular pathogens, we used class I tetramer reagents to directly examine Ag-specific T cells during lymphocytic choriomeningitis virus and Listeria monocytogenes infections. We found that the majority of pathogen-specific CD8+ T cells initiated expression of the inhibitory CD94/NKG2A heterodimer, the KLRG1 receptor, and a novel murine NK cell marker (10D7); conversely, very few Ag-specific T cells expressed Ly49 family members. The up-regulation of these receptors was independent of IL-15 and persisted long after clearance of the pathogen. The expression of CD94/NKG2A was rapidly initiated in naive CD8+ T cells responding to peptide Ags in vitro and on many of the naive T cells that proliferate when transferred into lymphopenic (Rag-1−/−) hosts. Thus, CD94/NKG2A expression is a common consequence of CD8+ T cell activation. Binding of the CD94/NKG2A receptor by its ligand (Qa-1b) did not significantly inhibit CD8+ T cell effector functions. However, expression of CD94 and NKG2A transgenes partially inhibited early events of T cell activation. These subtle effects suggest that CD94/NKG2A-mediated inhibition of T cells may be limited to particular circumstances or may synergize with other receptors that are similarly up-regulated.
The Importance of IL-1β and TNF-α, and the Noninvolvement of IL-6, in the Development of Monoclonal Antibody-Induced Arthritis
Authors: Kagari Takashi , Doi Hiromi , Shimozato Takaichi
Published: April 2014

Injection of anti-type II collagen Ab and LPS induces arthritis in mice. The levels of IL-1β, IL-6, and chemokines (macrophage inflammatory protein (MIP)-1α, MIP-2, and monocyte chemoattractant protein-1) in the hind paws increased with the onset of arthritis and correlated highly with arthritis scores. The level of TNF-α was also elevated, but only transiently. Quantitative real-time PCR analysis revealed increases in cytokine and chemokine mRNA. To elucidate the contribution of inflammatory cytokines and chemokines in arthritis development more directly, recombinant proteins, neutralizing Abs, and knockout mice were used. The injection of rIL-1β or TNF-α, but not IL-6 or chemokines, induced arthritis when mice were i.v. preinjected with anti-type II collagen Ab. However, a single injection of recombinant cytokines or chemokines into the hind paws did not induce swelling. Arthritis development was inhibited by neutralizing Ab against IL-1β, TNF-α, or MIP-1α. In contrast, the inhibitory effect by anti-MIP-2 Ab was partial and, surprisingly, Abs to IL-6 and monocyte chemoattractant protein-1 showed no inhibitory effect. Furthermore, arthritis development in IL-1R−/− mice and TNFR−/− mice was not observed at all, but severe arthritis was developed in IL-6−/− mice. These results suggest that IL-1β and TNF-α play more crucial roles than IL-6 or chemokines in this model. Because arthritis was also developed in SCID mice, the development of arthritis in the Ab-induced mice model is due to a mechanism that does not involve T or B cells.
Apical, But Not Basolateral, Endotoxin Preincubation Protects Alveolar Epithelial Cells Against Hydrogen Peroxide-Induced Loss of Barrier Function: The Role of Nitric Oxide Synthesis
Authors: Rose Frank , Guthmann Bernd , Tenenbaum Tobias , Fink Ludger , Ghofrani Ardeschir , Weissmann Norbert , König Peter , Ermert Leander , Dahlem Gabriele , Haenze Joerg , Kummer Wolfgang , Seeger Werner , Grimminger Friedrich
Published: April 2014

The influence of LPS preincubation on hydrogen peroxide (H2O2)-induced loss of epithelial barrier function was investigated in rat alveolar epithelial type II cells (ATII). Both apical and basolateral H2O2 administration caused a manyfold increase in transepithelial [3H]mannitol passage. Apical but not basolateral preincubation of ATII with LPS did not influence control barrier properties but fully abrogated the H2O2-induced leakage response. The effect of apical LPS was CD14 dependent and was accompanied by a strong up-regulation of NO synthase II mRNA and protein and NO release. Inhibition of NO by NG-monomethyl-l-arginine suppressed the LPS effect, whereas it was reproduced by exogenous application of gaseous NO or NO donor agents. Manipulation of the glutathione homeostasis (buthionine-(S,R)-sulfoximine) and the cGMP pathway (1H-(1,2,4)oxadiazolo[4,3-α]quinoxaline-1-one; zaprinast) did not interfere with the protective effect of LPS. Superoxide (O⨪2) generation by ATII cells was reduced by exogenous NO and LPS preincubation. O⨪2 scavenging with exogenous superoxide dismutase, the intracellular superoxide dismutase analog Mn(III)tetrakis(4-benzoic acid) porphyrin, and the superoxide scavenger nitroblue tetrazolium and, in particular, hydroxyl radical scavenging with hydroxyl radical scavenger 1,3-dimethyl-thiourea inhibited the H2O2-induced epithelial leakage response. In conclusion, apical but not basolateral LPS preincubation of ATII cells provides strong protection against H2O2-induced transepithelial leakage, attributable to an up-regulation of epithelial NO synthesis. It is suggested that the LPS-induced NO formation is effective via interaction with reactive oxygen species, including superoxide and hydroxyl radicals. The polarized epithelial response to LPS may be part of the lung innate immune system, activated by inhaled endotoxin or under conditions of pneumonia.
Leukocyte Infiltration, But Not Neurodegeneration, in the CNS of Transgenic Mice with Astrocyte Production of the CXC Chemokine Ligand 10
Authors: Boztug Kaan , Carson Monica J. , Pham-Mitchell Ngan , Asensio Valérie C. , DeMartino Julie , Campbell Iain L.
Published: April 2014

The CXC chemokine ligand (CXCL)10 is induced locally in the CNS in diverse pathologic states. The impact of CXCL10 production in the CNS was examined in transgenic mice with astrocyte-directed production of this chemokine. These glial fibrillary acidic protein (GF)-CXCL10 transgenic mice spontaneously developed transgene dose- and age-related leukocyte infiltrates in perivascular, meningeal, and ventricular regions of the brain that were composed of, surprisingly, mainly neutrophils and, to a lesser extent, T cells. No other overt pathologic or physical changes were evident. In addition, the cerebral expression of a number of inflammation-related genes (e.g., cytokines) was not significantly altered in the transgenic mice. The extent of leukocyte recruitment to the brain could be enhanced markedly by peripheral immunization of GF-CXCL10 mice with CFA and pertussis toxin. This was paralleled by a modest, transient increase in the expression of some cytokine and chemokine genes. Analysis of the expression of the CXCL10 receptor, CXCR3, by the brain-infiltrating leukocytes from immunized GF-CXCL10 transgenic mice revealed a significant enrichment for CXCR3-positive cells in the CNS compared with spleen. The majority of cells positive for CXCR3 coexpressed CD3, whereas Gr1-positive granulocytes were negative for CXCR3 expression. Thus, while astrocyte production of CXCL10 can promote spontaneous and potentiate immune-induced recruitment of leukocytes to the CNS, this is not associated with activation of a degenerative immune pathology. Finally, the accumulation of neutrophils in the brain of GF-CXCL10 transgenic mice is apparently independent of CXCR3 and involves an unknown mechanism.
Evidence for Immune Responses to a Self-Antigen in Lung Transplantation: Role of Type V Collagen-Specific T Cells in the Pathogenesis of Lung Allograft Rejection
Authors: Haque M. Azizul , Mizobuchi Teruaki , Yasufuku Kazuhiro , Fujisawa Takehiko , Brutkiewicz Randy R. , Zheng Yan , Woods Kena , Smith Gerald N. , Cummings Oscar W. , Heidler Kathleen M. , Blum Janice S. , Wilkes David S.
Published: April 2014

We have reported that lung allograft rejection involves an immune response to a native protein in the lung, type V collagen (col(V)), and that col(V)-induced oral tolerance prevented acute and chronic rejection. In support of these findings col(V) fragments were detected in allografts during rejection, but not in normal lungs. The purpose of the current study was to isolate and characterize col(V)-specific allograft-infiltrating T cells and to determine their contribution to the rejection response in vivo. Two col(V)-specific T cell lines, LT1 and LT3, were isolated from F344 (RT1lv1) rat lung allografts during rejection that occurred after transplantation into WKY (RT1l) recipients. Both cell lines, but not normal lung lymphocytes, proliferated in response to col(V). Neither LT1 nor LT3 proliferated in response to alloantigens. LT1 and LT3 were CD4+CD25− and produced IFN-γ in response to col(V). Compared with normal CD4+ T cells, both cell lines expressed a limited V-β TCR repertoire. Each cell strongly expressed V-β 9 and 16, but differed in expression of other V-βs. Adoptive transfer of each cell line did not induce pathology in lungs of normal WKY rats. In contrast, adoptive transfer of LT1, but not LT3, caused marked peribronchiolar and perivascular inflammation in isograft (WKY) lungs and abrogated col(V)-induced oral tolerance to allograft (F344) lungs. Collectively, these data show that lung allograft rejection involves both allo- and autoimmune responses, and graft destruction that occurs during the rejection response may expose allograft-infiltrating T cells to potentially antigenic epitopes in col(V).
Differential Expression of the IFN-γ-Inducible CXCR3-Binding Chemokines, IFN-Inducible Protein 10, Monokine Induced by IFN, and IFN-Inducible T Cell α Chemoattractant in Human Cardiac Allografts: Association with Cardiac Allograft Vasculopathy and Acute Rejection
Authors: Zhao David Xiao-Ming , Hu Yenya , Miller Geraldine G. , Luster Andrew D. , Mitchell Richard N. , Libby Peter
Published: April 2014

CXCR3 chemokines exert potent biological effects on both immune and vascular cells. The dual targets suggest their important roles in cardiac allograft vasculopathy (CAV) and rejection. Therefore, we investigated expression of IFN-inducible protein 10 (IP-10), IFN-inducible T cell α chemoattractant (I-TAC), monokine induced by IFN (Mig), and their receptor CXCR3 in consecutive endomyocardial biopsies (n = 133) from human cardiac allografts and corresponding normal donor hearts (n = 11) before transplantation. Allografts, but not normal hearts, contained IP-10, Mig, and I-TAC mRNA. Persistent elevation of IP-10 and I-TAC was associated with CAV. Allografts with CAV had an IP-10-GAPDH ratio 3.7 ± 0.8 compared with 0.8 ± 0.2 in those without CAV (p = 0.004). Similarly, I-TAC mRNA levels were persistently elevated in allografts with CAV (6.7 ± 1.9 in allografts with vs 1.5 ± 0.3 in those without CAV, p = 0.01). In contrast, Mig mRNA was induced only during rejection (2.4 ± 0.9 with vs 0.6 ± 0.2 without rejection, p = 0.015). In addition, IP-10 mRNA increased above baseline during rejection (4.1 ± 2.3 in rejecting vs 1.8 ± 1.2 in nonrejecting biopsies, p = 0.038). I-TAC did not defer significantly with rejection. CXCR3 mRNA persistently elevated after cardiac transplantation. Double immunohistochemistry revealed differential cellular distribution of CXCR3 chemokines. Intragraft vascular cells expressed high levels of IP-10 and I-TAC, while Mig localized predominantly in infiltrating macrophages. CXCR3 was localized in vascular and infiltrating cells. CXCR3 chemokines are induced in cardiac allografts and differentially associated with CAV and rejection. Differential cellular distribution of these chemokines in allografts indicates their central roles in multiple pathways involving CAV and rejection. This chemokine pathway may serve as a monitor and target for novel therapies to prevent CAV and rejection.
The Molecular Basis of Complete Complement C4A and C4B Deficiencies in a Systemic Lupus Erythematosus Patient with HomozygousC4AandC4BMutant Genes
Authors: Rupert Kristi L. , Moulds Joann M. , Yang Yan , Arnett Frank C. , Warren Robert W. , Reveille John D. , Myones Barry L. , Blanchong Carol A. , Yu C. Yung
Published: April 2014

The disease course of a complete C4-deficient patient in the U.S. was followed for 18 years. The patient experienced multiple episodes of infection, and he was diagnosed with systemic lupus erythematosus at age 9 years. The disease progressed to WHO class III mild lupus nephritis and to fatal CNS vasculitis at age 23 years. Immunochemical experiments showed that the patient and his sibling had complete absence of C4A and C4B proteins and were negative for the Rodgers and Chido blood group Ags. Segregation and definitive RFLP analyses demonstrated that the patient and his sibling inherited two identical haplotypes, HLA A2 B12 DR6, each of which carries a defective long C4A gene and a defective short C4B gene. PCR and DNA sequencing revealed that the mutant C4A contained a 2-bp insertion in exon 29 at the sequence for codon 1213. The identical mutation was absent in the mutant C4B. The C4B mutant gene was selectively amplified by long range PCR, and its 41 exons were completely sequenced. The C4B mutant had a novel single C nucleotide deletion at the sequence for codon 522 in exon 13, leading to frame-shift mutation and premature termination. Thus, a multiplex PCR is designed by which known mutations in C4A and C4B can be elucidated conveniently. Among the 28 individuals reported with complete C4 deficiency, 75–96% of the subjects (dependent on the inclusion criteria) were afflicted with autoimmune or immune complex disorders. Hence, complete C4 deficiency is one of the most penetrant genetic risk factors for human systemic lupus erythematosus.
Muscle Responds to an Antibody Reactive with the Acetylcholine Receptor by Up-Regulating Monocyte Chemoattractant Protein 1: A Chemokine with the Potential to Influence the Severity and Course of Experimental Myasthenia Gravis
Authors: Reyes-Reyna Sara , Stegall Timothy , Krolick Keith A.
Published: April 2014

Autoantibodies with reactivity against the postjunctional muscle receptor for acetylcholine receptor are able to interfere with contractile function of skeletal muscles and cause the symptoms of myasthenia gravis (MG) in humans, as well as in experimental animal models of MG. In the study described below using a rat model of MG, it was observed that exposure to acetylcholine receptor-reactive Abs also induced increased levels of chemokine (i.e., monocyte chemoattractant protein 1) production by skeletal muscle cells. This was true of both cultured rat myocytes exposed in vitro and rat muscle exposed in vivo following passive Ab transfer. Increased monocyte chemoattractant protein 1 production may explain the increased trafficking of leukocytes through muscle following Ab transfer described in this and other reports. These observations may also be relevant to the induction of disease symptoms in experimental animal models of MG, since numerous reports from this and other laboratories indicate that the cytokine environment provided by leukocytes trafficking through muscle may play a pivotal role in disease progression.
Epstein-Barr Nuclear Antigen 1-Specific CD4+ Th1 Cells Kill Burkitt’s Lymphoma Cells
Authors: Paludan Casper , Bickham Kara , Nikiforow Sarah , Tsang Ming L. , Goodman Kiera , Hanekom Willem A. , Fonteneau Jean-Francois , Stevanović Stefan , Münz Christian
Published: April 2014

The γ-herpesvirus, EBV, is reliably found in a latent state in endemic Burkitt’s lymphoma. A single EBV gene product, Epstein-Barr nuclear Ag 1 (EBNA1), is expressed at the protein level. Several mechanisms prevent immune recognition of these tumor cells, including a block in EBNA1 presentation to CD8+ killer T cells. Therefore, no EBV-specific immune response has yet been found to target Burkitt’s lymphoma. We now find that EBNA1-specific, Th1 CD4+ cytotoxic T cells recognize Burkitt’s lymphoma lines. CD4+ T cell epitopes of EBNA1 are predominantly found in the C-terminal, episome-binding domain of EBNA1, and ∼0.5% of peripheral blood CD4+ T cells are specific for EBNA1. Therefore, adaptive immunity can be directed against Burkitt’s lymphoma, and perhaps this role for CD4+ Th1 cells extends to other tumors that escape MHC class I presentation.
T Cell Subset Patterns That Predict Resistance to Spontaneous Lymphoma, Mammary Adenocarcinoma, and Fibrosarcoma in Mice
Authors: Miller Richard A. , Chrisp Clarence
Published: April 2014

Aging leads to changes in the proportion of several T cell subsets in peripheral blood, but it is not yet known whether these changes have prognostic significance for late-life diseases. To examine this question, levels of T cell subsets were measured at 8 and 18 mo of age in the peripheral blood of mice of a genetically heterogeneous stock, and the mice were then subsequently evaluated for life span and for cause of death. The results indicate that mice whose T cell subset patterns look like those of old mice tend to die at earlier ages, regardless of the specific cause of death. At 18 mo, 39% of the variance within the set of seven measured subsets could be combined statistically into a single number, whose correlation with individual subsets suggested that it could be interpreted as an index of immunological aging. T cell subset pattern, as represented by this index, was a predictor of life span in mice dying of lymphoma, fibrosarcoma, mammary adenocarcinoma, or of all other causes considered together. Even as early as 8 mo of age, T cell subset patterns are significant predictors of all three forms of cancer, although at this age the association is stronger in mated female mice than in virgin mice. These results support two controversial hypotheses, which are not mutually exclusive: 1) early immune senescence might predispose to early death from cancer and 2) differences in aging rate, as monitored by tests of immune status, might accelerate or decelerate a wide range of late life neoplastic diseases.
Cutting Edge: Oral Type I IFN-τ Promotes a Th2 Bias and Enhances Suppression of Autoimmune Encephalomyelitis by Oral Glatiramer Acetate
Authors: Soos Jeanne M. , Stüve Olaf , Youssef Sawsan , Bravo Manuel , Johnson Howard M. , Weiner Howard L. , Zamvil Scott S.
Published: April 2014

IFN-τ, a novel type I IFN that possesses immunomodulatory properties, lacks toxicity normally associated with other type I IFNs. We examined the effects of oral IFN-τ alone and in combination with oral glatiramer acetate in experimental allergic encephalomyelitis (EAE). By comparison of oral administration of IFN-α, -β, and -τ to myelin basic protein-specific TCR-transgenic mice, we demonstrate these type I IFNs promote secretion of the Th2 cytokine IL-10 with similar efficiency. Whereas IFN-α and -β induced IFN-γ secretion, a Th1 cytokine, IFN-τ did not. Oral IFN-τ alone suppressed EAE. When suboptimal doses were administered orally in combination to wild-type mice, IFN-τ and glatiramer acetate had a synergistic beneficial effect in suppression of EAE. This combination was associated with TGF-β secretion and enhanced IL-10 production. Thus, IFN-τ is a potential candidate for use as a single agent or in combination therapy for multiple sclerosis.
Cutting Edge: Regulation of T Cell Activation Threshold by CD28 Costimulation Through Targeting Cbl-b for Ubiquitination
Authors: Zhang Jian , Bárdos Tamás , Li Dongdong , Gál István , Vermes Csaba , Xu Jianye , Mikecz Katalin , Finnegan Alison , Lipkowitz Stan , Glant Tibor T.
Published: April 2014

Optimal T cell activation requires signaling through the TCR and CD28 costimulatory receptor. CD28 costimulation is believed to set the threshold for T cell activation. Recently, Cbl-b, a ubiquitin ligase, has been shown to negatively regulate CD28-dependent T cell activation. In this report, we show that CD28 costimulation selectively induces greater ubiquitination and degradation of Cbl-b in wild-type T cells than CD3 stimulation alone, and TCR-induced Cbl-b ubiquitination and degradation are significantly reduced in CD28-deficient T cells. Stimulation of CD28-deficient T cells with higher doses of anti-CD3 results in increased ubiquitination of Cbl-b, which correlates with enhanced T cell responses. Our results demonstrate that CD28 costimulation regulates the threshold for T cell activation, at least in part, by promoting Cbl-b ubiquitination and degradation.
Granulocyte-Macrophage Colony-Stimulating Factor Induces an Expression Program in Neonatal Microglia That Primes Them for Antigen Presentation
Authors: Re Fabio , Belyanskaya Svetlana L. , Riese Richiard J. , Cipriani Barbara , Fischer Falko R. , Granucci Francesca , Ricciardi-Castagnoli Paola , Brosnan Celia , Stern Lawrence J. , Strominger Jack L. , Santambrogio Laura
Published: April 2014

Neonatal microglial cells respond to GM-CSF and M-CSF by acquiring different morphologies and phenotypes. To investigate the extent and consequences of this process, a global gene expression analysis was performed, with significant changes in transcript levels confirmed by biochemical analyses. Primary murine microglial cells underwent substantial expression reprogramming after treatment with GM-CSF or M-CSF with many differentially expressed transcripts important in innate and adaptive immunity. In particular, many gene products involved in Ag presentation were induced by GM-CSF, but not M-CSF, thus potentially priming relatively quiescent microglia cells for Ag presentation. This function of GM-CSF is distinct from its primary function in cell proliferation and survival.
Quantitative Differences in Chemokine Receptor Engagement Generate Diversity in Integrin-Dependent Lymphocyte Adhesion
Authors: D’Ambrosio Daniele , Albanesi Cristina , Lang Rosmarie , Girolomoni Giampiero , Sinigaglia Francesco , Laudanna Carlo
Published: April 2014

Chemokines control the specificity of lymphocyte homing. Numerous chemokines have been identified but the significance of redundancy in chemokine networks is unexplained. Here we investigated the biological significance of distinct chemokines binding to the same receptor. Among CCR4 ligands, skin vessels endothelial cells present C-C chemokine ligand (CCL) 17 but not CCL22 consistent with CCL17 involvement in T lymphocyte arrest on endothelial cells. However, CCL22 is much more powerful than CCL17 in the induction of rapid integrin-dependent T cell adhesion on VCAM-1 under conditions of physiological flow. The dominance of CCL22 over CCL17 extends to other CCR4-mediated phenomena such as receptor desensitization and internalization and correlates with the peculiar kinetics of CCR4 engagement by the two ligands. A similar phenomenological pattern is also shown for CXC chemokine ligand 9 and CXC chemokine ligand 11, which share binding to CXCR3. Our analysis shows how quantitative variations in chemokine receptor expression level and ligand engagement may alter the selectivity of integrin-dependent lymphocyte adhesive responses, suggesting a mechanism by which chemokine networks may either generate or break the specificity of lymphocyte subset recruitment.
Enhancement of MHC Class II-Restricted Responses by Receptor-Mediated Uptake of Peptide Antigens
Authors: Zaliauskiene Lolita , Kang Sunghyun , Sparks Kerri , Zinn Kurt R. , Schwiebert Lisa M. , Weaver Casey T. , Collawn James F.
Published: April 2014

Peptides, either as altered peptide ligands, competitors, or vaccines, offer an outstanding potential for regulating immune responses because of their exquisite specificity. However, a major problem associated with peptide therapies is that they are poorly taken up by APCs. Because of poor bioavailability, high concentrations and repeated treatments are required for peptide therapies in vivo. To circumvent this problem, we tested whether covalently coupling a peptide T cell determinant, OVA323–339, to transferrin (Tf) enhances APC uptake and presentation as monitored by Th cell activation. Functional analysis of the Tf-peptide conjugates revealed that the conjugates were presented 10,000- and 100-fold more effectively by B cells than intact Ag and free peptide, respectively. Furthermore, we demonstrate that the Tf-peptide conjugates are taken up by B cells through a receptor-mediated process and subsequently delivered to the lysosomal compartment. Using an adoptive transfer assay, we show that that the Tf-peptide complexes are 100-fold more effective in vivo than the free peptide in activating CD4+ T cells by following an early activation marker, CD69. Our results demonstrate that coupling peptides to Tf enhances peptide presentation, thereby making it possible to take full advantage of peptide-specific therapies in modulating T cell responses.
1,25 (OH)2 Vitamin D3-Stimulated Osteoclast Formation in Spleen-Osteoblast Cocultures Is Mediated in Part by Enhanced IL-1α and Receptor Activator of NF-κB Ligand Production in Osteoblasts
Authors: Lee Sun-Kyeong , Kalinowski Judy , Jastrzebski Sandra , Lorenzo Joseph A.
Published: April 2014

We examined the ability of 1,25 (OH)2 vitamin D3 (Vit D) to stimulate osteoclast-like cell (OCL) formation in cocultures of spleen cells and primary calvarial osteoblasts from wild-type (WT) and IL-1R type 1-deficient (knockout; KO) mice. Vit D dose dependently increased OCL in cocultures containing WT osteoblasts. In contrast, there was a 90% reduction in OCL numbers in cocultures containing KO osteoblasts. In cocultures with either WT or KO osteoblasts, treatment with Vit D increased receptor activator of NF-κB ligand mRNA by 17-, 19-, or 3.5-fold, respectively. Vit D decreased osteoprotegerin mRNA to undetectable in all groups. Intracellular IL-1α protein increased after Vit D treatment in cocultures containing WT, but not KO osteoblasts. We also examined direct effects of Vit D, IL-1α, and their combination on gene expression in primary osteoblasts. In WT cells, Vit D and IL-1 stimulated receptor activator of NF-κB ligand mRNA expression by 3- and 4-fold, respectively, and their combination produced a 7-fold increase. Inhibition of osteoprotegerin mRNA in WT cells was partial with either agent alone and greatest with their combination. In KO cells, only Vit D stimulated a response. IL-1 alone increased IL-1α protein expression in WT osteoblasts. However, in combination with Vit D, there was a synergistic response (100-fold increase). In KO cultures, there were no effects of IL-1, Vit D, or their combination on IL-1α protein. These results demonstrate interactions between IL-1 and Vit D in primary osteoblasts that appear important in both regulation of IL-1α production and the ability of Vit D to support osteoclastogenesis.
FDC-SP, a Novel Secreted Protein Expressed by Follicular Dendritic Cells
Authors: Marshall Aaron J. , Du Quijiang , Draves Kevin E. , Shikishima Yasufumi , HayGlass Kent T. , Clark Edward A.
Published: April 2014

To define better the molecular basis for follicular dendritic cell (FDC) function, we used PCR-based cDNA subtraction to identify genes specifically expressed in primary FDC isolated from human tonsils. In this work we report the discovery of a novel gene encoding a small secreted protein, which we term FDC-SP (FDC secreted protein). The FDC-SP gene lies on chromosome 4q13 adjacent to clusters of proline-rich salivary peptides and C-X-C chemokines. Human and mouse FDC-SP proteins are structurally unique and contain a conserved N-terminal charged region adjacent to the leader peptide. FDC-SP has a very restricted tissue distribution and is expressed by activated FDCs from tonsils and TNF-α-activated FDC-like cell lines, but not by B cell lines, primary germinal center B cells, or anti-CD40 plus IL-4-activated B cells. Strikingly, FDC-SP is highly expressed in germinal center light zone, a pattern consistent with expression by FDC. In addition, FDC-SP is expressed in leukocyte-infiltrated tonsil crypts and by LPS- or Staphylococcus aureus Cowan strain 1-activated leukocytes, suggesting that FDC-SP can also be produced in response to innate immunity signals. We provide evidence that FDC-SP is posttranslationally modified and secreted and can bind to the surface of B lymphoma cells, but not T lymphoma cells, consistent with a function as a secreted mediator acting upon B cells. Furthermore, we find that binding of FDC-SP to primary human B cells is markedly enhanced upon activation with the T-dependent activation signals such as anti-CD40 plus IL-4. Together our data identify FDC-SP as a unique secreted peptide with a distinctive expression pattern within the immune system and the ability to specifically bind to activated B cells.
Solid-Phase Epitope Recovery: A High Throughput Method for Antigen Identification and Epitope Optimization
Authors: Lawendowski Carla A. , Giurleo Gina M. , Huang Yin Yin , Franklin G. Joseph , Kaplan Johanne M. , Roberts Bruce L. , Nicolette Charles A.
Published: April 2014

Self tolerance to MHC class I-restricted nonmutated self Ags is a significant hurdle to effective cancer immunotherapy. Compelling evidence is emerging that altered peptide ligands can be far more immunogenic than their corresponding native epitopes; however, there is no way to reliably predict which modifications will lead to enhanced native epitope-specific immune responses. We reasoned that this limitation could be overcome by devising an empirical screen in which the nearly complete combinatorial spectrum of peptides of optimal length can be rapidly assayed for reactivity with a MHC class I-restricted cytotoxic T cell clone. This method, solid-phase epitope recovery, quantitatively ranks all reactive peptides in the library and allows selection of altered peptide ligands having desirable immunogenic properties of interest. In contrast to rationally designed MHC anchor-modified peptides, peptides identified by the present method are highly substituted in predicted TCR contact residues and can reliably activate and expand effector cell populations in vitro which lyse target cells presenting the wild-type epitope. We demonstrate that solid-phase epitope recovery peptides corresponding to a poorly immunogenic epitope of the melanoma Ag, gp100, can reliably induce wild-type peptide-specific CTL using normal donor T cells in vitro. Furthermore, these peptides can complement one another to induce these responses in an overwhelming majority of normal individuals in vitro. These data provide a rationale for the design of superior vaccines comprising a mixture of structurally diverse yet functionally convergent peptides.
The Subpopulation of CD4+CD25+ Splenocytes That Delays Adoptive Transfer of Diabetes Expresses L-Selectin and High Levels of CCR7
Authors: Szanya Veronika , Ermann Joerg , Taylor Cariel , Holness Claire , Fathman C. Garrison
Published: April 2014

Recently, CD4+CD25+ T cells have been implicated in the control of diabetes, suggesting that the inflamed islets of Langerhans in prediabetic NOD mice are under peripheral immune surveillance. Here we show that CD4+CD25+ splenocytes inhibit diabetes in cotransfer with islet-infiltrating cells. Furthermore, CD62L expression is necessary for this disease-delaying effect of CD4+CD25+ cells in vivo, but not for their suppressor function in vitro. We demonstrate that the CD4+CD25+CD62L+ splenocytes express CCR7 at high levels and migrate toward secondary lymphoid tissue chemokine and ELC (macrophage-inflammatory protein-3β), lymphoid chemokines, whereas CD4+CD25+CD62L− splenocytes preferentially express CCR2, CCR4, and CXCR3 and migrate toward the corresponding inflammatory chemokines. These data demonstrate that CD4+CD25+CD62L+, but not CD4+CD25+CD62L−, splenocytes delay diabetes transfer, and that CD4+CD25+ suppressor T cells are comprised of at least two subpopulations that behave differently in cotransfer in vivo and express distinct chemokine receptor and chemotactic response profiles despite demonstrating equivalent suppressor functions in vitro.