bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for the Murine IgH μ Locus Acting as a Hot Spot for Intrachromosomal Homologous Recombination
Authors: Raynard Steven J. , Read Leah R. , Baker Mark D.
Published: April 2014

Homologous recombination accomplishes the exchange of genetic information between two similar or identical DNA duplexes. It can occur either by gene conversion, a process of unidirectional genetic exchange, or by reciprocal crossing over. Homologous recombination is well known for its role in generating genetic diversity in meiosis and, in mitosis, as a DNA repair mechanism. In the immune system, the evidence suggests a role for homologous recombination in Ig gene evolution and in the diversification of Ab function. Previously, we reported the occurrence of homologous recombination between repeated, donor and recipient alleles of the Ig H chain μ gene C (Cμ) region residing at the Ig μ locus in mouse hybridoma cells. In this study, we constructed mouse hybridoma cell lines bearing Cμ region heteroalleles to learn more about the intrachromosomal homologous recombination process. A high frequency of homologous recombination (gene conversion) was observed for markers spanning the entire recipient Cμ region, suggesting that recombination might initiate at random sites within the Cμ region. The Cμ region heteroalleles were equally proficient as either conversion donors or recipients. Remarkably, when the same Cμ heteroalleles were tested for recombination in ectopic genomic positions, the mean frequency of gene conversion was reduced by at least 65-fold. These results are consistent with the murine IgH μ locus behaving as a hot spot for intrachromosomal homologous recombination.
Xenopus laevis Stromal Cell-Derived Factor 1: Conservation of Structure and Function During Vertebrate Development
Authors: Braun Mike , Wunderlin Markus , Spieth Kathrin , Knöchel Walter , Gierschik Peter , Moepps Barbara
Published: April 2014

Transmembrane signaling of the CXC chemokine stromal cell-derived factor-1 (SDF-1) is mediated by CXCR4, a G protein-coupled receptor initially identified in leukocytes and shown to serve as a coreceptor for the entry of HIV into lymphocytes. Characterization of SDF-1- and CXCR4-deficient mice has revealed that SDF-1 and CXCR4 are of vital developmental importance. To study the role of the SDF-1/CXCR4-chemokine/receptor system as a regulator of vertebrate development, we isolated and characterized a cDNA encoding SDF-1 of the lower vertebrate Xenopus laevis (xSDF-1). Recombinant xSDF-1 was produced in insect cells, purified, and functionally characterized. Although xSDF-1 is only 64–66% identical with its mammalian counterparts, it is indistinguishable from human (h)SDF-1α in terms of activating both X. laevis CXCR4 and hCXCR4. Thus, both xSDF-1 and hSDF-1α promoted CXCR4-mediated activation of heterotrimeric Gi2 in a cell-free system and induced release of intracellular calcium ions in and chemotaxis of intact lymphoblastic cells. Analysis of the time course of xSDF-1 mRNA expression during Xenopus embryogenesis revealed a tightly coordinated regulation of xSDF-1 and X. laevis CXCR4. xSDF-1 mRNA was specifically detected in the developing CNS, incipient sensory organs, and the embryonic heart. In Xenopus, CXCR4 mRNA appears to be absent from the heart anlage, but present in neural crest cells. This observation suggests that xSDF-1 expressed in the heart anlage may attract cardiac neural crest cells expressing CXCR4 to migrate to the primordial heart to regulate both septation of the cardiac outflow tract and differentiation of the myocardium during early heart development.
Transient Association of Ku with Nuclear Substrates Characterized Using Fluorescence Photobleaching
Authors: Rodgers William , Jordan Stephen J. , Capra J. Donald
Published: April 2014

The autoantigen Ku, composed of subunits Ku70 and Ku86, is necessary for repair of DNA double-strand breaks by nonhomologous end joining. Similarly, Ku participates in repair of DNA double-strand breaks that occur during V(D)J recombination, and it is therefore required for the development of B and T lymphocytes. Although previous studies have identified the DNA-binding activities of Ku, little is known concerning its dynamics, such as the mobility of Ku in the nucleus and its rate of association with substrates. To address this question, fluorescence photobleaching experiments were performed using HeLa cells and B cells expressing a green fluorescent protein (GFP) fusion construct of either Ku70 or Ku86. The results show that Ku moves rapidly throughout the nucleus even following irradiation of the cells. However, the rate of diffusion of Ku was ∼100-fold slower than that predicted from its size. Association of Ku-GFP with a filamentous nuclear structure was also evident, and nuclear extraction experiments suggest that this represents nuclear matrix. A central domain of Ku70 containing its DNA-binding and heterodimerization regions and its nuclear localization signal shows that this alone is sufficient for the observed mobility of Ku70-GFP and its association with nuclear matrix. These data suggest the mobility of Ku is characterized by a transient, high flux association with nuclear substrates that includes both DNA and the nuclear matrix and may represent a mechanism for repair of double-strand breaks using the nuclear matrix as a scaffold.
Epitope Recognition by Diverse Antibodies Suggests Conformational Convergence in an Antibody Response
Authors: Nair Deepak T. , Singh Kavita , Siddiqui Zaved , Nayak Bishnu P. , Rao Kanury V. S. , Salunke Dinakar M.
Published: April 2014

Crystal structures of distinct mAbs that recognize a common epitope of a peptide Ag have been determined and analyzed in the unbound and bound forms. These Abs display dissimilar binding site structures in the absence of the Ag. The dissimilarity is primarily expressed in the conformations of complementarity-determining region H3, which is responsible for defining the epitope specificity. Interestingly, however, the three Abs exhibit similar complementarity-determining region conformations in the Ag binding site while recognizing the common epitope, indicating that different pathways of binding are used for Ag recognition. The epitope also exhibits conformational similarity when bound to each of these Abs, although the peptide Ag was otherwise flexible. The observed conformational convergence in the epitope and the Ag binding site was facilitated by the plasticity in the nature of interactions.
IL-10 Is Critical for Host Resistance and Survival During Gastrointestinal Helminth Infection
Authors: Schopf Lisa R. , Hoffmann Karl F. , Cheever Allen W. , Urban Joseph F. , Wynn Thomas A.
Published: April 2014

Resistance to many intestinal nematodes is dependent on the induction of polarized type 2 cytokine responses, whereas type 1 responses can exacerbate these infections. The contributions of IL-4 and IL-13 to the development of resistance have been well described for a variety of intestinal parasites; however, the role of IL-10 has not been previously investigated. In this study we infected IL-10-, IL-10/IL-4-, IL-10/IL-12-, IL-4-, and IL-12-deficient mice with Trichuris muris to determine whether IL-10 contributes to the development of immunity. Interestingly, T. muris-infected IL-10-, IL-4-, and IL-10/IL-4-deficient mice failed to expel the parasite, and animals deficient in IL-10 displayed marked morbidity and mortality. In contrast, double IL-10/IL-12-deficient mice were completely resistant and mounted a highly polarized type 2 cytokine response, demonstrating that the increased susceptibility of IL-10-deficient mice was dependent on IL-12. Further study suggested that the susceptibility of IL-10- and IL-10/IL-4-deficient mice was probably attributable to a marked increase in type 1 cytokine production in those animals. The mortality observed in T. muris-infected IL-10- and IL-10/IL-4-deficient mice correlated with increased inflammation, loss of Paneth cells, and absence of mucus in the cecum. Interestingly, survival was enhanced in T. muris-infected IL-10/IL-4-deficient mice if a broad spectrum antibiotic was administered, suggesting that an outgrowth of opportunistic bacteria was contributing to the high degree of morbidity and mortality. Overall, these studies reveal a critical role for IL-10 in the polarization of Th2 responses, development of resistance during T. muris infection, and maintenance of barrier function in the colon.
Identification of BING-4 Cancer Antigen Translated From an Alternative Open Reading Frame of a Gene in the Extended MHC Class II Region Using Lymphocytes From a Patient With a Durable Complete Regression Following Immunotherapy
Authors: Rosenberg Steven A. , Tong-On Panida , Li Yong , Riley John P. , El-Gamil Mona , Parkhurst Maria R. , Robbins Paul F.
Published: April 2014

Multiple human cancer Ags have been identified, although little is known concerning which would be most effectively used in cancer immunotherapy. To gain insight into the selection of appropriate Ags, the immunologic reactivity of a patient who had a durable complete regression of melanoma metastases was measured. PBMCs were directly cloned using the monoclonal anti-CD3 Ab OKT3 and IL-2 without any bias introduced by previous culture. A lymphocyte clone recognized a previously unknown shared melanoma Ag that was identified as the BING-4 protein encoded in a gene-rich region of the extended class II MHC. The HLA-A2-restricted BING-4 immunodominant peptide was translated from a 10-aa-long alternative open reading frame. In vitro sensitization against this peptide generated lymphocytes reactive against HLA-A2+ melanomas. Real-time semiquantitative RT-PCR analysis revealed that 8 of 15 melanoma cell lines overexpressed BING-4, and this correlated with recognition by lymphocytes. Overexpression was not found in normal tissues or other tumor types. Thus, BING-4 represents another candidate Ag for possible use in the immunotherapy of patients with melanoma.
A Unique Combination of Inflammatory Cytokines Enhances Apoptosis of Thyroid Follicular Cells and Transforms Nondestructive to Destructive Thyroiditis in Experimental Autoimmune Thyroiditis
Authors: Wang Su He , Bretz James D. , Phelps Ellen , Mezosi Emese , Arscott Patricia L. , Utsugi Saho , Baker James R.
Published: April 2014

Treatment of cultured primary human thyroid cells with IFN-γ and TNF-α uniquely allows the induction of Fas-mediated apoptosis. To investigate the role of this cytokine combination in vivo, CBA/J mice were immunized with thyroglobulin and then injected with IFN-γ and TNF-α. Compared with control animals, mice treated with IFN-γ and TNF-α showed significantly sustained lymphocytic infiltration in the thyroid, which was associated with the destruction of portions of the follicular architecture at wk 6 after initial immunization. Furthermore, the number of apoptotic thyroid follicular cells was increased only in the thyroids from mice treated with the IFN-γ and TNF-α. We also analyzed the function of the Fas pathway in vivo in cytokine-treated mice by using an agonist anti-Fas Ab injected directly into the thyroid. Minimal apoptosis of thyroid epithelial cells was observed unless the mice were pretreated with IFN-γ and TNF-α. These data demonstrate that this unique combination of inflammatory cytokines facilitates the apoptotic destruction of thyroid follicular cells in experimental autoimmune thyroiditis, in a manner similar to what is observed in Hashimoto’s thyroiditis in humans.
A Single-Chain Class II MHC-IgG3 Fusion Protein Inhibits Autoimmune Arthritis by Induction of Antigen-Specific Hyporesponsiveness
Authors: Zuo Li , Cullen Constance M. , DeLay Monica L. , Thornton Sherry , Myers Linda K. , Rosloniec Edward F. , Boivin Gregory P. , Hirsch Raphael
Published: April 2014

T cells play a central role in many autoimmune diseases. A method to specifically target the function of autoreactive T cell clones would avoid the global immunosuppression associated with current therapies. To develop a molecule capable of inhibiting autoreactive T cell responses in vivo, single-chain peptide-I-A-IgG3 fusion proteins were constructed and expressed in both mammalian and insect cells. The fusion proteins were designed with an IgG3 Fc moiety to make them divalent, allowing TCR cross-linking, while lacking FcR binding and costimulation. The fusion proteins stimulated T cell hybridomas in vitro in a peptide-specific, MHC-restricted manner but failed to do so in soluble form. In vivo administration of an I-Aq fusion protein, containing an immunodominant collagen II peptide, significantly delayed the onset and reduced the severity of collagen-induced arthritis in DBA/1 mice by induction of Ag-specific hyporesponsiveness. Such fusion proteins may be useful to study novel therapeutic approaches for T cell-mediated autoimmune diseases.
Cutting Edge: DNA Polymerases μ and λ Are Dispensable for Ig Gene Hypermutation
Authors: Bertocci Barbara , De Smet Annie , Flatter Eric , Dahan Auriel , Bories Jean-Christophe , Landreau Catherine , Weill Jean-Claude , Reynaud Claude-Agnès
Published: April 2014

Mutations arising in Ig V genes during an immune response are most likely introduced by one or several error-prone DNA polymerases. Many of the recently described nonreplicative DNA polymerases have an intrinsic fidelity compatible with such an activity, the strongest candidates being polymerase (pol) η, pol ι, pol ζ, and pol μ. We report in this work that mice inactivated for either of the two polymerases related to pol β (i.e., pol μ and pol λ) are viable and fertile and display a normal hypermutation pattern.
Lymphoepithelial Interactions Trigger Specific Regulation of Gene Expression in the M Cell-Containing Follicle-Associated Epithelium of Peyer’s Patches
Authors: El Bahi Sophia , Caliot Elise , Bens Marcelle , Bogdanova Anna , Kernéis Sophie , Kahn Axel , Vandewalle Alain , Pringault Eric
Published: April 2014

In the intestine, the follicle-associated epithelium (FAE) of Peyer’s patches (PP) performs Ag sampling as the first step in developing immune responses. Depending on the species, this epithelium contains 10–50% of M cells, which act as regulated gates in epithelial barriers that can be used opportunistically by pathogens to invade their host. However, the mechanisms involved in the differentiation and uptake processes of M cells are not known, in part because their limited number in the intestinal mucosa has hampered molecular and biochemical studies. In this work we provide evidence that PP lymphocytes can themselves modulate gene expression in PP in vivo and in an in vitro model of FAE. Transgenic mice carrying a reporter gene under the control of a modified l-pyruvate kinase promoter (SVPK) exhibit strong transgene expression in PP and FAE, but not in the adjacent villous cells. We used the mouse intestinal epithelial cell line m-ICcl2 transfected with the SVPK promoter fused to β-galactosidase to investigate the direct effect of PP lymphocytes on SVPK promoter activity. β-Galactosidase expression was 4.4-fold higher in transfected m-ICcl2 cells when they were cultured with PP lymphocytes. Conversely, green fluorescent protein expression was 1.8-fold lower in stably transfected differentiated intestinal Caco-2cl1 cells with the sucrase isomaltase promoter fused to green fluorescent protein cDNA when they were cultured with PP lymphocytes, indicating that the in vivo FAE down-regulation of sucrase isomaltase promoter is transcriptionally regulated.
I-κB Kinases α and β Have Distinct Roles in Regulating Murine T Cell Function
Authors: Ren Hong , Schmalstieg Aurelia , van Oers Nicolai S. C. , Gaynor Richard B.
Published: April 2014

NF-κB is a transcription factor that regulates a variety of genes involved in the control of the immune and inflammatory responses. Activation of NF-κB is mediated by an inducible I-κB kinase (IKK) complex comprised of two catalytic subunits, IKKα and IKKβ. In this study, the role of these kinases in the development and function of T lymphocytes was explored using transgenic mice expressing the dominant-negative forms of one or both kinases under the control of a T cell-specific promoter. Activation of the NF-κB pathway in thymocytes isolated from these transgenic mice following treatment with either PMA and ionomycin or anti-CD3 was markedly inhibited. Although inhibition of IKKα and/or IKKβ function did not alter T cell development in these transgenic mice, the proliferative response to anti-CD3 was reduced in thymocytes isolated from mice expressing dominant-negative IKKβ. However, inhibition of both IKKα and IKKβ was required to markedly reduce cytokine production in thymocytes isolated from these transgenic mice. Finally, we demonstrated that IKKα and IKKβ have opposite roles on the regulation of anti-CD3-induced apoptosis of double-positive thymocytes. These results suggest that IKKα and IKKβ have distinct roles in regulating thymocyte function.
Selective Requirement for CD40-CD154 in Drug-Induced Type 1 Versus Type 2 Responses to Trinitrophenyl-Ovalbumin
Authors: Nierkens Stefan , van Helden Pauline , Bol Marianne , Bleumink Rob , van Kooten Peter , Ramdien-Murli Seema , Boon Louis , Pieters Raymond
Published: April 2014

CD154 is transiently expressed by activated T cells and interacts with CD40 on B cells, dendritic cells, macrophages, and monocytes. This costimulatory receptor-ligand couple seems decisive in Ag-driven immune responses but may be differentially involved in type 1 vs type 2 responses. We studied the importance of CD40-CD154 in both responses using the reporter Ag popliteal lymph node assay in which selectively acting drugs generate clearly polarized type 1 (streptozotocin) or type 2 (D-penicillamine, diphenylhydantoin) responses to a constant coinjected Ag in the same mouse strain. Treatment of mice with anti-CD154 reduced characteristic immunological parameters in type 2 responses (B and CD4+ T cell proliferation, IgG1 and IgE Abs, and IL-4 secretion) and only slightly affected the type 1 response (small decrease in IFN-γ production, influx of CD11c+ and F4/80+ cells, and prevention of architectural disruption of the lymph node, but no effect on IgG2a Ab and TNF-α secretion or B and CD4+ T cell proliferation). The findings indicate that the CD40-CD154 costimulatory interaction is a prerequisite in drug-induced type 2 responses and is only marginally involved in type 1 responses. The observed expression patterns of CD80 and CD86 on different APC (B cells in type 2 and dendritic cells in type 1) may be responsible for this discrepancy.
Cells Expressing Indoleamine 2,3-Dioxygenase Inhibit T Cell Responses
Authors: Mellor Andrew L. , Keskin Derin B. , Johnson Theodore , Chandler Phillip , Munn David H.
Published: April 2014

Pharmacological inhibition of indoleamine 2,3-dioxygenase (IDO) activity during murine gestation results in fetal allograft rejection and blocks the ability of murine CD8+ dendritic cells to suppress delayed-type hypersensitivity responses to tumor-associated peptide Ags. These observations suggest that cells expressing IDO inhibit T cell responses in vivo. To directly evaluate the hypothesis that cells expressing IDO inhibit T cell responses, we prepared IDO-transfected cell lines and transgenic mice overexpressing IDO and assessed allogeneic T cell responses in vitro and in vivo. T cells cocultured with IDO-transfected cells did not proliferate but expressed activation markers. The potency of allogeneic T cell responses was reduced significantly when mice were preimmunized with IDO-transfected cells. In addition, adoptive transfer of alloreactive donor T cells yielded reduced numbers of donor T cells when injected into IDO-transgenic recipient mice. These outcomes suggest that genetically enhanced IDO activity inhibited T cell proliferation in vitro and in vivo. Genetic manipulation of IDO activity may be of therapeutic utility in suppressing undesirable T cell responses.
In Vivo CD86 Blockade Inhibits CD4+ T Cell Activation, Whereas CD80 Blockade Potentiates CD8+ T Cell Activation and CTL Effector Function
Authors: Lang Thomas J. , Nguyen Phuong , Peach Robert , Gause William C. , Via Charles S.
Published: April 2014

To address whether a functional dichotomy exists between CD80 and CD86 in naive T cell activation in vivo, we administered anti-CD80 or CD86 blocking mAb alone or in combination to mice with parent-into-F1 graft-vs-host disease (GVHD). In this model, the injection of naive parental T cells into unirradiated F1 mice results in either a Th1 cytokine-driven, cell-mediated immune response (acute GVHD) or a Th2 cytokine-driven, Ab-mediated response (chronic GVHD) in the same F1 recipient. Combined CD80/CD86 blockade beginning at the time of donor cell transfer mimicked previous results seen with CTLA4Ig and completely abrogated either acute or chronic GVHD by preventing the activation and maturation of donor CD4+ T cells as measured by a block in acquisition of memory marker phenotype and cytokine production. Similar results were seen with selective CD86 blockade; however, the degree of CD4 inhibition was always less than that seen with combined CD80/CD86 blockade. A more striking effect was seen with selective CD80 blockade in that chronic GVHD was converted to acute GVHD. This effect was associated with the induction of Th1 cytokine production, donor CD8+ T cell activation, and development of antihost CTL. The similarity of this effect to that reported for selective CTLA4 blockade suggests that CD80 is a critical ligand for CTLA4 in mediating the down-regulation of Th1 responses and CD8+ T cell activation. In contrast, CD86 is critical for the activation of naive CD4+ T cells in either a Th1 or a Th2 cytokine-mediated response.
Suppression of IL-12 Production by Soluble CD40 Ligand: Evidence for Involvement of the p44/42 Mitogen-Activated Protein Kinase Pathway
Authors: Wittmann Miriam , Kienlin Petra , Mommert Susanne , Kapp Alexander , Werfel Thomas
Published: April 2014

IL-12 is a key cytokine in skewing immune responses toward Th1-like reactions. Human monocytes/macrophages produce high amounts of bioactive IL-12 when a priming signal (IFN-γ or GM-CSF) precedes a second signal (e.g., LPS). We and others have previously shown that preincubation with LPS before this stimulation procedure can efficiently and selectively suppress the production of IL-12 by human monocytes. In this study, we show that an almost complete suppression of IL-12 production can also be observed after preincubation of monocytes with costimulatory cell surface molecules that bind to members of the TNFR superfamily (CD40 ligand, TNF-related activation-induced cytokine (TRANCE)). The suppression of IL-12 was observable on the mRNA and protein levels and was not due to endogenous production of known IL-12 antagonists (i.e., IL-10, IL-4, and PGE2), to an increased number of cells undergoing apoptosis, nor to down-regulation of the IFN-γ or CD40 receptor. Cell surface expression of the costimulatory molecules CD80 and CD86 was not reduced by the preincubation procedure, and only a moderate reduction of IL-6 production was observed. Several studies have identified signal transduction pathways that are activated by CD40 signaling, including activation of mitogen-activated protein kinases. The presence of the extracellular signal-related kinase-specific mitogen-activated protein kinase kinase 1/2-specific inhibitors PD98059 and U0126 abrogated suppression induced by sCD40 ligand or other second signals. This indicates that activation of extracellular signal-regulated kinase 1/2 contributes to the underlying mechanism of IL-12 suppression. This mechanism may be relevant in other inflammatory responses and may help to develop therapeutic strategies in Th1-mediated diseases.
A Molecular Marker for Thymocyte-Positive Selection: Selection of CD4 Single-Positive Thymocytes with Shorter TCRB CDR3 During T Cell Development
Authors: Yassai Maryam , Ammon Kristin , Goverman Joan , Marrack Phillipa , Naumov Yuri , Gorski Jack
Published: April 2014

The generation of the naive T cell repertoire is a direct result of maturation and selection events in the thymus. Although maturation events are judged predominantly on the expression of surface markers, molecular markers, more intimately involved in the selection process, can be informative. We have identified a molecular marker for selection in later stages of maturation in humans. Thymocytes are selected for the expression of TCR β-chains with shorter CDR3 at the double-positive to single-positive (SP) transition. Here we extend these studies to the mouse and show that the selection phenotype is not related to α-chain pairing but is a function of the MHC haplotype. Interestingly, the selection is much more apparent in CD4 SP thymocytes than in CD8 SP cells. This is in contrast to human thymocytes, where the selection is equally apparent in both lineages. The involvement of MHC in the process argues that this is a positive selection stage. The difference in the extent of this selection between the two SP lineages may indicate a class difference in the nature of the TCR-MHC interaction, the role of coreceptors in the selection process, or both.
Human NK Cell-Mediated Cytotoxicity Triggered by CD86 and Galα1,3-Gal Is Inhibited in Genetically Modified Porcine Cells
Authors: Costa Cristina , Barber Domingo F. , Fodor William L.
Published: April 2014

Delayed xenograft rejection is a major hurdle that needs to be addressed to prolong graft survival in pig-to-primate xenotransplantation. NK cell activation has been implicated in delayed xenograft rejection. Both Ab-dependent and independent mechanisms are responsible for the high susceptibility of porcine cells to human NK cell-mediated cytotoxicity. Previous reports demonstrated a role of Galα1,3-Gal Ag in triggering the Ab-independent responses. We hypothesize that expression of CD80 and/or CD86 on porcine cells may also play a role in NK cell activation as human NK cells express a variant of CD28. Our initial analysis showed that porcine endothelial cells and fibroblasts express CD86, but not CD80. Genetic engineering of these cells to express hCD152-hCD59, a chimeric molecule designed to block CD86 in cis, was accompanied by a reduction in susceptibility to human NK cell-mediated cytotoxicity. The use of a specific anti-porcine CD86-blocking Ab and the NK92 and YTS cell lines further confirmed the involvement of CD86 in triggering NK cell-mediated lysis of porcine cells. Maximal protection was achieved when hCD152-hCD59 was expressed in H transferase-transgenic cells, which show reduced Galα1,3-Gal expression. In this work, we describe two mechanisms of human NK cell-mediated rejection of porcine cells and demonstrate that genetically modified cells resist Ab-independent NK cell-mediated cytotoxicity.
The Potency of TCR Signaling Differentially Regulates NFATc/p Activity and Early IL-4 Transcription in Naive CD4+ T Cells
Authors: Brogdon Jennifer L. , Leitenberg David , Bottomly Kim
Published: April 2014

The potency of TCR signaling can regulate the differentiation of naive CD4+ T cells into Th1 and Th2 subsets. In this work we demonstrate that TCR signaling by low-affinity, but not high-affinity, peptide ligands selectively induces IL-4 transcription within 48 h of priming naive CD4+ T cells. This early IL-4 transcription is STAT6 independent and occurs before an increase in GATA-3. Furthermore, the strength of the TCR signal differentially affects the balance of NFATp and NFATc DNA binding activity, thereby regulating IL-4 transcription. Low-potency TCR signals result in high levels of nuclear NFATc and low levels of NFATp, which are permissive for IL-4 transcription. These data provide a model for how the strength of TCR signaling can influence the generation of Th1 and Th2 cells.
Molecular Mechanisms Underlying Differential Contribution of CD28 Versus Non-CD28 Costimulatory Molecules to IL-2 Promoter Activation
Authors: Zhou Xu-Yu , Yashiro-Ohtani Yumi , Nakahira Masakiyo , Park Woong Ryeon , Abe Ryo , Hamaoka Toshiyuki , Naramura Mayumi , Gu Hua , Fujiwara Hiromi
Published: April 2014

T cell costimulation via CD28 and other (non-CD28) costimulatory molecules induces comparable levels of [3H]TdR incorporation, but fundamentally differs in the contribution to IL-2 production. In this study, we investigated the molecular basis underlying the difference between CD28 and non-CD28 costimulation for IL-2 gene expression. Resting T cells from a mutant mouse strain generated by replacing the IL-2 gene with a cDNA encoding green fluorescent protein were stimulated with a low dose of anti-CD3 plus anti-CD28 or anti-non-CD28 (CD5 or CD9) mAbs. CD28 and non-CD28 costimulation capable of inducing potent [3H]TdR uptake resulted in high and marginal levels of green fluorescent protein expression, respectively, indicating their differential IL-2 promoter activation. CD28 costimulation exhibited a time-dependent increase in the binding of transcription factors to the NF-AT and NF-κB binding sites and the CD28-responsive element of the IL-2 promoter, whereas non-CD28 costimulation did not. Particularly, a striking difference was observed for the binding of NF-κB to CD28-responsive element and the NF-κB binding site. Decreased NF-κB activation in non-CD28 costimulation resulted from the failure to translocate a critical NF-κB member, c-Rel, to the nuclear compartment due to the lack of IκBβ inactivation. These observations suggest that unlike CD28 costimulation, non-CD28 costimulation fails to sustain IL-2 promoter activation and that such a failure is ascribed largely to the defect in the activation of c-Rel/NF-κB.
Identification of a Candidate Regulatory Region in the HumanCD8Gene Complex by Colocalization of DNase I Hypersensitive Sites and Matrix Attachment Regions Which Bind SATB1 and GATA-3
Authors: Kieffer Lynda J. , Greally John M. , Landres Inna , Nag Shanta , Nakajima Yuko , Kohwi-Shigematsu Terumi , Kavathas Paula B.
Published: April 2014

To locate elements regulating the human CD8 gene complex, we mapped nuclear matrix attachment regions (MARs) and DNase I hypersensitive (HS) sites over a 100-kb region that included the CD8B gene, the intergenic region, and the CD8A gene. MARs facilitate long-range chromatin remodeling required for enhancer activity and have been found closely linked to several lymphoid enhancers. Within the human CD8 gene complex, we identified six DNase HS clusters, four strong MARs, and several weaker MARs. Three of the strong MARs were closely linked to two tissue-specific DNase HS clusters (III and IV) at the 3′ end of the CD8B gene. To further establish the importance of this region, we obtained 19 kb of sequence and screened for potential binding sites for the MAR-binding protein, SATB1, and for GATA-3, both of which are critical for T cell development. By gel shift analysis we identified two strong SATB1 binding sites, located 4.5 kb apart, in strong MARs. We also detected strong GATA-3 binding to an oligonucleotide containing two GATA-3 motifs located at an HS site in cluster IV. This clustering of DNase HS sites and MARs capable of binding SATB1 and GATA-3 at the 3′ end of the CD8B gene suggests that this region is an epigenetic regulator of CD8 expression.
Detailed Analysis of CD4+ Th Responses to Envelope and Gag Proteins of Simian Immunodeficiency Virus Reveals an Exclusion of Broadly Reactive Th Epitopes from the Glycosylated Regions of Envelope
Authors: Sarkar Surojit , Kalia Vandana , Murphey-Corb Michael , Montelaro Ronald C.
Published: April 2014

Ag-specific CD4+ Th cells play a key role in the development, maturation, and maintenance of pathogen-specific humoral and cellular immune responses. To define the fine specificity of broadly reactive Th responses associated with mature immunity in a lentiviral system, we analyzed peptide-specific Th responses in eight macaques chronically infected with a reference live attenuated SIV at 12–14 mo postinoculation. All macaques had stable immunocompetent Th cells at the time of analysis, and a unique array of Th responses to 20-mer overlapping peptides from envelope (Env) and Gag was identified for each macaque, which were then used to define a set of 31 broadly reactive peptide epitopes. Only 5 of the 31 broadly reactive Th epitope peptides mapped to the surface (SU) domain of Env. Interestingly, these were all confined to two conserved nonglycosylated regions toward the carboxyl terminus of SU, suggesting a structural influence of glycosylation on development of Th responses. Gag and the Env transmembrane proteins contained the majority of broadly reactive peptide epitopes (12 and 14 peptides, respectively), which were uniformly distributed throughout their sequence. This study defines for the first time broadly reactive Th epitope peptides of SIV Env and Gag proteins that are associated with enduring broadly protective vaccine immunity to attenuated SIV, which may be used for the design and evaluation of experimental vaccines. Moreover, the data suggest that extensive glycosylation of SU may provide yet another immune escape mechanism developed by lentiviruses to restrict the breadth of Th repertoire to SU, a major immunologically exposed protein of the virus.
Two Lipoproteins Extracted fromEscherichia coliK-12 LCD25 Lipopolysaccharide Are the Major Components Responsible for Toll-Like Receptor 2-Mediated Signaling
Authors: Lee Hyun-Ku , Lee Jongdae , Tobias Peter S.
Published: April 2014

Toll-like receptor 2 (TLR2)-mediated cell activation induced by commercial preparations of LPS was recently shown to arise from impurities whose identities are not known. In this work, we determined the molecules responsible for TLR2-mediated cell activation in LPS derived from Escherichia coli K-12 strain LCD25. When LCD25 LPS was phenol extracted, two proteins capable of TLR2-mediated cell activation were purified and identified as E. coli lipoproteins. We cloned, expressed, and purified these two lipoproteins, Lip19 and Lip12. Lip19 or Lip12 activated TNF-α production from RAW264.7 and THP-1 cells in a TLR2-dependent manner. However, neither Lip19 nor Lip12 activated HUVECs, which lack endogenous TLR2. Additionally, IκB kinase β and c-Jun N-terminal kinase 1 activation in THP-1 cells induced by Lip19 or Lip12 was observed. TLR2 activation by Lip19 and Lip12 in HEK293 cells was blocked by inhibitory anti-TLR2 mAbs. The unlipidated mutants, Lip19-C19S and Lip12-C21S, in which the NH2-terminal cysteine was substituted by serine, lost their ability to activate TLR2-transfected HEK 293 cells. Taken together, these results demonstrate that two lipoproteins constitute the major contaminants responsible for TLR2-mediated cell activation in E. coli LCD25 LPS.
Leptin-Deficient Mice Exhibit Impaired Host Defense in Gram-Negative Pneumonia
Authors: Mancuso Peter , Gottschalk Andrew , Phare Susan M. , Peters-Golden Marc , Lukacs Nicholas W. , Huffnagle Gary B.
Published: April 2014

Leptin is an adipocyte-derived hormone that is secreted in correlation with total body lipid stores. Serum leptin levels are lowered by the loss of body fat mass that would accompany starvation and malnutrition. Recently, leptin has been shown to modulate innate immune responses such as macrophage phagocytosis and cytokine synthesis in vitro. To determine whether leptin plays a role in the innate host response against Gram-negative pneumonia in vivo, we compared the responses of leptin-deficient and wild-type mice following an intratracheal challenge of Klebsiella pneumoniae. Following K. pneumoniae administration, we observed increased leptin levels in serum, bronchoalveolar lavage fluid, and whole lung homogenates. In a survival study, leptin-deficient mice, as compared with wild-type mice, exhibited increased mortality following K. pneumoniae administration. The increased susceptibility to K. pneumoniae in the leptin-deficient mice was associated with reduced bacterial clearance and defective alveolar macrophage phagocytosis in vitro. The exogenous addition of very high levels of leptin (500 ng/ml) restored the defect in alveolar macrophage phagocytosis of K. pneumoniae in vitro. While there were no differences between wild-type and leptin-deficient mice in lung homogenate cytokines TNF-α, IL-12, or macrophage-inflammatory protein-2 after K. pneumoniae administration, leukotriene synthesis in lung macrophages from leptin-deficient mice was reduced. Leukotriene production was restored by the addition of exogenous leptin (500 ng/ml) to macrophages in vitro. This study demonstrates for the first time that leptin-deficient mice display impaired host defense in bacterial pneumonia that may be due to a defect in alveolar macrophage phagocytosis and leukotriene synthesis.
Activation of the Neutrophil Nicotinamide Adenine Dinucleotide Phosphate Oxidase by Galectin-1
Authors: Almkvist Jenny , Dahlgren Claes , Leffler Hakon , Karlsson Anna
Published: April 2014

Galectins are a group of lactose-binding proteins widely distributed in nature. Twelve mammalian galectins have so far been identified, but their functions are to a large extent unknown. In this work we study galectin-1 in its interaction with human neutrophils, with regard to both cell surface binding and activation of the superoxide-producing NADPH-oxidase. We show that galectin-1 is able to activate the neutrophil NADPH-oxidase, provided that the cells have been primed by extravasation from the blood into the tissue, an activation pattern that is similar to that of galectin-3. Using in vitro priming protocols, the galectin-1 responsiveness was found to correlate to granule mobilization and galectin-1 binding to the cells, suggesting the presence of granule-localized receptors that are up-regulated to the cell surface upon priming. By galectin-1 overlay of fractionated neutrophils we identified potential galectin-1 receptor candidates localized in the membranes of the secretory vesicle and gelatinase granules. The binding of galectin-1 and galectin-3 to neutrophil proteins was compared, as were the dose dependencies for activation by the two lectins. The results suggest that, although similarities are found between the two galectins, they appear to activate the NADPH-oxidase using different receptors. In conclusion, galectin-1 appears to have proinflammatory functions, mediated through activation of the neutrophil respiratory burst.
Monocyte Chemoattractant Protein-1 and 5-Lipoxygenase Products Recruit Leukocytes in Response to Platelet-Activating Factor-Like Lipids in Oxidized Low-Density Lipoprotein
Authors: Silva Adriana R. , de Assis Edson F. , Caiado Lara F. C. , Marathe Gopal K. , Bozza Marcelo T. , McIntyre Thomas M. , Zimmerman Guy A. , Prescott Stephen M. , Bozza Patricia T. , Castro-Faria-Neto Hugo C.
Published: April 2014

Oxidized low-density lipoprotein (LDL) contains inflammatory agents, including oxidatively fragmented phospholipids that activate the platelet-activating factor (PAF) receptor, but in vivo events caused by these pathologically generated agents are not well defined. Injection of PAF-like lipids derived from oxidized LDL, or C4-PAF that is a major PAF-like lipid in these particles, into the pleural cavity of mice resulted in rapid monocyte, neutrophil, and eosinophil accumulation. Increased numbers of intracellular lipid bodies in these cells show they were in an inflammatory environment. Leukocyte recruitment was abolished by a PAF receptor antagonist, as expected. PAF-like lipids induced 5-lipoxygenase expression in leukocytes, mRNA expression for monocyte chemoattractant protein-1 (MCP-1) and other chemokines, synthesis of MCP-1, and leukotriene B4. The 5-lipoxygenase inhibitor zileuton impaired neutrophil influx, while MCP-1 had a more global role, as determined with MCP-1−/− mice. The lack of MCP-1 abrogated leukocyte accumulation and lipid body formation both in vivo and in vitro and chemokine transcription in vivo, and reduced in vivo leukotriene B4 production. Thus, PAF-like phospholipids in oxidized LDL induce an inflammatory infiltrate through the PAF receptor, chemokine transcription, lipid body formation, and 5-lipoxygenase expression in leukocytes. MCP-1 has a key role in this inflammatory response, and 5-lipoxygenase products are essential for neutrophil recruitment into the inflamed pleural cavity.
IFN-γ Production by Intrinsic Renal Cells and Bone Marrow-Derived Cells Is Required for Full Expression of Crescentic Glomerulonephritis in Mice
Authors: Timoshanko Jennifer R. , Holdsworth Stephen R. , Kitching A. Richard , Tipping Peter G.
Published: April 2014

The contribution of IFN-γ from bone marrow (BM) and non-BM-derived cells to glomerular and cutaneous delayed-type hypersensitivity (DTH) was studied in mice. Chimeric IFN-γ mice (IFN-γ+/+ BM chimera), in which IFN-γ production was restricted to BM-derived cells, were created by transplanting normal C57BL/6 (wild-type (WT)) BM into irradiated IFN-γ-deficient mice. BM IFN-γ-deficient chimeric mice (IFN-γ−/− BM chimera) were created by transplanting WT mice with IFN-γ-deficient BM. WT and sham chimeric mice (WT mice transplanted with WT BM) developed crescentic glomerulonephritis (GN) with features of DTH (including glomerular T cell and macrophage infiltration) in response to an Ag planted in their glomeruli and skin DTH following subdermal Ag challenge. IFN-γ-deficient mice showed significant protection from crescentic GN and reduced cutaneous DTH. IFN-γ+/+ BM chimeric and IFN-γ−/− BM chimeric mice showed similar attenuation of crescentic GN as IFN-γ-deficient mice, whereas cutaneous DTH was reduced only in IFN-γ−/− BM chimeras. In crescentic GN, IFN-γ was expressed by tubular cells and occasional glomerular cells and was colocalized with infiltrating CD8+ T cells, but not with CD4+ T cells or macrophages. Renal MHC class II expression was reduced in IFN-γ+/+ BM chimeric mice and was more severely reduced in IFN-γ-deficient mice and IFN-γ−/− BM chimeric mice. These studies show that IFN-γ expression by both BM-derived cells and intrinsic renal cells is required for the development of crescentic GN, but IFN-γ production by resident cells is not essential for the development of cutaneous DTH.
Gene Expression Profiling in DQA1*0501+ Children with Untreated Dermatomyositis: A Novel Model of Pathogenesis
Authors: Tezak Zivana , Hoffman Eric P. , Lutz Jennica L. , Fedczyna Tamara O. , Stephan Dietrich , Bremer Eric G. , Krasnoselska-Riz Irina , Kumar Ajit , Pachman Lauren M.
Published: April 2014

Juvenile dermatomyositis (JDM), the most common pediatric inflammatory myopathy, is a systemic vasculopathy affecting young children. Epidemiology studies documenting an antecedent illness in the 3 mo before the first definite symptom (rash and/or weakness) of JDM are supported by immunologic data that suggest that the disease pathophysiology is Ag driven. The purpose of this study was to compare the gene expression profiles in muscle biopsies of four untreated DQA1*0501+ JDM children with profiles from children with a known necrotizing myopathy (Duchenne muscular dystrophy), as well as an in vitro antiviral model (NF90), and healthy pediatric controls. Nearly half (47%) of the dysregulated genes in JDM were associated with the immune response. In particular, increased expression of IFN-αβ-inducible genes 6-16, myxovirus resistance protein p78, latent cytosolic transcription factor, LMP2, and TAP1 was observed. This profile is consistent with an IFN-αβ transcription cascade seen in the in vitro viral resistance model. The IFN-αβ-inducible profile was superimposed on transcription profiles reflective of myofiber necrosis and regeneration shared with Duchenne muscular dystrophy. Expressed genes were confirmed by quantitative real-time PCR (6-16), immunofluorescence (thrombospondin 4), and immunolocalization (IFN-γ, p21). We hypothesize that these data support a model of Ag (?viral) induction of an apparent autoimmune disease based on dynamic interaction between the muscle, vascular, and immune systems in the genetically susceptible (DQA1*0501+) child.
Depletion of Collagen II-Reactive T Cells and Blocking of B Cell Activation Prevents Collagen II-Induced Arthritis in DBA/1j Mice
Authors: Zhang Huang-Ge , Yang PingAr , Xie Jinfu , Liu Zhongyu , Liu Di , Xiu Liang , Zhou Tong , Wang Yongming , Hsu Hui-Chen , Mountz John D.
Published: April 2014

Collagen II (CII)-induced arthritis in DBA/1j mice is mediated by both CII-reactive T cells and anti-CII Ab-producing B cells. To determine the relative role of these processes in the development of arthritis, we specifically eliminated CII-reactive T cells by treating the mice with CII-pulsed syngeneic macrophages that had been transfected with a binary adenovirus system. These macrophages express murine Fas ligand in a doxycycline-inducible manner with autocrine suicide inhibited by concomitant expression of p35. The mice were treated i.v. with four doses of CII-APC-AdFasLp35Tet or a single dose of AdCMVsTACI (5 × 109 PFU), or both simultaneously, beginning 2 wk after priming with CII in CFA. Treatment with CII-APC-AdFasLp35Tet alone or in combination with a single dose of AdCMVsTACI prevented the development of CII-induced arthritis and T cell infiltration in the joint. The elimination of T cells was specific in that a normal T cell response was observed on stimulation with OVA after treatment with CII-APC-AdFasLp35Tet. Treatment with AdCMVsTACI alone prevented production of detectable levels of circulating anti-CII autoantibodies and reduced the severity of arthritis but did not prevent its development. These results indicate that the CII-reactive T cells play a crucial role in the development of CII-induced arthritis and that the anti-CII Abs act to enhance the development of CII-induced arthritis.
A Role for IL-15 in Driving the Onset of Spontaneous Autoimmune Thyroiditis?
Authors: Kaiser Pete , Rothwell Lisa , Vašíček Dusan , Hala Karel
Published: April 2014

The obese strain (OS) of chickens, which suffers from spontaneous autoimmune thyroiditis, is an excellent animal model for Hashimoto’s thyroiditis and provides a unique opportunity to investigate the mechanisms underlying and driving the onset of the disease. Following recent advances in cloning chicken cytokines, we can now begin to investigate the role of cytokines in driving the lymphoid infiltration of the thyroid seen in these birds from day 7 posthatch. Using real-time quantitative RT-PCR, we characterized the expression of IFN-γ, IL-1β, IL-2, IL-6, IL-8, IL-15, and IL-18 in thyroids from OS birds and control CB line birds, both in the embryo just before hatch (embryonic day 20) and at 3 and 5 days posthatch. All of these cytokines were up-regulated compared with levels in thyroids from CB birds, at least at some time points, with some evidence for coordination of regulation, e.g., for the proinflammatory cytokines IL-1β and IL-8. Only IL-15 was up-regulated at all time points. IL-15 was also shown to be up-regulated in spleens of OS birds at embryonic day 20 and 5 days posthatch, suggesting that IL-15 is constitutively up-regulated in this line of birds. This could explain the general immune system hyperreactivity exhibited by OS chickens and may be a factor driving the lymphoid infiltration of the thyroid.