bluuebunny/crossref_metadata_2025 huggingface dataset:
Pertussis Toxin by Inducing IL-6 Promotes the Generation of IL-17-Producing CD4 Cells
Authors: Chen Xin , Howard O. M. Zack , Oppenheim Joost J.
Published: April 2014

Compelling evidence has now demonstrated that IL-17-producing CD4 cells (Th17) are a major contributor to autoimmune pathogenesis, whereas CD4+CD25+ T regulatory cells (Treg) play a major role in suppression of autoimmunity. Differentiation of proinflammatory Th17 and immunosuppressive Treg from naive CD4 cells is reciprocally related and contingent upon the cytokine environment. We and others have reported that in vivo administration of pertussis toxin (PTx) reduces the number and function of mouse Treg. In this study, we have shown that supernatants from PTx-treated mouse splenic cells, which contained IL-6 and other proinflammatory cytokines, but not PTx itself, overcame the inhibition of proliferation seen in cocultures of Treg and CD4+CD25− T effector cells. This stimulatory effect could be mimicked by individual inflammatory cytokines such as IL-1β, IL-6, and TNF-α. The combination of these cytokines synergistically stimulated the proliferation of CD4+CD25− T effector cells despite the presence of Treg with a concomitant reduction in the percentage of FoxP3+ cells and generation of IL-17-expressing cells. PTx generated Th17 cells, while inhibiting the differentiation of FoxP+ cells, from naive CD4 cells when cocultured with bone marrow-derived dendritic cells from wild-type mice, but not from IL-6−/− mice. In vivo treatment with PTx induced IL-17-secreting cells in wild-type mice, but not in IL-6−/− mice. Thus, in addition to inhibiting the development of Treg, the immunoadjuvant activity of PTx can be attributable to the generation of IL-6-dependent IL-17-producing CD4 cells.
NK Cells Negatively Regulate Antigen Presentation and Tumor-Specific CTLs in a Syngeneic Lymphoma Model
Authors: Barber Melissa A. , Zhang Tong , Gagne Bethany A. , Sentman Charles L.
Published: April 2014

NK cells are known to kill tumor cells and produce proinflammatory cytokines that lead to the generation of tumor-specific CTLs. Many studies have used MHC class I-deficient tumor cells and/or adjuvants that induce NK cell responses. In this study, the focus was on less-immunogenic lymphoma cells that express MHC class I as a model to study NK cell responses to tumors that do not directly stimulate NK cell activation. When RMA tumor cells that expressed a truncated version of OVA, or RMA cells alone, were injected into mice that were depleted of NK cells, the mice developed an increased number of tumor-specific CTLs, increased IFN-γ responses, and a higher amount of Ag presentation in draining LNs compared with mice with intact NK cells. These data suggest that NK cells can inhibit the development of effective adaptive immunity in the absence of signals that trigger NK cell activation.
Up-Regulation of Gene Related to Anergy in Lymphocytes Is Associated with Notch-Mediated Human T Cell Suppression
Authors: Kostianovsky Alex M. , Maier Lisa M. , Baecher-Allan Clare , Anderson Ana C. , Anderson David E.
Published: April 2014

A growing body of literature indicates that the Notch pathway can influence the activation and differentiation of peripheral murine T cells, though comparatively little is known about the effects of Notch signaling in human T cells. In the present report we demonstrate that Jagged-1-induced Notch signaling (using immobilized Jagged-1 fusion protein) during stimulation of purified human CD4+ and CD8+ T cells potently inhibits T cell proliferation and effector function, including both Th1- and Th2-associated cytokines. Inhibition of T cell activation is not due to apoptosis or disruption of proximal TCR signaling, but is associated with up-regulation of GRAIL (gene related to anergy in lymphocytes) in CD4+ T cells, with modest effects on other E3 ubiquitin ligases such as c-Cbl and Itch. When evaluated for its effects on CD4+ T cell differentiation, Jagged-1-mediated signaling inhibits T cell cytokine secretion with no significant effect on proliferative responses. Collectively, these data demonstrate that Notch signaling in human T cells induced by Jagged-1 promotes a novel form of T cell hyporesponsiveness that differs from anergy, whereby primary T cell proliferation and cytokine secretion are potently inhibited, and effector function but not proliferative capacity are ameliorated upon secondary stimulation.
Airway Epithelial STAT3 Is Required for Allergic Inflammation in a Murine Model of Asthma
Authors: Simeone-Penney Marina C. , Severgnini Mariano , Tu Powen , Homer Robert J. , Mariani Thomas J. , Cohn Lauren , Simon Amy R.
Published: April 2014

The STAT3 transcription factor is critical for cytokine signaling and the acute phase response, but its role in allergic asthma is largely undefined. To investigate the role of STAT3 in mediating allergic inflammation, we used chemical and genetic approaches to inactivate STAT3 in the airway epithelium of mice. In a murine model of chronic asthma, we demonstrate that the administration of house dust mite (HDM) leads to robust STAT3 activation in the airway epithelium, smooth muscle, and immune cells in the lungs of C57BL/6 mice. To investigate the role of STAT3 in HDM-induced airway inflammation, a conditional knockout of STAT3 in the airway epithelium was generated, e-STAT3−/−. We determined that e-STAT3−/− mice had a significant decrease in HDM-induced airway eosinophilia, lung Th2 accumulation, and chemokines compared with wild-type animals. Importantly, the e-STAT3−/− mice had a significant decrease in airway hyperresponsiveness to methacholine. The administration of two STAT kinase inhibitors diminished STAT3 activation and markedly abrogated the HDM-induced lung inflammation. These findings suggest that STAT3 acts as a novel epithelial regulator of the allergic response by altering Th2 cell recruitment and effector function, and thus, targeting this molecule may provide the basis for a novel asthma therapy.
Sensitization to TLR7 Agonist in IFN-β-Preactivated Dendritic Cells
Authors: Severa Martina , Remoli Maria Elena , Giacomini Elena , Annibali Viviana , Gafa Valerie , Lande Roberto , Tomai Mark , Salvetti Marco , Coccia Eliana M.
Published: April 2014

TLRs interact with a growing list of pathogen-derived products and these interactions drive the activation of innate and adaptive immune responses. Dendritic cells (DC) play a key role in these events expressing a heterogeneous repertoire of TLRs. We have previously demonstrated the production of type I IFNs in DC following bacterial infections and TLR triggering. In this study, we sought to characterize the transcriptome specifically induced in human DC by IFN-β production stimulated upon LPS treatment. To this aim, by using cDNA microarrays, we compared the transcriptome of DC following LPS treatment in the absence or presence of neutralizing anti-type I IFN Abs. Interestingly, we found that the expression of TLR7 was induced during LPS-induced maturation of DC in a type I IFN-dependent manner. The induction of TLR7 in maturing DC was mainly a consequence of the transcriptional activity of IRF-1, whose binding site was located within TLR7 promoter. Moreover, we also demonstrated that “priming” of immature DC, that usually express TLR8 but not TLR7, with exogenous IFN-β induced a functionally active TLR7. In fact, treatment with the TLR7-specific ligand 3M-001 up-regulated the expression of CD83, CD86, and CD38 in IFN-β-primed DC but not in immature DC. Therefore, a robust enhancement in proinflammatory as well as regulatory cytokines was observed. These data suggest that TLR4-mediated type I IFN release activates specific transcription programs in DC amplifying the expression of pathogen sensors to correctly and combinatorially respond to a bacterial as well as viral infection.
Fcγ Receptor IIB on Dendritic Cells Enforces Peripheral Tolerance by Inhibiting Effector T Cell Responses
Authors: Desai Dharmesh D. , Harbers Stephanie O. , Flores Marcella , Colonna Lucrezia , Downie Matthew P. , Bergtold Amy , Jung Steffen , Clynes Raphael
Published: April 2014

The uptake of immune complexes by FcRs on APCs augments humoral and cellular responses to exogenous Ag. In this study, CD11c+ dendritic cells are shown to be responsible in vivo for immune complex-triggered priming of T cells. We examine the consequence of Ab-mediated uptake of self Ag by dendritic cells in the rat insulin promoter-membrane OVA model and identify a role for the inhibitory FcγRIIB in the maintenance of peripheral CD8 T cell tolerance. Effector differentiation of diabetogenic OT-I CD8+ T cells is enhanced in rat insulin promoter-membrane OVA mice lacking FcγRIIB, resulting in a high incidence of diabetes. FcγRIIB-mediated inhibition of CD8 T cell priming results from suppression of both DC activation and cross-presentation through activating FcγRs. Further FcγRIIB on DCs inhibited the induction of OVA-specific Th1 effectors, limiting Th1-type differentiation and memory T cell accumulation. In these MHC II-restricted responses, the presence of FcγRIIB only modestly affected initial CD4 T cell proliferative responses, suggesting that FcγRIIB limited effector cell differentiation primarily by inhibiting DC activation. Thus, FcγRIIB can contribute to peripheral tolerance maintenance by inhibiting DC activation alone or by also limiting processing of exogenously acquired Ag.
Basal B Cell Receptor-Directed Phosphatidylinositol 3-Kinase Signaling Turns Off RAGs and Promotes B Cell-Positive Selection
Authors: Verkoczy Laurent , Duong Bao , Skog Patrick , Aït-Azzouzene Djemel , Puri Kamal , Vela José Luis , Nemazee David
Published: April 2014

PI3K plays key roles in cell growth, differentiation, and survival by generating the second messenger phosphatidylinositol-(3,4,5)-trisphosphate (PIP3). PIP3 activates numerous enzymes, in part by recruiting them from the cytosol to the plasma membrane. We find that in immature B lymphocytes carrying a nonautoreactive Ag receptor, PI3K signaling suppresses RAG expression and promotes developmental progression. Inhibitors of PI3K signaling abrogate this positive selection. Furthermore, immature primary B cells from mice lacking the p85α regulatory subunit of PI3K suppress poorly RAG expression, undergo an exaggerated receptor editing response, and, as in BCR-ligated cells, fail to progress into the G1 phase of cell cycle. Moreover, immature B cells carrying an innocuous receptor have sustained elevation of PIP3 levels and activation of the downstream effectors phospholipase C (PLC)γ2, Akt, and Bruton’s tyrosine kinase. Of these, PLCγ2 appears to play the most significant role in down-regulating RAG expression. It therefore appears that when the BCR of an immature B cell is ligated, PIP3 levels are reduced, PLCγ2 activation is diminished, and receptor editing is promoted by sustained RAG expression. Taken together, our results provide evidence that PI3K signaling is an important cue required for fostering development of B cells carrying a useful BCR.
Peritoneal Cell-Derived Mast Cells: An In Vitro Model of Mature Serosal-Type Mouse Mast Cells
Authors: Malbec Odile , Roget Karine , Schiffer Cécile , Iannascoli Bruno , Dumas Antoine Ribadeau , Arock Michel , Daëron Marc
Published: April 2014

Bone marrow-derived mast cells (BMMC) have been used extensively as a mast cell model. BMMC, however, are immature cells that have no known physiological equivalent in tissues. They do not respond to IgG immune complexes. They may therefore not be appropriate for studying the physiopathology of IgE-induced allergies or IgG-induced tissue-specific inflammatory diseases which both depend on mature mast cells. Resident peritoneal mast cells are a minor population of differentiated cells that are not readily purified. They, however, can be expanded in culture to generate large numbers of homogeneous cells. We show here that these peritoneal cell-derived mast cells (PCMC) are mature serosal-type mouse mast cells which retain most morphological, phenotypic, and functional features of peritoneal mast cells. Like peritoneal mast cells, PCMC respond to IgG Abs. IgG immune complex-induced responses depended on FcγRIIIA and were negatively regulated by FcγRIIB. We found that a moderate FcγRIIB-dependent negative regulation, due not to a higher FcγRIIIA/FcγRIIB ratio, but to a relatively inefficient use of the lipid phosphatase SHIP1, determines this property of PCMC. PCMC also respond to IgE Abs. IgE-induced PCMC responses, however, differed quantitatively and qualitatively from BMMC responses. PCMC secreted no or much lower amounts of lipid mediators, chemokines, and cytokines, but they contained and released much higher amounts of preformed granular mediators. PCMC, but not BMMC, also contained and, upon degranulation, released molecules with a potent proteolytic activity. These properties make PCMC a useful new model for understanding the physiopathology of mast cells in IgE- and IgG-dependent tissue inflammation.
Type I Interferon Signaling and B Cells Maintain Hemopoiesis during Pneumocystis Infection of the Lung
Authors: Meissner Nicole , Rutkowski Melanie , Harmsen Ann L. , Han Soo , Harmsen Allen G.
Published: April 2014

Loss of CD4 T cells is the hallmark of HIV infection. However, type I IFN-producing plasmacytoid dendritic cells may also be lost. This results in susceptibility to an opportunistic infection such as Pneumocystis pneumonia. In addition, regenerative bone marrow failure resulting in pancytopenia is another common problem in advanced stage AIDS. This may be linked to both the failing immune system and recurrent opportunistic infections. We generated lymphocyte-deficient type I IFN receptor-deficient mice (IFrag−/−) to study the effects on Pneumocystis infection of the lung. When IFrag−/− animals were infected with Pneumocystis they died between days 16 and 21 postinfection with minimal pneumonia but severe anemia due to complete bone marrow failure. This included the loss of uncommitted hemopoietic precursor cells. Bone marrow failure was prevented by the reconstitution of IFrag−/− mice with wild-type lymphocytes, especially B cells. T and B cells lacking type I IFN receptor signaling could only partially prevent bone marrow failure in response to Pneumocystis infection. However, the presence of T and B cells lacking type I IFN signaling resulted in compensatory extramedullary hemopoiesis in the liver and spleen. Lymphocyte support of the regenerative capacity of the bone marrow was provided by both type I IFN-dependent and -independent mechanisms that acted synergistically. Our findings point to the requirement of both type I IFNs and lymphocytes in the regenerative capabilities of the hemopoietic system under the pressure of Pneumocystis infection, but not during steady-state hemopoiesis. This may have implications in the management of pancytopenia in AIDS.
Cutting Edge: Direct Action of Thymic Stromal Lymphopoietin on Activated Human CD4+ T Cells
Authors: Rochman Irina , Watanabe Norihiko , Arima Kazuhiko , Liu Yong-Jun , Leonard Warren J.
Published: April 2014

Thymic stromal lymphopoietin (TSLP) is a cytokine that promotes CD4+ T cell homeostasis and contributes to allergic and inflammatory responses. TSLP can act directly on mouse CD4+ T cells, but in humans, the available data have indicated that TSLP receptors are not expressed on CD4+ T cells and that TSLP instead activates dendritic cells, which in turn promote the proliferation and differentiation of CD4+ T cells. We now unexpectedly demonstrate the presence of TSLP receptors on activated human CD4+ T cells. Strikingly, whereas freshly isolated peripheral blood human T cells show little if any response to TSLP, TCR stimulation allows a potent response to this cytokine. Moreover, TSLP increases the sensitivity of human CD4+ T cells to low doses of IL-2, augmenting responsiveness of these cells to TCR engagement. Our results establish that human CD4+ T cells are direct targets for TSLP.
Comparative Prime-Boost Vaccinations Using Semliki Forest Virus, Adenovirus, and ALVAC Vectors Demonstrate Differences in the Generation of a Protective Central Memory CTL Response against the P815 Tumor
Authors: Näslund Tanja I. , Uyttenhove Catherine , Nordström Eva K. L. , Colau Didier , Warnier Guy , Jondal Mikael , Van den Eynde Benoı̂t J. , Liljeström Peter
Published: April 2014

Tumor-specific Ags are potential target molecules in the therapeutic treatment of cancer. One way to elicit potent immune responses against these Ags is to use recombinant viruses, which activate both the innate and the adaptive arms of the immune system. In this study, we have compared Semliki Forest virus (SFV), adenovirus, and ALVAC (poxvirus) vectors for their capacity to induce CD8+ T cell responses against the P1A tumor Ag and to elicit protection against subsequent challenge injection of P1A-expressing P815 tumor cells in DBA/2 mice. Both homologous and heterologous prime-boost regimens were studied. In most cases, both higher CD8+ T cell responses and better tumor protections were observed in mice immunized with heterologous prime-boost regimens, suggesting that the combination of different viral vectors is beneficial for the induction of an effective immune response. However, homologous immunization with SFV provided potent tumor protection despite a rather moderate primary CD8+ T cell response as compared with mice immunized with recombinant adenovirus. SFV-immunized mice showed a rapid and more extensive expansion of P1A-specific CD8+ T cells in the tumor-draining lymph node after tumor challenge and had a higher frequency of CD62L+ P1A-specific T cells in the blood, spleen, and lymph nodes as compared with adenoimmunized mice. Our results indicate that not only the magnitude but in particular the quality of the CD8+ T cell response correlates with tumor protection.
Lack of Essential Role of NF-κB p50, RelA, and cRel Subunits in Virus-Induced Type 1 IFN Expression
Authors: Wang Xingyu , Hussain Sofia , Wang Emilie-Jeanne , Wang Xiuyan , Li Ming O. , García-Sastre Adolfo , Beg Amer A.
Published: April 2014

Type 1 IFNs (IFN-αβ) play pivotal roles in the host antiviral response and in TLR-induced signaling. IFN regulatory factor (IRF) and NF-κB transcription factors are thought to be crucial for virus-induced mRNA expression of IFN-β. Although recent studies have demonstrated essential roles for IRF3 and IRF7, the definitive role of NF-κB factors in IFN-β (or IFN-α) expression remains unknown. Using mice deficient in distinct members of the NF-κB family, we investigated NF-κB function in regulating type 1 IFN expression in response to Sendai virus and Newcastle disease virus infection. Surprisingly, IFN-β and IFN-α expression was strongly induced following virus infection of mouse embryonic fibroblasts (MEFs) from p50−/−, RelA/p65−/−, cRel−/−, p50−/−cRel−/−, and p50−/−RelA−/− mice. Compared with wild-type MEFs, only RelA−/− and p50−/−RelA−/− MEFs showed a modest reduction in IFN-β expression. To overcome functional redundancy between different NF-κB subunits, we expressed a dominant-negative IκBα protein in p50−/−RelA−/− MEFs to inhibit activation of remaining NF-κB subunits. Although viral infection of these cells failed to induce detectable NF-κB activity, both Sendai virus and Newcastle disease virus infection led to robust IFN-β expression. Virus infection of dendritic cells or TLR9-ligand CpG-D19 treatment of plasmacytoid dendritic cells from RelA−/− or p50−/−cRel−/− mice also induced robust type 1 IFN expression. Our findings therefore indicate that NF-κB subunits p50, RelA, and cRel play a relatively minor role in virus-induced type 1 IFN expression.
Th17 Augmentation in OTII TCR Plus T Cell-Selective Type 1 Sphingosine 1-Phosphate Receptor Double Transgenic Mice
Authors: Huang Mei-Chuan , Watson Susan R. , Liao Jia-Jun , Goetzl Edward J.
Published: April 2014

Sphingosine 1-phosphate (S1P) in blood and lymph controls lymphoid traffic and tissue migration of T cells through signals from the type 1 S1PR (S1P1), but less is known of effects of the S1P-S1P1 axis on nonmigration functions of T cells. CD4 T cells from a double transgenic (DTG) mouse express OTII TCRs specific for OVA peptide 323–339 (OVA) and a high level of transgenic S1P1, resistant to suppression by T cell activation. OVA-activated DTG CD4 T cells respond as expected to S1P by chemotactic migration and reduction in secretion of IFN-γ. In addition, DTG CD4 T cells stimulated by OVA secrete a mean of 2.5-fold more IL-17 than those from OTII single transgenic mice with concomitantly higher levels of mRNA encoding IL-17 by real-time PCR and of CD4 T cells with intracellular IL-17 detected by ELISPOT assays. OVA challenge of s.c. air pockets elicited influx of more OTII TCR-positive T cells producing a higher level of IL-17 in DTG mice than OTII control mice. Augmentation of the number and activity of Th17 cells by the S1P-S1P1 axis may thus enhance host defense against microbes and in other settings increase host susceptibility to autoimmune diseases.
Adaptation of TCR Repertoires to Self-Peptides in Regulatory and Nonregulatory CD4+ T Cells
Authors: Wong Jamie , Obst Reinhard , Correia-Neves Margarida , Losyev Grigoriy , Mathis Diane , Benoist Christophe
Published: April 2014

Currently, it is not understood how the specificity of the TCR guides CD4+ T cells into the conventional lineage (Tconv) vs directing them to become regulatory (Treg) cells defined by the Foxp3 transcription factor. To address this question, we made use of the “Limited” (LTD) mouse, which has a restricted TCR repertoire with a fixed TCRβ chain and a TCRα chain minilocus. The TCR repertoires of Tconv and Treg cells were equally broad, were distinct, yet overlapped significantly, representing a less strict partition than previously seen between CD4 and CD8 T cells. As a group, the CDR3α motifs showed a significant trend to higher positive charge in Treg than in Tconv cells. The Tconv and Treg repertoires were both reshaped between thymus and periphery. Reducing the array of peptides presented by MHC class II molecules by introducing the H2-DMo/o mutation into the LTD mouse led to parallel shifts in the repertoires of Tconv and Treg cells. In both cases, the CDR3α elements were entirely different and strikingly shortened, relative to normal LTD mice. These peculiar sequences conferred reactivity to wild-type MHC class II complexes and were excluded from the normal repertoire, even among Treg cells, indicating that some forms of self-reactivity are incompatible with selection into the Treg lineage. In conclusion, the Treg repertoire is broad, with distinct composition and characteristics, yet significantly overlapping and sharing structural constraints with the repertoire of conventional CD4+ T cells.
The Central Leucine-Rich Repeat Region of Chicken TLR16 Dictates Unique Ligand Specificity and Species-Specific Interaction with TLR2
Authors: Keestra A. Marijke , de Zoete Marcel R. , van Aubel Rémon A. M. H. , van Putten Jos P. M.
Published: April 2014

The ligand specificity of human TLR (hTLR) 2 is determined through the formation of functional heterodimers with either hTLR1 or hTLR6. The chicken carries two TLR (chTLR) 2 isoforms, type 1 and type 2 (chTLR2t1 and chTLR2t2), and one putative TLR1/6/10 homologue (chTLR16) of unknown function. In this study, we report that transfection of HeLa cells with the various chicken receptors yields potent NF-κB activation for the receptor combination of chTLR2t2 and chTLR16 only. The sensitivity of this complex was strongly enhanced by human CD14. The functional chTLR16/chTLR2t2 complex responded toward both the hTLR2/6-specific diacylated peptide S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) and the hTLR2/1 specific triacylated peptide tripalmitoyl-S-(bis(palmitoyloxy)propyl)-Cys-Ser-(Lys)3-Lys (Pam3CSK4), indicating that chTLR16 covers the functions of both mammalian TLR1 and TLR6. Dissection of the species specificity of TLR2 and its coreceptors showed functional chTLR16 complex formation with chTLR2t2 but not hTLR2. Conversely, chTLR2t2 did not function in combination with hTLR1 or hTLR6. The use of constructed chimeric receptors in which the defined domains of chTLR16 and hTLR1 or hTLR6 had been exchanged revealed that the transfer of leucine-rich repeats (LRR) 6–16 of chTLR16 into hTLR6 was sufficient to confer dual ligand specificity to the human receptor and to establish species-specific interaction with chTLR2t2. Collectively, our data indicate that diversification of the central LRR region of the TLR2 coreceptors during evolution has put constraints on both their ligand specificity and their ability to form functional complexes with TLR2.
The Neutralization Properties of a HIV-Specific Antibody Are Markedly Altered by Glycosylation Events Outside the Antigen-Binding Domain
Authors: Miranda Luis R. , Duval Mark , Doherty Heather , Seaman Michael S. , Posner Marshall R. , Cavacini Lisa A.
Published: April 2014

Neutralizing Abs constitute a pivotal mechanism of the adaptive immune response against HIV-1 infection. Yet, most of the Abs that appear in the circulation during HIV infection are nonneutralizing. In this study, we report a dramatic change of the neutralizing properties of a human Ab reactive with the nonneutralizing epitope termed cluster I on the HIV-1 transmembrane protein gp41 when the Ab was produced in Chinese hamster ovary (CHO)-K1 cells. Our laboratory has previously reported that the Ab F240, when produced in a hybridoma, is nonneutralizing as assessed by standard neutralization assays. The F240 IgG1 Ab expressed in CHO cells acquired a strong neutralization activity against a broad range of HIV isolates without a change in immunoreactivity. Sequencing of the F240 mRNAs produced in the parental hybridoma and CHO cells revealed identical sequences, suggesting that acquired neutralization resulted from cell-specific posttranslational modifications. We found that the Ab produced by CHO cells is glycosylated to a greater extent than the parental Ab produced by the hybridoma. Moreover, treatment with peptide N-glycosidase F abrogated F240 neutralization, in an isolate-specific manner, but not Ab b12 neutralization. Interestingly, the F240 isotype-switched variants IgG3 and IgG4, also expressed in CHO cells, exhibited identical immunoreactivity to IgG1 isotypes but had clear differences in viral neutralization. These results suggest that structural features of the Ig molecule other than the primary sequence of the variable regions play a more prominent role in HIV neutralization than anticipated.
Defining In Vivo Transcription Factor Complexes of the Murine CD21 and CD23 Genes
Authors: Debnath Irina , Roundy Kirstin M. , Weis Janis J. , Weis John H.
Published: April 2014

The expression of the CD21 and CD23 genes is coincident with differentiation from transition 1 B cells (T1) to transition 2 B cells (T2). To define constituents controlling CD21 and CD23 expression, we conducted chromatin immunoprecipitation analyses for candidate transcription factors. We found constitutive binding of Oct-1, NFAT species, YY1, NF-κB-p52, Pax5, E2A, and RBP-Jκ to CD21 sequences and NF-κB-p52, Pax5, NFAT species, E2A, and RBP-Jκ to CD23 promoter sequences. Splenic T and B cell subsets displayed constitutive binding of YY1, NF-κB-p52, Pax5, and Oct-1 proteins to CD21 sequences in B cells but no specific binding of NFATc3 or Pax5 in T cells. Similarly, CD23 sequences demonstrated constitutive binding of NF-κB-p52 in splenic T and B cells but only Pax5 in B cells. Of the various NFAT species, only a subset were found forming constitutive DNA/protein complexes with the CD21, CD23, and IL-2 gene sequences. Maturing B cells in the marrow possess stable Pax5 complexes on CD19, CD21, and CD23 gene promoters in the nuclei of such cells, even though only CD19 is expressed. The similarity of genetic controlling elements between the CD21 and CD23 genes does not suggest a mechanism for alternative regulation of these genes; however, separation of splenic B cell subsets into T1, T2, marginal zone (MZ), and mature follicular B cells, followed by quantitative RT-PCR, demonstrated the lack of appreciable CD23 transcripts in CD21+ MZ cells. We propose an alternative derivation of MZ cells as maturing directly from T1 cells, leaving CD23 transcriptionally inactive in that lineage of cells.
Genomics and Diversity of the Common Marmoset Monkey NK Complex
Authors: Averdam Anne , Kuhl Heiner , Sontag Mario , Becker Tamara , Hughes Austin L. , Reinhardt Richard , Walter Lutz
Published: April 2014

The common marmoset monkey (Callithrix jacchus) is a New World primate that is increasingly used in biomedical research as a model organism. Due to the occurrence of natural bone marrow chimerism, it represents a particularly useful primate model in immunological research. In this study, we describe the genomic organization of the CD94, NKG2, and LY49L genes in the NK complex (NKC) of the common marmoset based on complete sequencing of a bacterial artificial chromosome clonal contig. This region of the marmoset NKC is 1.5 times smaller than its human counterpart, but the genes are colinear and orthologous. One exception is the activating NKG2CE gene, which is probably an ancestral form of the NKG2C- and NKG2E-activating receptor genes of humans and great apes. The two completely sequenced marmoset bacterial artificial chromosome clones are derived from distinct haplotypes, which differ by 200 sites in the overlapping sequence. Analyses of NKC genes in nine additional marmoset individuals revealed a moderate degree of polymorphism of the CD94, NKG2A, NKG2CE, and NKG2D genes. Furthermore, expression analyses identified several alternatively spliced transcripts, particularly of the CD94 gene. Several products of alternative splicing of NKC genes are highly conserved among primates. Alternative transcriptional start sites were found, but these probably do not lead to a change of the translational start site or result in longer or shorter cytoplasmic regions of these type II membrane receptors.
Extended Gene Map Reveals Tripartite Motif, C-Type Lectin, and Ig Superfamily Type Genes within a Subregion of the Chicken MHC-B Affecting Infectious Disease
Authors: Shiina Takashi , Briles W. Elwood , Goto Ronald M. , Hosomichi Kazuyoshi , Yanagiya Kazuyo , Shimizu Sayoko , Inoko Hidetoshi , Miller Marcia M.
Published: April 2014

MHC haplotypes have a remarkable influence on whether tumors form following infection of chickens with oncogenic Marek’s disease herpesvirus. Although resistance to tumor formation has been mapped to a subregion of the chicken MHC-B region, the gene or genes responsible have not been identified. A full gene map of the subregion has been lacking. We have expanded the MHC-B region gene map beyond the 92-kb core previously reported for another haplotype revealing the presence of 46 genes within 242 kb in the Red Jungle Fowl haplotype. Even though MHC-B is structured differently, many of the newly revealed genes are related to loci typical of the MHC in other species. Other MHC-B loci are homologs of genes found within MHC paralogous regions (regions thought to be derived from ancient duplications of a primordial immune defense complex where genes have undergone differential silencing over evolutionary time) on other chromosomes. Still others are similar to genes that define the NK complex in mammals. Many of the newly mapped genes display allelic variability and fall within the MHC-B subregion previously shown to affect the formation of Marek’s disease tumors and hence are candidates for genes conferring resistance.
Viral Interference with Antigen Presentation Does Not Alter Acute or Chronic CD8 T Cell Immunodominance in Murine Cytomegalovirus Infection
Authors: Munks Michael W. , Pinto Amelia K. , Doom Carmen M. , Hill Ann B.
Published: April 2014

Both human CMV and murine CMV (MCMV) elicit large CD8 T cell responses, despite the potent effects of viral genes that interfere with the MHC class I (MHC I) pathway of Ag presentation. To investigate the impact of immune evasion on CD8 T cell priming, we infected mice with wild-type (wt) MCMV or a mutant lacking its MHC I immune evasion genes, Δm4+m6+m152 MCMV. In acute infection, the two viruses elicited a CD8 T cell response to 26 peptide epitopes that was virtually identical in total size, kinetics, and immunodominance hierarchy. This occurred despite results demonstrating that primary DCs are susceptible to the effects of MCMV’s MHC I immune evasion genes. Eight months later, responses to both wt and mutant MCMV displayed the same CD8 T cell “memory inflation” and altered immunodominance that characterize the transition to chronic MCMV infection in C57BL/6 mice. Taken together, these findings suggest either that cross-priming dominates over direct CD8 T cell priming in both acute and chronic MCMV infection, or else that the MHC I immune evasion genes of MCMV are unable to alter direct CD8 T cell priming in vivo. At 2 years postinfection, differences in CD8 T cell immunodominance emerged between individual mice, but on average there were only slight differences between wt and mutant virus infections. Overall, the data indicate that the presence or absence of MHC I immune evasion genes has remarkably little impact on the size or specificity of the MCMV-specific CD8 T cell response over an entire lifetime of infection.
Cutting Edge: A Common Polymorphism Impairs Cell Surface Trafficking and Functional Responses of TLR1 but Protects against Leprosy
Authors: Johnson Christopher M. , Lyle Elizabeth A. , Omueti Katherine O. , Stepensky Vitaly A. , Yegin Olcay , Alpsoy Erkan , Hamann Lutz , Schumann Ralf R. , Tapping Richard I.
Published: April 2014

TLRs constitute an essential family of pattern recognition molecules that, through direct recognition of conserved microbial components, initiate inflammatory responses following infection. In this role, TLR1 enables host responses to a variety of bacteria, including pathogenic species of mycobacteria. In this study, we report that I602S, a common single nucleotide polymorphism within TLR1, is associated with aberrant trafficking of the receptor to the cell surface and diminished responses of blood monocytes to bacterial agonists. When expressed in heterologous systems, the TLR1 602S variant, but not the TLR1 602I variant, exhibits the expected deficiencies in trafficking and responsiveness. Among white Europeans, the 602S allele represents the most common single nucleotide polymorphism affecting TLR function identified to date. Surprisingly, the 602S allele is associated with a decreased incidence of leprosy, suggesting that Mycobacterium leprae subverts the TLR system as a mechanism of immune evasion.
Impaired Accumulation of Antigen-Specific CD8 Lymphocytes in Chemokine CCL25-Deficient Intestinal Epithelium and Lamina Propria
Authors: Wurbel Marc-André , Malissen Marie , Guy-Grand Delphine , Malissen Bernard , Campbell James J.
Published: April 2014

CCL25 and CCR9 constitute a chemokine/receptor pair involved in T cell development and in gut-associated immune responses. In this study, we generated CCL25−/− mice to answer questions that could not be addressed with existing CCR9−/− mice. Similar phenotypes were observed for both CCL25−/− and CCR9−/− mice, consistent with the notion that CCL25 and CCR9 interact with each other exclusively. We assessed the requirement for CCL25 in generating CCR9high CD8 intestinal memory-phenotype T cells and the subsequent accumulation of these cells within effector sites. TCR-transgenic naive CD8 T cells were transferred into wild-type or CCL25-deficient hosts. Oral sensitization with Ag allowed these naive donor cells to efficiently differentiate into CCR9high memory-phenotype cells within the mesenteric lymph nodes of wild-type hosts. This differentiation event occurred with equal efficiency in the MLN of CCL25-deficient hosts, demonstrating that CCL25 is not required to induce the CCR9high memory phenotype in vivo. However, we found that CCL25 deficiency severely impaired the Ag-dependent accumulation of donor-derived CD8 T cells within both lamina propria and epithelium of the small intestine. Thus, although CCL25 is not necessary for generating memory-phenotype CD8 T cells with “gut-homing” properties, this chemokine is indispensable for their trafficking to the small intestine.
Krüppel-Like Factor 2 Controls T Cell Trafficking by Activating L-Selectin (CD62L) and Sphingosine-1-Phosphate Receptor 1 Transcription
Authors: Bai Ailin , Hu Hui , Yeung Mandy , Chen Jianzhu
Published: April 2014

Krüppel-like factor 2 (KLF2) is a member of zinc-finger transcription factors. Based on its expression in naive and memory T cells and the activated phenotype of few T cells in mice lacking KLF2 in the lymphoid lineage, KLF2 is postulated to regulate T cell homeostasis by promoting cell quiescence. In this study, we show that in reporter gene assays KLF2 directly activates the promoters of both CD62L and sphingosine-1-phosphate receptor 1 (S1P1), whose expression is critical for T cell egress from the thymus and homing to the lymph nodes. Correspondingly, exogenous KLF2 expression in primary T cells significantly up-regulates both CD62L and S1P1. Following adoptive transfer, KLF2-transduced T cells are much more efficient in homing to lymphoid organs than nontransduced T cells. These findings suggest that KLF2 regulates T cell homeostasis at least partly by controlling CD62L and S1P1 expression, and therefore T cell egress from the thymus and circulation in the periphery.
Acute Alcohol Exposure Exerts Anti-Inflammatory Effects by Inhibiting IκB Kinase Activity and p65 Phosphorylation in Human Monocytes
Authors: Mandrekar Pranoti , Jeliazkova Valentina , Catalano Donna , Szabo Gyongyi
Published: April 2014

Acute alcohol use is associated with impaired immune responses and decreased proinflammatory cytokine production. Our earlier studies have shown that acute alcohol intake inhibits NF-κB DNA binding in an IκBα-independent manner. We report using human peripheral blood monocytes and Chinese hamster ovary cells transfected with CD14 cells that acute alcohol treatment in vitro exerts NF-κB inhibition by disrupting phosphorylation of p65. Immunoprecipitation of p65 and IκBα revealed that acute alcohol exposure for 1 h decreased NF-κB-IκBα complexes in the cytoplasm. Phosphorylation of p65 at Ser536 is mediated by IκB kinase (IKK)β and is required for NF-κB-dependent cellular responses. We show that acute alcohol treatment decreased LPS-induced IKKα and IKKβ activity resulting in decreased phosphorylation of p65 at Ser536. Furthermore, nuclear expression of IKKα increased after alcohol treatment, which may contribute to inhibition of NF-κB. Decreased phosphorylation of nuclear p65 at Ser276 was likely not due to alcohol-induced inhibition of protein kinase A and mitogen- and stress-activated protein kinase-1 activity. Although decreased IκBα phosphorylation after acute alcohol treatment was attributable to reduced IKKβ activity, degradation of IκBα during alcohol exposure was IKKβ-independent. Alcohol-induced degradation of IκBα in the presence of a 26S proteasome inhibitor suggested proteasome-independent IκBα degradation. Collectively, our studies suggest that acute alcohol exposure modulates IκBα-independent NF-κB activity primarily by affecting phosphorylation of p65. These findings further implicate an important role for IKKβ in the acute effects of alcohol in immune cells.
The Loss of Telomerase Activity in Highly Differentiated CD8+CD28−CD27− T Cells Is Associated with Decreased Akt (Ser473) Phosphorylation
Authors: Plunkett Fiona J. , Franzese Ornella , Finney Helene M. , Fletcher Jean M. , Belaramani Lavina L. , Salmon Mike , Dokal Inderjeet , Webster David , Lawson Alastair D. G. , Akbar Arne N.
Published: April 2014

The enzyme telomerase is essential for maintaining the replicative capacity of memory T cells. Although CD28 costimulatory signals can up-regulate telomerase activity, human CD8+ T cells lose CD28 expression after repeated activation. Nevertheless, telomerase is still inducible in CD8+CD28− T cells. To identify alternative costimulatory pathways that may be involved, we introduced chimeric receptors containing the signaling domains of CD28, CD27, CD137, CD134, and ICOS in series with the CD3 zeta (ζ) chain into primary human CD8+ T cells. Although CD3 ζ-chain signals alone were ineffective, triggering of all the other constructs induced proliferation and telomerase activity. However, not all CD8+CD28− T cells could up-regulate this enzyme. The further fractionation of CD8+CD28− T cells into CD8+CD28− CD27+ and CD8+CD28−CD27− subsets showed that the latter had significantly shorter telomeres and extremely poor telomerase activity. The restoration of CD28 signaling in CD8+CD28−CD27− T cells could not reverse the low telomerase activity that was not due to decreased expression of human telomerase reverse transcriptase, the enzyme catalytic subunit. Instead, the defect was associated with decreased phosphorylation of the kinase Akt, that phosphorylates human telomerase reverse transcriptase to induce telomerase activity. Furthermore, the defective Akt phosphorylation in these cells was specific for the Ser473 but not the Thr308 phosphorylation site of this molecule. Telomerase down-regulation in highly differentiated CD8+CD28−CD27− T cells marks their inexorable progress toward a replicative end stage after activation. This limits the ability of memory CD8+ T cells to be maintained by continuous proliferation in vivo.
Requirements for T Lymphocyte Migration in Explanted Lymph Nodes
Authors: Huang Julie H. , Cárdenas-Navia L. Isabel , Caldwell Charles C. , Plumb Troy J. , Radu Caius G. , Rocha Paulo N. , Wilder Tuere , Bromberg Jonathan S. , Cronstein Bruce N. , Sitkovsky Michail , Dewhirst Mark W. , Dustin Michael L.
Published: April 2014

Although the requirements for T lymphocyte homing to lymph nodes (LNs) are well studied, much less is known about the requirements for T lymphocyte locomotion within LNs. Imaging of murine T lymphocyte migration in explanted LNs using two-photon laser-scanning fluorescence microscopy provides an opportunity to systematically study these requirements. We have developed a closed system for imaging an intact LN with controlled temperature, oxygenation, and perfusion rate. Naive T lymphocyte locomotion in the deep paracortex of the LN required a perfusion rate of >13 μm/s and a partial pressure of O2 (pO2) of >7.4%. Naive T lymphocyte locomotion in the subcapsular region was 38% slower and had higher turning angles and arrest coefficients than naive T lymphocytes in the deep paracortex. T lymphocyte activation decreased the requirement for pO2, but also decreased the speed of locomotion in the deep paracortex. Although CCR7−/− naive T cells displayed a small reduction in locomotion, systemic treatment with pertussis toxin reduced naive T lymphocyte speed by 59%, indicating a contribution of Gαi-mediated signaling, but involvement of other G protein-coupled receptors besides CCR7. Receptor knockouts or pharmacological inhibition in the adenosine, PG/lipoxygenase, lysophosphatidylcholine, and sphingosine-1-phosphate pathways did not individually alter naive T cell migration. These data implicate pO2, tissue architecture, and G-protein coupled receptor signaling in regulation of naive T lymphocyte migration in explanted LNs.
Expression of the Voltage-Gated Sodium Channel NaV1.5 in the Macrophage Late Endosome Regulates Endosomal Acidification
Authors: Carrithers Michael D. , Dib-Hajj Sulayman , Carrithers Lisette M. , Tokmoulina Gouzel , Pypaert Marc , Jonas Elizabeth A. , Waxman Stephen G.
Published: April 2014

Voltage-gated sodium channels expressed on the plasma membrane activate rapidly in response to changes in membrane potential in cells with excitable membranes such as muscle and neurons. Macrophages also require rapid signaling mechanisms as the first line of defense against invasion by microorganisms. In this study, our goal was to examine the role of intracellular voltage-gated sodium channels in macrophage function. We demonstrate that the cardiac voltage-gated sodium channel, NaV1.5, is expressed on the late endosome, but not the plasma membrane, in a human monocytic cell line, THP-1, and primary human monocyte-derived macrophages. Although the neuronal channel, NaV1.6, is also expressed intracellularly, it has a distinct subcellular localization. In primed cells, NaV1.5 regulates phagocytosis and endosomal pH during LPS-mediated endosomal acidification. Activation of the endosomal channel causes sodium efflux and decreased intraendosomal pH. These results demonstrate a functionally relevant intracellular voltage-gated sodium channel and reveal a novel mechanism to regulate macrophage endosomal acidification.
The Nod-Like Receptor Family Member Naip5/Birc1e Restricts Legionella pneumophila Growth Independently of Caspase-1 Activation
Authors: Lamkanfi Mohamed , Amer Amal , Kanneganti Thirumala-Devi , Muñoz-Planillo Raúl , Chen Grace , Vandenabeele Peter , Fortier Anne , Gros Philippe , Núñez Gabriel
Published: April 2014

Similar to Ipaf and caspase-1, the Nod-like receptor protein Naip5 restricts intracellular proliferation of Legionella pneumophila, the causative agent of a severe form of pneumonia known as Legionnaires’ disease. Thus, Naip5 has been suggested to regulate Legionella replication inside macrophages through the activation of caspase-1. In this study, we show that cytosolic delivery of recombinant flagellin activated caspase-1 in A/J macrophages carrying a mutant Naip5 allele, and in C57BL/6 (B6) macrophages congenic for the mutant Naip5 allele (B6-Naip5A/J), but not in Ipaf−/− cells. In line with these results, A/J and B6-Naip5A/J macrophages induced high levels of caspase-1 activation and IL-1β secretion when infected with Legionella. In addition, transgenic expression of a functional Naip5 allele in A/J macrophages did not alter Legionella-induced caspase-1 activation and IL-1β secretion. Notably, defective Naip5 signaling renders B6-Naip5A/J macrophages permissive for Legionella proliferation despite normal caspase-1 activation. These results indicate that the restriction of intracellular Legionella replication is more complex than previously appreciated and requires both Ipaf-dependent caspase-1 activation as well as functional Naip5 signaling.
STAT1 Activation Causes Translocation of Bax to the Endoplasmic Reticulum during the Resolution of Airway Mucous Cell Hyperplasia by IFN-γ
Authors: Stout Barbara A. , Melendez Karla , Seagrave JeanClare , Holtzman Michael J. , Wilson Bridget , Xiang Jialing , Tesfaigzi Yohannes
Published: April 2014

Disruption of the normal resolution process of inflammation-induced mucous cell hyperplasia may lead to sustained mucous hypersecretion in chronic diseases. During prolonged exposure of mice to allergen, IFN-γ reduces mucous cell hyperplasia, but the signaling responsible for the cell death is largely unknown. A brief phosphorylation of STAT1 by IFN-γ was required for cell death in airway epithelial cells (AEC), and during prolonged exposure to allergen, mucous cell hyperplasia remained elevated in STAT1−/− but was resolved in STAT1+/+ mice. Although IFN-γ treatment of primary human AECs and other airway cell lines left Bax protein levels unchanged, it caused translocation of Bax from the cytosol to the endoplasmic reticulum (ER) but not to the mitochondria. Localization of Bax to the ER was observed in IFN-γ-treated primary AECs isolated from STAT1+/+ mice but not in cells from STAT1−/− mice. In addition, ER Bax was detected in mucous cells of STAT1+/+ but not STAT1−/− airways of mice exposed to allergen for prolonged periods. IFN-γ did not release cytochrome c from mitochondria but reduced ER calcium stores and dilated the ER, confirming that the IFN-γ-induced cell death is mediated through changes localized in the ER. Collectively, these observations suggest that STAT1-dependent translocation of Bax to the ER is crucial for IFN-γ-induced cell death of AECs and the resolution of allergen-induced mucous cell hyperplasia.