bluuebunny/crossref_metadata_2025 huggingface dataset:
Compromised Humoral and Delayed-Type Hypersensitivity Responses in IL-23-Deficient Mice
Authors: Ghilardi Nico , Kljavin Noelyn , Chen Qi , Lucas Sophie , Gurney Austin L. , de Sauvage Frederic J.
Published: April 2014

The heterodimeric cytokine IL-23 consists of a private cytokine-like p19 subunit and a cytokine receptor-like subunit, p40, which is shared with IL-12. Previously reported IL-12p40-deficient mice have profound immune defects resulting from combined deficiency in both IL-12 and IL-23. To address the effects of specific IL-23 deficiency, we generated mice lacking p19 by gene targeting. These mice display no overt abnormalities but mount severely compromised T-dependent humoral immune responses. IL-23p19−/− mice produce strongly reduced levels of Ag-specific Igs of all isotypes, but mount normal T-independent B cell responses. In addition, delayed type hypersensitivity responses are strongly impaired in the absence of IL-23, indicating a defect at the level of memory T cells. T cells stimulated with IL-23-deficient APCs secrete significantly reduced amounts of the proinflammatory cytokine IL-17, and IL-23-deficient mice phenotypically resemble IL-17-deficient animals. Thus, IL-23 plays a critical role in T cell-dependent immune responses, and our data provide further support for the existence of an IL-23/IL-17 axis of communication between the adaptive and innate parts of the immune system.
CD4 T Cell-Dependent CD8 T Cell Maturation
Authors: Khanolkar Aaruni , Fuller Michael J. , Zajac Allan J.
Published: April 2014

We have investigated the contribution of CD4 T cells to the optimal priming of functionally robust memory CD8 T cell subsets. Intranasal infection of CD4 T cell-deficient (CD4−/−) mice with lymphocytic choriomeningitis virus resulted in the elaboration of virus-specific CD8 T cell responses that cleared the infection. However, by comparison with normal mice, the virus-specific CD8 T cells in CD4−/− mice were quantitatively and qualitatively different. In normal mice, lymphocytic choriomeningitis virus-specific memory CD8 T cells are CD44high, many are CD122high, and a majority of these cells regain expression of CD62L overtime. These cells produce IFN-γ and TNF-α, and a subset also produces IL-2. In the absence of CD4 T cell help, a distinct subset of memory CD8 T cells develops that remains CD62Llow up to 1 year after infection and exhibits a CD44intCD122low phenotype. These cells are qualitatively different from their counterparts in normal hosts, as their capacity to produce TNF-α and IL-2 is diminished. In addition, although CD4-independent CD8 T cells can contain the infection following secondary viral challenge, their ability to expand is impaired. These findings suggest that CD4 T cell responses not only contribute to the optimal priming of CD8 T cells in chronically infected hosts, but are also critical for the phenotypic and functional maturation of CD8 T cell responses to Ags that are more rapidly cleared. Moreover, these data imply that the development of CD62Lhigh central memory CD8 T cells is arrested in the absence of CD4 T cell help.
A Role for Toll-Like Receptor 4 in Dendritic Cell Activation and Cytolytic CD8+ T Cell Differentiation in Response to a Recombinant Heat Shock Fusion Protein
Authors: Palliser Deborah , Huang Qian , Hacohen Nir , Lamontagne Steven P. , Guillen Eduardo , Young Richard A. , Eisen Herman N.
Published: April 2014

Recombinant heat shock fusion proteins (Hsfp) injected into mice without added adjuvants can stimulate production of CD8 cytolytic T cells. Because initiation of productive immune responses generally requires dendritic cell (DC) activation, the question arises as to whether the Hsfp can activate DC independently of contaminating LPS. Using microarray analyses of DC from LPS-insensitive mice having a point mutation in Toll-like receptor 4 (Tlr4) (C3H/HeJ), or lacking Tlr4 (B10/ScNCr), we show here that unlike a LPS standard, Hsfp activated DC from HeJ mice almost as well as DC from wild-type mice. Consistent with the microarray analysis, the Hsfp’s ability to activate DC was not eliminated by polymyxin B but was destroyed by proteinase K. The Hsfp did not, however, stimulate DC from mice lacking Tlr4. In vivo the CD8 T cell response to the Hsfp in mice lacking Tlr4 was impaired: the responding CD8 cells initially proliferated vigorously but their development into cytolytic effector cells was diminished. Overall, the results indicate that this Hsfp can activate DC independently of LPS but still requires Tlr4 for an optimal CD8 T cell response.
Different Potentials of γδ T Cell Subsets in Regulating Airway Responsiveness: Vγ1+ Cells, but Not Vγ4+ Cells, Promote Airway Hyperreactivity, Th2 Cytokines, and Airway Inflammation
Authors: Hahn Youn-Soo , Taube Christian , Jin Niyun , Sharp Laura , Wands J. M. , Aydintug M. Kemal , Lahn Michael , Huber Sally A. , O’Brien Rebecca L. , Gelfand Erwin W. , Born Willi K.
Published: April 2014

Allergic airway inflammation and hyperreactivity are modulated by γδ T cells, but different experimental parameters can influence the effects observed. For example, in sensitized C57BL/6 and BALB/c mice, transient depletion of all TCR-δ+ cells just before airway challenge resulted in airway hyperresponsiveness (AHR), but caused hyporesponsiveness when initiated before i.p. sensitization. Vγ4+ γδ T cells strongly suppressed AHR; their depletion relieved suppression when initiated before challenge, but not before sensitization, and they suppressed AHR when transferred before challenge into sensitized TCR-Vγ4−/−/6−/− mice. In contrast, Vγ1+ γδ T cells enhanced AHR and airway inflammation. In normal mice (C57BL/6 and BALB/c), enhancement of AHR was abrogated only when these cells were depleted before sensitization, but not before challenge, and with regard to airway inflammation, this effect was limited to C57BL/6 mice. However, Vγ1+ γδ T cells enhanced AHR when transferred before challenge into sensitized B6.TCR-δ−/− mice. In this study Vγ1+ cells also increased levels of Th2 cytokines in the airways and, to a lesser extent, lung eosinophil numbers. Thus, Vγ4+ cells suppress AHR, and Vγ1+ cells enhance AHR and airway inflammation under defined experimental conditions. These findings show how γδ T cells can be both inhibitors and enhancers of AHR and airway inflammation, and they provide further support for the hypothesis that TCR expression and function cosegregate in γδ T cells.
A Novel Anti-Inflammatory Role of Simvastatin in a Murine Model of Allergic Asthma
Authors: McKay Anne , Leung Bernard P. , McInnes Iain B. , Thomson Neil C. , Liew Foo Y.
Published: April 2014

Statins, the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, are effective serum cholesterol-lowering agents in clinical practice, and they may also have anti-inflammatory properties. Asthma is characterized by chronic eosinophilic inflammation in the airways, which is thought to be regulated by the activity of T lymphocytes. We therefore examined the anti-inflammatory activity of simvastatin in a murine model of allergic asthma. In mice previously sensitized to OVA, simvastatin treatment, either orally or i.p., reduced the total inflammatory cell infiltrate and eosinophilia in bronchoalveolar lavage fluid in response to inhaled OVA challenge. Simvastatin therapy i.p. was also associated with a reduction in IL-4 and IL-5 levels in bronchoalveolar lavage fluid and, at higher doses, a histological reduction in inflammatory infiltrates in the lungs. OVA-induced IL-4, IL-5, IL-6, and IFN-γ secretion was reduced in thoracic lymph node cultures from simvastatin-treated mice. Simvastatin treatment did not alter serum total IgE or OVA-specific IgG1 and IgG2a levels. These data demonstrate the therapeutic potential of statin-sensitive pathways in allergic airways disease.
IL-12 Inhibits Thymic Involution by Enhancing IL-7- and IL-2-Induced Thymocyte Proliferation
Authors: Li Lina , Hsu Hui-Chen , Stockard Cecil R. , Yang PingAr , Zhou Juling , Wu Qi , Grizzle William E. , Mountz John D.
Published: April 2014

IL-12 has been reported to affect thymic T cell selection, but the role of IL-12 in thymic involution has not been studied. We found that in vivo, IL-12b knockout (IL-12b−/−) mice exhibited accelerated thymic involution compared with wild-type (WT) B6 mice. This is characterized by an increase in thymocytes with the early development stage phenotype of CD25−CD44+CD4−CD8− in aged IL-12b−/− mice. Histologically, there were accelerated degeneration of thymic extracellular matrix and blood vessels, a significantly decreased thymic cortex/medulla ratio, and increased apoptotic cells in aged IL-12b−/− mice compared with WT mice. There was, however, no apparent defect in thymic structure and thymocyte development in young IL-12−/− mice. These results suggest the importance of IL-12 in maintaining thymic integrity and function during the aging process. Surprisingly, in WT B6 mice, there was no age-related decrease in the levels of IL-12 produced from thymic dendritic cells. Stimulation of thymocytes with IL-12 alone also did not enhance the thymocyte proliferative response in vitro. IL-12, however, provided a strong synergistic effect to augment the IL-7 or IL-2 induced thymocyte proliferative response, especially in aged WT and IL-12b−/− mice. Our data strongly support the role of IL-12 as an enhancement cytokine, which acts through its interactions with other cytokines to maintain thymic T cell function and development during aging.
Differential Segmental Flexibility and Reach Dictate the Antigen Binding Mode of Chimeric IgD and IgM: Implications for the Function of the B Cell Receptor
Authors: Løset Geir Å. , Roux Kenneth H. , Zhu Ping , Michaelsen Terje E. , Sandlie Inger
Published: April 2014

Mature, naive B cells coexpress IgD and IgM with identical binding sites. In this study, the binding properties of such IgM and IgD are compared to determine how size and shape may influence their ability to bind Ag and thus function as receptors. To dissect their intrinsic binding properties, recombinant IgM and IgD were produced in soluble form as monomers of the basic H2L2 Ab architecture, each with two Ag binding sites. Since these sites are connected with a hinge region in IgD and structural Ig domains in IgM, the two molecules differ significantly in this region. The results show that IgD exhibited the larger angle and longer distance between its binding sites, as well as having the greater flexibility. Relative functional affinity was assessed on two antigenic surfaces with high or low epitope density, respectively. At high epitope density, IgM had a higher functional affinity for the Ag compared with IgD. The order was reversed at low epitope density due to a decrease in the functional affinity of IgM. Studies of binding kinetics showed similar association rates for both molecules. The dissociation rate, however, was slower for IgM at high epitope density and for IgD at low epitope density. Taken together, the results show that IgM and IgD with identical Ag binding regions have different Ag binding properties.
Posttranscriptional Inhibition of Gene Expression byMycobacterium tuberculosisOffsets Transcriptional Synergism with IFN-γ and Posttranscriptional Up-Regulation by IFN-γ
Authors: Qiao Yaming , Prabhakar Savita , Canova Antony , Hoshino Yoshihiko , Weiden Michael , Pine Richard
Published: April 2014

Host defense against Mycobacterium tuberculosis requires the cytokine IFN-γ and IFN regulatory factor 1 (IRF-1), a transcription factor that is induced to high levels by IFN-γ. Therefore, we chose to study regulation of IRF-1 expression as a model for effects of M. tuberculosis on response to IFN-γ. We found that IRF-1 mRNA abundance increased far more than transcription rate in human monocytic THP-1 cells stimulated by IFN-γ, but less than transcription rate in cells infected by M. tuberculosis. IFN-γ stimulation of infected cells caused a synergistic increase in IRF-1 transcription, yet IRF-1 mRNA abundance was similar in uninfected and infected cells stimulated by IFN-γ, as was the IRF-1 protein level. Comparable infection by Mycobacterium bovis bacillus Calmette-Guérin failed to induce IRF-1 expression and had no effect on the response to IFN-γ. We also examined the kinetics of transcription, the mRNA t1/2, and the distribution of IRF-1 transcripts among total nuclear RNA, poly(A) nuclear RNA, and poly(A) cytoplasmic RNA pools in cells that were infected by M. tuberculosis and/or stimulated by IFN-γ. Our data suggest that infection by M. tuberculosis inhibits RNA export from the nucleus. Moreover, the results indicate that regulated entry of nascent transcripts into the pool of total nuclear RNA affects IRF-1 expression and that this process is stimulated by IFN-γ and inhibited by M. tuberculosis. The ability of infection by M. tuberculosis to limit the increase in IRF-1 mRNA expression that typically follows transcriptional synergism may contribute to the pathogenicity of M. tuberculosis.
Toward a Definition of Self: Proteomic Evaluation of the Class I Peptide Repertoire
Authors: Hickman Heather D. , Luis Angela D. , Buchli Rico , Few Steven R. , Sathiamurthy Muthuraman , VanGundy Rodney S. , Giberson Christopher F. , Hildebrand William H.
Published: April 2014

MHC class I molecules present host- and pathogen-derived peptides for immune surveillance. Much attention is given to the search for viral and tumor nonself peptide epitopes, yet the question remains, “What is self?” Analyses of Edman motifs and of small sets of individual peptides suggest that the class I self repertoire consists of thousands of different peptides. However, there exists no systematic characterization of this self-peptide backdrop, causing the definition of class I-presented self to remain largely hypothetical. To better understand the breadth and nature of self proteins sampled by class I HLA, we sequenced >200 endogenously loaded HLA-B*1801 peptides from a human B cell line. Peptide-source proteins, ranging from actin-related protein 6 to zinc finger protein 147, possessed an assortment of biological and molecular functions. Major categories included binding proteins, catalytic proteins, and proteins involved in cell metabolism, growth, and maintenance. Genetically, peptides encoded by all chromosomes were presented. Statistical comparison of proteins presented by class I vs the human proteome provides empiric evidence that the range of proteins sampled by class I is relatively unbiased, with the exception of RNA-binding proteins that are over-represented in the class I peptide repertoire. These data show that, in this cell line, class I-presented self peptides represent a comprehensive and balanced summary of the proteomic content of the cell. Importantly, virus- and tumor-induced changes in virtually any cellular compartment or to any chromosome can be expected to be presented by class I molecules for immune recognition.
All Known In Vivo Functions of the Oct-2 Transcription Factor Require the C-Terminal Protein Domain
Authors: Corcoran Lynn M. , Koentgen Frank , Dietrich Wendy , Veale Margaret , Humbert Patrick O.
Published: April 2014

Oct-2, a transcription factor expressed in the B lymphocyte lineage and in the developing CNS, functions through of a number of discrete protein domains. These include a DNA-binding POU homeodomain flanked by two transcriptional activation domains. In vitro studies have shown that the C-terminal activation domain, a serine-, threonine- and proline-rich sequence, possesses unique qualities, including the ability to activate transcription from a distance in a B cell-specific manner. In this study, we describe mice in which the endogenous oct-2 gene has been modified through gene targeting to create a mutated allele, oct-2ΔC, which encodes Oct-2 protein isoforms that lack all sequence C-terminal to the DNA-binding domain. Surprisingly, despite the retention of the DNA-binding domain and the glutamine-rich N-terminal activation domain, the truncated protein(s) encoded by the oct-2ΔC allele are unable to rescue any of the previously described defects exhibited by oct-2 null mice. Homozygous oct-2ΔC/ΔC mice die shortly after birth, and B cell maturation, B-1 cell self renewal, serum Ig levels, and B lymphocyte responses to in vitro stimulation are all reduced or absent, to a degree equivalent to that seen in oct-2 null mice. We conclude that the C-terminal activation domain of Oct-2 is required to mediate the unique and indispensable functions of the Oct-2 transcription factor in vivo.
Relevance of Posttranslational Modifications for the Arthritogenicity of Type II Collagen
Authors: Myers Linda K. , Myllyharju Johanna , Nokelainen Minna , Brand David D. , Cremer Michael A. , Stuart John M. , Bodo Michael , Kivirikko Kiri I. , Kang Andrew H.
Published: April 2014

To establish the role of posttranslational modification in modulating the immune response to collagen, recombinant human type II collagen (rCII) was produced using a yeast expression system (rCIIpic) and a baculovirus expression system (rCIIbac). The biosynthesis of CII requires extensive posttranslational modification including the hydroxylation of prolyl and lysyl residues and glycosylation of selected hydroxylysyl residues. Amino acid analyses indicated that the rCIIbac was adequately hydroxylated at prolyl residues but underhydroxylated at lysyl residues and underglycosylated compared with tissue-derived CII, whereas rCIIpic was adequately hydroxylated at prolyl residues but unhydroxylated at lysyl residues and had no glycosylation. When DBA/1 mice were immunized with rCII, rCIIpic induced a lower incidence of arthritis than tissue-derived CII, whereas rCIIbac induced an intermediate level of arthritis. The severity of the arthritis was significantly lower in mice immunized with rCIIpic compared with mice immunized with tissue-derived CII, whereas that of rCIIbac was intermediate. These data indicate that the degree of lysine hydroxylation and glycosylation plays a role in the induction of arthritis. The recombinant collagens were then compared with tissue-derived CII when given as i.v. or oral tolerogens to suppress arthritis. Both recombinant collagens were less potent than tissue-derived CII, and this decrease in arthritis was associated with a decrease in Ab response to CII. These data suggest that the degree of glysosylation affects the immune response to CII, so that underglycosylated CII is less effective in the induction of arthritis and in its ability to suppress collagen-induced arthritis.
The Ig-Like Domain of Tapasin Influences Intermolecular Interactions
Authors: Turnquist Heth R. , Petersen Jason L. , Vargas Shanna E. , McIlhaney Mary M. , Bedows Elliott , Mayer Werner E. , Grandea Andres G. , Van Kaer Luc , Solheim Joyce C.
Published: April 2014

Presentation of antigenic peptides to T lymphocytes by MHC class I molecules is regulated by events involving multiple endoplasmic reticulum proteins, including tapasin. By studying the effects of substitutions in the tapasin Ig-like domain, we demonstrated that H-2Ld/tapasin association can be segregated from reconstitution of folded Ld surface expression. This finding suggests that peptide acquisition by Ld is influenced by tapasin functions that are independent of Ld binding. We also found that the presence of a nine-amino acid region in the Ig-like domain of mouse or human tapasin is required for association with Ld, and certain point substitutions in this sequence abrogate human, but not mouse, tapasin association with Ld. These data are consistent with a higher overall affinity between Ld and mouse tapasin compared with human tapasin. In addition, we found that other point mutations in the same region of the tapasin Ig-like domain affect MHC class I surface expression and Ag presentation. Finally, we showed that the cysteine residues in the Ig-like domain of tapasin influence tapasin’s stability, its interaction with the MHC class I H chain, and its stabilization of TAP. Mutagenesis of these cysteines decreases tapasin’s electrophoretic mobility, suggesting that these residues form an intramolecular disulfide bond. Taken together, these results reveal a critical role for the tapasin Ig-like domain in tapasin function.
Toxoplasma gondii Interferes with Lipopolysaccharide-Induced Mitogen-Activated Protein Kinase Activation by Mechanisms Distinct from Endotoxin Tolerance
Authors: Kim Leesun , Butcher Barbara A. , Denkers Eric Y.
Published: April 2014

We show in this study that Toxoplasma gondii infection induces rapid activation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2, and stress-activated protein kinase/c-Jun N-terminal kinase MAPK, followed promptly by their deactivation in mouse macrophages. Nevertheless, when infected cells were subsequently subjected to LPS triggering, MAPK activation was severely defective, in particular in the case of p38 MAPK, which is required for LPS-triggered TNF-α and IL-12 production. Similar effects occurred during endotoxin tolerance, but the phenomena were distinct. LPS pretriggering failed to activate the major p38 MAPK kinase, MAPK kinase 3/6. Toxoplasma infection, in contrast, resulted in sustained activation of this kinase. Furthermore, endotoxin pre-exposure blocked IκBα degradation upon subsequent LPS triggering, but this was not the case for Toxoplasma preinfection. Endotoxin-mediated down-regulation of the LPS receptor, Toll-like receptor 4, has been suggested as one possible mechanism contributing to tolerance, and we found in this study that LPS down-modulated Toll-like receptor 4 expression. In contrast, Toxoplasma infection induced up-regulation of this pattern recognition receptor. Our results show that T. gondii blocks LPS-triggered cytokine production in part through MAPK inactivation, and that this occurs through pathways distinct from endotoxin-induced tolerance.
Essential Involvement of IFN-γ in Clostridium difficile Toxin A-Induced Enteritis
Authors: Ishida Yuko , Maegawa Tsuneo , Kondo Toshikazu , Kimura Akihiko , Iwakura Yoichiro , Nakamura Shinichi , Mukaida Naofumi
Published: April 2014

Clostridium difficile has emerged as the important causative agent of antibiotics-associated pesudomembranous colitis; especially its toxin A is presumed to be responsible for the colitis. We examined the pathophysiological roles of IFN-γ in toxin A-induced enteritis using IFN-γ knockout (KO) mice. When toxin A of C. difficile was injected into the ileal loops of BALB/c wild-type (WT) mice, massive fluid secretion, disruption of intestinal epithelial structure, and massive neutrophil infiltration developed within 4 h after the injection. IFN-γ protein was faintly detected in some CD3-positive lymphocytes in the lamina propria and submucosa of the ileum of untreated WT mice. On the contrary, at 2 and 4 h after toxin A injection, IFN-γ protein was detected in infiltrating neutrophils and to a lesser degree in CD3-positive lymphocytes. In the ileum of WT mice, toxin A treatment markedly enhanced the gene expression of TNF-α, macrophage inflammatory protein-1α and -2, KC, and ICAM-1 >2 h after treatment. In contrast, the histopathological changes were marginal, without enhanced fluid secretion in the ileum of toxin A-treated IFN-γ KO mice. Moreover, toxin A-induced gene expression of TNF-α, neutrophil chemotactic chemokines, and ICMA-1 was remarkably attenuated in IFN-γ KO mice. Furthermore, pretreatment of WT mice with a neutralizing anti-IFN-γ Ab prevented toxin A-induced enteritis. These observations indicate that IFN-γ is the crucial mediator of toxin A-induced acute enteritis and suggest that IFN-γ is an important molecular target for the control of C. difficile-associated pseudomembranous colitis.
NK Cells, but Not NKT Cells, Are Involved in Pseudomonas aeruginosa Exotoxin A-Induced Hepatotoxicity in Mice
Authors: Mühlen Katrin A. , Schümann Jens , Wittke Frederick , Stenger Steffen , van Rooijen Nico , van Kaer Luc , Tiegs Gisa
Published: April 2014

Pseudomonas aeruginosa exotoxin A (PEA) causes T cell- and Kupffer cell (KC)-dependent liver injury in mice. TNF-α as well as IL-18 and perforin are important mediators of liver damage following PEA injection. In this study, we focus on the role of NK and NKT cells in PEA-induced liver toxicity. Depletion of both NK and NKT cells by injection of anti-NK1.1 Ab as well as depletion of NK cells alone by anti-asialo GM1 Ab protected mice from PEA-induced hepatotoxicity, whereas mice lacking only NKT cells were susceptible. Additionally, we observed infiltration of NK cells, T cells, and neutrophils into liver parenchyma after injection of PEA. The number of NKT cells, however, remained unchanged. The increase in intrahepatic NK cells depended on KCs and the TNF-α-dependent up-regulation of the adhesion molecule VCAM-1 in the liver, but not on NKT cells. PEA also augmented the cytotoxicity of hepatic NK cells against typical NK target cells (YAC-1 cells). This effect depended on KCs, but not on TNF-α or NKT cells. Furthermore, only weak expression of MHC class I was detected on hepatocytes, which was further down-regulated in PEA-treated mice. This could explain the susceptibility of hepatocytes to NK cell cytolytic activity in this model. Our results demonstrate that NK cells, activated and recruited independently of NKT cells, contribute to PEA-induced T cell-dependent liver injury in mice.
Role of Nicotinamide Adenine Dinucleotide Phosphate Oxidase 1 in Oxidative Burst Response to Toll-Like Receptor 5 Signaling in Large Intestinal Epithelial Cells
Authors: Kawahara Tsukasa , Kuwano Yuki , Teshima-Kondo Shigetada , Takeya Ryu , Sumimoto Hideki , Kishi Kyoichi , Tsunawaki Shohko , Hirayama Toshiya , Rokutan Kazuhito
Published: April 2014

The NADPH oxidase 1 (Nox1) is a gp91phox homologue preferentially expressed in the colon. We have established primary cultures of guinea pig large intestinal epithelial cells giving 90% purity of surface mucous cells. These cells spontaneously released superoxide anion (O2−) of 160 nmol/mg protein/h and expressed the Nox1, p22phox, p67phox, and Rac1 mRNAs, but not the gp91phox, Nox4, p47phox, p40phox, and Rac2 mRNAs. They also expressed novel homologues of p47phox and p67phox (p41nox and p51nox, respectively). Human colon cancer cell lines (T84 and Caco2 cells) expressed the Nox1, p22phox, p51nox, and Rac1 mRNAs, but not the other NADPH component mRNAs, and secreted only small amounts of O2− (<2 nmol/mg protein/h). Cotransfection of p41nox and p51nox cDNAs in T84 cells enhanced PMA-stimulated O2− release 5-fold. Treatment of the transfected T84 cells with recombinant flagellin (rFliC) from Salmonella enteritidis further augmented the O2− release in association with the induction of Nox1 protein. The enhanced O2− production by cotransfection of p41nox and p51nox vectors further augmented the rFliC-stimulated IL-8 release from T84 cells. T84 cells expressed the Toll-like receptor 5, and rFliC rapidly phosphorylated TGF-β-activated kinase 1 and TGF-β-activated kinase 1-binding protein 1. A potent inhibitor for NF-κB (pyrrolidine dithiocarbamate) significantly blocked the rFliC-primed increase in O2− production and induction of Nox1 protein. These results suggest that p41nox and p51nox are involved in the Nox1 activation in surface mucous cells of the colon, and besides that, epithelial cells discern pathogenicities among bacteria to appropriately operate Nox1 for the host defense.
T Cell Reactivity during Infectious Mononucleosis and Persistent Gammaherpesvirus Infection in Mice
Authors: Flaño Emilio , Hardy Charles L. , Kim In-Jeong , Frankling Claire , Coppola Michael A. , Nguyen Phuong , Woodland David L. , Blackman Marcia A.
Published: April 2014

Intranasal infection of mice with murine gammaherpesvirus 68 causes a dramatic increase in numbers of activated CD8+ T cells in the blood, analogous in many respects to EBV-induced infectious mononucleosis in humans. In the mouse model, this lymphocytosis has two distinct components: an early, conventional virus-specific CD8+ T cell response, and a later response characterized by a dramatic increase among CD8+ T cells that bear Vβ4+ TCRs. We previously demonstrated that Vβ4+CD8+ T cells recognize an uncharacterized ligand expressed on latently infected B cells in an MHC-independent manner. The frequency of Vβ4+CD8+ T cells increases dramatically following the peak of viral latency in the spleen. In the current studies, we show that elevated Vβ4+CD8+ T cell levels are sustained long-term in persistently infected mice, apparently a consequence of continued ligand expression. In addition, we show that Vβ4+CD8+ T cells can acquire effector functions, including cytotoxicity and the capacity to secrete IFN-γ, although they have an atypical activation profile compared with well-characterized CD8+ T cells specific for conventional viral epitopes. The characteristics of Vβ4+CD8+ T cells (potential effector function, stimulation by latently infected B cells, and kinetics of expansion) suggested that this dominant T cell response plays a key role in the immune control of latent virus. However, Ab depletion and adoptive transfer studies show that Vβ4+CD8+ T cells are not essential for this function. This murine model of infection may provide insight into the role of unusual populations of activated T cells associated with persistent viral infections.
Very Diverse CD8 T Cell Clonotypic Responses after Virus Infections
Authors: Pewe Lecia L. , Netland Jason M. , Heard Stephen B. , Perlman Stanley
Published: April 2014

We measured CD8 T cell clonotypic diversity to three epitopes recognized in C57BL/6 mice infected with mouse hepatitis virus, strain JHM, or lymphocytic choriomeningitis virus. We isolated epitope-specific T cells with an IFN-γ capture assay or MHC class I/peptide tetramers and identified different clonotypes by Vβ chain sequence analysis. In agreement with our previous results, the number of different clonotypes responding to all three epitopes fit a log-series distribution. From these distributions, we estimated that >1000 different clonotypes responded to each immunodominant CD8 T cell epitope; the response to a subdominant CD8 T cell epitope was modestly less diverse. These results suggest that T cell response diversity is greater by 1–2 orders of magnitude than predicted previously.
IL-10 Inhibits FcεRI Expression in Mouse Mast Cells
Authors: Gillespie Sheila R. , DeMartino Randall R. , Zhu Jingfang , Chong Hey Jin , Ramirez Carlos , Shelburne Christopher P. , Bouton L. Andrew , Bailey Daniel P. , Gharse Anita , Mirmonsef Paria , Odom Sandra , Gomez Gregorio , Rivera Juan , Fischer-Stenger Krista , Ryan John J.
Published: April 2014

FcεRI expression and function is a central aspect of allergic disease. Using bone marrow-derived mouse mast cell populations, we have previously shown that the Th2 cytokine IL-4 inhibits FcεRI expression and function. In the current study we show that the Th2 cytokine IL-10 has similar regulatory properties, and that it augments the inhibitory effects of IL-4. FcεRI down-regulation was functionally significant, as it diminished inflammatory cytokine production and IgE-mediated FcεRI up-regulation. IL-10 and IL-4 reduced FcεRI β protein expression without altering the α or γ subunits. The ability of IL-4 and IL-10 to alter FcεRI expression by targeting the β-chain, a critical receptor subunit known to modulate receptor expression and signaling, suggests the presence of a Th2 cytokine-mediated homeostatic network that could serve to both initiate and limit mast cell effector function.
HIV-Infected Lymphocytes Regulate Fibronectin Synthesis by TGFβ1 Secretion
Authors: Pal Sampa , Schnapp Lynn M.
Published: April 2014

Alterations in lymph node architecture occur with HIV infection and contribute to immunological derangements. We previously showed that matrix fibronectin stabilized HIV and increased HIV infection of PBL. We showed increased fibronectin deposition in lymph nodes of HIV-infected patients. However, we did not detect a difference in fibronectin synthesis between uninfected and infected PBL. Therefore, we hypothesized that interactions of HIV-infected cells with fibroblasts resulted in increased fibronectin deposition. We detected increased fibronectin deposition by immunofluorescence on fibroblasts cocultured with HIV-infected PBL. We also found a 6-fold increase in fibronectin mRNA levels in fibroblasts cocultured with HIV-infected PBL by real-time PCR. Furthermore, when HIV-infected PBL were added to reporter fibroblasts stably transfected with a fibronectin promoter, we found a 1.5- to 2-fold increase in promoter activity. Since conditioned medium from HIV-infected PBL also increased fibronectin promoter activity, we hypothesized that a soluble factor such as TGFβ was responsible for increased fibronectin secretion. Pretreatment of supernatant from HIV-infected PBL with a neutralizing Ab to TGFβ1 abrogated the increased fibronectin promoter activity. We confirmed that HIV-infected PBL produced increased TGFβ1 by ELISA. Using Mv1Lu reporter cells, we found a 2- to 3-fold increase in biologically active TGFβ in supernatants of HIV-infected PBL. Finally, we determined that HIV infection did not change the percentage of active TGFβ. Our data suggest that HIV-infected lymphocytes indirectly contribute to lymph node remodeling by secretion of TGFβ1, which increases fibronectin synthesis by fibroblasts.
Recruitment of IFN-γ-Producing (Th1-Like) Cells into the Inflamed Retina In Vivo Is Preferentially Regulated by P-Selectin Glycoprotein Ligand 1:P/E-Selectin Interactions
Authors: Xu Heping , Manivannan Ayyakkannu , Jiang Hui-Rong , Liversidge Janet , Sharp Peter F. , Forrester John V. , Crane Isabel J.
Published: April 2014

Although there is evidence that altering the Th1/Th2 balance toward Th2 cells may be important in the resolution of Th1-type autoimmune disease, adoptive transfer of Th2 cells is not effective in protecting against Th1-type disease and may cause disease. Therefore, we examined the recruitment of Th1- and Th2-like cells into the retina in the murine autoimmune disease experimental autoimmune uveoretinitis. CD4 T cells were polarized in vitro to IFN-γ-producing Th1-like cells and non-IFN-γ-producing Th2-like cells, labeled, and adoptively transferred. Trafficking to the retina in vivo was evaluated by scanning laser ophthalmoscopy and infiltration by confocal microscopy. There were more rolling and adherent Th1-like cells and they rolled more slowly than did Th2-like cells. Th1-like cells were preferentially recruited into the retinal parenchyma at both initiation and resolution. Surface P-selectin glycoprotein ligand 1 (PSGL-1) and LFA-1 were up-regulated on both populations but were expressed at higher levels on Th1-like cells. Up-regulation of CD44 expression was higher on Th2-like cells. P-selectin, E-selectin, and ICAM-1 are up-regulated on postcapillary venules in the retina. Pretreatment of Th1-like cells with anti-PSGL-1 inhibited rolling and infiltration of Th1-like cells but not Th2-like cells, providing direct in vivo evidence for the inability of Th2 to respond to P/E-selectin despite increased expression of PSGL-1. Anti-LFA-1 pretreatment inhibited infiltration of both Th1- and Th2-like cells, but more so Th-1. We suggest that random trafficking of activated T cells (both Th1 and Th2) across the blood-retina barrier is mediated by CD44:CD44R and LFA-1:ICAM-1, whereas preferential recruitment of Th1 cells is mediated by PSGL-1:P/E-selectin.
Vaccine-Induced CD4+ T Cell Responses to MAGE-3 Protein in Lung Cancer Patients
Authors: Atanackovic Djordje , Altorki Nasser K. , Stockert Elisabeth , Williamson Barbara , Jungbluth Achim A. , Ritter Erika , Santiago Darren , Ferrara Cathy A. , Matsuo Mitsutoshi , Selvakumar Annamalai , Dupont Bo , Chen Yao-Tseng , Hoffman Eric W. , Ritter Gerd , Old Lloyd J. , Gnjatic Sacha
Published: April 2014

MAGE-3 is the most commonly expressed cancer testis Ag and thus represents a prime target for cancer vaccines, despite infrequent natural occurrence of MAGE-3-specific immune responses in vivo. We report in this study the successful induction of Ab, CD8+, and CD4+ T cells in nonsmall cell lung cancer patients vaccinated with MAGE-3 recombinant protein. Two cohorts were analyzed: one receiving MAGE-3 protein alone, and one receiving MAGE-3 protein with adjuvant AS02B. Of nine patients in the first cohort, three developed marginal Ab titers and another one had a CD8+ T cell response to HLA-A2-restricted peptide MAGE-3 271–279. In contrast, of eight patients from the second cohort vaccinated with MAGE-3 protein and adjuvant, seven developed high-titered Abs to MAGE-3, and four had a strong concomitant CD4+ T cell response to HLA-DP4-restricted peptide 243–258. One patient simultaneously developed CD8+ T cells to HLA-A1-restricted peptide 168–176. The novel monitoring methodology used in this MAGE-3 study establishes that protein vaccination induces clear CD4+ T cell responses that correlate with Ab production. This development provides the framework for further evaluating integrated immune responses in vaccine settings and for optimizing these responses for clinical benefit.
Effects of Sustained HIV-1 Plasma Viremia on HIV-1 Gag-Specific CD4+ T Cell Maturation and Function
Authors: Palmer Brent E. , Boritz Eli , Wilson Cara C.
Published: April 2014

An in vitro proliferative defect has been observed in HIV-1-specific CD4+ T cells from infected subjects with high-level plasma HIV-1 viremia. To determine the mechanism of this defect, HIV-1 Gag-specific CD4+ T cells from treated and untreated HIV-1-infected subjects were analyzed for cytokine profile, proliferative capacity, and maturation state. Unexpectedly high frequencies of HIV-1-specific, IL-2-producing CD4+ T cells were measured in subjects with low or undetectable plasma HIV-1 loads, regardless of treatment status, and IL-2 frequencies correlated inversely with viral loads. IL-2-producing CD4+ T cells also primarily displayed a central memory (TCm; CCR7+CD45RA−) maturation phenotype, whereas IFN-γ-producing cells were mostly effector memory (TEm, CCR7−CD45RA−). Among Gag-specific, IFN-γ-producing CD4+ T cells, higher TEm frequencies and lower TCm frequencies were observed in untreated, high viral load subjects than in subjects with low viral loads. The percentage of HIV-1 Gag-specific CD4+ TCm correlated inversely with HIV-1 viral load and directly with Gag-specific CD4+ T cell proliferation, whereas the opposite relationships were observed for HIV-1-specific CD4+ TEm. These results suggest that HIV-1 viremia skews Gag-specific CD4+ T cells away from an IL-2-producing TCm phenotype and toward a poorly proliferating TEm phenotype, which may limit the effectiveness of the HIV-1-specific immune response.
Glu496 to Ala Polymorphism in the P2X7 Receptor Impairs ATP-Induced IL-1β Release from Human Monocytes
Authors: Sluyter Ronald , Shemon Anne N. , Wiley James S.
Published: April 2014

Priming of monocytes with LPS produces large quantities of intracellular, biologically inactive IL-1β that can be processed and released by subsequent activation of the P2X7 receptor by extracellular ATP. We examined whether a loss-of-function polymorphism of the human P2X7 receptor (Glu496Ala) impairs this process. Both ATP-induced ethidium+ uptake and ATP-induced shedding of L-selectin (CD62L) were nearly absent in monocytes from four subjects homozygous for Glu496Ala confirming that this polymorphism impairs P2X7 function. The level of ATP-induced IL-1β released in 2 h from LPS-activated whole blood from homozygous subjects was 50% of that from wild-type samples. A more marked defect in IL-1β release was observed from LPS-activated monocytes of homozygous subjects which was only 22% of that released from wild-type monocytes after a 30-min incubation with ATP. However, after a 60-min incubation with ATP, the amount of IL-1β released from homozygous monocytes was 70% of that released from wild-type monocytes. Incubation of monocytes of either genotype with nigericin resulted in a similar release of IL-1β. Western blotting demonstrated that ATP induced the release of mature 17-kDa IL-1β from monocytes, and confirmed that this process was impaired in homozygous monocytes. Finally, ATP-induced 86Rb+ efflux was 9-fold lower from homozygous monocytes than from wild-type monocytes. The results indicate that ATP-induced release of IL-1β is slower in monocytes from subjects homozygous for the Glu496Ala polymorphism in the P2X7 receptor and that this reduced rate of IL-1β release is associated with a lower ATP-induced K+ efflux.
Murine CD93 (C1qRp) Contributes to the Removal of Apoptotic Cells In Vivo but Is Not Required for C1q-Mediated Enhancement of Phagocytosis
Authors: Norsworthy Peter J. , Fossati-Jimack Liliane , Cortes-Hernandez Josefina , Taylor Philip R. , Bygrave Anne E. , Thompson Richard D. , Nourshargh Sussan , Walport Mark J. , Botto Marina
Published: April 2014

Human CD93 (known as C1qRp) has been shown to be a phagocytic receptor involved in the in vitro C1q-dependent enhancement of phagocytosis. However, binding of CD93 to C1q and its function remain controversial. In this study, we have generated CD93-deficient mice (CD93−/−) to investigate its biological role(s). The CD93−/− mice were viable and showed no gross abnormalities in their development. Thioglycolate-elicited peritoneal macrophages deficient in CD93 showed a similar enhancement in complement- and FcγR-dependent uptake of RBC to the wild-type macrophages when plated on C1q-coated surfaces suggesting that the lack of this receptor had no effect on these C1q-mediated events. There was no impairment in either complement- or FcγR-dependent phagocytic assays in vivo. By contrast, the CD93−/− mice had a significant phagocytic defect in the clearance of apoptotic cells in vivo (human Jurkat T cells and murine thymocytes: p = 0.0006 and p = 0.0079, respectively) compared with strain-matched controls. However, in vitro, the CD93−/− macrophages showed similar engulfment of apoptotic cells to wild-type macrophages. Furthermore, no supporting evidence for a role of CD93 as an adhesion molecule was found using intravital microscopy or analyzing peritoneal cell recruitment in response to three different inflammatory stimuli (thioglycolate, zymosan A, and IL-1β). Thus, our findings indicate that murine CD93 is expressed on the peritoneal macrophage, especially on thioglycolate-elicited cells, but does not appear to play a key role in C1q-mediated enhancement of phagocytosis or in the intercellular adhesion events tested. However, our results suggest that it may contribute to the in vivo clearance of dying cells.
Distinct Profiles of Human B Cell Effector Cytokines: A Role in Immune Regulation?
Authors: Duddy Martin E. , Alter Andrea , Bar-Or Amit
Published: April 2014

There is growing interest in the fundamental roles that B cells may play in regulating immune responses. Emerging animal studies point to an important contribution of B cell effector cytokines to immune modulation, yet little is known about the factors regulating such cytokine production. We report that the profile of human B cell cytokine production is context dependent, being critically influenced by the balance of signals through the B cell receptor and CD40. B cells appropriately stimulated by sequential B cell receptor and CD40 stimulation proliferate and secrete TNF-α, lymphotoxin, and IL-6, which can act not only as autocrine growth and differentiation factors, but also serve to amplify the ongoing immune response. In contrast, CD40 stimulation alone, a mimic of a B cell receiving bystander T cell help in the absence of specific Ag recognition, induces negligible proinflammatory cytokines, but significant production of IL-10 that serves to suppress inappropriate immune responses. We thus describe a novel paradigm of reciprocal regulation of B cell effector cytokines, and ascribe active roles for human B cells in either promoting or suppressing local immune responses through context-dependent cytokine production.
CD1d1-Dependent Control of the Magnitude of an Acute Antiviral Immune Response
Authors: Roberts Tonya J. , Lin Yinling , Spence Philip M. , Van Kaer Luc , Brutkiewicz Randy R.
Published: April 2014

CD1d1-restricted NK T (NKT) cells rapidly secrete both Th1 and Th2 cytokines upon activation and are therefore thought to play a regulatory role during an immune response. In this study we examined the role of CD1d1 molecules and NKT cells in regulating virus-induced cytokine production. CD1d1-deficient (CD1KO) mice, which lack NKT cells, were infected with lymphocytic choriomeningitis virus, and spontaneous cytokine release from splenocytes was measured. We found that CD1KO mice produce significantly higher amounts of IL-2, IL-4, and IFN-γ compared with wild-type controls postinfection. Depletion studies of individual lymphocyte subpopulations suggested that CD4+ T cells are required; however, isolation of specific lymphocyte populations indicated that CD4+ T cells alone are not sufficient for the increase in cytokine production in CD1KO mice. Splenocytes from lymphocytic choriomeningitis virus-infected CD1KO mice continued to produce enhanced cytokine levels long after viral clearance and cleared viral RNA faster than wild-type mice. There was no difference in the number of splenocytes between uninfected wild-type and CD1KO mice, whereas the latter knockout mice had an increased number of splenocytes after infection. Collectively, these data provide clear evidence that the expression of CD1d1 molecules controls the magnitude of the cell-mediated immune response to an acute viral infection.
Memory T Cells Originate from Adoptively Transferred Effectors and Reconstituting Host Cells after Sequential Lymphodepletion and Adoptive Immunotherapy
Authors: Wang Li-Xin , Kjaergaard Jorgen , Cohen Peter A. , Shu Suyu , Plautz Gregory E.
Published: April 2014

Adoptive transfer of tumor-specific effector T cells induces regression of advanced tumors and induces a long term memory response; however, the origin of this response has not been clearly defined. In this study Thy1.2+ mice bearing advanced MCA-205 tumors were treated with sublethal total body irradiation, followed by adoptive transfer of congenic Thy1.1+ T cells that had been sensitized to tumor in vivo and then activated ex vivo with anti-CD3, IL-2, and IL-7. Splenocytes were recovered >140 days after the initial therapy, and the L-selectinlow memory cell subset was separated into host Thy1.2+ and transferred Thy1.1+ cells and restimulated ex vivo. Both adoptively transferred Thy1.1+ cells as well as reconstituted host Thy1.2+ cells could specifically eliminate MCA-205 pulmonary metastases. Interestingly, hosts with partial responses followed by tumor recurrence nevertheless harbored memory cells that could be isolated and numerically amplified ex vivo to regenerate potent effector function. Memory cells were recovered after adoptive transfer into lymphodepleted nontumor-bearing hosts, indicating that they were not dependent on continued Ag exposure. These experiments establish that rapid ex vivo expansion of tumor Ag-primed T cells does not abrogate their capacity to become long-lived memory cells. Moreover, immune-mediated tumor regression coincident with lymphoid reconstitution produces another wave of host memory cells. These data suggest an approach to rescuing antitumor immune function even in hosts with long-standing progressive tumor through restorative ex vivo activation.