bluuebunny/crossref_metadata_2025 huggingface dataset:
A Change in the Structure of Vβ Chromatin Associated with TCR β Allelic Exclusion
Authors: Tripathi Rajkamal , Jackson Annette , Krangel Michael S.
Published: April 2014

To investigate chromatin control of TCR β rearrangement and allelic exclusion, we analyzed TCR β chromatin structure in double negative (DN) thymocytes, which are permissive for TCR β recombination, and in double positive (DP) thymocytes, which are postallelic exclusion and nonpermissive for Vβ to DβJβ recombination. Histone acetylation mapping and DNase I sensitivity studies indicate Vβ and DβJβ segments to be hyperacetylated and accessible in DN thymocytes. However, they are separated from each other by hypoacetylated and inaccessible trypsinogen chromatin. The transition from DN to DP is accompanied by selective down-regulation of Vβ acetylation and accessibility. The level of DP acetylation and accessibility is minimal for five of six Vβ segments studied but remains substantial for one. Hence, the observed changes in Vβ chromatin structure appear sufficient to account for allelic exclusion of many Vβ segments. They may contribute to, but not by themselves fully account for, allelic exclusion of others.
Txk, a Member of Nonreceptor Tyrosine Kinase of Tec Family, Acts as a Th1 Cell-Specific Transcription Factor and Regulates IFN-γ Gene Transcription
Authors: Takeba Yuko , Nagafuchi Hiroko , Takeno Mitsuhiro , Kashiwakura Jun-ichi , Suzuki Noboru
Published: April 2014

Precise mechanisms responsible for Th1 cell activation and differentiation are not fully elucidated. We have recently reported that Txk, a member of Tec family nonreceptor tyrosine kinase, is expressed on Th1/Th0 cells, and Txk regulates specifically IFN-γ gene expression. In this study, we found that Txk bound to IFN-γ promoter region. Txk transfection increased transcriptional activity of IFN-γ promoter plus luciferase constructs severalfold, including IFN-γ promoter −538, −208, and −53. IFN-γ promoter −39 was refractory to the Txk transfection. The actual site to which Txk bound was the element consisting of −53 and −39 bp from the transcription start site of human IFN-γ gene, a site distinct from several previously characterized binding sites. We found that the entire −53/−39 region was necessary for the binding to and function of Txk, because mutant promoter oligoDNA that contained contiguous five base substitutions dispersed throughout the −53/−39 inhibited the binding, and the mutant promoters did not respond to the Txk transfection. Similar sequences of this element are found within the 5′ flanking regions of several Th1 cell-associated protein genes. Thus, Txk is expressed on Th1/Th0 cells with the IFN-γ production and acts as a Th1 cell-specific transcription factor.
Follicular Dendritic Cells and the Persistence of HIV Infectivity: The Role of Antibodies and Fcγ Receptors
Authors: Smith-Franklin Beverly A. , Keele Brandon F. , Tew John G. , Gartner Suzanne , Szakal Andras K. , Estes Jacob D. , Thacker Tyler C. , Burton Gregory F.
Published: April 2014

Large quantities of HIV are found trapped on the surface of follicular dendritic cells (FDCs), and virus persists on these cells until they ultimately die. We recently found that FDCs maintain HIV infectivity for long periods in vivo and in vitro. Because FDCs trap Ags (and virus) in the form of immune complexes and are rich in FcγRs, we reasoned that Ab and FcγRs may be required for FDC-mediated maintenance of HIV infectivity. To investigate this hypothesis, HIV immune complexes were formed in vitro and incubated for increasing times with or without FDCs, after which the remaining infectious virus was determined by HIV-p24 production in rescue cultures. FDCs maintained HIV infectivity in vitro in a dose-dependent manner but required the presence of specific Ab for this activity regardless of whether laboratory-adapted or primary X4 and R5 isolates were tested. In addition, Abs against either virally or host-encoded proteins on the virion permitted FDC-mediated maintenance of HIV infectivity. We found that the addition of FDCs to HIV immune complexes at the onset of culture gave optimal maintenance of infectivity. Moreover, blocking FDC-FcγRs or killing the FDCs dramatically reduced their ability to preserve virus infectivity. Finally, FDCs appeared to decrease the spontaneous release of HIV-1 gp120, suggesting that FDC-virus interactions stabilize the virus particle, thus contributing to the maintenance of infectivity. Therefore, optimal maintenance of HIV infectivity requires both Ab against particle-associated determinants and FDC-FcγRs.
Serpin 2a Is Induced in Activated Macrophages and Conjugates to a Ubiquitin Homolog
Authors: Hamerman Jessica A. , Hayashi Fumitaka , Schroeder Lea A. , Gygi Steven P. , Haas Arthur L. , Hampson Lynne , Coughlin Paul , Aebersold Ruedi , Aderem Alan
Published: April 2014

After i.p. infection of mice with the intracellular bacterium Mycobacterium bovis bacillus Calmette-Guérin, macrophages recovered from the peritoneal cavity display classical signs of immune activation. We have identified a member of the serine protease inhibitor (serpin) family which is highly induced in macrophages during bacillus Calmette-Guérin infection. Serpin 2a (spi2a) expression is also induced in macrophages in vivo during infection with Salmonella typhimurium and Listeria monocytogenes, and in vitro by a variety of bacteria and bacterial products. The cytokine IFN-γ also induces spi2a expression in macrophages, and this induction is synergistic with bacterial products. We also demonstrate here that a ubiquitin homolog, IFN-stimulated gene of 15-kDa (ISG15), is strongly induced during in vitro and in vivo activation of macrophages and that it conjugates to spi2a in activated macrophages. The ISG15-spi2a conjugates were identified by tandem mass spectrometry and contained spi2a conjugated to either one or two molecules of ISG15. Whereas spi2a was induced by either bacterial products or IFN-γ, ISG15 was induced only by bacterial products. Although many protein targets have been described for ubiquitin conjugation, spi2a is the first ISG15-modified protein to be reported. Macrophage activation is accompanied by the activation of a variety of proteases. It is of interest that a member of the serine protease inhibitor family is concomitantly induced and modified by a ubiquitin-like protein.
Susceptibility of IFN Regulatory Factor-1 and IFN Consensus Sequence Binding Protein-Deficient Mice to Brucellosis
Authors: Ko Jinkyung , Gendron-Fitzpatrick Annette , Splitter Gary A.
Published: April 2014

IFN-γ is a key cytokine controlling Brucella infection, and the diverse functions of this cytokine are mediated by IFN regulatory factors (IRFs) such as IRF-1, IRF-2, and IFN consensus sequence binding protein (ICSBP). However, the roles of these three IRFs in Brucella infection have not been investigated. The infection of each IRF-deficient mouse strain provides an opportunity to determine not only the significance of each IRF molecule but also the crucial immune components necessary for host defense during in vivo infection, because respective IRF-deficient mouse strains contain unique immunodeficient phenotypes. Brucella abortus S2308-infected IRF-1−/− mice were dead within 2 wk postinfection, while IRF-2−/− mice contained less splenic Brucella CFU than wild-type mice at the early stage of infection. Infected ICSBP−/− mice maintained a plateau of splenic Brucella CFU throughout the infection. Additional infection of IL-12p40-, NO synthase 2-, and gp91phox-deficient mice indicates that these immune components are crucial for Brucella immunity and may contribute to the susceptibility of IRF-1−/− and ICSBP−/− mice. Immunologic and histopathological analyses of infected IRF-1−/− mice indicate that the absence of IL-12p40 induction and serious hepatic damage are involved in the death of IRF-1−/− mice. These results indicate that 1) IRF-1 and ICSBP are essential transcriptional factors for IFN-γ-mediated protection against Brucella; 2) IL-12, reactive nitrogen intermediates, and reactive oxygen intermediates are crucial immune components against Brucella, and their absence may contribute to the susceptibility of IRF-1−/− and ICSBP−/− mice; and 3) hepatic damage caused by Brucella virulence contributes to the death of IRF-1−/− mice.
Activation of Macrophage Promatrix Metalloproteinase-9 by Lipopolysaccharide-Associated Proteinases
Authors: Min Danqing , Moore Anthony G. , Bain Michael A. , Breit Samuel N. , Lyons J. Guy
Published: April 2014

LPS induces an up-regulation of promatrix metalloproteinase-9 (proMMP9) gene expression in cells of the monocyte/macrophage lineage. We demonstrate here that LPS preparations are also able to activate proMMP9 made by human macrophages or THP-1 cells via LPS-associated proteinases, which cleave the N-terminal propeptide at a site or sites close to the one cleaved upon activation with organomercurial compounds. LPS-associated proteinases are serine proteinases that are able to cleave denatured collagens (gelatin) and the mammalian serine proteinase inhibitor, α1-proteinase inhibitor, thereby pushing the balance of extracellular matrix turnover even further toward degradation. A low molecular mass, low affinity inhibitor of MMP9, possibly derived from the propeptide, is generated during proMMP9 activation. However, inhibition of the LPS-associated proteinases had no effect on proMMP9 synthesis, indicating that their proteolytic activity was not required for signaling the up-regulation of the proMMP9 gene.
IL-5 Promotes Eosinophil Trafficking to the Esophagus
Authors: Mishra Anil , Hogan Simon P. , Brandt Eric B. , Rothenberg Marc E.
Published: April 2014

Eosinophil infiltration into the esophagus occurs in a wide range of diseases; however, the underlying pathophysiological mechanisms involved are largely unknown. We now report that the Th2 cytokine, IL-5, is necessary and sufficient for the induction of eosinophil trafficking to the esophagus. We show that transgenic mice overexpressing IL-5 under the control of a T cell (CD2) or a small intestinal enterocyte (fatty acid-binding protein) promoter have markedly increased eosinophil numbers in the esophagus. For example, esophageal eosinophil levels are 1.9 ± 0.9 and 121 ± 14 eosinophils/mm2 in wild-type and CD2-IL-5-transgenic mice, respectively. Consistent with this effect being mediated by a systemic mechanism, pharmacological administration of IL-5 via a miniosmotic pump in the peritoneal cavity resulted in blood and esophageal eosinophilia. To examine the role of IL-5 in oral Ag-induced esophageal eosinophilia, eosinophilic esophagitis was induced by allergen exposure in IL-5-deficient and wild-type mice. Importantly, IL-5-deficient mice were resistant to eosinophilic esophagitis. Finally, we examined the role of eotaxin when IL-5 was overproduced in vivo. Esophageal eosinophil levels in CD2-IL-5-transgenic mice were found to decrease 15-fold in the absence of the eotaxin gene; however, esophageal eosinophil numbers in eotaxin-deficient IL-5-transgenic mice still remained higher than wild-type mice. In conclusion, these studies demonstrate a central role for IL-5 in inducing eosinophil trafficking to the esophagus.
Nonhematopoietic Expression of Janus Kinase 3 Is Required for Efficient Recruitment of Th2 Lymphocytes and Eosinophils in OVA-Induced Airway Inflammation
Authors: Verbsky James W. , Randolph David A. , Shornick Laurie P. , Chaplin David D.
Published: April 2014

Tyrosine kinases of the Janus kinase (Jak) family transduce signals from the type I and type II cytokine receptors. Jak3 is unique in this family because its expression must be induced and is predominantly limited to cells of the lymphoid and myeloid lineages. Deficient expression of Jak3 interferes with normal development and function of T, B, and NK cells. Using irradiated Jak3-deficient (Jak3−/−) mice reconstituted with normal bone marrow (Jak3−/− chimeric mice), we have investigated possible actions of Jak3 outside of the hematopoietic system. We show that efficient recruitment of inflammatory cells to the airways of OVA-sensitized mice challenged with aerosolized OVA requires the expression of Jak3 in radioresistant nonhematopoietic cells. Failure to develop eosinophil-predominant airway inflammation in Jak3−/− chimeric mice is not due to failure of T cell sensitization, because Jak3−/− chimeric mice showed delayed-type hypersensitivity responses indistinguishable from wild-type chimeric mice. Jak3−/− chimeric mice, however, express less endothelial-associated VCAM-1 after airway Ag challenge. Given the key role of VCAM-1 in recruitment of Th2 cells and eosinophils, our data suggest that Jak3 in airway-associated endothelial cells is required for the expression of eosinophilic airway inflammation. This requirement for nonhematopoietic expression of Jak3 represents the first demonstration of a physiological function of Jak3 outside of the lymphoid lineages.
Inflammatory Mediators in Uveitis: Differential Induction of Cytokines and Chemokines in Th1- Versus Th2-Mediated Ocular Inflammation
Authors: Foxman Ellen F. , Zhang Meifen , Hurst Stephen D. , Muchamuel Tony , Shen Defen , Wawrousek Eric F. , Chan Chi-Chao , Gery Igal
Published: April 2014

Ocular inflammation leads to vision loss through the destruction and scarring of delicate tissues along the visual axis. To identify inflammatory mediators involved in this process, we used real time RT-PCR to quantify the expression of mRNA transcripts of 34 cytokines, 26 chemokines, and 14 chemokine receptors at certain time points during T cell-mediated ocular inflammation. We induced disease by adoptive transfer of Ag-specific Th1 or Th2 cells into recipients expressing the target Ag in their eyes. We also compared the mediator expression patterns seen in adoptive transfer-induced inflammation with that seen in mouse eyes developing experimental autoimmune uveoretinitis. In addition, we used laser capture microdissection to examine chemokine mRNA production by both retinal pigment epithelium cells and infiltrating leukocytes in inflamed eyes. Major findings included the following: 1) Three patterns of expression of the inflammation-related molecules were seen in recipients of adoptively transferred Th cells: preferential expression in Th1 recipients, or in Th2 recipients, or similar expression in both recipient groups. 2) In experimental autoimmune uveoretinitis, the inflammatory mediator expression pattern largely paralleled that seen in Th1-induced disease. 3) Both retinal pigment epithelium and infiltrating leukocytes expressed chemokine transcripts in distinct, but overlapping patterns in inflamed eyes. 4) Interestingly, trancripts of multiple cytokines, chemokines, and chemokine receptors were constitutively expressed in high levels in mouse eyes. Seven of these molecules have not been previously associated with the eye. These data underscore the multiplicity of mediators that participate in the pathogenesis of eye inflammation and point to upstream cytokines as potential therapeutic targets.
Depletion of Dendritic Cells, But Not Macrophages, in Patients with Sepsis
Authors: Hotchkiss Richard S. , Tinsley Kevin W. , Swanson Paul E. , Grayson Mitchell H. , Osborne Dale F. , Wagner Tracey H. , Cobb J. Perren , Coopersmith Craig , Karl Irene E.
Published: April 2014

Dendritic cells (DCs) are a group of APCs that have an extraordinary capacity to interact with T and B cells and modulate their responses to invading pathogens. Although a number of defects in the immune system have been identified in sepsis, few studies have examined the effect of sepsis on DCs, which is the purpose of this study. In addition, this study investigated the effect of sepsis on macrophages, which are reported to undergo apoptosis, and MHC II expression, which has been noted to be decreased in sepsis. Spleens from 26 septic patients and 20 trauma patients were evaluated by immunohistochemical staining. Although sepsis did not decrease the number of macrophages, sepsis did cause a dramatic reduction in the percentage area of spleen occupied by FDCs, i.e., 2.9 ± 0.4 vs 0.7 ± 0.2% in trauma and septic patients, respectively. The number of MHC II-expressing cells, including interdigitating DCs, was decreased in septic, compared with trauma, patients. However, sepsis did not appear to induce a loss of MHC II expression in those B cells, macrophages, or DCs that were still present. The dramatic loss of DCs in sepsis may significantly impair B and T cell function and contribute to the immune suppression that is a hallmark of the disorder.
The Role of IL-12 in the Induction of Intravenous Tolerance in Experimental Autoimmune Encephalomyelitis
Authors: Zhang Guang-Xian , Xu Hui , Kishi Masahiko , Calida Divina , Rostami Abdolmohamad
Published: April 2014

Intravenous administration of autoantigen is an effective method to induce Ag-specific tolerance against experimental autoimmune encephalomyelitis (EAE). IL-12 is a potent Th1 stimulator and an essential cytokine in the induction of EAE. The role of IL-12 in the induction of i.v. tolerance is not clear. In this study, we induced tolerance by i.v. administering myelin basic protein (MBP) peptide Ac1–11 (MBP1–11) in EAE. We observed significant suppression of IL-12 production by the lymph node cells of MBP1–11-injected mice. To see whether the low level of IL-12 is the cause or effect of tolerance, we administered IL-12 to the EAE mice at the time of i.v. MBP1–11 injection. Exogenous IL-12 abrogated the suppression of clinical and pathological EAE by i.v. tolerance. IL-12 blocked the suppressive effect of i.v. tolerance on the proliferative response to MBP1–11 and MBP1–11-induced production of IL-12 and IFN-γ. Furthermore, IL-12 completely blocked the i.v. tolerance-induced type 1 T regulatory cell response. These data suggest that i.v. administration of autoantigen results in the suppression of endogenous IL-12 and the consequent switching of the immune response from an immunogenic to a tolerogenic form.
An Anti-Idiotype Vaccine Elicits a Specific Response to N-Glycolyl Sialic Acid Residues of Glycoconjugates in Melanoma Patients
Authors: Alfonso Mauro , Díaz Alain , Hernández Ana María , Pérez Alexis , Rodríguez Edmundo , Bitton Roberto , Pérez Rolando , Vázquez Ana María
Published: April 2014

We generated the 1E10 γ-type anti-idiotype mAb (Ab2) specific to an Ab1 mAb able to react specifically with N-glycolyl-containing gangliosides and with Ags expressed on human melanoma and breast carcinoma cells. This Ab2 mAb induced an Ab response in animal models sharing immunochemically defined idiotopes with the Ab1. The treatment of tumor-bearing mice with 1E10 mAb induced a strong antitumor activity. A clinical trial was conducted in 20 patients with advanced malignant melanoma. Patients were treated with six intradermal injections of aluminum hydroxide-precipitated 1E10 anti-Id mAb given at 2-wk intervals. Sixteen of the 17 patients who received at least four doses of the anti-Id vaccine develop Ab3 Abs capable of inhibiting Ab2 binding to Ab1 (Ab3Id+). In contrast to the incapacity of 1E10 mAb to generate Ab3 Abs with the same antigenic specificity as the Ab1 mAb in mice, a very specific and strong Ab3 response against N-glycolyl-containing gangliosides was induced in 16 patients (Ab3Ag+). No evidence of serious or unexpected adverse effects has been observed in this clinical trial. 1E10 anti-Id vaccine was safe, well tolerated, and immunologically effective, with most patients being able to generate a specific immune response against 1E10 and Neu-glycolyl-GM3 ganglioside.
C1q Deficiency and Autoimmunity: The Effects of Genetic Background on Disease Expression
Authors: Mitchell Daniel A. , Pickering Matthew C. , Warren Joanna , Fossati-Jimack Liliane , Cortes-Hernandez Josefina , Cook H. Terence , Botto Marina , Walport Mark J.
Published: April 2014

Gene-targeted C1q-deficient mice have been shown to develop a syndrome reminiscent of human systemic lupus erythematosus with antinuclear Abs and proliferative glomerulonephritis. Initial phenotypic analysis conducted in (129 × C57BL/6) hybrid mice showed that background genes were a significant factor for the full expression of the autoimmune disease. To assess the contribution of background genes in the expression of the autoimmune phenotype, the disrupted C1qa gene was backcrossed for seven generations onto C57BL/6 and MRL/Mp+/+ strains. These were intercrossed with C57BL/6.lpr/lpr and MRL/Mp-lpr/lpr strains to generate C1q-deficient substrains. In C1q-deficient C57BL/6 mice, no evidence of an autoimmune phenotype was found, and C1q deficiency in both the C57BL/6.lpr/lpr and MRL/Mp-lpr/lpr strains did not modify the autoimmune phenotype observed in wild-type controls. However, in C1q-deficient MRL/Mp+/+ animals an acceleration of both the onset and the severity of antinuclear Abs and glomerulonephritis was seen. Disease was particularly pronounced in females, which developed severe crescentic glomerulonephritis accompanied by heavy proteinuria. In addition, the C1q-deficient MRL/Mp+/+ mice had an impairment in the phagocytic clearance of apoptotic cells in vivo. These data demonstrate that the expression of autoimmunity in C1q-deficient mice is strongly influenced by other background genes. The work also highlights the potential value of the C1q-deficient MRL/Mp+/+ strain as a tool with which to dissect further the underlying mechanisms of the autoimmune syndrome associated with C1q deficiency.
Cutting Edge: Biasing Immune Responses by Directing Antigen to Macrophage Fcγ Receptors
Authors: Anderson Charles F. , Mosser David M.
Published: April 2014

An immune response can deviate toward either a Th1- or Th2-like response. In this work we examine the contribution that activated macrophages and IgG Abs make toward this deviation. The use of activated macrophages as APCs resulted in a strong polarized T cell response that was predominated by IFN-γ. However, when Ag was targeted to FcγRs on these macrophages, the T cell response was reversed and biased toward a Th2-like response. This Th2-like phenotype was stable and was retained when the T cells were subsequently restimulated under nonbiasing conditions. The T cell biasing and its reversal via FcγR was also observed in vivo. Mice vaccinated with IgG-opsonized OVA made high levels of IgG Ab of the IgG1 isotype. These studies demonstrate that the ligation of FcγR on activated macrophages can reverse the Th1 biasing that occurs as a result of innate immune responses to microbial products.
Cutting Edge: Anti-Inflammatory Properties of Low Levels of IFN-γ
Authors: Flaishon Liat , Topilski Ian , Shoseyov David , Hershkoviz Rami , Fireman Elizabeth , Levo Yoram , Marmor Sylvia , Shachar Idit
Published: April 2014

Activation of naive T and B cells occurs only within the context of organized lymphoid tissue. Thus, the continuous recirculation of mature lymphocytes is crucial for the development of primary immune response to foreign Ags. We have previously shown that low levels of IFN-γ inhibit homing of B cells to the secondary lymphoid organs. In this study, we demonstrate that similarly low doses of IFN-γ down-regulate integrin-mediated adhesion and migration of naive T and Th2 cells, and have a profound effect on the in vivo homing of naive T cells to the lymph nodes. Moreover, we show that these low doses of IFN-γ have anti-inflammatory effects in an in vivo asthma model. Thus, in contrast to the proinflammatory effects of IFN-γ at relatively high concentrations, low dose IFN-γ appears to exert global suppressory effects on T cell trafficking and may have clinical application as an anti-inflammatory agent.
Polar Redistribution of the Sialoglycoprotein CD43: Implications for T Cell Function
Authors: Savage Nigel D. L. , Kimzey Stephanie L. , Bromley Shannon K. , Johnson Kenneth G. , Dustin Michael L. , Green Jonathan M.
Published: April 2014

Contact between T cells and APCs results in the orchestrated segregation of molecules at the cell-cell interface and formation of a specialized structure termed the immunological synapse. This model predicts the topological seclusion of large molecules such as CD43 from the site of closest contact between the T cell and APC, allowing for the close apposition of cell membranes and effective TCR engagement. Similarly, during T cell migration segregation of CD43 to the uropod is thought to aid integrin adhesion at the leading edge of the cell by removing steric hindrance. We show in this work that CD43 distribution on T cells is regulated by a membrane proximal ezrin binding site and that failure to displace CD43 from the immunological synapse has no inhibitory effects on primary T cell activation. We also report that CD43 expression at the contact zone between T cells and matrix does not negatively regulate motility but may regulate LFA-1 de-adhesion. These results suggest that the steric barrier model of CD43 is inadequate and that alternative mechanisms account for the negative regulatory properties of CD43.
Activating Immunity in the Liver. II. IFN-β Attenuates NK Cell-Dependent Liver Injury Triggered by Liver NKT Cell Activation
Authors: Trobonjaca Zlatko , Kröger Andrea , Stober Detlef , Leithäuser Frank , Möller Peter , Hauser Hansjörg , Schirmbeck Reinhold , Reimann Jörg
Published: April 2014

Dendritic cell (DC)-dependent activation of liver NKT cells triggered by a single i.v. injection of a low dose (10–100 ng/mouse) of α-galactosyl ceramide (αGalCer) into mice induces liver injury. This response is particularly evident in HBs-tg B6 mice that express a transgene-encoded hepatitis B surface Ag in the liver. Liver injury following αGalCer injection is suppressed in mice depleted of NK cells, indicating that NK cells play a role in NK T cell-initiated liver injury. In vitro, liver NKT cells provide a CD80/86-dependent signal to αGalCer-pulsed liver DC to release IL-12 p70 that stimulates the IFN-γ response of NKT and NK cells. Adoptive transfer of NKT cell-activated liver DC into the liver of nontreated, normal (immunocompetent), or immunodeficient (RAG−/− or HBs-tg/RAG−/−) hosts via the portal vein elicited IFN-γ responses of liver NK cells in situ. IFN-β down-regulates the pathogenic IL-12/IFN-γ cytokine cascade triggered by NKT cell/DC/NK cell interactions in the liver. Pretreating liver DC in vitro with IFN-β suppressed their IL-12 (but not IL-10) release in response to CD40 ligation or specific (αGalCer-dependent) interaction with liver NKT cells and down-regulated the IFN-γ response of the specifically activated liver NKT cells. In vivo, IFN-β attenuated the NKT cell-triggered induction of liver immunopathology. This study identifies interacting subsets of the hepatic innate immune system (and cytokines that up- and down-regulate these interactions) activated early in immune-mediated liver pathology.
Temporal Segregation of 4-1BB Versus CD28-Mediated Costimulation: 4-1BB Ligand Influences T Cell Numbers Late in the Primary Response and Regulates the Size of the T Cell Memory Response Following Influenza Infection
Authors: Bertram Edward M. , Lau Peggy , Watts Tania H.
Published: April 2014

In this report, we demonstrate that CD28−/− mice are severely impaired in the initial expansion of Db/NP366-374-specific CD8 T cells in response to influenza virus infection, whereas 4-1BB ligand (4-1BBL)−/− mice show no defect in primary T cell expansion to influenza virus. In contrast, 4-1BBL−/− mice show a decrease in Db/NP366-374-specific T cells late in the primary response. Upon secondary challenge with influenza virus, 4-1BBL−/− mice show a decrease in the number of Db/NP366-374-specific T cells compared to wild-type mice such that the level of the CD8 T cell expansion during the in vivo secondary response is reduced to the level of a primary response, with concomitant reduction of CTL effector function. In contrast, Ab responses, as well as secondary CD4 T cell responses, to influenza are unaffected by 4-1BBL deficiency. Thus, CD28 is critical for initial T cell expansion, whereas 4-1BB/4-1BBL signaling affects T cell numbers much later in the response and is essential for the survival and/or responsiveness of the memory CD8 T cell pool.
Continuous Exposure of Mice to Superantigenic Toxins Induces a High-Level Protracted Expansion and an Immunological Memory in the Toxin-Reactive CD4+ T Cells
Authors: Chen Luqiu , Koyanagi Madoka , Fukada Kenji , Imanishi Ken’ichi , Yagi Junji , Kato Hidehito , Miyoshi-Akiyama Tohru , Zhang Ruihua , Miwa Keishi , Uchiyama Takehiko
Published: April 2014

We analyzed the responses of several T cell fractions reactive with superantigenic toxins (SAGTs), staphylococcal enterotoxin A (SEA), or Yersinia pseudotuberculosis-derived mitogen (YPM) in mice implanted with mini-osmotic pumps filled with SEA or YPM. In mice implanted with the SEA pump, SEA-reactive Vβ3+CD4+ T cells exhibited a high-level protracted expansion for 30 days, and SEA-reactive Vβ11+CD4+ T cells exhibited a low-level protracted expansion. SEA-reactive CD8+ counterparts exhibited only a transient expansion. A similar difference in T cell expansion was also observed in YPM-reactive T cell fractions in mice implanted with the YPM pump. Vβ3+CD4+ and Vβ11+CD4+ T cells from mice implanted with the SEA pump exhibited cell divisions upon in vitro restimulation with SEA and expressed surface phenotypes as memory T cells. CD4+ T cells from mice implanted with the SEA pump exhibited high IL-4 production upon in vitro restimulation with SEA, which was due to the enhanced capacity of the SEA-reactive CD4+ T cells to produce IL-4. The findings in the present study indicate that, in mice implanted with a specific SAGT, the level of expansion of the SAGT-reactive CD4+ T cell fractions varies widely depending on the TCR Vβ elements expressed and that the reactive CD4+ T cells acquire a capacity to raise a memory response. CD8+ T cells are low responders to SAGTs.
The State of CD4+ T Cell Activation Is a Major Factor for Determining the Kinetics and Location of T Cell Responses to Oral Antigen
Authors: Lee Hae-ock , Cooper Cristine J. , Choi Jung-hee , Alnadjim Ziad , Barrett Terrence A.
Published: April 2014

Current models suggest that inductive immune responses to enteric Ag are initiated in Peyer’s patches (PP) and mesenteric lymph nodes (MLN) followed by migration of activated, memory-like CD4+ T cells to extralymphoid sites in the intestinal lamina propria (LP). The resultant immune system contains both naive and activated T cells. To examine the differential responses of naive and memory-like T cells to oral Ag, bone marrow chimeras (BMC) were generated. Irradiated BALB/c hosts were reconstituted with a mix of DO11.10 × RAG-1−/− and BALB/c bone marrow. In unprimed DO11.10 and BMC models, LP and PP DO11.10 T cells responded to oral Ag with similar kinetics. Responses of activated, memory-like T cells to oral Ag were examined in thymectomized BMC 60 days after i.p. immunization with OVA peptide in Freund’s adjuvant (OVA323–339/CFA). Results indicate that i.p. OVA323–339/CFA generated a high proportion of memory-like CD45RBlow DO11.10 T cells in peripheral lymphoid (40%) and intestinal LP (70%) tissue. Previously activated DO11.10 T cells in the LP responded to oral Ag earlier and at 50% higher levels compared with memory CD4+ T cells localized to PP tissue. These data indicate that responses to oral Ag in antigenically naive animals are initiated in PP whereas in Ag-experienced animals LP T cells respond earlier and more vigorously than cells in PP. Taken together, these data suggest that previous activation alters the hierarchy of T cell responses to oral Ag by enhancing the efficiency of LP T cell activation.
Formation of the Killer Ig-Like Receptor Repertoire on CD4+CD28null T Cells
Authors: Snyder Melissa R. , Muegge Lars-Olof , Offord Chetan , O’Fallon William M. , Bajzer Zeljko , Weyand Cornelia M. , Goronzy Jörg J.
Published: April 2014

Killer Ig-like receptors (KIRs) are expressed on CD4+CD28null T cells, a highly oligoclonal subset of T cells that is expanded in patients with rheumatoid arthritis. It is unclear at what stage of development these T cells acquire KIR expression. To determine whether KIR expression is a consequence of clonal expansion and replicative senescence, multiple CD4+CD28null T cell clones expressing the in vivo dominant TCR β-chain sequences were identified in three patients and analyzed for their KIR gene expression pattern. Based on sharing of TCR sequences, the clones were grouped into five clone families. The repertoire of KIRs was diverse, even within each clone family; however, the gene expression was not random. Three particular receptors, KIR2DS2, KIR2DL2, and KIR3DL2, had significant differences in gene expression frequencies between the clone families. These data suggest that KIRs are successively acquired after TCR rearrangement, with each clone family developing a dominant expression pattern. The patterns did not segregate with the individual from whom the clones were derived, indicating that peripheral selection in the host environment was not a major shaping force. Several models were examined using a computer algorithm that was designed to simulate the expression of KIRs at various times during T cell proliferation. The computer simulations favored a model in which KIR gene expression is inducible for a limited time during the initial stages of clonal expansion.
Differential Roles for Extracellularly Regulated Kinase-Mitogen-Activated Protein Kinase in B Cell Antigen Receptor-Induced Apoptosis and CD40-Mediated Rescue of WEHI-231 Immature B Cells
Authors: Gauld Stephen B. , Blair Derek , Moss Catriona A. , Reid Steven D. , Harnett Margaret M.
Published: April 2014

One of the major unresolved questions in B cell biology is how the B cell Ag receptor (BCR) differentially signals to transduce anergy, apoptosis, proliferation, or differentiation during B cell maturation. We now report that extracellularly regulated kinase-mitogen-activated protein kinase (Erk-MAP kinase) can play dual roles in the regulation of the cell fate of the immature B cell lymphoma, WEHI-231, depending on the kinetics and context of Erk-MAP kinase activation. First, we show that the BCR couples to an early (≤2 h) Erk-MAP kinase signal which activates a phospholipase A2 pathway that we have previously shown to mediate collapse of mitochondrial membrane potential, resulting in depletion of cellular ATP and cathepsin B execution of apoptosis. Rescue of BCR-driven apoptosis by CD40 signaling desensitizes such early extracellularly regulated kinase (Erk) signaling and hence uncouples the BCR from the apoptotic mitochondrial phospholipase A2 pathway. A second role for Erk-MAP kinase in promoting the growth and proliferation of WEHI-231 immature B cells is evidenced by data showing that proliferating and CD40-stimulated WEHI-231 B cells exhibit a sustained cycling pattern (8–48 h) of Erk activation that correlates with cell growth and proliferation. This growth-promoting role for Erk signaling is supported by three key pieces of evidence: 1) signaling via the BCR, under conditions that induce growth arrest, completely abrogates sustained Erk activation; 2) CD40-mediated rescue from growth arrest correlates with restoration of cycling Erk activation; and 3) sustained inhibition of Erk prevents CD40-mediated rescue of BCR-driven growth arrest of WEHI-231 immature B cells. Erk-MAP kinase can therefore induce diverse biological responses in WEHI-231 cells depending on the context and kinetics of activation.
Negligible Class II MHC Presentation of B Cell Receptor-Derived Peptides by High Density Resting B Cells
Authors: Snyder Christopher M. , Zhang Xianghua , Wysocki Lawrence J.
Published: April 2014

Resting B lymphocytes have been credited with inducing T cell tolerance to Ig-derived and monovalent self-Ags that are internalized via the B cell receptor (BCR). These conclusions are predicated upon the assumptions that resting B cells display BCR-associated peptides in class II MHC and that the cells remain quiescent during the course of experimental manipulation. To determine whether resting B cells display BCR-associated epitopes in class II MHC, we devised a sensitive assay that averted potential activation of B cells by Ag and minimized activation by prolonged culture. Ex vivo, Percoll-fractionated B cells expressing a κ transgene encoding a T cell epitope were cultured with a reactive T cell hybridoma for 12 h. Whereas low density, LPS-activated, and BCR-activated B cells elicited significant IL-2 from the T cell hybridoma, resting high density B cells did not. Parallel results were obtained with normal B cells expressing a second epitope encoded by an endogenous VH gene. Anergic B cells, which are uniformly low density, also significantly stimulated the T cell hybridoma. Finally, longer culture periods with normal B cells resulted in a higher degree of B cell activation and significant stimulation of reactive T cell hybridomas. Our results provide evidence that activation of B cells profoundly enhances the processing and presentation of BCR-associated Ags.
The Complementarity-Determining Region-Like Loops of CD8α Interact Differently with β2-Microglobulin of the Class I Molecules H-2Kb and Thymic Leukemia Antigen, While Similarly with Their α3 Domains
Authors: Devine Lesley , Rogozinski Linda , Naidenko Olga V. , Cheroutre Hilde , Kavathas Paula B.
Published: April 2014

The murine CD8 glycoprotein interacts with both classical MHC class I molecules and some nonclassical molecules, including the thymic leukemia Ag (TL). TL binds preferentially to CD8αα homodimers with a 10-fold higher affinity than H-2Kb class I molecules. To understand the molecular basis for this difference, we created a panel of CD8α mutants and tested the ability of the CD8αα homodimers to bind to H-2Kb tetramers and TL tetramers. Mutations in three CD8 residues located on the complementarity-determining region-like loops contacting the negatively charged loop in the α3 domain of MHC class I greatly reduced binding to both tetramers. Because TL and H-2Kb class I sequences are highly conserved in the α3 domain of MHC class I, this suggests that CD8 contacts the α3 domain of TL and H-2Kb in a similar manner. In contrast, mutations in residues on the A and B β strands of CD8 that are involved in contact with β2-microglobulin affected interaction with the H-2Kb tetramer, but not the TL tetramer. Therefore, the orientation of interaction of TL with CD8 appears to be different from that of H-2Kb. The unique high affinity binding of TL with CD8αα is most likely a result of amino acid differences in the α3 domain between TL and H-2Kb, particularly at positions 198 (K to D) and 228 (M to T), which are contact residues in the CD8αα-H-2Kb cocrystal.
Complementarity-Determining Region 1 Sequence Requirements Drive Limited Vα Usage in Response to Influenza Hemagglutinin 307–319 Peptide
Authors: Brawley James V. , Concannon Patrick
Published: April 2014

We have developed a T cell activation-based system that allows for the selection of TCRs with defined peptide/MHC specificities from libraries in which complementarity-determining region (CDR) sequences have been randomized by in vitro mutagenesis. Using this system, we have explored the sequence requirements for CDR1 and CDR2 of the TCR α-chain in a human T cell response characterized by restricted Vα and Vβ usage. Libraries of T cells expressing receptors built on the framework of a TCR specific for the influenza virus peptide hemagglutinin 307–319 presented by HLA-DR4, but with random sequences inserted at CDR1α or CDR2α, were selected for response to the same peptide/MHC ligand. A wide variety of CDR2α sequences were found to be permissive for recognition. Indeed, >25% of T cell clones chosen at random displayed a significant response. In contrast, a similar challenge of a randomized CDR1α library yielded only the parental sequence, and then only after multiple rounds of selection. T cell clones cross-reactive on closely related HLA alleles (subtypes of DR4) could be isolated from randomized libraries, but not clones restricted by more distantly related alleles such as HLA-DR1. These results indicate that, in the context of this T cell response, the structural requirements for recognition at CDR1α are significantly more restricted than at CDR2α. This system for mutation and selection of TCRs in vitro may be of use in engineering T cells with defined specificities for therapeutic applications.
Mitochondria-Dependent Caspase-9 Activation Is Necessary for Antigen Receptor-Mediated Effector Caspase Activation and Apoptosis in WEHI 231 Lymphoma Cells
Authors: Herold Marco J. , Kuss Andreas W. , Kraus Christa , Berberich Ingolf
Published: April 2014

Engagement of the B cell Ag receptor (BCR) on immature B cells leads to growth arrest followed by apoptosis. Concomitant signaling through CD40 sustains proliferation and rescues the cells from apoptosis. Previously, we have shown that cross-linking CD40 on B cells stimulates the expression of A1, an antiapoptotic member of the Bcl-2 family, and that transduction of the murine B lymphoma line WEHI 231, a model for immature B cells, with A1 protected the cells against BCR-induced apoptosis. Here we demonstrate that A1 strongly interferes with activation of caspase-7, the major effector caspase activated after BCR cross-linking on WEHI 231 lymphoma cells. The pathway leading to activation of the effector caspase cascade including caspase-7 is unclear. Using retrovirally transduced WEHI 231 cell populations, we show that a catalytically inactive mutant of caspase-7 is cleaved almost as efficiently as the wild-type form, arguing against autocatalysis as the sole activating process. In contrast, overexpression of catalytically inactive caspase-9 strongly interferes with caspase-7 processing, poly(ADP-ribose) polymerase cleavage, and DNA laddering, suggesting a role for caspase-9 and hence for the mitochondrial pathway. The importance of the mitochondrial/caspase-9 pathway for BCR-triggered apoptosis is highlighted by our finding that both A1 and the mutant caspase-9 attenuate BCR-induced apoptosis. Thus, our data suggest that the BCR-mediated apoptotic signal in immature B cells spreads via a mitochondrial/caspase-9 pathway.
Regulation of IL-1 Receptor-Associated Kinases by Lipopolysaccharide
Authors: Hu Jean , Jacinto Randy , McCall Charles , Li Liwu
Published: April 2014

IL-1R-associated kinase (IRAK) plays a pivotal role in IL-1R/Toll-like receptor (TLR)-mediated signaling and NF-κB activation. IRAK from leukocytes undergoes rapid activation and inactivation/degradation following IL-1 or LPS stimulation. The rapid degradation of IRAK may serve as a negative feedback mechanism of down-regulating IL-1R/TLR-mediated signaling and cytokine gene transcription. Although IL-1/IL-1R-triggered IRAK degradation has been studied in detail, the mechanism of LPS-induced IRAK activation and degradation is not clearly defined. In this study, we demonstrate that the IRAK N-terminal 186-aa region is required for LPS-induced degradation. The N-terminally truncated IRAK protein expressed in human monocytic THP-1 cells remains stable upon LPS challenge. In comparison, IRAK as well as the IRAK mutant with C-terminal truncation undergo degradation with LPS stimulation. We demonstrate that pretreatment with protein kinase C inhibitor calphostin inhibits LPS-induced IRAK degradation. Furthermore, we observe coimmunoprecipitation of endogenous IRAK and protein kinase C-ζ protein. We show that functional TLR4 is required for LPS-mediated IRAK degradation. IRAK protein in the murine GG2EE cells harboring a mutated TLR4 gene does not undergo degradation upon LPS treatment. In sharp contrast, we observe that the IRAK homolog, IRAK2, does not undergo degradation upon prolonged LPS treatment, suggesting complex regulation of the innate immunity network upon microbial challenge.
An Analysis of T Cell Intrinsic Roles ofE2Aby Conditional Gene Disruption in the Thymus
Authors: Pan Lihua , Hanrahan Jenifer , Li Jie , Hale Laura P. , Zhuang Yuan
Published: April 2014

The importance of E2A transcription factors in T cell development has been demonstrated in studies of E2A-deficient mice, which display abnormal T cell development and a high frequency of T cell lymphomas. Because E2A expression is not restricted to the T cell lineage, the primary cause of the T cell phenotype in E2A-deficient mice was not fully determined. To further investigate the role of E2A in T cell lineage, we generated mice with the E2A gene disrupted exclusively during thymocyte development using the Cre-lox system. We show that this system allows E2A gene disruption to occur throughout the double-negative stage of thymocyte development. E2A deletion appears to be completed before development reaches the double-positive stage. Consistent with the gene disruption, these mice reveal a T cell intrinsic role for E2A during the transition from the double-negative stage to the double-positive stage of thymocyte development. In contrast to germline E2A knockout mice, conditional E2A knockout mice do not develop T cell lymphoma. This work establishes a new model for further investigating E2A function in T cell development and leukemiogenesis.
Fine Specificity of TCR Complementarity-Determining Region Residues and Lipid Antigen Hydrophilic Moieties in the Recognition of a CD1-Lipid Complex
Authors: Grant Ethan P. , Beckman Evan M. , Behar Samuel M. , Degano Massimo , Frederique Daphney , Besra Gurdyal S. , Wilson Ian A. , Porcelli Steven A. , Furlong Stephen T. , Brenner Michael B.
Published: April 2014

αβ TCR can recognize peptides presented by MHC molecules or lipids and glycolipids presented by CD1 proteins. Whereas the structural basis for peptide/MHC recognition is now clearly understood, it is not known how the TCR can interact with such disparate molecules as lipids. Recently, we demonstrated that the αβ TCR confers specificity for both the lipid Ag and CD1 isoform restriction, indicating that the TCR is likely to recognize a lipid/CD1 complex. We hypothesized that lipids may bind to CD1 via their hydrophobic alkyl and acyl chains, exposing the hydrophilic sugar, phosphate, and other polar functions for interaction with the TCR complementarity-determining regions (CDRs). To test this model, we mutated the residues in the CDR3 region of the DN1 TCR β-chain that were predicted to project between the CD1b α helixes in a model of the TCR/CD1 complex. In addition, we tested the requirement for the negatively charged and polar functions of mycolic acid for Ag recognition. Our findings indicate that the CDR loops of the TCR form the Ag recognition domain of CD1-restricted TCRs and suggest that the hydrophilic domains of a lipid Ag can form a combinatorial epitope recognized by the TCR.
Generation of Cytotoxic T Cells Against Virus-Infected Human Brain Macrophages in a Murine Model of HIV-1 Encephalitis
Authors: Poluektova Larisa Y. , Munn David H. , Persidsky Yuri , Gendelman Howard E.
Published: April 2014

HIV-1 encephalitis (HIVE) and its associated dementia can occur in up to 20% of infected individuals, usually when productive viral replication in brain mononuclear phagocytes (macrophages and microglia) and depletion of CD4+ T lymphocytes are most significant. T cells control viral replication through much of HIV-1 disease, but how this occurs remains incompletely understood. With this in mind, we studied HIV-1-specific CTL responses in a nonobese diabetic (NOD)-SCID mouse model of HIVE. HIV-1-infected monocyte-derived macrophages (MDM) were injected into the basal ganglia after syngeneic immune reconstitution by HLA-A*0201-positive human PBL to generate a human PBL-NOD-SCID HIVE mouse. Engrafted T lymphocytes produced HIV-1gag- and HIV-1pol-specific CTL against virus-infected brain MDM within 7 days. This was demonstrated by tetramer staining of human PBL in mouse spleens and by IFN-γ ELISPOT. CD8, granzyme B, HLA-DR, and CD45R0 Ag-reactive T cells and CD79α-positive B cells migrated to and were in contact with human MDM in brain areas where infected macrophages were abundant. The numbers of productively infected MDM were markedly reduced (>85%) during 2 wk of observation. The human PBL-NOD-SCID HIVE mouse provides a new tool for studies of cellular immune responses against HIV-1-infected brain mononuclear phagocytes during natural disease and after vaccination.