bluuebunny/crossref_metadata_2025 huggingface dataset:
Autocrine Type I IFN and Contact with Endothelium Promote the Presentation of Influenza A Virus by Monocyte-Derived APC
Authors: Qu Chunfeng , Moran Thomas M. , Randolph Gwendalyn J.
Published: April 2014

Purified monocytes infected with influenza A virus do not become mature dendritic cells (DCs) and they present viral peptides poorly to autologous memory T cells. In this study, we investigated whether influenza A-infected monocytes matured to DCs with a high capacity to stimulate T cells when they were infected with influenza A virus in a model tissue setting wherein they were cocultured with endothelium grown on a type I collagen matrix. Intercellular interactions with endothelium strongly promoted the Ag-presenting capacity of monocyte-derived cells infected with influenza A virus, and the heterologous coculture system also enhanced production of IFN-α by monocytes in the absence of plasmacytoid cells. Production of IFN-α in the presence of endothelium correlated with monocyte differentiation to mature DCs and their ability to stimulate proliferation and IFN-γ production by autologous T cells. Monocyte-derived cells that developed into migratory DCs promoted proliferation of influenza A virus-specific CD4+ and CD8+ cells, whereas those that developed into macrophages promoted proliferation of CD8+ T cells only. This onset of APC activity could be partially blocked with Ab to the IFN-αβ receptor when monocytes were infected with UV-treated virus, but neutralizing this pathway was inconsequential when monocytes were infected with live virus. Thus, type I IFN and direct contact with endothelium promote development of influenza A virus-presenting activity in monocyte-derived cells in a setting in which this differentiation does not depend on plasmacytoid cells. However, when infected with live influenza virus, the role of type I IFN in mediating differentiation and Ag-presenting capacity is expendable, apparently due to other mechanisms of virus-mediated activation.
Acceleration of Human Neutrophil Apoptosis by TRAIL
Authors: Renshaw Stephen A. , Parmar Jasvir S. , Singleton Vanessa , Rowe Sarah J. , Dockrell David H. , Dower Steven K. , Bingle Colin D. , Chilvers Edwin R. , Whyte Moira K. B.
Published: April 2014

Neutrophil granulocytes have a short lifespan, with their survival limited by a constitutive program of apoptosis. Acceleration of neutrophil apoptosis following ligation of the Fas death receptor is well-documented and TNF-α also has a transient proapoptotic effect. We have studied the role of the death receptor ligand TRAIL in human neutrophils. We identified the presence of mRNAs for TRAIL, TRAIL-R2, and TRAIL-R3, and cell surface expression of TRAIL-R2 and -R3 in neutrophil populations. Neutrophil apoptosis is specifically accelerated by exposure to a leucine zipper-tagged form of TRAIL, which mimics cell surface TRAIL. Using blocking Abs to TRAIL receptors, specifically TRAIL-R2, and a TRAIL-R1:FcR fusion protein, we have excluded a role for TRAIL in regulating constitutive neutrophil apoptosis. No additional proapoptotic effect of leucine zipper TRAIL was identified following TRAIL treatment of neutrophils in the presence of gliotoxin, an inhibitor of NF-κB, suggesting TRAIL does not activate NF-κB in human neutrophils. TRAIL treatment of human neutrophils did not induce a chemotactic response. The susceptibility of neutrophils to TRAIL-mediated apoptosis suggests a role for TRAIL in the regulation of inflammation and may provide a mechanism for clearance of neutrophils from sites of inflammation.
Enhanced Th2 Cell-Mediated Allergic Inflammation in Tyk2-Deficient Mice
Authors: Seto Yohei , Nakajima Hiroshi , Suto Akira , Shimoda Kazuya , Saito Yasushi , Nakayama Keiichi I. , Iwamoto Itsuo
Published: April 2014

Allergic inflammation is mediated by Th2 cell-derived cytokines, including IL-4, IL-5, and IL-13, and down-regulated by IFN-γ and IL-12. Tyk2 is a member of the Janus family of protein tyrosine kinases and is activated by a variety of cytokines: IFN-αβ, IL-6, IL-10, IL-12, and IL-13. In this study, we investigated the role of Tyk2 in the regulation of Ag-induced Th cell differentiation and Ag-induced allergic inflammation in the airways using Tyk2-deficient (Tyk2−/−) mice. When splenocytes were stimulated with antigenic peptide, IL-12-mediated Th1 cell differentiation was decreased, but IL-4-mediated Th2 cell differentiation was increased in Tyk2−/− mice. In vivo, Ag-specific IgE and IgG1 production was increased, but Ag-specific IgG2a production was decreased in Tyk2−/− mice as compared with those in control mice. In addition, Ag-induced eosinophil and CD4+ T cell recruitment, as well as the production of Th2 cytokines in the airways, was increased in Tyk2−/− mice. Adoptive transfer experiments revealed that CD4+ T cells were responsible for the enhanced Ag-induced eosinophil recruitment in Tyk2−/− mice. In contrast, although the level of IL-13 was increased in the airways of Tyk2−/− mice after Ag inhalation, the number of goblet cells, as well as Muc5ac mRNA expression, was decreased in Tyk2−/− mice. Together, these results indicate that Tyk2 plays a bilateral role in the regulation of allergic inflammation in the airways: Tyk2 plays a role in the down-regulation of Th2 cell-mediated Ab production and eosinophil recruitment in the airways by regulating Th1/Th2 balance toward Th1-type, while Tyk2 is necessary for the induction of IL-13-mediated goblet cell hyperplasia in the airways.
Cutting Edge: Proliferating Fibroblasts Respond to Collagenous C1q with Phosphorylation of p38 Mitogen-Activated Protein Kinase and Apoptotic Features
Authors: Bordin Sandra , Whitfield Douglas
Published: April 2014

Interactions of C1q collagen tails with human fibroblasts induce G1 mitotic arrest. The hypothesis tested in this study is that the antiproliferative effect of C1q tails is mediated through activation of stress responsive pathway(s). Upon C1q treatment, proliferating fibroblasts were examined by immunoblotting with a panel of Abs to the mitogen-activated protein kinase (MAPK) superfamily. The cells selectively increased phosphorylation of p38 MAPK, upstream dual activator MAPK kinase 3/6, and downstream transcription factors activating transcription factor 2, ETS domain transcription factor 1, and C/EBP homologous protein in a time-dependent manner. Phosphorylations were mediated, in part, by ligation of surface C1q tail-binding calreticulin. These events correlated with the appearance of apoptotic nuclei and DNA fragmentation in the cultures, which increased with a time response curve. The apoptotic features were linked to p38 activities because the selective inhibitor SB203580 prevented both phosphorylation of the pathway and DNA fragmentation. Hence, p38 activation might provide a molecular basis for linking mitotic arrest and apoptosis of fibroblasts by C1q tails.
Inhibition of Metalloproteinase Cleavage Enhances the Cytotoxicity of Fas Ligand
Authors: Knox Pauline G. , Milner Anne E. , Green Nicky K. , Eliopoulos Aristides G. , Young Lawrence S.
Published: April 2014

The Fas ligand (FasL)/Fas receptor (CD95) pathway is an important mediator of apoptosis in the immune system and can also mediate cancer cell death. Soluble FasL (sFasL), shed from the membrane-bound form of the molecule by a putative metalloproteinase (MP), may function to locally regulate the activity of membrane-bound FasL. Using a replication-defective recombinant adenovirus-expressing FasL (RAdFasL), we identified a variable ability of different carcinoma cells to respond to FasL-induced cytotoxicity and to shed sFasL. Blockade of FasL cleavage with an MP inhibitor significantly enhanced RAdFasL-induced apoptosis suggesting that sFasL may antagonize the effect of membrane-bound FasL. In support of this concept, a recombinant adenovirus expressing a noncleavable form of FasL (RAdD4) was found to be a potent inducer of apoptosis even at very low virus doses. Our results highlight the therapeutic potential of noncleavable FasL as an antitumor agent and emphasize the important role of MP via the production of sFasL in regulating the response of the Fas pathway. Moreover, these findings have general implications for the therapeutic exploitation of TNF family ligands and for the possible impact of MP-based therapies on the normal physiology of Fas/TNF pathways.
Impaired Lymphopoiesis and Altered B Cell Subpopulations in Mice Overexpressing Lnk Adaptor Protein
Authors: Takaki Satoshi , Tezuka Yoshinari , Sauer Karsten , Kubo Chiyomi , Kwon Sang-Mo , Armstead Erin , Nakao Kazuki , Katsuki Motoya , Perlmutter Roger M. , Takatsu Kiyoshi
Published: April 2014

Lnk is an adaptor protein expressed primarily in lymphocytes and hemopoietic precursor cells. Marked expansion of B lineage cells occurs in lnk−/− mice, indicating that Lnk regulates B cell production by negatively controlling pro-B cell expansion. In addition, lnk−/− hemopoietic precursors have an advantage in repopulating the hemopoietic system of irradiated host animals. In this study, we show that Lnk overexpression results in impaired expansion of lymphoid precursor cells and altered mature B cell subpopulations. The representation of both B lineage and T lineage cells was reduced in transgenic mice overexpressing Lnk under the control of a lymphocyte-specific expression vector. Whereas the overall number of B and T cells was correlated with Lnk protein expression levels, marginal zone B cells in spleen and B1 cells in the peritoneal cavity were relatively resistant to Lnk overexpression. The C-terminal tyrosine residue, conserved among Lnk family adaptor proteins, was dispensable for the negative regulatory roles of Lnk in lymphocyte development. Our results illuminate the novel negative regulatory mechanism mediated by the Lnk adaptor protein in controlling lymphocyte production and function.
CD154 Plays a Central Role in Regulating Dendritic Cell Activation During Infections That Induce Th1 or Th2 Responses
Authors: Straw Amy D. , MacDonald Andrew S. , Denkers Eric Y. , Pearce Edward J.
Published: April 2014

We compared splenic DC activation during infection with either the Th2 response-inducing parasite Schistosoma mansoni or with the Th1 response-inducing parasite Toxoplasma gondii. CD8α+ DC from schistosome-infected mice exhibited a 2- to 3-fold increase in the expression of MHC class II, CD80, and CD40 (but not CD86) compared with DC from uninfected control animals, while CD8α− DC exhibited a 2- to 3-fold increase in the expression of MHC class II and CD80 and no alteration, compared with DC from uninfected mice, in the expression of CD86 or CD40. Intracellular staining revealed that DC did not produce IL-12 during infection with S. mansoni. In contrast, infection with T. gondii resulted in a more pronounced increase in the expression of activation-associated molecules (MHC class II, CD80, CD86, and CD40) on both CD8α− and CD8α+ splenic DC and promoted elevated IL-12 production by DC. Analysis of MHC class I and of additional costimulatory molecules (ICOSL, ICAM-1, OX40L, 4-1BBL, and B7-DC) revealed a generally similar pattern, with greater indication of activation in T. gondii-infected mice compared with S. mansoni-infected animals. Strikingly, the activation of DC observed during infection with either parasite was not apparent in DC from infected CD154−/− mice, indicating that CD40/CD154 interactions are essential for maintaining DC activation during infection regardless of whether the outcome is a Th1 or a Th2 response. However, the ability of this activation pathway to induce IL-12 production by DC is restrained in S. mansoni-infected, but not T. gondii-infected, mice by Ag-responsive CD11c− cells.
Mechanisms of Enhanced Macrophage-Mediated Prostaglandin E2 Production and Its Suppressive Role in Th1 Activation in Th2-Dominant BALB/c Mice
Authors: Kuroda Etsushi , Yamashita Uki
Published: April 2014

PGE2 has been known to suppress Th1 responses. We studied the difference in strains of mice in PGE2 production by macrophages and its relation to Th1 activation. Macrophages from BALB/c mice produced greater amounts of PGE2 than those from any other strains of mice, including C57BL/6, after LPS stimulation. In accordance with the amount of PGE2 produced, macrophage-derived IL-12 and T cell-derived IFN-γ production were more strongly suppressed in BALB/c macrophages than in C57BL/6 macrophages. When macrophages were treated with indomethacin or EP4 antagonist, Th1 cytokines were more markedly increased in cells from BALB/c mice than in those from C57BL/6 mice. Although cyclooxygenase-2 was expressed similarly after LPS stimulation in these mouse strains, the release of arachidonic acid and the expression of type V secretory phospholipase A2 mRNA were greater in BALB/c macrophages. However, exogenous addition of arachidonic acid did not reverse the lower production of PGE2 by C57BL/6 macrophages. The expression of microsomal PGE synthase, a final enzyme of PGE2 synthesis, was also greater in BALB/c macrophages. These results indicate that the greater production of PGE2 by macrophages, which is regulated by secretory phospholipase A2 and microsomal PGE synthase but not by cyclooxygenase-2, is related to the suppression of Th1 cytokine production in BALB/c mice.
Nonmyelin-Specific T Cells Accelerate Development of Central Nervous System APC and Increase Susceptibility to Experimental Autoimmune Encephalomyelitis
Authors: Jones Richard E. , Kay Thomas , Keller Thomas , Bourdette Dennis
Published: April 2014

Previously we demonstrated that both myelin-specific and nonmyelin-specific rat T cells were capable of accelerating the development of transplanted rat BM-derived APC in the CNS of SCID C.B-17/scid (SCID) mice. This suggested that nonmyelin-specific T cells might be capable of increasing susceptibility to EAE by increasing the number and function of APC in the CNS before disease induction. To assess this possibility, we evaluated disease incidence, day of onset, duration, mean peak severity, cumulative disease index, and histopathology in the presence or absence of nonmyelin-specific T cells. The results demonstrate an association between T cell responses to nonmyelin Ags, accelerated development of BM-derived CNS APC before disease induction, and heightened susceptibility to CNS inflammation mediated by myelin-specific T cells. This suggests that T cell responses to nonmyelin Ags can potentiate CNS inflammation by elevating the functional presence of CNS APC.
IL-6 and Matrix Metalloproteinase-1 Are Regulated by the Cyclin-Dependent Kinase Inhibitor p21 in Synovial Fibroblasts
Authors: Perlman Harris , Bradley Kathleen , Liu Hongtao , Cole Shawn , Shamiyeh Eli , Smith Roy C. , Walsh Kenneth , Fiore Stefano , Koch Alisa E. , Firestein Gary S. , Haines G. Kenneth , Pope Richard M.
Published: April 2014

During the pathogenesis of rheumatoid arthritis (RA), the synovial fibroblasts increase in number and produce proinflammatory cytokines and matrix metalloproteinases (MMPs) that function to promote inflammation and joint destruction. Recent investigations have suggested that cell cycle activity and inflammation may be linked. However, little is known about the mechanisms responsible for the coordinate regulation of proliferation and the expression of proinflammatory molecules in RA synovial fibroblasts. Here, we demonstrate a 50 ± 10% decrease in the expression of p21, a cell cycle inhibitor, in the synovial fibroblast population from RA compared with osteoarthritis (OA) synovial tissue. Moreover, p21 positivity in the synovial fibroblasts inversely correlates with medium synovial lining thickness (r = −0.76; p < 0.02). The expression of p21 is also reduced in isolated RA synovial fibroblasts compared with OA synovial fibroblasts. Adenovirus-mediated delivery of p21 (Ad-p21) arrests both RA and OA synovial fibroblasts in the G0/G1 phase of the cell cycle without inducing cytotoxicity. However, the spontaneous production of IL-6 and MMP-1 is suppressed only in the Ad-p21-infected RA synovial fibroblasts, indicating a novel role for p21 in RA. Analyses of p21-deficient mouse synovial fibroblasts reveal a 100-fold increase in IL-6 protein and enhance IL-6 and MMP-3 mRNA. Restoration of p21, but not overexpression of Rb, which also induces G0/G1 cell cycle arrest, decreases IL-6 synthesis in p21-null synovial fibroblasts. Furthermore, in RA synovial fibroblasts the ectopic expression of p21 reduces activation of the AP-1 transcription factor. Additionally, p21-null synovial fibroblasts display enhanced activation of AP-1 compared with wild-type synovial fibroblasts. These data suggest that alterations in p21 expression may activate AP-1 leading to enhanced proinflammatory cytokine and MMP production and development of autoimmune disease.
The GTP-Binding Domain of Class II Transactivator Regulates Its Nuclear Export
Authors: Raval Aparna , Weissman Jocelyn D. , Howcroft T. Kevin , Singer Dinah S.
Published: April 2014

The transcriptional coactivator class II transactivator (CIITA), although predominantly localized in the nucleus, is also present in the cytoplasm. The subcellular distribution of CIITA is actively regulated by the opposing actions of nuclear export and import. In this study, we show that nuclear export is negatively regulated by the GTP-binding domain (GBD; aa 421–561) of CIITA: mutation or deletion of the GBD markedly increased export of CIITA from the nucleus. Remarkably, a CIITA GBD mutant binds CRM1/exportin significantly better than does wild-type CIITA, leading to the conclusion that GTP is a negative regulator of CIITA nuclear export. We also report that, in addition to the previously characterized N- and C-terminal nuclear localization signal elements, there is an additional N-terminal nuclear localization activity, present between aa 209 and 222, which overlaps the proline/serine/threonine-rich domain of CIITA. Thus, fine-tuning of the nucleocytoplasmic distribution of coactivator proteins involved in transcription is an active and dynamic process that defines a novel mechanism for controlling gene regulation.
Identification of IFN Regulatory Factor-1 Binding Site in IL-12 p40 Gene Promoter
Authors: Maruyama Saho , Sumita Kohsuke , Shen Hua , Kanoh Makoto , Xu Xin , Sato Mitsuharu , Matsumoto Masahito , Shinomiya Hiroto , Asano Yoshihiro
Published: April 2014

IL-12 is a heterodimer composed of p40 and p35 and is a key cytokine that functions to protect the host from viral and microbial infections. IL-12 links the innate immune system with the acquired immune system during infection, and induces differentiation of type 1 T cells that play an important role in the eradication of microbes. The induction of the IL-12 p40 gene is regulated by NF-κB in the presence of IFN-γ. IFN-γ induces IFN regulatory factor-1 (IRF-1), which in turn induces the transcription of the IL-12 p40 gene. However, the IRF-1 binding site in the promoter region of the IL-12 p40 gene has not yet been formally determined. In the present study, we demonstrated that IRF-1 directly binds to the IL-12 p40 gene promoter and identified its binding site. The IRF-1 binding site in the promoter region of the IL-12 p40 gene is shown to be in the −72 to −58 area of the 5′-upstream region. The −63 to −61 position is the critical site within this region for the binding of IRF-1 to the IL-12 p40 gene promoter. While IFN-γ must be present for IL-12 p40 gene induction, the p35 gene is strongly induced by LPS, even in the absence of IFN-γ, and therefore the induction of the p35 gene is IRF-1 independent.
Cutting Edge: The G-U Mismatch Glycosylase Methyl-CpG Binding Domain 4 Is Dispensable for Somatic Hypermutation and Class Switch Recombination
Authors: Bardwell Philip D. , Martin Alberto , Wong Edmund , Li Ziqiang , Edelmann Winfried , Scharff Matthew D.
Published: April 2014

Affinity maturation of the humoral response is accomplished by somatic hypermutation and class switch recombination (CSR) of Ig genes. Activation-induced cytidine deaminase likely initiates these processes by deamination of cytidines in the V and switch regions of Ig genes. This activity is expected to produce G-U mismatches that can be substrates for MutS homolog 2/MutS homolog 6 heterodimers and for uracil DNA glycosylase. However, G-T and G-U mismatches are also substrates of the methyl-CpG binding domain 4 (Mbd4) glycosylase. To determine whether Mbd4 functions downstream of activation-induced cytidine deaminase activity, we examined somatic hypermutation and CSR in Mbd4−/− mice. In this study, we report that CSR, as analyzed by an in vitro switch assay and by in vivo immunizations, is unaffected in Mbd4−/− mice. In addition, the hypermutated JH2 to JH4 region in Peyer’s patch B cells showed no effects as a result of Mbd4 deficiency. These data indicate that the Mbd4 glycosylase does not significantly contribute to mechanisms of Ab diversification.
Targeting Apoptotic Tumor Cells to FcγR Provides Efficient and Versatile Vaccination Against Tumors by Dendritic Cells
Authors: Akiyama Kenichi , Ebihara Shin , Yada Ayumi , Matsumura Kimio , Aiba Setsuya , Nukiwa Toshihiro , Takai Toshiyuki
Published: April 2014

Dendritic cells (DCs) loaded with tumor-associated Ags (TAAs) act as potent adjuvant that initiates antitumor immune responses in vivo. However, TAA-based DC vaccination requires prior identification of TAAs. Apoptotic tumor cells (ATCs) can be an excellent source for DC loading because their potential uncharacterized Ags would be efficiently presented to T cells without any prior characterization and isolation of these Ags. However, ATCs alone are considered to be inefficient for activating antitumor immunity, possibly because of their inability to induce DC maturation. In this study, the aim was to enhance antitumor immune response by taking advantage of ATCs that have been opsonized with IgG (ATC-immune complexes, ATC-ICs) so as to target them to FcR for IgG (FcγRs) on DCs. It was found that when compared with ATCs, ATC-ICs were efficiently internalized by DCs via FcγRs, and this process induced maturation of DCs, which was more efficient than that of ATCs. Importantly, ATC-IC loading was shown to be more efficient than ATCs alone in its capacity for inducing antitumor immunity in vivo, in terms of cytotoxic T cell induction and tumor rejection. These results show that using ATC-ICs may overcome the limitations and may enhance the immune response of current ATC-based DC vaccination therapy.
Th1 and Th2 Pancreatic Inflammation Differentially Affects Homing of Islet-Reactive CD4 Cells in Nonobese Diabetic Mice
Authors: Hill Natasha J. , Van Gunst Kurt , Sarvetnick Nora
Published: April 2014

The control of lymphocyte recruitment to the site of inflammation is an important component determining the pathogenicity of an autoimmune response. Progression from insulitis to diabetes in the nonobese diabetic mouse is typically associated with Th1 pancreatic inflammation, whereas Th2 inflammation can seemingly be controlled indefinitely. We show that a Th1 (IFN-γ) pancreatic environment greatly accelerates the recruitment of adoptively transferred islet-specific CD4 T cells to the islets and also accelerates the onset of diabetes. The increased number of islet-reactive T cells in the pancreas does not result from increased proliferation or a decreased rate of apoptosis; instead, it appears to be caused by a greatly facilitated rate of entry to the pancreas. In contrast, a Th2 (IL-4) pancreatic environment does act to enhance Ag-specific proliferation and decrease the rate of apoptosis in islet-specific CD4 T cells. Nonpathogenic/regulatory cells are not preferentially expanded by the presence of IL-4. Increased recruitment to the islets was also observed in the presence of IL-4, but to a lesser extent than in the presence of IFN-γ, and this lesser increase in the rate of recruitment did not accelerate diabetes onset within the time period examined. Therefore, the production of Th1 cytokines by initial islet-infiltrating cells may cause a greater increase than Th2 cytokines in the rate of recruitment of activated T cells. This difference in rate of recruitment may be critical in determining whether the initial infiltrate proceeds to diabetes or whether a steady state insulitis develops that can be maintained.
CD4+CD25− T Cells in Aged Mice Are Hyporesponsive and Exhibit Suppressive Activity
Authors: Shimizu Jun , Moriizumi Eiko
Published: April 2014

Studies on humans and rodents have established that functional deterioration of CD4 T cells occurs with aging. We report in this study that ∼70% of CD4+CD25− T cell preparations from individual 24-mo-old mice are hyporesponsive to in vitro stimulation with anti-CD3 Ab. The remaining 30% of CD4+CD25− T cell preparations showing the intermediate or normal responsiveness in the primary stimulation also exhibit the hyporesponsive properties after primary stimulation. Both of these hyporesponsive aged CD4+CD25− T cells could inhibit the proliferation of cocultured CD4+CD25− T cells from young mice, like CD4+CD25+ T cells, which have recently been demonstrated as an immune regulator in young mice. Another experiment revealed that hyporesponsive aged CD4+CD25− T cells arrest the cell division of cocultured young CD4+CD25− T cells. The suppressive activity observed in aged CD4+CD25− T cells is aging-dependent, not mediated by humoral factors, cell-contact dependent, and broken by the addition of IL-2 or anti-GITR Ab, but not by anti-CTLA-4 Ab. These studies show that aging not only leads to a decline in the ability to mount CD4+CD25− T cell responses, but at the same time, also renders these aged CD4+CD25− T cells suppressive.
Lack of the Transcriptional Coactivator OBF-1 Prevents the Development of Systemic Lupus Erythematosus-Like Phenotypes in Aiolos Mutant Mice
Authors: Sun Jian , Matthias Gabriele , Mihatsch Michael J. , Georgopoulos Katia , Matthias Patrick
Published: April 2014

Here we show that mice lacking the zinc finger transcription factor Aiolos develop the symptoms of human systemic lupus erythematosus (SLE), which is characterized by the production of anti-dsDNA Ab and immune complex-mediated glomerulonephritis. This finding indicates that normal Aiolos function is necessary to maintain immune homeostasis and suppress the development of systemic autoimmune disease and implicates Aiolos as a possible candidate gene for SLE. Interestingly, Aiolos-null mice can no longer mount autoimmune reactions and completely fail to develop SLE when they are deficient for the B cell-specific transcription coactivator OBF-1. The lack of OBF-1 reverses several Aiolos mutant mouse phenotypes, such as B cell hyperproliferation, high expression of activation marker on B cells, and spontaneous germinal center formation. Unexpectedly, B cell development at the immature B cell stage is severely impaired in the bone marrow of Aiolos/OBF-1 double-deficient mice, demonstrating the key role of these factors in the transition from pre-B to immature B cells. Our results indicate that B cells play a crucial role in the development of SLE in Aiolos mutant mice and might be useful for the strategy of SLE treatment.
B Cell Developmental Requirement for the Gαi2 Gene
Authors: Dalwadi Harnisha , Wei Bo , Schrage Matthew , Su Tom T. , Rawlings David J. , Braun Jonathan
Published: April 2014

Null mutation of the Gαi2 trimeric G protein results in a discrete and profound mucosal disorder, including inflammatory bowel disease (IBD), attenuation of IL-10 expression, and immune function polarized to Th1 activity. Genetic and adoptive transfer experiments have established a role for B cells and IL-10 in mucosal immunologic homeostasis and IBD resistance. In this study, we addressed the hypothesis that Gαi2 is required for the development of IL-10-producing B cells. Gαi2−/− mice were reduced in the relative abundance of marginal zone (MZ), transitional type 2 (T2), and B-1a B cells and significantly increased in follicular mature and B-1b B cells. Reconstitution of RAG2−/− mice with Gαi2−/− bone marrow induced an IBD-like colitis and a deficiency in absolute numbers of MZ, T2, and B-1 B cells. Thus, the Gαi2−/− genotype in colitis susceptibility and B cell development involved a cis effect within the hemopoietic compartment. In vitro, the B cell population of Gαi2−/− mice was functionally deficient in LPS-induced proliferation and IL-10 production, consistent with the exclusive capacity of T2 and MZ cell subpopulations for LPS responsiveness. In vivo, Gαi2−/− mice were selectively impaired for the IgM response to T-independent type II, consistent with the relative depletion of MZ and peritoneal B-1 subpopulations. Collectively, these results reveal a selective role for Gαi2 in MZ and B-1 B cell development. Disorders of this Gαi2-dependent process in B cell development may represent a mechanism for IBD susceptibility.
Preferential Survival of CD8 T and NK Cells Expressing High Levels of CD94
Authors: Gunturi Anasuya , Berg Rance E. , Forman James
Published: April 2014

The Qa-1b/Qdm tetramer binds to CD94/NKG2 receptors expressed at high levels on ∼50% of murine NK cells. Although very few CD8 T cells from naive mice express CD94/NKG2 receptors, ∼50% of CD8 T cells taken from mice undergoing a secondary response against Listeria monocytogenes (LM) are CD94high and bind the tetramer. Although CD94int NK cells do not bind the tetramer, CD94int CD8 T cells do, and this binding is dependent on the CD8 coreceptor. We found that the extent of apoptosis in CD8 T and NK cells was inversely related to the expression of CD94, with lower levels of apoptosis seen in CD94high cells after 1–3 days of culture. The difference in CD8 T cell survival was evident as early as 6 h after culture and persisted until nearly all the CD94neg/int cells were apoptotic by 48 h. In contrast, expression of inhibitory Ly-49A,G2,C/I molecules was associated with higher levels of apoptosis. Cross-linking CD94/NKG2 receptors on CD8 T cells from a mouse undergoing an LM infection further reduced the percentage of apoptotic cells on the CD94-expressing populations, while cross-linking Ly-49I had no effect on CD8 T cells expressing Ly-49I. Cross-linking CD3 on CD8 T cells from a mouse undergoing a secondary LM infection increases the extent of apoptosis, but this is prevented by cross-linking CD94/NKG2 receptors at the same time. Similar results were observed with NK cells in that the CD94high population displayed less apoptosis than CD94int cells after 1–3 days in culture. Therefore, the expression of CD94/NKG2 is correlated with a lower level of apoptosis and may play an important role in the maintenance of CD8 T and NK cells.
Expression of IFN-γ Upon Triggering of Activating Ly49D NK Receptors In Vitro and In Vivo: Costimulation with IL-12 or IL-18 Overrides Inhibitory Receptors
Authors: Ortaldo John R. , Young Howard A.
Published: April 2014

NK cells can express both activating and inhibitory Ly49 receptors on their cell surface. When cells expressing both receptors are presented with a ligand, inhibition dominates the functional outcome. In this report we demonstrate that costimulation of the activating Ly49D murine NK cell receptor with IL-12 or IL-18 is capable of over-riding the inhibitory Ly49G2 receptor blockade for cytokine production both in vitro and in vivo. This synergy is mediated by and dependent upon Ly49D-expressing NK cells and results in significant systemic expression of IFN-γ. This would place NK cells and their activating Ly-49 receptors as important initiators of microbial, antiviral, and antitumor immunity and provide a mechanism for the release of activating Ly49 receptors from inhibitory receptor blockade.
Antigen Density Presented By Dendritic Cells In Vivo Differentially Affects the Number and Avidity of Primary, Memory, and Recall CD8+ T Cells
Authors: Bullock Timothy N. J. , Mullins David W. , Engelhard Victor H.
Published: April 2014

We studied the size and avidity of primary and recall CD8+ T cell responses in vivo in mice immunized with dendritic cells presenting different densities of a MHC class I-restricted peptide. Increasing the epitope density on a fixed number of dendritic cells increased the size of the primary response, yet had no influence on the avidity of the effector cells. However, epitope density-based selection of cells with different avidities was evident in the subsequent memory population, and in recall responses. Additionally, mice primed with different peptide densities had similarly sized quiescent memory and recall responses. Our findings provide evidence for an important role for epitope density in the selection of T cells in vivo.
Temperature Effect on IgE Binding to CD23 Versus FcεRI
Authors: Chen Bing-Hung , Kilmon Michelle A. , Ma Check , Caven Timothy H. , Chan-Li Yee , Shelburne Anne E. , Tombes Robert M. , Roush Eric , Conrad Daniel H.
Published: April 2014

A chimeric soluble CD23, consisting of the extracellular domain of mouse CD23 and a modified leucine zipper (lz-CD23), has been shown to inhibit IgE binding to the FcεRI. A similar human CD23 construct was also shown to inhibit binding of human IgE to human FcεRI. In both systems, the inhibition was found to be temperature dependent; a 10-fold molar excess of lz-CD23 gave 90–98% inhibition at 4°C, dropping to 20–30% inhibition at 37°C. Surface plasmon resonance analysis of lz-CD23 binding to an IgE-coated sensor chip suggested that the effective concentration of lz-CD23 was lower at the higher temperatures. Analysis of 125I-IgE binding to CD23+-Chinese hamster ovary cells also indicated that increased temperature resulted in a lower percentage of IgE capable of interacting with CD23. In contrast, IgE interacts more effectively with FcεRI+-rat basophilic leukemia cells at 37°C compared with 4°C. The results support the concept that the open and closed IgE structures found by crystallography interact differently with the two IgE receptors and suggest that temperature influences the relative percentage of IgE in the respective structural forms. Changes in CD23 oligomerization also plays a role in the decreased binding seen at physiological temperatures.
Immunoglobulin Superantigen Protein L Induces IL-4 and IL-13 Secretion from Human FcεRI+ Cells Through Interaction with the κ Light Chains of IgE
Authors: Genovese Arturo , Borgia Guglielmo , Björck Lars , Petraroli Angelica , de Paulis Amato , Piazza Marcello , Marone Gianni
Published: April 2014

Peptostreptococcus magnus protein L is a multidomain bacterial surface protein that correlates with virulence. It consists of up to five homologous Ig-binding domains (B1–B5) that interact with the variable domain of Ig κ L chains. Intact protein L stimulates the synthesis and the release of IL-4 and IL-13 from human basophils in vitro. A protein L fragment covering the Ig-binding domains B1–B4 also induced IL-4 and IL-13 release from basophils. There was an excellent correlation (rs = 0.82; p < 0.001) between the maximal percent IL-4 release induced by protein L and that induced by anti-IgE and between intact protein L and the B1–B4 fragment (rs = 0.90; p < 0.01). Removal of IgE bound to basophils markedly reduced the IL-4 release induced by anti-IgE, protein L, and B1–B4. Preincubation of basophils with protein L or anti-IgE caused complete cross-desensitization to subsequent challenge with the heterologous stimulus. IgE purified from myeloma patients PS and PP (λ chains) blocked anti-IgE-induced IL-4 release, but not the releasing activity of protein L. In contrast, IgE purified from myeloma patient ADZ (κ chains) blocked both anti-IgE- and protein L-induced secretion. Cyclosporin A, but not cyclosporin H, inhibited protein L-induced release of IL-4 and IL-13 from basophils. Thus, protein L acts as a bacterial Ig superantigen to induce the synthesis and release of IL-4 and IL-13 from basophils by interacting with κ L chains of the IgE isotype.
Characterization of Thymic Progenitors in Adult Mouse Bone Marrow
Authors: Perry S. Scott , Pierce L. Jeanne , Slayton William B. , Spangrude Gerald J.
Published: April 2014

Thymic cellularity is maintained throughout life by progenitor cells originating in the bone marrow. In this study, we describe adult mouse bone cells that exhibit several features characteristic of prothymocytes. These include 1) rapid thymic engraftment kinetics following i.v. transplantation, 2) dramatic expansion of thymic progeny, and 3) limited production of hemopoietic progeny other than thymocytes. The adult mouse bone marrow population that is depleted of cells expressing any of a panel of lineage-specific Ags, stem cell Ag-1 positive, and not expressing the Thy1.1 Ag (Thy1.1−) (Thy1.1− progenitors) can repopulate the thymus 9 days more rapidly than can hemopoietic stem cells, a rate of thymic repopulation approaching that observed with transplanted thymocytes. Additionally, Thy1.1− progenitors expand prolifically to generate thymocyte progeny comparable in absolute numbers to those observed from parallel hemopoietic stem cell transplants, and provide a source of progenitors that spans multiple waves of thymic seeding. Nevertheless, the Thy1.1− population yields relatively few B cells and rare myeloid progeny posttransplant. These observations describe the phenotype of an adult mouse bone marrow population highly enriched for rapidly engrafting, long-term thymocyte progenitors. Furthermore, they note disparity in B and T cell expansion from this lymphoid progenitor population and suggest that it contains the progenitor primarily responsible for seeding the thymus throughout life.
Restoration of Ig Secretion: Mutation of Germline-Encoded Residues in T15L Chains Leads to Secretion of Free Light Chains and Assembled Antibody Complexes Bearing Secretion-Impaired Heavy Chains
Authors: Whitcomb Elizabeth A. , Martin Tammy M. , Rittenberg Marvin B.
Published: April 2014

We previously described T15H chain mutants that were impaired in assembly with L chain and in ability to be secreted from the cell. The unmutated T15L chain is unusual in that it is secretion-impaired in the absence of assembly with H chain. The T15L chain preferentially pairs with T15H in vivo, suggesting that if we introduced mutations that would allow secretion of free T15L chain, they might also lead to the secretion of the complex with the defective H chain. We mutated four positions in the germline T15L that had amino acids infrequently found in other κ-chains. Mutation to the most frequently occurring amino acid at three of the four positions allowed secretion of free L chain, while the combination of two secretion-restoring mutations was synergistic. Coexpression of secretion-restored mutant L chains with the secretion-defective mutant H chains rescued secretion of the assembled H2L2 complex, suggesting that during somatic hypermutation in vivo, deleterious mutations at the H chain may be compensated by mutations on the L chain. To our knowledge, this is the first example of mutations in IgL chains that are able to restore secretion-defective H chains to secretion competence in mammalian cells.
Crystal Structure of a Hydrophobic Immunodominant Antigenic Site on Hepatitis C Virus Core Protein Complexed to Monoclonal Antibody 19D9D6
Authors: Ménez Renée , Bossus Marc , Muller Bruno H. , Sibaï Geneviève , Dalbon Pascal , Ducancel Frédéric , Jolivet-Reynaud Colette , Stura Enrico A.
Published: April 2014

The first crystal structure of a complex between a hepatitis C virus (HCV) core protein-derived peptide (residues 13–40) and the Ab fragment of a murine mAb (19D9D6) has been solved, allowing determination of the recognized epitope and elucidation of its conformation. This Ab, raised against the first 120 residues of the core protein, recognizes core particles and strongly competes with anticore human Abs, suggesting that it is highly representative of the human anti-HCV core response. Its epitope lies within the first 45 aa of the protein, the major antigenic segment of core recognized both by murine and human Abs. Surprisingly, the recognized epitope (29–37: QIVGGVYLL) has an unusual preponderance of hydrophobic residues, some of which are buried in a small hydrophobic core in the nuclear magnetic resonance structure of the peptide (2–45) in solution, suggesting that the Ab may induce a structural rearrangement upon recognition. The flexibility may reside entirely within the Ag, since the Fab′-peptide complex structure at 2.34 Å shows that the Ab binding site is hardly perturbed by complexation. Given that the recognized residues are unlikely to be solvent exposed, we are left with the interesting possibility that Ab-core interactions may take place in a nonaqueous environment.
Associations of MHC Ancestral Haplotypes with Resistance/Susceptibility to AIDS Disease Development
Authors: Flores-Villanueva Pedro O. , Hendel Houria , Caillat-Zucman Sophie , Rappaport Jay , Burgos-Tiburcio Alberto , Bertin-Maghit Sebastien , Ruiz-Morales Jorge A. , Teran Maria E. , Rodriguez-Tafur Juan , Zagury Jean-François
Published: April 2014

We tested the association of MHC ancestral haplotypes with rapid or slow progression to AIDS by comparing their frequencies in the French genetics of resistance/susceptibility to immunodeficiency virus cohort with that reported in a control French population. Seven ancestral haplotypes were identified in the genetics of resistance/susceptibility to immunodeficiency virus cohort with a frequency >1%. The 8.1 (odds ratio (OR) = 3, p = 0.006), 35.1 (OR = 5.7, p = 0.001), and 44.2 (OR = 3.4, p = 0.007) ancestral haplotypes were associated with rapid progression, whereas the 35.2 (OR = 3.6, p = 0.001), 44.1 (OR = 5.4, p < 10−4), and 57.1 (OR = 5.8, p < 10−4) ancestral haplotypes were associated with slow progression to AIDS. Although the frequency of each ancestral haplotype is low in the population, the OR were quite higher than those previously obtained for single HLA allele associations, with some p values as low as 10−4. The analysis of the recombinant fragments of these haplotypes allowed the identification of the MHC regions in the 35.1, 35.2, and 44.2 haplotypes associated with rapid progression to AIDS and the MHC regions of the 44.1 and 57.1 haplotypes associated with slow progression to AIDS. Previous studies have identified single HLA alleles associated with disease progression. Our results on recombinant fragments confirm the direct role of HLA-B35 in rapid progression. Associations with HLA-A29 and -B57 might be due to linkage disequilibrium with other causative genes within the MHC region.
Constrained Intracellular Survival ofMycobacterium tuberculosisin Human Dendritic Cells
Authors: Tailleux Ludovic , Neyrolles Olivier , Honoré-Bouakline Stéphanie , Perret Emmanuelle , Sanchez Françoise , Abastado Jean-Pierre , Lagrange Philippe Henri , Gluckman Jean Claude , Rosenzwajg Michelle , Herrmann Jean-Louis
Published: April 2014

Dendritic cells (DCs) are likely to play a key role in immunity against Mycobacterium tuberculosis, but the fate of the bacterium in these cells is still unknown. Here we report that, unlike macrophages (Mφs), human monocyte-derived DCs are not permissive for the growth of virulent M. tuberculosis H37Rv. Mycobacterial vacuoles are neither acidic nor fused with host cell lysosomes in DCs, in a mode similar to that seen in mycobacterial infection of Mφs. However, uptake of the fluid phase marker dextran, and of transferrin, as well as accumulation of the recycling endosome-specific small GTPase Rab11 onto the mycobacterial phagosome, are almost abolished in infected DCs, but not in Mφs. Moreover, communication between mycobacterial phagosomes and the host-cell biosynthetic pathway is impaired, given that <10% of M. tuberculosis vacuoles in DCs stained for the endoplasmic reticulum-specific proteins Grp78/BiP and calnexin. This correlates with the absence of the fusion factor N-ethylmaleimide-sensitive factor onto the vacuolar membrane in this cell type. Trafficking between the vacuoles and the host cell recycling and biosynthetic pathways is strikingly reduced in DCs, which is likely to impair access of intracellular mycobacteria to essential nutrients and may thus explain the absence of mycobacterial growth in this cell type. This unique location of M. tuberculosis in DCs is compatible with their T lymphocyte-stimulating functions, because M. tuberculosis-infected DCs have the ability to specifically induce cytokine production by autologous T lymphocytes from presensitized individuals. DCs have evolved unique subcellular trafficking mechanisms to achieve their Ag-presenting functions when infected by intracellular mycobacteria.
The Induction and Kinetics of Antigen-Specific CD8 T Cells Are Defined by the Stage Specificity and Compartmentalization of the Antigen in Murine Toxoplasmosis
Authors: Kwok Lai-Yu , Lütjen Sonja , Soltek Sabine , Soldati Dominique , Busch Dirk , Deckert Martina , Schlüter Dirk
Published: April 2014

Toxoplasma gondii forms different life stages, fast-replicating tachyzoites and slow-growing bradyzoites, in mammalian hosts. CD8 T cells are of crucial importance in toxoplasmosis, but it is unknown which parasite stage is recognized by CD8 T cells. To analyze stage-specific CD8 T cell responses, we generated various recombinant Toxoplasma gondii expressing the heterologous Ag β-galactosidase (β-gal) and studied whether 1) secreted or cytoplasmic Ags and 2) tachyzoites or bradyzoites, which persist intracerebrally, induce CD8 T cells. We monitored the frequencies and kinetics of β-gal-specific CD8 T cells in infected mice by MHC class I tetramer staining. Upon oral infection of B6C (H-2bxd) mice, only β-gal-secreting tachyzoites induced β-gal-specific CD8 T cells. However, upon secondary infection of mice that had received a primary infection with tachyzoites secreting β-gal, β-gal-secreting tachyzoites and bradyzoites transiently increased the frequency of intracerebral β-gal-specific CD8 T cells. Frequencies of splenic and cerebral β-gal-specific CD8 T cells peaked at day 23 after infection, thereafter persisting at high levels in the brain but declining in the spleen. Splenic and cerebral β-gal-specific CD8 T cells produced IFN-γ and were cytolytic upon specific restimulation. Thus, compartmentalization and stage specificity of an Ag determine the induction of CD8 T cells in toxoplasmosis.
CD4+ T Cells Mediate Abscess Formation in Intra-abdominal Sepsis by an IL-17-Dependent Mechanism
Authors: Chung Doo Ryeon , Kasper Dennis L. , Panzo Ronald J. , Chtinis Tanuja , Grusby Michael J. , Sayegh Mohamed H. , Tzianabos Arthur O.
Published: April 2014

Abscess formation associated with intra-abdominal sepsis causes severe morbidity and can be fatal. Previous studies have implicated T cells in the pathogenesis of abscess formation, and we have recently shown that CD4+ T cells activated in vitro by zwitterionic capsular polysaccharides from abscess-inducing bacteria such as Staphylococcus aureus and Bacteroides fragilis initiate this host response when transferred to naive rats. In this study, we show that mice deficient in αβTCR-bearing T cells or CD4+ T cells fail to develop abscesses following challenge with B. fragilis or abscess-inducing zwitterionic polysaccharides, compared with CD8−/− or wild-type animals. Transfer of CD4+ T cells from wild-type mice to αβTCR−/− animals reconstituted this ability. The induction of abscesses required T cell costimulation via the CD28-B7 pathway, and T cell transfer experiments with STAT4−/− and STAT6−/− mice demonstrated that this host response is dependent on STAT4 signaling. Significantly higher levels of IL-17, a proinflammatory cytokine produced almost exclusively by activated CD4+ T cells, were associated with abscess formation in Th2-impaired (STAT6−/−) mice, while STAT4−/− mice had significantly lower levels of this cytokine than control animals. The formation of abscesses was preceded by an increase in the number of activated CD4+ T cells in the peritoneal cavity 24 h following bacterial challenge. Confocal laser-scanning microscopy analysis revealed that CD4+ T cells comprise the abscess wall in these animals and produce IL-17 at this site. Administration of a neutralizing Ab specific for IL-17 prevented abscess formation following bacterial challenge in mice. These data delineate the specific T cell response necessary for the development of intra-abdominal abscesses and underscore the role of IL-17 in this disease process.