bluuebunny/crossref_metadata_2025 huggingface dataset:
Lipopolysaccharide-Stimulated or Granulocyte-Macrophage Colony-Stimulating Factor-Stimulated Monocytes Rapidly Express Biologically Active IL-15 on Their Cell Surface Independent of New Protein Synthesis
Authors: Neely Graham G. , Robbins Stephen M. , Amankwah Ernest K. , Epelman Slava , Wong Howard , Spurrell Jason C. L. , Jandu Kiran K. , Zhu Weibin , Fogg Darin K. , Brown Christopher B. , Mody Christopher H.
Published: April 2014

Although IL-15 shares many of the biological activities of IL-2, IL-2 expression is primarily under transcriptional regulation, while the mechanisms involved in the regulation of IL-15 are complex and not completely understood. In the current study, we found that CD14+ monocytes constitutively exhibit both IL-15 mRNA and protein. IL-15 protein was found stored intracellularly and stimulation of CD14+ monocytes with either LPS or GM-CSF resulted in mobilization of IL-15 stores to the plasma membrane. This rapidly induced surface expression was the result of a translocation of preformed stores, confirming that posttranslational regulatory stages limit IL-15, because it was not accompanied by an increase in IL-15 mRNA and occurred independent of de novo protein synthesis. After fixation, activated monocytes, but not resting monocytes, were found to support T cell proliferation, and this effect was abrogated by the addition of an IL-15-neutralizing Ab. The presence of preformed IL-15 stores and the ability of stimulated monocytes to mobilize these stores to their surface in an active form is a novel mechanism of regulation for IL-15.
Essential Role of Stat5 for IL-5-Dependent IgH Switch Recombination in Mouse B Cells
Authors: Horikawa Keisuke , Kaku Hiroaki , Nakajima Hiroshi , Davey Helen W. , Henninghausen Lothar , Iwamoto Itsuo , Yasue Tokutaro , Kariyone Ai , Takatsu Kiyoshi
Published: April 2014

IL-5 stimulation of CD38-activated murine splenic B cells induces μ-γ1 CSR at the DNA level leading to a high level of IgG1 production. Further addition of IL-4 in the system enhances IL-5-dependent μ-γ1 CSR. Although some of the postreceptor signaling events initiated by IL-5 in activated B cells have been characterized, the involvement of Stat in IL-5 signaling has not been thoroughly evaluated. In this study, we examined the activation of Stat5 and activation-induced cytidine deaminase (AID) in CD38-activated murine splenic B cells by IL-5. The role of Stat5a and Stat5b in IL-5-induced μ-γ1 CSR and also IgG1 and IgM production was documented, as IL-5 does not act on CD38-stimulated splenic B cells from Stat5a−/− and Stat5b−/− mice. Expression levels of CD38-induced germline γ1 transcripts and AID in Stat5a−/− and Stat5b−/− B cells upon IL-5 stimulation were comparable to those of wild-type B cells. The impaired μ-γ1 CSR by Stat5b−/− B cells, but not by Stat5a−/− B cells, was rescued in part by IL-4, as the addition of IL-4 to the culture of CD38- and IL-5-stimulated B cells induced μ-γ1 CSR leading to IgG1 production. Analysis of cell division cycle number of wild-type B cells revealed that μ-γ1 CSR was observed after five or six cell divisions. Stat5a−/− and Stat5b−/− B cells showed similar cell division cycles, but they did not undergo μ-γ1 CSR. Our data support the notion that both Stat5a and Stat5b are essential for IL-5-dependent μ-γ1 CSR and Ig secretion; however, their major target may not be AID. Stat5a and Stat5b are not redundant, but rather are at least partially distinctive in their function.
Mannose Receptor Ligand-Positive Cells Express the Metalloprotease Decysin in the B Cell Follicle
Authors: Mueller Chris G. F. , Cremer Isabelle , Paulet Pierre E. , Niida Shumpei , Maeda Norihiko , Lebeque Serge , Fridman Wolf H. , Sautès-Fridman Catherine
Published: April 2014

Decysin, a gene encoding a disintegrin metalloprotease, is transcribed in human dendritic cells (DC) and germinal centers (GC). We have cloned its murine homologue and show that it is processed by the endoprotease furin before secretion of the catalytic domain. We have defined the cell types that express decysin in mouse spleen in the course of an immune response to T cell-dependent Ags. Like in humans, decysin is transcribed by activated CD11c+ DC that enter the T cell zone from the marginal zone (MZ). In the GC, decysin is expressed by follicular DC and tingible body macrophages. In addition, a MZ cell population expresses decysin and appears to migrate into the B cell follicle. The majority of these follicle-homing cells express the mannose receptor ligand, a marker for the macrophage-like MZ metallophils. The follicle-homing cells are M-CSF dependent, as they are absent in op/op mice that lack functional M-CSF. This suggests that mannose receptor ligand+ MZ metallophils differentiate into cells that migrate from the MZ into the B cell follicle. Decysin represents the first marker for this previously unrecognized cell population of the mouse spleen, which may represent a precursor for GCDC and may be specialized in the transport of unprocessed Ag from the MZ into developing GC.
Transcriptional Suppression of Matrix Metalloproteinase-9 Gene Expression by IFN-γ and IFN-β: Critical Role of STAT-1α
Authors: Ma Zhendong , Qin Hongwei , Benveniste Etty N.
Published: April 2014

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that play crucial roles in proteolytic degradation of the extracellular matrix. Aberrant expression of the 92-kDa type IV collagenase (MMP-9) is implicated in the invasion and angiogenesis process of malignant tumors and in inflammatory diseases of the CNS. We investigated the effects of IFN-γ and IFN-β, cytokines used for treating some cancers and multiple sclerosis, on MMP-9 expression in human astroglioma and fibrosarcoma cell lines and primary astrocytes. Our results demonstrate that IFN-γ and IFN-β significantly inhibit MMP-9 enzymatic activity and protein expression that is induced by PMA and the cytokine TNF-α. The inhibitory effects of IFN-γ and IFN-β on MMP-9 expression correlate with decreased steady state MMP-9 mRNA levels and suppression of MMP-9 promoter activity. IFN-γ- and IFN-β-mediated inhibition of MMP-9 gene expression is dependent on the transcription factor STAT-1α, since IFN-γ and IFN-β fail to suppress MMP-9 expression in STAT-1α-deficient primary astrocytes and human fibrosarcoma cells. Reconstitution of human STAT-1α successfully restores the inhibitory effects of IFN-γ and IFN-β on MMP-9 gene expression. Thus, these data demonstrate the critical role of STAT-1α in IFN-γ and IFN-β suppression of MMP-9 gene expression.
Antibody Is Required for Clearance of Infectious Murine Hepatitis Virus A59 from the Central Nervous System, But Not the Liver
Authors: Matthews Amy E. , Weiss Susan R. , Shlomchik Mark J. , Hannum Lynn G. , Gombold James L. , Paterson Yvonne
Published: April 2014

Intracerebral inoculation with mouse hepatitis virus strain A59 results in viral replication in the CNS and liver. To investigate whether B cells are important for controlling mouse hepatitis virus strain A59 infection, we infected muMT mice who lack membrane-bound IgM and therefore mature B lymphocytes. Infectious virus peaked and was cleared from the livers of muMT and wild-type mice. However, while virus was cleared from the CNS of wild-type mice, virus persisted in the CNS of muMT mice. To determine how B cells mediate viral clearance, we first assessed CD4+ T cell activation in the absence of B cells as APC. CD4+ T cells express wild-type levels of CD69 after infection in muMT mice. IFN-γ production in response to viral Ag in muMT mice was also normal during acute infection, but was decreased 31 days postinfection compared with that in wild-type mice. The role of Ab in viral clearance was also assessed. In wild-type mice plasma cells appeared in the CNS around the time that virus is cleared. The muMT mice that received A59-specific Ab had decreased virus, while mice with B cells deficient in Ab secretion did not clear virus from the CNS. Viral persistence was not detected in FcR or complement knockout mice. These data suggest that clearance of infectious mouse hepatitis virus strain A59 from the CNS requires Ab production and perhaps B cell support of T cells; however, virus is cleared from the liver without the involvement of Abs or B cells.
Hepatitis C Virus Core Protein Inhibits Human T Lymphocyte Responses by a Complement-Dependent Regulatory Pathway
Authors: Yao Zhi Qiang , Nguyen Duong Tony , Hiotellis Apostolos I. , Hahn Young S.
Published: April 2014

Complement proteins are involved in early innate immune responses against pathogens and play a role in clearing circulating viral Ags from the blood of infected hosts. We have previously demonstrated that hepatitis C virus (HCV) core, the first protein to be expressed and circulating in the blood of infected individuals, inhibited human T cell proliferative response through interaction with the complement receptor, globular domain of C1q receptor (gC1qR). To investigate the mechanisms of HCV core/gC1qR-induced inhibition of T cell proliferation, we examined the effect of core protein on the early events in T cell activation. We found that HCV core inhibited phosphorylation of extracellular signal-regulated kinase (ERK) and mitogen-activated ERK kinase (MEK). HCV core-induced impairment of ERK/MEK mitogen-activated protein kinase resulted in the inhibition of IL-2 and IL-2Rα gene transcription, which led to the inhibition of IL-2 production and high-affinity IL-2R expression. Importantly, the ability of anti-gC1qR Ab treatment to reverse HCV core-induced inhibition of ERK/MEK phosphorylation reveals that the interaction between HCV core and gC1qR is linked to the interference of ERK/MEK mitogen-activated protein kinase activation. These results imply that HCV core-induced blockage of intracellular events in T cell activation by a complement-dependent regulatory pathway may play a critical role in the establishment of HCV persistence during the acute phase of viral infection.
IL-12p40-Dependent Agonistic Effects on the Development of Protective Innate and Adaptive Immunity AgainstSalmonellaEnteritidis
Authors: Lehmann Jörg , Bellmann Steffi , Werner Christoph , Schröder Regina , Schütze Nicole , Alber Gottfried
Published: April 2014

To study a potential IL-12p40-dependent but IL-12p75-independent agonistic activity regulating the immune response against Salmonella Enteritidis, the course of infection in IL-12p35-deficient mice (IL-12p35−/−, capable of producing IL-12p40) was compared with that of IL-12p40−/− mice. Mice lacking IL-12p40 revealed a higher mortality rate and higher bacterial organ burden than mice capable of producing IL-12p40. This phenotype was found in both genetically susceptible (BALB/c, Itys) and resistant mice (129Sv/Ev, Ityr) indicating Ity-independent mechanisms. The more effective control of bacteria in the IL-12p35−/− mice was associated with elevated serum IFN-γ and TNF-α levels. In contrast, IL-12p40−/− mice showed reduced IFN-γ production, which was associated with significantly elevated serum IgE levels. Early during infection (days 3 and 4 postinfection), as well as late (day 20 postinfection), the number of infected phagocytes was strongly increased in the absence of IL-12p40 indicating impaired bactericidal activity when IL-12p40 was missing. Liver histopathology revealed a decreased number of mononuclear granulomas in IL-12p40−/− mice. Depletion of CD4+ or CD8+ T lymphocytes in vivo suggested that both T cell subpopulations contribute to the IL-12p40-dependent protective functions. Analysis of IL-12p40 vs IL-23p19 mRNA expression revealed an up-regulation of only IL-12p40 mRNA during Salmonella infection. Together these data indicate that IL-12p40 can induce protective mechanisms during both the innate and the adaptive type 1 immune response in Salmonella infection. This novel activity of IL-12p40 complements the well described dominant and essential role of IL-12p75 in protective immunity to Salmonella infection.
Monocyte-Fibronectin Interactions, Via α5β1 Integrin, Induce Expression of CXC Chemokine-Dependent Angiogenic Activity
Authors: White Eric S. , Livant Donna L. , Markwart Sonja , Arenberg Douglas A.
Published: April 2014

Monocyte-derived macrophages are important sources of angiogenic factors in cancer and other disease states. Upon extravasation from vasculature, monocytes encounter the extracellular matrix. We hypothesized that interaction with extracellular matrix proteins leads monocytes to adopt an angiogenic phenotype. We performed endothelial cell chemotaxis assays on conditioned medium (CM) from monocytes that had been cultured in vitro on various matrix substrates (collagen I, laminin, Matrigel, fibronectin), in the presence of autologous serum, or on tissue culture plastic alone. Monocytes cultured on Matrigel and on fibronectin were the most potent inducers of angiogenic activity compared with tissue culture plastic or autologous serum-differentiated monocytes. This increased angiogenic activity was associated with increased expression of angiogenic CXC chemokines (IL-8, epithelial neutrophil-activating peptide-78, growth-related oncogene α, and growth-related oncogene γ) but not of vascular endothelial growth factor. Additionally, CM from monocytes cultured on fibronectin-depleted Matrigel (MGFN−) induced significantly less angiogenic activity than CM from monocytes cultured on control-depleted Matrigel. ELISA analysis of CM from monocytes cultured on MGFN− revealed a significant decrease in GRO-α and GRO-γ compared with CM from monocytes cultured on MG. Incubation of monocytes before adherence on fibronectin with PHSCN (a competitive peptide inhibitor of the PHSRN sequence of fibronectin binding via α5β1 integrin) results in diminished expression of angiogenic activity and CXC chemokines compared with control peptide. These data suggest that fibronectin, via α5β1 integrin, promotes CXC chemokine-dependent angiogenic activity from monocytes.
Chemokines Regulate IL-6 and IL-8 Production by Fibroblast-Like Synoviocytes from Patients with Rheumatoid Arthritis
Authors: Nanki Toshihiro , Nagasaka Kenji , Hayashida Kenji , Saita Yuji , Miyasaka Nobuyuki
Published: April 2014

Rheumatoid arthritis (RA) is characterized by proliferation of synoviocytes that produce inflammatory cytokines and chemokines. The expressed chemokines are thought to be involved in the migration of inflammatory cells into the synovium. In this study we show that CCL2/monocyte chemotactic protein-1, CCL5/RANTES, and CXCL12/stromal cell-derived factor-1 enhanced IL-6 and IL-8 production by fibroblast-like synoviocytes (FLS) from patients with RA, and their corresponding receptors, CCR2, CCR5, and CXCR4, respectively, were expressed by RA FLS. The chemokines stimulated RA FLS more effectively than skin fibroblasts. Culture with CCL2 enhanced phosphorylation of extracellular signal-related kinase 1 (ERK1) and ERK2, but not phosphorylation of p38 or Src. Moreover, activation of ERK1/2 was inhibited by pertussis toxin, a Gi-coupled protein inhibitor, and RS-504393, CCR2 antagonist, suggesting that ERK1/2 was activated by CCL2 via CCR2 and Gi-coupled protein. On the other hand, CCL2, CCL5, and CXCL12 were expressed on RA FLS, and their production was regulated by TNF-α, IL-1β, and TGF-β1. Our results indicate that the chemokines not only play a role in inflammatory cell migration, but are also involved in the activation of FLS in RA synovium, possibly in an autocrine or paracrine manner.
Antigen Processing Defects in Cervical Carcinomas Limit the Presentation of a CTL Epitope from Human Papillomavirus 16 E6
Authors: Evans Mererid , Borysiewicz Leszek K. , Evans Alan S. , Rowe Martin , Jones Matthew , Gileadi Uzi , Cerundolo Vincenzo , Man Stephen
Published: April 2014

Human papillomavirus (HPV) infection, particularly type 16, is causally associated with the development of cervical cancer. The E6 and E7 proteins of HPV are constitutively expressed in cervical carcinoma cells making them attractive targets for CTL-based immunotherapy. However, few studies have addressed whether cervical carcinomas can process and present HPV E6/E7-derived Ags for recognition by CTL. We generated HLA-A*0201-restricted CTL clones against HPV16 E629–38 that recognized HPV16 E6 Ags transfected into B lymphoblastoid cells. These CTL were unable to recognize HLA-A*0201+ HPV16 E6+ cervical carcinoma cell lines even when the level of endogenous HPV16 E6 in these cells was increased by transfection. This defect in presentation of HPV16 E629–38 correlated with low level expression of HLA class I, proteasome subunits low molecular mass protein 2 and 7, and the transporter proteins TAP1 and TAP2 in the cervical carcinoma cell lines. The expression of all of these proteins could be up-regulated by IFN-γ, but this was insufficient for CTL recognition unless the level of HPV16 E6 Ag was also increased by transfection. CTL recognition of the HPV16 E629–38 epitope in 721.174 B cells was dependent on TAP expression but independent of immunoproteasome expression. Collectively, these findings suggest that presentation of the HPV16 E629–38 epitope in cervical carcinoma cell lines is limited both by the level of TAP expression and by the low level or availability of the source HPV E6 oncoprotein. These observations place constraints on the use of this, and potentially other, HPV-derived CTL epitopes for the immunotherapy of cervical cancer.
Highly Activated CD8+ T Cells in the Brain Correlate with Early Central Nervous System Dysfunction in Simian Immunodeficiency Virus Infection
Authors: Marcondes Maria Cecilia Garibaldi , Burudi E. M. E. , Huitron-Resendiz Salvador , Sanchez-Alavez Manuel , Watry Debbie , Zandonatti Michelle , Henriksen Steven J. , Fox Howard S.
Published: April 2014

One of the consequences of HIV infection is damage to the CNS. To characterize the virologic, immunologic, and functional factors involved in HIV-induced CNS disease, we analyzed the viral loads and T cell infiltrates in the brains of SIV-infected rhesus monkeys whose CNS function (sensory evoked potential) was impaired. Following infection, CNS evoked potentials were abnormal, indicating early CNS disease. Upon autopsy at 11 wk post-SIV inoculation, the brains of infected animals contained over 5-fold more CD8+ T cells than did uninfected controls. In both infected and uninfected groups, these CD8+ T cells presented distinct levels of activation markers (CD11a and CD95) at different sites: brain > CSF > spleen = blood > lymph nodes. The CD8+ cells obtained from the brains of infected monkeys expressed mRNA for cytolytic and proinflammatory molecules, such as granzymes A and B, perforin, and IFN-γ. Therefore, the neurological dysfunctions correlated with increased numbers of CD8+ T cells of an activated phenotype in the brain, suggesting that virus-host interactions contributed to the related CNS functional defects.
Dual Role of the IL-12/IFN-γ Axis in the Development of Autoimmune Myocarditis: Induction by IL-12 and Protection by IFN-γ
Authors: Eriksson Urs , Kurrer Michael O. , Sebald Wolfgang , Brombacher Frank , Kopf Manfred
Published: April 2014

IL-12 and IFN-γ positively regulate each other and type 1 inflammatory responses, which are believed to cause tissue damage in autoimmune diseases. We investigated the role of the IL-12/IFN-γ (Th1) axis in the development of autoimmune myocarditis. IL-12p40-deficient mice on a susceptible background resisted myocarditis. In the absence of IL-12, autospecific CD4+ T cells proliferated poorly and showed increased Th2 cytokine responses. However, IFN-γ-deficient mice developed fatal autoimmune disease, and blockade of IL-4R signaling did not confer susceptibility to myocarditis in IL-12p40-deficient mice, demonstrating that IL-12 triggers autoimmunity by a mechanism independent of the effector cytokines IFN-γ and IL-4. In conclusion, our results suggest that the IL-12/IFN-γ axis is a double-edged sword for the development of autoimmune myocarditis. Although IL-12 mediates disease by induction/expansion of Th1-type cells, IFN-γ production from these cells limits disease progression.
Characterization of CCR9 Expression and CCL25/Thymus-Expressed Chemokine Responsiveness During T Cell Development: CD3highCD69+ Thymocytes and γδTCR+ Thymocytes Preferentially Respond to CCL25
Authors: Uehara Shoji , Song Kaimei , Farber Joshua M. , Love Paul E.
Published: April 2014

CCR9 mediates chemotaxis of thymocytes in response to CCL25/thymus-expressed chemokine, and its mRNA is selectively expressed in thymus and small intestine, the two known sites of T lymphopoiesis. To examine the expression of CCR9 during lymphocyte development, we generated polyclonal Ab that recognizes murine CCR9. CCR9 was expressed on the majority of immature CD4+CD8+ (double-positive) thymocytes, but not on immature CD4−CD8− (double-negative) thymocytes. CCR9 was down-regulated during the transition of double-positive thymocytes to the CD4+ or CD8+ (single-positive) stage, and only a minor subset of CD8+ lymph node T cells expressed CCR9. All CCR9+ thymocyte subsets migrated in response to CCL25; however, CD69+ thymocytes demonstrated enhanced CCL25-induced migration compared with CD69− thymocytes. Ab-mediated TCR stimulation also enhanced CCL25 responsiveness, indicating that CCL25-induced thymocyte migration is augmented by TCR signaling. Approximately one-half of all γδTCR+ thymocytes and peripheral γδTCR+ T cells expressed CCR9 on their surface, and these cells migrated in response to CCL25. These findings suggest that CCR9 may play an important role in the development and trafficking of both αβTCR+ and γδTCR+ T cells.
Staphylokinase-Specific Cell-Mediated Immunity in Humans
Authors: Warmerdam Petra A. M. , Vanderlick Kristel , Vandervoort Petra , De Smedt Heidi , Plaisance Stéphane , De Maeyer Marc , Collen Désiré
Published: April 2014

Staphylokinase is a highly fibrin-specific clot-dissolving agent that constitutes a promising drug for clinical development. It is of bacterial origin, and the majority of patients develop neutralizing Ab after its administration. Several antigenic regions, recognized by these Ab, have been identified, but the underlying immunogenic features of staphylokinase remain unknown. In this study, we show that staphylokinase is a T cell-dependent Ag, and that an immunological memory may be acquired, even without administration of staphylokinase. Thrombolysis with staphylokinase provokes the proliferation of staphylokinase-specific T lymphocytes, which remain elevated over 10 mo posttreatment. Interestingly, analysis of a large number of staphylokinase-specific T cell clones isolated from 10 unrelated donors revealed only six distinct immunogenic regions in the molecule. Moreover, five of the six regions are recognized by T lymphocytes from several individuals, indicating that these regions are not restricted to a single HLA-DR allele. Therefore, these new insights can guide the design of variants with a lower immunogenic profile in humans.
Classical MHC Class I Genes Composed of Highly Divergent Sequence Lineages Share a Single Locus in Rainbow Trout (Oncorhynchus mykiss)
Authors: Aoyagi Kazuhiko , Dijkstra Johannes M. , Xia Chun , Denda Ikuo , Ototake Mitsuru , Hashimoto Keiichiro , Nakanishi Teruyuki
Published: April 2014

The classical MHC class I genes have been known to be highly polymorphic in various vertebrates. To date, putative allelic sequences of the classical MHC class I genes in teleost fish have been reported in several studies. However, the establishment of their allelic status has been hampered in most cases by the lack of appropriate genomic information. In the present study, using heterozygous and homozygous fish, we obtained classical-type MHC class I sequences of rainbow trout (Oncorhynchus mykiss) and investigated their allelic relationship by gene amplification and Southern and Northern hybridization analyses. The results indicated that all MHC class I sequences we obtained were derived from a single locus. Based on this, a unique polymorphic nature of the MHC class I locus of rainbow trout has been revealed. The mosaic combination of highly divergent ancient sequences in the peptide-binding domains is notable, and the variable nature around the boundary between the α3 and transmembrane domains is unprecedented.
Development and Homeostasis of T Cell Memory in Rhesus Macaque
Authors: Pitcher Christine J. , Hagen Shoko I. , Walker Joshua M. , Lum Richard , Mitchell Bridget L. , Maino Vernon C. , Axthelm Michael K. , Picker Louis J.
Published: April 2014

The rhesus macaque (RM) is a critical animal model for studies of viral pathogenesis and immunity, yet fundamental aspects of their cellular immune response remain poorly defined. One such deficiency is the lack of validated phenotypic signatures for their naive and memory T cell subsets, and the resultant unavailability of accurate information on their memory T cell development, homeostasis, and function. In this study, we report a phenotypic paradigm allowing definitive characterization of these subsets and their comprehensive functional analysis. Naive T cells are optimally delineated by their homogeneous CD95lowCD28highβ7 integrinint (CD4+) or CD95lowCD28intCD11alow (CD8+) phenotypes. This subset 1) was present in blood and secondary lymph tissues, but not effector sites; 2) vastly predominated in the fetal/neonatal immune system, but rapidly diminished with postnatal age; 3) lacked IFN-γ production capability, and specific responses to RM CMV; and 4) demonstrated low in vivo proliferative activity. CD4+ and CD8+ memory subsets were CD95high, but otherwise phenotypically heterogeneous and included all IFN-γ production, RM CMV-specific responses, effector site T cells, and demonstrated high in vivo proliferative activity (∼10 times the naive subset). These analyses also revealed the RM “effector memory” subset within the overall memory population. This population, best defined by lack of CD28 expression, contained the majority of RM CMV-specific cells, was highly enriched in extralymphoid effector sites, and comprised an increasing proportion of total memory cells with age. The effector memory subset demonstrated similar in vivo proliferative activity and survival as CD28+ “central memory” T cells, consistent with independent homeostatic regulation.
Role of STAT6 in IgE Receptor/FcεRI-Mediated Late Phase Allergic Responses of Mast Cells
Authors: Malaviya Ravi , Uckun Fatih M.
Published: April 2014

In this study we show that IgE receptor engagement triggers activation of STAT6 in mast cells. We sought to determine the role of STAT6 activation in IgE receptor-mediated mast cell responses using STAT6 knockout mice. After IgE receptor engagement, bone marrow mast cells from STAT6−/− mice exhibited normal histamine and leukotriene C4 release, but their cytokine release was markedly reduced. In accordance with these in vitro data, IgE/Ag-challenged STAT6−/− mice showed normal early phase, but severely impaired late phase, allergic reactions. These findings provide unprecedented evidence that STAT6 plays a pivotal role in mast cell responses to IgE/Ag st imulation.
Generation of Diversity in the Innate Immune System: Macrophage Heterogeneity Arises from Gene-Autonomous Transcriptional Probability of Individual Inducible Genes
Authors: Ravasi Timothy , Wells Christine , Forest Alistair , Underhill David M. , Wainwright Brandon J. , Aderem Alan , Grimmond Sean , Hume David A.
Published: April 2014

Microbial products such as LPS stimulate macrophages to produce a wide diversity of inducible gene products needed for immediate host defense and priming of an appropriate acquired immune response. In this study, we have examined LPS-inducible gene expression in subclones of a mouse macrophage cell line, RAW264, using cDNA microarrays. Even archetypal target genes such as TNF-α were not induced in all subclones, and there was no absolute correlation between expression of pairs of genes. Nevertheless, the array analysis revealed clusters of genes that were more likely to be coexpressed. RAW264 cells stably transfected with luciferase reporter genes driven by LPS-responsive promoters revealed the same kind of clonal heterogeneity. The results indicate that each LPS-inducible gene has its own inherent probability of activation in response to LPS.
The Membrane Attack Complex of Complement Causes Severe Demyelination Associated with Acute Axonal Injury
Authors: Mead Richard J. , Singhrao Sim K. , Neal James W. , Lassmann H. , Morgan B. Paul
Published: April 2014

Complement is implicated in pathology in the human demyelinating disease multiple sclerosis and in animal models that mimic the demyelination seen in multiple sclerosis. However, the components of the complement system responsible for demyelination in vivo remain unidentified. In this study, we show that C6-deficient (C6−) PVG/c rats, unable to form the membrane attack complex (MAC), exhibit no demyelination and significantly reduced clinical score in the Ab-mediated experimental autoimmune encephalomyelitis model when compared with matched C6-sufficient (C6+) rats. In C6+ rats, perivenous demyelination appeared, accompanied by abundant mononuclear cell infiltration and axonal injury. Neither demyelination nor axonal damage was seen in C6− rats, whereas levels of mononuclear cell infiltration were equivalent to those seen in C6+ rats. Reconstitution of C6 to C6− rats yielded pathology and clinical disease indistinguishable from that in C6+ rats. We conclude that demyelination and axonal damage occur in the presence of Ab and require activation of the entire complement cascade, including MAC deposition. In the absence of MAC deposition, complement activation leading to opsonization and generation of the anaphylatoxins C5a and C3a is insufficient to initiate demyelination.
Prolonged Allograft Survival in TNF Receptor 1-Deficient Recipients Is Due to Immunoregulatory Effects, Not to Inhibition of Direct Antigraft Cytotoxicity
Authors: McKee Charlotte M. , Defina Rachel , He Hongzhen , Haley Kathleen J. , Stone James R. , Perkins David L.
Published: April 2014

TNF-α and lymphotoxin (LT)α have been shown to be important mediators of allograft rejection. TNF-R1 is the principal receptor for both molecules. Mice with targeted genetic deletions of TNF-R1 demonstrate normal development of T and B lymphocytes but exhibit functional defects in immune responses. However, the role of TNF-R1-mediated signaling in solid organ transplant rejection has not been defined. To investigate this question, we performed vascularized heterotopic allogeneic cardiac transplants in TNF-R1-deficient (TNF-R1−/−) and wild-type mice. Because all allografts in our protocol expressed TNF-R1, direct antigraft effects of TNF-α and LTα were not prevented. However, immunoregulatory effects on recipient inflammatory cells by TNF-R1 engagement was eliminated in TNF-R1−/− recipients. In our study, cardiac allograft survival was significantly prolonged in TNF-R1−/− recipients. Despite this prolonged allograft survival, we detected increased levels of CD8 T cell markers in allografts from TNF-R1−/− recipients, suggesting that effector functions, but not T cell recruitment, were blocked. We also demonstrated the inhibition of multiple chemokines and cytokines in allografts from TNF-R1−/− recipients including RANTES, IFN-inducible protein-10, lymphotactin, and IL-1R antagonist, as well as altered levels of chemokine receptors. We correlated gene expression with the physiologic process of allograft rejection using self-organizing maps and identified distinct patterns of gene expression in allografts from TNF-R1−/− recipients. These findings indicate that in our experimental system TNF-α and LTα exert profound immunoregulatory effects through TNF-R1.
Requirement for NK Cells in CD40 Ligand-Mediated Rejection of Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia Cells
Authors: Gruber Tanja A. , Skelton Dianne C. , Kohn Donald B.
Published: April 2014

We have previously developed a murine model of Philadelphia chromosome-positive acute lymphoblastic leukemia by i.v. injection of a pre-B ALL cell line (BM185) derived from Bcr-Abl-transformed BALB/c bone marrow. We are studying the potential to elicit autologous antileukemic immune responses by introducing genes encoding immunomodulators (CD40 ligand (CD40L), CD80, and GM-CSF) into leukemia cells. BM185 cells expressing CD40L or CD80 alone, when injected into BALB/c mice, were rejected in approximately 25% of mice, whereas cohorts receiving BM185 cells expressing two or more immunomodulator genes rejected challenge 50–76% of the time. The greatest protection was conferred in mice receiving BM185 cells expressing all three immunomodulators. Addition of murine rIL-12 treatments in conjunction with BM185/CD80/CD40L/GM-CSF vaccination allowed rejection of preestablished leukemia. BM185 cell lines expressing CD40L were rejected in BALB/c nu/nu (nude) mice, in contrast to cell lines expressing CD80 and/or GM-CSF. Nude mice depleted of NK cells were no longer protected when challenged with BM185/CD40L, demonstrating a requirement for NK cells. Similarly, NK cell depletion in immunocompetent BALB/c mice resulted in a loss of protection when challenged with BM185/CD40L, confirming the data seen in nude mice. The ability of CD40L to act in a T cell-independent manner may be important for clinical applications in patients with depressed cellular immunity following chemotherapy.
Impaired Development of T Lymphoid Precursors from Pluripotent Hematopoietic Stem Cells in New Zealand Black Mice
Authors: Hashimoto Yoshiko , Montecino-Rodriguez Encarnacion , Gershwin M. Eric , Dorshkind Kenneth
Published: April 2014

Bone marrow cells from autoimmune-prone New Zealand Black (NZB) mice are less efficient at colonizing fetal thymic lobes than cells from normal strains. This study demonstrates that the reduced capacity of NZB bone marrow cells to repopulate the thymus does not result from their inability to migrate to or enter the thymus. Rather, the T lymphopoietic defect of NZB mice is due to an impaired ability of pluripotent hematopoietic stem cells (PHSCs) to generate more committed lymphoid progeny, which could include common lymphoid precursors and/or other T cell-committed progenitors. Although PHSCs from NZB mice were not as efficient at thymic repopulation as comparable numbers of PHSCs from control strains, the ability of common lymphoid precursors from NZB mice to repopulate the thymus was not defective. Similarly, more differentiated NZB T cell precursors included in the intrathymic pool of CD4−CD8− cells also exhibited normal T lymphopoietic potential. Taken together, the results identify an unappreciated defect in NZB mice and provide further evidence that generation of lymphoid progeny from the PHSCs is a regulated event.
Human Anergic CD4+ T Cells Can Act as Suppressor Cells by Affecting Autologous Dendritic Cell Conditioning and Survival
Authors: Frasca Loredana , Scottà Cristiano , Lombardi Giovanna , Piccolella Enza
Published: April 2014

T cell suppression exerted by regulatory T cells represents a well-established phenomenon, but the mechanisms involved are still a matter of debate. Recent data suggest that anergic T cells can suppress responder T cell activation by inhibiting Ag presentation by dendritic cells (DC). In this study, we focused our attention on the mechanisms that regulate the susceptibility of DC to suppressive signals and analyzed the fate of DC and responder T cells. To address this issue, we have cocultured human alloreactive or Ag-specific CD4+ T cell clones, rendered anergic by incubation with immobilized anti-CD3 Ab, with autologous DC and responder T cells. We show that anergic T cells affect either Ag-presenting functions or survival of DC, depending whether immature or mature DC are used as APC. Indeed, MHC and costimulatory molecule expression on immature DC activated by responder T cells is inhibited, while apoptotic programs are induced in mature DC and in turn in responder T cells. Ligation of CD95 by CD95L expressed on anergic T cells in the absence of CD40-CD40L (CD154) interaction are critical parameters in eliciting apoptosis in both DC and responder T cells. In conclusion, these findings indicate that the defective activation of CD40 on DC by CD95L+ CD154-defective anergic T cells could be the primary event in determining T cell suppression and support the role of CD40 signaling in regulating both conditioning and survival of DC.
CD2 Facilitates Differentiation of CD4 Th Cells Without Affecting Th1/Th2 Polarization
Authors: Sasada Tetsuro , Yang Hailin , Reinherz Ellis L.
Published: April 2014

The role of CD2 in murine CD4 helper T cell differentiation and polarization was examined using TCR-Cyt-5CC7-I transgenic recombination activating gene-2−/− H-2a mice on CD2+/+ or CD2−/− backgrounds. In the absence of CD2, thymic development was abnormal as judged by reduction in the steady state number of total, double-positive, and CD4 single-positive (SP) thymocytes, as well as a defect in their restorative dynamics after peptide-induced negative selection in vivo. In addition, in CD2−/− animals, lymph node CD4 SP T cells manifest a 10- to 100-fold attenuated activation response to cytochrome c (CytC) agonist peptides as judged by induction of CD25 and CD69 cell surface expression or [3H]TdR incorporation; differences in the magnitude of responsiveness and requisite molar peptide concentrations were even greater for altered peptide ligands. Although the presence or absence of CD2 did not impact the final Th1 or Th2 polarization outcome, CD2 expression reduced the CytC peptide concentration threshold necessary to facilitate both Th1 and Th2 differentiation. In vivo administration of CytC peptide to CD2−/− animals yielded an impaired CD4 SP T cell effector/memory phenotype compared with similarly treated CD2+/+ mice. Analysis of TCR-Cyt-5CC7-I human CD2 double-transgenic mice similarly failed to reveal a preferential Th1 vs Th2 polarization. Collectively, these results indicate that CD2 is important for the efficient development of CD4 SP thymocytes and TCR-dependent activation of mature CD4 lymph node T cells, but does not direct a particular helper T cell subset polarity.
Viral IFN-Regulatory Factors Inhibit Activation-Induced Cell Death Via Two Positive Regulatory IFN-Regulatory Factor 1-Dependent Domains in the CD95 Ligand Promoter
Authors: Kirchhoff Sabine , Sebens Thorsten , Baumann Sven , Krueger Andreas , Zawatzky Rainer , Li-Weber Min , Meinl Edgar , Neipel Frank , Fleckenstein Bernhard , Krammer Peter H.
Published: April 2014

The CD95 (also called APO-1/Fas) system plays a major role in the induction of apoptosis in lymphoid and nonlymphoid tissues. The CD95 ligand (CD95L) is induced in response to a variety of signals, including IFN-γ and TCR/CD3 stimulation. Here we report the identification of two positive regulatory IFN-regulatory factor-dependent domains (PRIDDs) in the CD95L promoter and its 5′ untranslated region, respectively. EMSAs demonstrate specific binding of IFN-γ-induced IFN-regulatory factor 1 (IRF-1) to the PRIDD sequences. Ectopic IRF-1 expression induces CD95L promoter activity. Furthermore, we demonstrate that PRIDDs play an important role in TCR/CD3-mediated CD95L induction. Most interestingly, viral IRFs of human herpes virus 8 (HHV8) totally abolish IRF-1-mediated and strongly reduce TCR/CD3-mediated CD95L induction. We demonstrate here for the first time that viral IRFs inhibit activation-induced cell death. Thus, these results demonstrate an important mechanism of HHV8 to modulate the immune response by down-regulation of CD95L expression. Inhibition of CD95-dependent T cell function might contribute to the immune escape of HHV8.
FcαRI/CD89 Circulates in Human Serum Covalently Linked to IgA in a Polymeric State
Authors: van der Boog Paul J. M. , van Zandbergen Ger , de Fijter Johan W. , Klar-Mohamad Ngaisah , van Seggelen Anneke , Brandtzaeg Per , Daha Mohamed R. , van Kooten Cees
Published: April 2014

The FcR for IgA CD89/FcαRI, is a type I receptor glycoprotein, expressed on myeloid cells, with important immune effector functions. In vitro CD89 can be released from CD89-expressing cells upon activation. Little information is available on the existence of this soluble molecule in vivo. Using specific and sensitive ELISA techniques (detection limit 50 pg/ml), we were not able to detect circulating CD89 in human sera. However, using Western blotting, a 30-kDa soluble CD89 molecule was demonstrated in both serum and plasma. Moreover, using a specific semiquantitative dot-blot system, we found CD89 in all human sera tested (mean concentration 1900 ng/ml). Size fractionation of human serum using gel filtration chromatography showed that the CD89 molecule was predominantly present in larger molecular mass fractions. Direct complexes between IgA and CD89 were demonstrated by anti-IgA affinity purification, and when analyzed under nonreducing conditions appeared to be covalently linked. Size fractionation of affinity-purified IgA showed the presence of soluble CD89 only in the high molecular mass fractions of IgA, but not in monomeric IgA. High molecular mass complexes of CD89-IgA could be distinguished from J chain containing dimeric IgA. These data show that CD89 circulates in complex with IgA, and suggest that CD89 might contribute to the formation of polymeric serum IgA.
CD40 Ligand-Deficient T Cells from X-Linked Hyper-IgM Syndrome Carriers Have Intrinsic Priming Capability
Authors: Lobo Francis M. , Scholl Paul R. , Fuleihan Ramsay L.
Published: April 2014

Deficiency in CD40 ligand (CD40L) expression is associated with impaired T cell immunity in mouse models and in humans. Previous studies have indicated that this is due to the failure of induction of extrinsic costimulatory molecules. However, other studies have suggested that CD40L is an intrinsic costimulatory molecule. The X-linked hyper-IgM syndrome (XHIM) is a primary immunodeficiency caused by mutations in CD40L, resulting in impaired Ab production and T cell immunity. CD4+ T cells from female carriers of XHIM express a variable degree of normal CD40L based on random X chromosome inactivation. We have examined T cells from XHIM carriers to investigate whether CD40L supports T cell function by acting as an intrinsic costimulator or by induction of other costimulatory molecules by examining coexpression of CD40L and markers of T lymphocyte priming. These carriers provide a unique model for comparison of CD40L-expressing and -nonexpressing lymphocytes in that all factors, including immunological experience, are equivalent between the two populations. Our results show that compared with CD40L-deficient T cells, T cells that express CD40L normally have a minimal advantage in becoming primed, as defined by CD45 RO isoform expression and production of IFN-γ and TNF-α. Conversely, CD40L-deficient T lymphocytes clearly were capable of becoming primed as defined by the same parameters. These findings imply that the intrinsic costimulatory activity of CD40L is not required for attaining primed status, and that CD40L primarily supports T cell function by inducing extrinsic factors that can be shared by CD40L-deficient cells.
The Role of T Cell Help in the Production of Antibodies Specific for Galα1–3Gal
Authors: Cretin Nathalie , Bracy Jennifer , Hanson Krista , Iacomini John
Published: April 2014

The majority of xenoreactive natural Abs in humans recognize the carbohydrate Ag present on pig tissue, Galα1–3Galβ1–4GlcNAc-R (αGal), synthesized by the enzyme UDP galactose:β-d-galactosyl-1,4-N-acetyl-d-glucosaminide α(1–3)galactosyltransferase or αGT. Using αGT knockout mice (GT0 mice), which like humans produce serum Abs that bind αGal, we examined the role of T cells in production of Abs specific for αGal. GT0 mice were crossed with TCR-β knockout mice (TCR-β0) to generate double-knockout mice (GT0/TCR-β0). While GT0/TCR-β+ mice exhibited an age-dependent increase in the serum titer of natural Abs specific for αGal, a similar increase was not observed in GT0/TCR-β0 mice, and the titer of αGal-specific Abs in double knockouts was significantly lower than in age-matched GT0/TCR-β+ mice. Immunization with pig cells resulted in a significant increase in the serum titer of αGal-specific Abs in GT0/TCR-β+ mice, but had no effect on the level of αGal-specific serum Abs in GT0/TCR-β0 mice. Treatment of GT0/TCR-β+ mice with anti-CD40L Abs before immunization with pig cells prevented sensitization to αGal. Our data suggest that the majority of αGal-specific Abs are T cell dependent and that production of αGal-specific Abs after sensitization can be prevented by blocking costimulatory pathways.
Cutting Edge: Precursor Frequency Affects the Helper Dependence of Cytotoxic T Cells
Authors: Mintern Justine D. , Davey Gayle M. , Belz Gabrielle T. , Carbone Francis R. , Heath William R.
Published: April 2014

Generation of CTL immunity often depends on the availability of CD4 T cell help. In this report, we show that CTL responses induced by cross-priming can be converted from CD4-dependent to CD4-independent by increasing the frequency of CTL precursors. In the absence of CD4 T cells, high numbers of CTL precursors were able to expand in number and become effector CTL. The ability of high frequencies of CD8 T cells to override help was not due to their ability to signal CD40 via expression of CD154. These findings suggest that when precursor frequencies are high, priming of CD8 T cell responses may not require CD4 T cell help.
Cutting Edge: Inducible Costimulator Protein Regulates Both Th1 and Th2 Responses to Cutaneous Leishmaniasis
Authors: Greenwald Rebecca J. , McAdam Alexander J. , Van der Woude Diane , Satoskar Abhay R. , Sharpe Arlene H.
Published: April 2014

The CD28 family member inducible costimulator protein (ICOS) has an important role in T cell differentiation and Ig class switching. To investigate the role of ICOS in vivo, ICOS−/− mice were infected s.c. with Leishmania mexicana. While wild-type mice developed large, cutaneous lesions, the growth of lesions and tissue histopathology was significantly delayed in ICOS−/− mice. ICOS−/− mice exhibited marked decreases in both Th1 and Th2 cytokine production and profound defects in L. mexicana-specific Ig isotype class switching to IgG1 and IgG2a and reduced total IgE levels. Our findings indicate that ICOS is a key regulator of both Th1 and Th2 responses and has a role in controlling cutaneous L. mexicana infection.
Cutting Edge: STAT6 Serves as a Positive and Negative Regulator of Gene Expression in IL-4-Stimulated B Lymphocytes
Authors: Schroder Andreas J. , Pavlidis Paul , Arimura Akinori , Capece Danielle , Rothman Paul B.
Published: April 2014

STAT6 plays an important role in IL-4-mediated B cell activation and differentiation. To identify primary and secondary target genes of STAT6, gene expression profiles of IL-4-stimulated B cells from STAT6+/+ vs STAT6−/− mice were compared. Statistical analysis revealed that 106 distinct probe sets including 70 known genes were differentially expressed between the 2 genotypes. These genes include transcription factors, kinases, and other enzymes, cell surface receptors, and Ig H chains. Surprisingly, although 31 genes were expressed at higher levels in STAT6+/+ B cells, 39 genes were expressed at higher abundance in STAT6−/− B cells. This result implies both positive and negative regulatory functions of STAT6 in IL-4-mediated gene expression. Furthermore, IL-4 induces expression of the transcription factor Krox20, which is required for maximal IL-4-induced transcription.