bluuebunny/crossref_metadata_2025 huggingface dataset:
A Role for Accessibility to Self-Peptide-Self-MHC Complexes in Intrathymic Negative Selection
Authors: Viret Christophe , Sant’Angelo Derek B. , He Xin , Ramaswamy Hemavathi , Janeway Charles A.
Published: April 2014

Whether intrathymic-positive and -negative selection of conventional αβ T cells occur in anatomically distinct sites is a matter of debate. By using a system composed of two distinct immune receptors, the Y-Ae mAb and the 1H3.1 (Vα1/Vβ6) TCR, both directed against the 52–68 fragment of the I-Eα-chain (Eα52–68) bound to I-Ab, we examined the occurrence of negative selection imposed in vivo by a self-peptide-self-MHC class II complex with differential tissue expression. 1H3.1 TCR-transgenic (Tg) mice were bred to mice having an I-Eα transgene with expression directed to all MHC class II-positive cells, restricted to thymic epithelial cells, or restricted to B cells, dendritic cells, and medullary thymic epithelial cells. All 1H3.1 TCR/I-Eα double-Tg mice revealed a severely diminished thymic cellularity. Their lymph node cells were depleted of Vβ6+CD4+ cells and were unresponsive to Eα52–68 in vitro. The absolute number of CD4+CD8+ thymocytes was drastically reduced in all combinations, indicating that negative selection caused by an endogenously expressed self-determinant can effectively occur in the thymic cortex in vivo. Moreover, both cortical epithelial cells and, interestingly, the few cortical dendritic cells were able to support negative selection of CD4+CD8+ thymocytes, albeit with a distinct efficiency. Collectively, these observations support a model where, in addition to the avidity of the thymocyte/stromal cell interaction, in vivo negative selection of autoreactive TCR-Tg T cells is determined by accessibility to self-peptide-self-MHC complexes regardless of the anatomical site.
Distinguishing Self from Nonself: Immunogenicity of the MurineH47Locus Is Determined by a Single Amino Acid Substitution in an Unusual Peptide
Authors: Mendoza Lisa M. , Villaflor Gilbert , Eden Peter , Roopenian Derry , Shastri Nilabh
Published: April 2014

Histocompatibility (H) Ags are responsible for chronic graft rejection and graft vs host disease in solid tissue and bone marrow transplantation among MHC-matched individuals. Here we defined the molecular basis of self-nonself discrimination for the murine chromosome 7 encoded H47 histocompatibility locus, known by its trait of graft-rejection for over 40 years. H47 encodes a novel, highly conserved cell surface protein containing the SCILLYIVI (SII9) nonapeptide in its transmembrane region. The p7 isoleucine-to-phenylalanine substitution in SII9 defined the antigenic polymorphism and T cell specificity. Despite absence of the canonical consensus motif and weak binding to Db MHC I, both H47 peptides were presented to CTLs. However, unlike all the other known H loci, the relative immunogenicity of both H47 alleles varied dramatically and was profoundly influenced by neighboring H loci. The results provide insights into the peptide universe that defines nonself and the basis of histoincompatibility.
Inducible Expression of Stat4 in Dendritic Cells and Macrophages and Its Critical Role in Innate and Adaptive Immune Responses
Authors: Fukao Taro , Frucht David M. , Yap George , Gadina Massimo , O’Shea John J. , Koyasu Shigeo
Published: April 2014

Autocrine activation of APC by IL-12 has recently been revealed; we demonstrate here that inducible expression of Stat4 in APC is central to this process. Stat4 is induced in dendritic cells (DC) in a maturation-dependent manner and in macrophages in an activation-dependent manner. Stat4 levels directly correlate with IL-12-dependent IFN-γ production by APC as well as IFN-γ production by DC during Ag presentation. The Th2 cytokines IL-4 and IL-10 suppress Stat4 induction in DC and macrophages when present during maturation and activation, respectively, diminishing IFN-γ production. In contrast, IL-4 has no effect on Stat4 levels in mature DC and actually augments IFN-γ production by DC during Ag presentation, indicating that IL-4 acts differently in a spatiotemporal manner. The functional importance of Stat4 is evident in Stat4−/− DC and macrophages, which fail to produce IFN-γ. Furthermore, Stat4−/− macrophages are defective in NO production in response to IL-12 and are susceptible to Toxoplasma. Autocrine IL-12 signaling is required for high-level IFN-γ production by APC at critical stages in both innate and adaptive immunity, and the control of Stat4 expression is likely an important regulator of this process.
Suppression of IL-12 Transcription in Macrophages Following Fcγ Receptor Ligation
Authors: Grazia Cappiello Maria , Sutterwala Fayyaz S. , Trinchieri Giorgio , Mosser David M. , Ma Xiaojing
Published: April 2014

Ligating FcγR on macrophages results in suppression of IL-12 production. We show that FcγR ligation selectively down-regulates IL-12 p40 and p35 gene expression at the level of transcription. The region responsive to this inhibition maps to the Ets site of the p40 promoter. PU.1, IFN consensus sequence binding protein, and c-Rel form a complex on this element upon macrophage activation. Receptor ligation abolishes the binding of this PU.1-containing activation complex, and abrogates p40 transcription. A dominant-negative construct of PU.1 diminishes IL-12 p40 promoter activity and endogenous IL-12 p40 protein secretion. Thus, the specificity of IL-12 down-regulation following receptor ligation lies in the inhibition of binding of a PU.1-containing complex to the Ets site of the IL-12 promoter. These findings provide evidence demonstrating for the first time the importance of PU.1 in the transcriptional regulation of IL-12 gene expression.
NF-κB and STAT5 Play Important Roles in the Regulation of Mouse Toll-Like Receptor 2 Gene Expression
Authors: Musikacharoen Tipayaratn , Matsuguchi Tetsuya , Kikuchi Takeshi , Yoshikai Yasunobu
Published: April 2014

Toll-like receptor 2 (TLR2) is involved in the innate immunity by recognizing various bacterial components. We have previously reported that TLR2 gene expression is rapidly induced by LPS or inflammatory cytokines in macrophages, and by TCR engagement or IL-2/IL-15 stimulation in T cells. Here, to investigate the mechanisms governing TLR2 transcription, we cloned the 5′ upstream region of the mouse TLR2 (mTLR2) gene and mapped its transcriptional start site. The 5′ upstream region of the mTLR2 gene contains two NF-κB, two CCAAT/enhancer binding protein, one cAMP response element-binding protein, and one STAT consensus sequences. In mouse macrophage cell lines, deletion of both NF-κB sites caused the complete loss of mTLR2 promoter responsiveness to TNF-α. NF-κB sites were also important but not absolutely necessary for LPS-mediated mTLR2 promoter activation. In T cell lines, mTLR2 responsiveness to IL-15 was abrogated by the 3′ NF-κB mutation, whereas 5′ NF-κB showed no functional significance. The STAT binding site also seemed to contribute, as the deletion of this sequence significantly reduced the IL-15-mediated mTLR2 promoter activation. EMSAs confirmed nuclear protein binding to both NF-κB sites in macrophages following LPS and TNF-α stimulation and to the 3′ NF-κB site in T cells after IL-15 treatment. Thus, NF-κB activation is important but differently involved in the regulation of mTLR2 gene expression in macrophages and T cells following LPS or cytokine stimulation.
Differential Expression of Fas Ligand in Th1 and Th2 Cells Is Regulated by Early Growth Response Gene and NF-AT Family Members
Authors: Dzialo-Hatton Robin , Milbrandt Jeffrey , Hockett Richard D. , Weaver Casey T.
Published: April 2014

Inducible expression of Fas ligand (CD95 ligand) by activated T cells and the resulting apoptosis of CD95-bearing cells is a critical component of peripheral T cell homeostasis and cytotoxic effector mechanisms. Transcriptional control of the expression of Fas ligand has been attributed to a number of factors, including early growth response gene 2 (Egr2), Egr3, Sp1, and NF-AT, although a direct contribution of NF-AT is controversial. The present study confirms a role for Egr factors and indicates that NF-AT is essential for optimal expression of murine Fas ligand through a direct interaction with an NF-AT consensus element. The role of these factors was further defined by studying the differential expression of Fas ligand in Th1 and Th2 lines derived from DO11.10 TCR transgenic mice. EMSA analyses of a composite Egr/NF-AT site showed recruitment of Sp1 to this site in Th2 cells, but not in Th1 cells. Furthermore, gel-shift analyses demonstrated the binding of Egr1, 2, and 3 in Th2 cells and Egr1 and 2, but not Egr3 in Th1 cells at a known Egr site. Northern analysis corroborated the lack of Egr3 in Th1 cells. Differential usage of these transcription factors by Th1 and Th2 cells suggests a potential mechanism underlying the differential expression of Fas ligand by distinct T cell lineages.
HIV-1 Tat Inhibits IL-2 Gene Transcription Through Qualitative and Quantitative Alterations of the Cooperative Rel/AP1 Complex Bound to the CD28RE/AP1 Composite Element of the IL-2 Promoter
Authors: González Esther , Punzón Carmen , González Manuel , Fresno Manuel
Published: April 2014

Dysregulation of cytokine secretion plays an important role in AIDS pathogenesis. Here, we demonstrate that expression of HIV-1 Tat protein in Jurkat cells induces a severe impairment of IL-2 but not TNF gene transcription. Interestingly, this inhibition correlates with the effect of the viral protein on the transactivation of the CD28RE/AP1 composite element (−164/−154), but not with that observed on the NFAT/AP1 site of the IL-2 gene promoter, neither with the effect on NF-κB- nor AP1-independent binding sites. Endogenous expression of Tat induced a decrease in the amount of the specific protein complex bound to the CD28RE/AP1 probe after PMA plus calcium ionophore stimulation. This effect was accompanied by qualitative alterations of the AP1 complex. Thus, in wild-type Jurkat cells, c-jun was absent from the complex, whereas in Tat-expressing cells, c-jun was increasingly recruited overtime. By contrast, similar amounts of c-rel and a small amount of NFAT1 were detected both in wild type and in Jurkat Tat+ cells. Furthermore, Tat not only induced the participation of c-jun in the cooperative complex but also a decrease in its transactivation activity alone or in combination with c-rel. Thus, the interaction of Tat with the components of this rel/AP1 cooperative complex seems to induce quantitative and qualitative alterations of this complex as activation progresses, resulting in a decrease of IL-2 gene transcription. Altogether our results suggest the existence of tuned mechanisms that allow the viral protein to specifically affect cooperative interactions between transcription factors.
Depletion of Alveolar Macrophages Exerts Protective Effects in Pulmonary Tuberculosis in Mice
Authors: Leemans Jaklien C. , Juffermans Nicole P. , Florquin Sandrine , van Rooijen Nico , Vervoordeldonk Margriet J. , Verbon Annelies , van Deventer Sander J. H. , van der Poll Tom
Published: April 2014

Mycobacterium tuberculosis bacilli are intracellular organisms that reside in phagosomes of alveolar macrophages (AMs). To determine the in vivo role of AM depletion in host defense against M. tuberculosis infection, mice with pulmonary tuberculosis induced by intranasal administration of virulent M. tuberculosis were treated intranasally with either liposome-encapsulated dichloromethylene diphosphonate (AM− mice), liposomes, or saline (AM+ mice). AM− mice were completely protected against lethality, which was associated with a reduced outgrowth of mycobacteria in lungs and liver, and a polarized production of type 1 cytokines in lung tissue, and by splenocytes stimulated ex vivo. AM− mice displayed deficient granuloma formation, but were more capable of attraction and activation of T cells into the lung and had increased numbers of pulmonary polymorphonuclear cells. These data demonstrate that depletion of AMs is protective during pulmonary tuberculosis.
Toll-Like Receptor 4 Mediates Intracellular Signaling Without TNF-α Release in Response to Cryptococcus neoformans Polysaccharide Capsule
Authors: Shoham Shmuel , Huang Chao , Chen Jian-Min , Golenbock Douglas T. , Levitz Stuart M.
Published: April 2014

Toll-like receptors (TLR) 2 and 4 are cell surface receptors that in association with CD14 enable phagocytic inflammatory responses to a variety of microbial products. Activation via these receptors triggers signaling cascades, resulting in nuclear translocation of NF-κB and a proinflammatory response including TNF-α production. We investigated whether TLRs participate in the host response to Cryptococcus neoformans glucuronoxylomannan (GXM), the major capsular polysaccharide of this fungus. Chinese hamster ovary fibroblasts transfected with human TLR2, TLR4, and/or CD14 bound fluorescently labeled GXM. The transfected Chinese hamster ovary cells were challenged with GXM, and activation of an NF-κB-dependent reporter construct was evaluated. Activation was observed in cells transfected with both CD14 and TLR4. GXM also stimulated nuclear NF-κB translocation in PBMC and RAW 264.7 cells. However, stimulation of these cells with GXM resulted in neither TNF-α secretion nor activation of the extracellular signal-regulated kinase 1/2, p38, and stress-activated protein kinase/c-Jun N-terminal kinase mitogen-activated protein kinase pathways. These findings suggest that TLRs, in conjunction with CD14, function as pattern recognition receptors for GXM. Furthermore, whereas GXM stimulates cells to translocate NF-κB to the nucleus, it does not induce activation of mitogen-activated protein kinase pathways or release of TNF-α. Taken together, these observations suggest a novel scenario whereby GXM stimulates cells via CD14 and TLR4, resulting in an incomplete activation of pathways necessary for TNF-α production.
Evidence for a Role for SAM68 in the Responses of Human Neutrophils to Ligation of CD32 and to Monosodium Urate Crystals
Authors: Gilbert Caroline , Barabé Frédéric , Rollet-Labelle Emmanuelle , Bourgoin Sylvain G. , McColl Shaun R. , Damaj Bassam B. , Naccache Paul H.
Published: April 2014

SAM68 (Src-associated in mitosis 68 kDa) is a member of the signal transduction of activator RNA novel gene family coding for proteins postulated to be involved in signal transduction and activation of RNA. It has been implicated through its phosphorylation status in the control of the transition from the G1 to the S phases during mitosis. However, the implication and role of SAM68 in nonproliferative cells are unknown. The present study was initiated to examine the role of SAM68 in the phagocytic responses of the terminally differentiated human neutrophils. The results obtained show that SAM68 is present in human neutrophils and that it is tyrosine phosphorylated in response to stimulation by monosodium urate crystals or by ligation of CD32. Stimulation of neutrophils by these agonists decreases the association of SAM68 with Sepharose-conjugated poly-U beads. Additionally, the amount of immunoprecipitable SAM68 was modulated differentially after stimulation by monosodium urate crystals or by CD32 engagement indicating that the posttranslational modifications and/or protein associations of SAM68 induced by these two agonists differed. The results of this study provide evidence for an involvement of SAM68 in signal transduction by phagocytic agonists in human neutrophils and indicate that SAM68 may play a role in linking the early events of signal transduction to the posttranscriptional modulation of RNA.
Two Proteins Modulating Transendothelial Migration of Leukocytes Recognize Novel Carboxylated Glycans on Endothelial Cells
Authors: Srikrishna Geetha , Panneerselvam Krishnasamy , Westphal Vibeke , Abraham Violet , Varki Ajit , Freeze Hudson H.
Published: April 2014

We recently showed that a class of novel carboxylated N-glycans was constitutively expressed on endothelial cells. Activated, but not resting, neutrophils expressed binding sites for the novel glycans. We also showed that a mAb against these novel glycans (mAbGB3.1) inhibited leukocyte extravasation in a murine model of peritoneal inflammation. To identify molecules that mediated these interactions, we isolated binding proteins from bovine lung by their differential affinity for carboxylated or neutralized glycans. Two leukocyte calcium-binding proteins that bound in a carboxylate-dependent manner were identified as S100A8 and annexin I. An intact N terminus of annexin I and heteromeric assembly of S100A8 with S100A9 (another member of the S100 family) appeared necessary for this interaction. A mAb to S100A9 blocked neutrophil binding to immobilized carboxylated glycans. Purified human S100A8/A9 complex and recombinant human annexin I showed carboxylate-dependent binding to immobilized bovine lung carboxylated glycans and recognized a subset of mannose-labeled endothelial glycoproteins immunoprecipitated by mAbGB3.1. Saturable binding of S100A8/A9 complex to endothelial cells was also blocked by mAbGB3.1. These results suggest that the carboxylated glycans play important roles in leukocyte trafficking by interacting with proteins known to modulate extravasation.
The Expression of Prostaglandin E Receptors EP2 and EP4 and Their Different Regulation by Lipopolysaccharide in C3H/HeN Peritoneal Macrophages
Authors: Ikegami Reiko , Sugimoto Yukihiko , Segi Eri , Katsuyama Masato , Karahashi Hisae , Amano Fumio , Maruyama Takayuki , Yamane Hana , Tsuchiya Soken , Ichikawa Atsushi
Published: April 2014

The expression and regulation of the PGE receptors, EP2 and EP4, both of which are coupled to the stimulation of adenylate cyclase, were examined in peritoneal resident macrophages from C3H/HeN mice. mRNA expression of EP4 but not EP2 was found in nonstimulated cells, but the latter was induced by medium change alone, and this induction was augmented by LPS. mRNA expression of EP4 was down-regulated by LPS but not by medium change. PGE2 increased the cAMP content of both LPS-treated and nontreated cells. ONO-604, an EP4 agonist, also increased cAMP content in nonstimulated cells and in cells treated with LPS for 3 h, but not for 6 h. Butaprost, an EP2 agonist, was effective only in the cells treated with LPS for 6 h. The inhibitory effects of ONO-604 on TNF-α and IL-12 production were equipotent with PGE2 at any time point, but the inhibitory effects of butaprost were only seen from 14 h after stimulation. PGE2 or dibutyryl cAMP alone, but not butaprost, reduced EP4 expression, and indomethacin reversed the LPS-induced down-regulation of EP4, indicating that the down-regulation of EP4 is mediated by LPS-induced PG synthesis and EP4 activation. Indeed, when we used C3H/HeJ (LPS-hyporesponsive) macrophages, such reduction in EP4 expression was found in the cells treated with PGE2 alone, but not in LPS-treated cells. In contrast, up-regulation of EP2 expression was again observed in LPS-treated C3H/HeJ macrophages. These results suggest that EP4 is involved mainly in the inhibition of cytokine release, and that the gene expression of EP2 and EP4 is differentially regulated during macrophage activation.
Expression and Characterization of the Chemokine Receptors CCR2 and CCR5 in Mice
Authors: Mack Matthias , Cihak Josef , Simonis Christopher , Luckow Bruno , Proudfoot Amanda E. I. , Plachý Jir̆í , Brühl Hilke , Frink Michael , Anders Hans-Joachim , Vielhauer Volker , Pfirstinger Jochen , Stangassinger Manfred , Schlöndorff Detlef
Published: April 2014

The chemokine receptors CCR2 and CCR5 play important roles in the recruitment of monocytes/macrophages and T cells. To better understand the role of both receptors in murine models of inflammatory diseases and to recognize potential problems when correlating these data to humans, we have generated mAbs against murine CCR2 and CCR5. In mice CCR2 is homogeneously expressed on monocytes and on 2–15% of T cells, closely resembling the expression pattern in humans. In contrast to humans, murine NK cells are highly CCR5 positive. In addition, CCR5 is expressed on 3–10% of CD4 and 10–40% of CD8-positive T cells and is weakly detectable on monocytes. Using a model of immune complex nephritis, we examined the effects of inflammation on chemokine receptor expression and found a 10-fold enrichment of CCR5+ and CCR2+ T cells in the inflamed kidneys. The activity of various chemokines and the antagonistic properties of the mAbs were measured by ligand-induced internalization of CCR2 and CCR5 on primary leukocytes. The Ab MC-21 (anti-CCR2) reduced the activity of murine monocyte chemotactic protein 1 by 95%, whereas the Ab MC-68 (anti-CCR5) blocked over 99% of the macrophage-inflammatory protein 1α and RANTES activity. MC-21 and MC-68 efficiently blocked the ligand binding to CCR2 and CCR5 with an IC50 of 0.09 and 0.6–1.0 μg/ml, respectively. In good correlation to these in vitro data, MC-21 almost completely prevented the influx of monocytes in thioglycollate-induced peritonitis. Therefore, both Abs appear as useful reagents to further study the role of CCR2 and CCR5 in murine disease models.
T Cells of Multiple Sclerosis Patients Target a Common Environmental Peptide that Causes Encephalitis in Mice
Authors: Winer Shawn , Astsaturov Igor , Cheung Roy K. , Schrade Katrin , Gunaratnam Lakshman , Wood Denise D. , Moscarello Mario A. , O’Connor Paul , McKerlie Colin , Becker Dorothy J. , Dosch Hans-Michael
Published: April 2014

Multiple sclerosis (MS) is a chronic autoimmune disease triggered by unknown environmental factors in genetically susceptible hosts. MS risk was linked to high rates of cow milk protein (CMP) consumption, reminiscent of a similar association in autoimmune diabetes. A recent rodent study showed that immune responses to the CMP, butyrophilin, can lead to encephalitis through antigenic mimicry with myelin oligodendrocyte glycoprotein. In this study, we show abnormal T cell immunity to several other CMPs in MS patients comparable to that in diabetics. Limited epitope mapping with the milk protein BSA identified one specific epitope, BSA193, which was targeted by most MS but not diabetes patients. BSA193 was encephalitogenic in SJL/J mice subjected to a standard protocol for the induction of experimental autoimmune encephalitis. These data extend the possible, immunological basis for the association of MS risk, CMP, and CNS autoimmunity. To pinpoint the same peptide, BSA193, in encephalitis-prone humans and rodents may imply a common endogenous ligand, targeted through antigenic mimicry.
Cutting Edge: Eotaxin Elicits Rapid Vesicular Transport-Mediated Release of Preformed IL-4 from Human Eosinophils
Authors: Bandeira-Melo Christianne , Sugiyama Kumiya , Woods Lesley J. , Weller Peter F.
Published: April 2014

IL-4 release is important in promoting Th2-mediated allergic and parasitic immune responses. Although human eosinophils are potential sources of IL-4, physiologic mechanisms to elicit its release have not been established. By flow cytometry and microscopy, eosinophils from normal donors uniformly contained preformed IL-4. In contrast to cytolytic IL-4 release from calcium ionophore-activated eosinophils, eotaxin and RANTES, but not IFN-γ, elicited IL-4 release by noncytotoxic mechanisms. With a dual Ab capture and detection immunofluorescent microscopic assay, IL-4 was released at discrete cell surface sites. IL-5 enhanced eotaxin-induced IL-4 release, which was mediated by G protein-coupled CCR3 receptors, detectable as early as 5 min and maximum within 1 h. IL-4 release was not diminished by transcription or protein synthesis inhibitors, but was suppressed by brefeldin A, an inhibitor of vesicle formation. Thus, CCR3-mediated signaling can rapidly mobilize IL-4 stored preformed in human eosinophils for release by vesicular transport to contribute to immune responses.
Cutting Edge: The Wiskott-Aldrich Syndrome Protein Is Required for Efficient Phagocytosis of Apoptotic Cells
Authors: Leverrier Yann , Lorenzi Roberto , Blundell Michael P. , Brickell Paul , Kinnon Christine , Ridley Anne J. , Thrasher Adrian J.
Published: April 2014

Phagocytosis of apoptotic cells by macrophages and dendritic cells is necessary for clearance of proinflammatory debris and for presentation of viral, tumor, and self Ags. While a number of receptors involved in the cognate recognition of apoptotic cells by phagocytes have been identified, the signaling events that result in internalization remain poorly understood. Here we demonstrate that clearance of apoptotic cells is accompanied by recruitment of the Wiskott-Aldrich syndrome (WAS) protein to the phagocytic cup and that it’s absence results in delayed phagocytosis both in vitro and in vivo. Therefore, we propose that WAS protein plays an important and nonredundant role in the safe removal of apoptotic cells and that deficiency contributes significantly to the immune dysregulation of WAS. The efficiency of apoptotic cell clearance may be a key determinant in the suppression of tissue inflammation and prevention of autoimmunity.
Isotype-Dependent Inhibition of Tumor Growth In Vivo by Monoclonal Antibodies to Death Receptor 4
Authors: Chuntharapai Anan , Dodge Kelly , Grimmer Katharine , Schroeder Kurt , Marsters Scot A. , Koeppen Hartmut , Ashkenazi Avi , Kim K. Jin
Published: April 2014

To explore an approach for death receptor targeting in cancer, we developed murine mAbs to human death receptor 4 (DR4). The mAb 4H6 (IgG1) competed with Apo2L/TNF-related apoptosis-inducing ligand (DR4’s ligand) for binding to DR4, whereas mAb 4G7 (IgG2a) did not. In vitro, both mAbs showed minimal intrinsic apoptosis-inducing activity, but each triggered potent apoptosis upon cross-linking. In a colon tumor nude mouse model in vivo, mAb 4H6 treatment without addition of exogenous linkers induced apoptosis in tumor cells and caused complete tumor regression, whereas mAb 4G7 partially inhibited tumor growth. An IgG2a isotype switch variant of mAb 4H6 was much less effective in vivo than the parent IgG1-4H6, despite similar binding affinities to DR4. The same conclusion was obtained by comparing other IgG1 and IgG2 mAbs to DR4 for their anti-tumor activities in vivo. Thus, the isotype of anti-DR4 mAb may be more important than DR4 binding affinity for tumor elimination in vivo. Anti-DR4 mAbs of the IgG1 isotype may provide a useful tool for investigating the therapeutic potential of death receptor targeting in cancer.
NF-κB RelA (p65) Is Essential for TNF-α-Induced Fas Expression but Dispensable for Both TCR-Induced Expression and Activation-Induced Cell Death
Authors: Zheng Ye , Ouaaz Fateh , Bruzzo Peter , Singh Veena , Gerondakis Steve , Beg Amer A.
Published: April 2014

The Fas death receptor plays a key role in the killing of target cells by NK cells and CTLs and in activation-induced cell death of mature T lymphocytes. These cytotoxic pathways are dependent on induction of Fas expression by cytokines such as TNF-α and IFN-γ or by signals generated after TCR engagement. Although much of our knowledge of the Fas death pathway has been generated from murine studies, little is known about regulatory mechanisms important for murine Fas expression. To this end, we have molecularly cloned a region of the murine Fas promoter that is responsible for mediating TNF-α and PMA/PHA-induced expression. We demonstrate here that induction of Fas expression by both stimuli is critically dependent on two sites that associate with RelA-containing NF-κB complexes. To determine whether RelA and/or other NF-κB subunits are also important for regulating Fas expression in primary T cells, we used CD4 T cells from RelA−/−, c-Rel−/−, and p50−/− mice. Although proliferative responses were significantly impaired, expression of Fas and activation-induced cell death was unaffected in T cells obtained from these different mice. Importantly, we show that unlike fibroblasts, which consist primarily of RelA-containing NF-κB complexes, T cells have high levels of both RelA and c-Rel complexes, suggesting that Fas expression in T cells may be dependent on redundant functions of these NF-κB subunits.
Bacterial CpG-DNA Triggers Activation and Maturation of Human CD11c−, CD123+ Dendritic Cells
Authors: Bauer Marc , Redecke Vanessa , Ellwart Joachim W. , Scherer Barbara , Kremer Jean-Pierre , Wagner Hermann , Lipford Grayson B.
Published: April 2014

Human plasmacytoid precursor dendritic cells (ppDC) are a major source of type I IFN upon exposure to virus and bacteria, yet the stimulus causing their maturation into DCs is unknown. After PBMC activation with immunostimulatory bacterial DNA sequences (CpG-DNA) we found that ppDC are the primary source of IFN-α. In fact, either CpG-DNA or dsRNA (poly(I:C)) induced IFN-α from purified ppDC. Surprisingly, only CpG-DNA triggered purified ppDC survival, maturation, and production of TNF, GM-CSF, IL-6, and IL-8, but not IL-10 or IL-12. Known DC activators such as CD40 ligation triggered ppDC maturation, but only IL-8 production, while bacterial LPS was negative for all activation criteria. An additional finding was that only CpG-DNA could counteract IL-4-induced apoptosis in ppDC. Therefore, CpG-DNA represents a pathogen-associated molecular pattern for ppDC. In contrast to these finding, CpG-DNA, like LPS, caused TNF, IL-6, and IL-12 release from PBMC and purified monocytes; however, differentiation of monocytes into DCs with GM-CSF and IL-4 unexpectedly resulted in refractoriness to CpG-DNA, but not LPS. Taken together, these results suggest that within a DC subset a multiplicity of responses can be generated by distinct environmental stimuli and that responses to a given stimulus may be dissimilar between DC subsets.
CXCR-4 Desensitization Is Associated with Tissue Localization of Hemopoietic Progenitor Cells
Authors: Shen Hongmei , Cheng Tao , Olszak Ivona , Garcia-Zepeda Eduardo , Lu Zhijian , Herrmann Steven , Fallon Robert , Luster Andrew D. , Scadden David T.
Published: April 2014

The chemokine stroma-derived factor (SDF)-1, and its receptor, CXCR-4, have been shown to be essential for the translocation of hemopoietic stem cells from the fetal liver to the bone marrow (BM). We hypothesized that if CXCR-4 plays a crucial role in the localization of human hemopoiesis, stem cells from distinct tissue sources should demonstrate distinct CXCR-4 expression or signaling profiles. CD34+ cells from BM were compared with blood: either mobilized peripheral blood or umbilical cord blood. Unexpectedly, significantly higher levels of CXCR-4 surface expression on CD34+ cells from blood sources, mobilized peripheral blood, or cord blood were observed compared with BM (p = 0.0005 and p = 0.002, respectively). However, despite lower levels of CXCR-4, responsiveness of the cells to SDF-1 as measured by either calcium flux or transmigration was proportionally greatest in cells derived from BM. Further, internalization of CXCR-4 in response to ligand, associated with receptor desensitization, was significantly lower on BM-derived cells. Therefore, preserved chemokine receptor signaling was highly associated with marrow rather than blood localization. To test the functional effects of perturbing CXCR-4 signaling, adult mice were exposed to the methionine-SDF-1β analog that induces prolonged down-regulation/desensitization of CXCR-4 and observed mobilization of Lin−, Sca-1+, Thy-1low, and c-kit+ hemopoietic progenitor cells to the peripheral blood with a >30-fold increase compared with PBS control (p = 0.0007 day 1 and p = 0.004 day 2). These data demonstrate that CXCR-4 expression and function can be dissociated in progenitor cells and that desensitization of CXCR-4 induces stem cell entry into the circulation.
Expression Cloning of the STRL33/BONZO/TYMSTR Ligand Reveals Elements of CC, CXC, and CX3C Chemokines
Authors: Wilbanks Alyson , Zondlo Susan Carr , Murphy Kristine , Mak Simona , Soler Dulce , Langdon Patricia , Andrew David P. , Wu Lijun , Briskin Michael
Published: April 2014

STRL33/BONZO/TYMSTR is an orphan chemokine and HIV/SIV coreceptor receptor that is expressed on activated T lymphocytes. We describe an expression cloning strategy whereby we isolated a novel chemokine, which we name CXCL16. CXCL16 is an α (CXC) chemokine but also has characteristics of CC chemokines and a structure similar to fractalkine (neurotactin) in having a transmembrane region and a chemokine domain suspended by a mucin-like stalk. A recombinant version of CXCL16 fails to mediate chemotaxis to all known chemokine receptor transfectants tested but does mediate robust chemotaxis, high affinity binding, and calcium mobilization to Bonzo receptor transfectants, indicating that this is a unique receptor ligand interaction. In vitro polarized T cell subsets including Th1, Th2, and Tr1 cells express functional Bonzo, suggesting expression of this receptor in chronic inflammation, which we further verified by demonstration of CXCL16-mediated migration of tonsil-derived CD4+ T lymphocytes. CXCL16 is expressed on the surface of APCs including subsets of CD19+ B cells and CD14+ monocyte/macrophages, and functional CXCL16 is also shed from macrophages. The combination of unique structural features of both Bonzo and CXCL16 suggest that this interaction may represent a new class of ligands for this receptor family. Additionally, this chemokine might play a unique dual role of attracting activated lymphocyte subsets during inflammation as well as facilitating immune responses via cell-cell contact.
Induction of Cross-Tolerance by Lipopolysaccharide and Highly Purified Lipoteichoic Acid Via Different Toll-Like Receptors Independent of Paracrine Mediators
Authors: Lehner Martin D. , Morath Siegfried , Michelsen Kathrin S. , Schumann Ralf R. , Hartung Thomas
Published: April 2014

Exposure of macrophages to LPS induces a state of hyporesponsiveness to subsequent stimulation with LPS termed LPS desensitization or tolerance. To date, it is not known whether similar mechanisms of macrophage refractoriness are induced on contact with components of Gram-positive bacteria. In the present study, we demonstrate that pretreatment with highly purified lipoteichoic acid (LTA) results in suppression of cytokine release on restimulation with LTA in vitro and in vivo in both C3H/HeN and C3H/HeJ mice, but not in macrophages from Toll-like receptor (TLR)-2-deficient mice. Furthermore, desensitization in response to LPS or LTA exposure also inhibits responses to the other stimulus (“cross-tolerance”), suggesting that signaling pathways shared by TLR2 and TLR4 are impaired during tolerance. Finally, we show that LPS- or LTA-induced cross-tolerance is not transferred to hyporesponsive cells cocultured with LPS/LTA-responsive macrophages, showing that soluble mediators do not suffice for tolerance induction in neighboring cells.
Therapeutic Effect of IL-13 Immunoneutralization During Chronic Experimental Fungal Asthma
Authors: Blease Kate , Jakubzick Claudia , Westwick John , Lukacs Nicholas , Kunkel Steven L. , Hogaboam Cory M.
Published: April 2014

IL-13 and IL-4 are key contributors to the asthmatic phenotype. The temporal role of these cytokines in airway function, inflammation, and remodeling were assessed in a chronic murine model of Asperigillus fumigatus-induced allergic asthma. IL-13 and IL-4 protein levels were significantly elevated by 30 days after conidia challenge in A. fumigatus-sensitized mice. Furthermore, IL-13Rα1 mRNA expression was significantly elevated 7 days after conidia challenge and remained elevated until day 21. In contrast, IL-13Rα2 mRNA expression, although constitutively expressed in naive lung, was absent in the lungs of A. fumigatus-sensitized mice both before and after conidia challenge. Membrane-bound IL-4R mRNA expression was significantly elevated 7 days after conidia challenge; however, soluble IL-4R mRNA expression was increased 30 days after conidia challenge. Immunoneutralization of IL-13 between days 14 and 30 or days 30 and 38 after fungal sensitization and challenge significantly attenuated airway hyperresponsiveness, collagen deposition, and goblet cell hyperplasia at day 38 after conidia challenge; however, the effects of IL-4 immunoneutralization during the same time periods were not as marked. IFN-γ and IL-12 release after Aspergillus Ag restimulation was elevated from spleen cells isolated from mice treated with IL-4 anti-serum compared with IL-13 anti-serum or normal rabbit serum-treated mice. This study demonstrates a pronounced therapeutic effect of IL-13-immunoneutralization at extended time points following the induction of chronic asthma. Most importantly, these therapeutic effects were not reversed following cessation of treatment, and IL-13 anti-serum treatment did not alter the systemic immune response to Ag restimulation, unlike IL-4 immunoneutralization. Therefore, IL-13 provides an attractive therapeutic target in allergic asthma.
Posttransplant Administration of Donor Leukocytes Induces Long-Term Acceptance of Kidney or Liver Transplants by an Activation-Associated Immune Mechanism
Authors: Yan Yiqun , Shastry Suma , Richards Craig , Wang Chuanmin , Bowen David G. , Sharland Alexandra F. , Painter Dorothy M. , McCaughan Geoffrey W. , Bishop G. Alex
Published: April 2014

Donor leukocytes play a dual role in rejection and acceptance of transplanted organs. They provide the major stimulus for rejection, and their removal from the transplanted organ prolongs its survival. Paradoxically, administration of donor leukocytes also prolongs allograft survival provided that they are administered 1 wk or more before transplantation. Here we show that administration of donor leukocytes immediately after transplantation induced long-term acceptance of completely MHC-mismatched rat kidney or liver transplants. The majority of long-term recipients of kidney transplants were tolerant of donor-strain skin grafts. Acceptance was associated with early activation of recipient T cells in the spleen, demonstrated by a rapid increase in IL-2 and IFN-γ at that site followed by an early diffuse infiltrate of activated T cells and apoptosis within the tolerant grafts. In contrast, IL-2 and IFN-γ mRNA were not increased in the spleens of rejecting animals, and the diffuse infiltrate of activated T cells appeared later but resulted in rapid graft destruction. These results define a mechanism of allograft acceptance induced by donor leukocytes that is associated with activation-induced cell death of recipient T cells. They demonstrate for the first time that posttransplant administration of donor leukocytes leads to organ allograft tolerance across a complete MHC class I plus class II barrier, a finding with direct clinical application.
Intermolecular Antigen Spreading Occurs During the Preclinical Period of Human Type 1 Diabetes
Authors: Brooks-Worrell Barbara , Gersuk Vivian H. , Greenbaum Carla , Palmer Jerry P.
Published: April 2014

Intra- and intermolecular spreading of T cell responses to autoantigens has been implicated in the pathogenesis of autoimmune diseases. Therefore, we questioned whether T cell responses from subjects identified as at-risk (positive for autoantibody reactivity to islet proteins) for the development of type 1 diabetes, a cell-mediated autoimmune disease, would demonstrate intermolecular Ag spreading of T cell responses to islet cell proteins. Previously, we have demonstrated that by the time subjects develop type 1 diabetes, they have T cell responses to numerous islet proteins, whereas T cells from normal controls respond to a limited number of islet proteins. Initial testing of PBMC responses from 25 nondiabetic at-risk subjects demonstrated that 16 of the 25 subjects have PBMC responses to islet proteins similar to controls. Fourteen of these 16 subjects were available for follow-up. Eleven of the 14 developed T cell responses to increasing numbers of islet proteins, and 6 of these subjects developed type 1 diabetes. In the nine subjects who already demonstrated T cell Ag spreading at the initial visit, four were available for follow-up. Of these four, two had increases in T cell reactivity to islet proteins, while two maintained their initial levels of T cell reactivity. We also observed Ag spreading in autoantibody reactivity to islet proteins in nine of the 18 at-risk subjects available for follow-up. Our data strongly support the conclusion that intermolecular spreading of T cell and Ab responses to islet proteins occurs during the preclinical period of type 1 diabetes.
Anti-Sm B Cell Differentiation in Ig Transgenic MRL/Mp-lpr/lprMice: Altered Differentiation and an Accelerated Response
Authors: Santulli-Marotto Sandra , Qian Ye , Ferguson Stacy , Clarke Stephen H.
Published: April 2014

To determine the regulation of B cells specific for the ribonucleoprotein Sm, a target of the immune system in human and mouse lupus, we have generated mice carrying an anti-Sm H chain transgene (2-12H). Anti-Sm B cells in nonautoimmune 2-12H-transgenic (Tg) mice are functional, but, in the absence of immunization, circulating anti-Sm Ab levels are not different from those of non-Tg mice. In this report, we compare the regulation of anti-Sm B cells in nonautoimmune and autoimmune MRL/Mp-lpr/lpr (MRL/lpr) and bcl-2-22-Tg mice. Activation markers are elevated on splenic and peritoneal anti-Sm B cells of both nonautoimmune and autoimmune genetic backgrounds indicating Ag encounter. Although tolerance to Sm is maintained in 2-12H/bcl-2-22-Tg mice, it is lost in 2-12H-Tg MRL/lpr mice, as the transgene accelerates and increases the prevalence of the anti-Sm response. The 2-12H-Tg MRL/lpr mice have transitional anti-Sm B cells in the spleen similar to nonautoimmune mice. However, in contrast to nonautoimmune mice, there are few if any peritoneal anti-Sm B-1 cells. These data suggest that a defect in B-1 differentiation may be a factor in the loss of tolerance to Sm and provide insight into the low prevalence of the anti-Sm response in lupus.
Cutting Edge: The Common γ-Chain Is an Indispensable Subunit of the IL-21 Receptor Complex
Authors: Asao Hironobu , Okuyama Chikara , Kumaki Satoru , Ishii Naoto , Tsuchiya Shigeru , Foster Don , Sugamura Kazuo
Published: April 2014

The common γ-chain (γc) is an indispensable subunit of the functional receptor complexes for IL-4, IL-7, IL-9, and IL-15 as well as IL-2. Here we show that the γc is also shared with the IL-21R complex. Although IL-21 binds to the IL-21R expressed on γc-deficient ED40515− cells, IL-21 is unable to transduce any intracytoplasmic signals. However, in EDγ-16 cells, a γc-transfected ED40515− cell line, IL-21 binds to the IL-21R and can activate Janus kinase (JAK)1, JAK3, STAT1, and STAT3. The chemical cross-linking study reveals the direct binding of IL-21 to the γc. These data clearly demonstrate that the γc is an indispensable subunit of the functional IL-21R complex.
Signal Thresholds and Modular Synergy During Expression of Costimulatory Molecules in B Lymphocytes
Authors: Natarajan Krishnamurthy , Sahoo Naresh C. , Rao Kanury V. S.
Published: April 2014

We analyzed intracellular pathways modulating surface densities of CD80 and CD86 in B cells activated through ligation of the Ag receptor, and the adhesion molecule CD54. Whereas B cell Ag receptor (BCR) cross-linking alone stimulated increased expression of CD86, up-regulation of CD80 required dual stimulation with anti-IgM and anti-CD54. The principal downstream component contributed by BCR signaling, toward both CD80 and CD86 induction, was the elevated concentration of free cytoplasmic Ca2+, recruited by way of capacitative influx. This alone was sufficient to generate an increase in CD86 levels. However, CD80 enhancement required the concerted action of both intracellular Ca2+ concentration and CD54-initiated pathways. The nexus between anti-IgM and anti-CD54 stimulation, in the context of CD80 regulation, was identified to involve a self-propagating process of sequential synergy. The first step involved amplified accumulation of intracellular cAMP, as a result of cross-talk between BCR-mobilized Ca2+ and CD54-derived signals. This then facilitated a second synergistic interaction between Ca2+ and cAMP, culminating in CD80 expression. Our findings of distinct signal transducer requirements, with the added consequences of cross-talk, offers an explanation for variable modulation of costimulatory molecule expression in response to diverse physiological stimuli. Importantly, these results also reveal how concentration threshold barriers for recruitment of individual second messengers can be overcome by constructive convergence of signaling modules.
IL-12 Augments CD8+ T Cell Development for Contact Hypersensitivity Responses and Circumvents Anti-CD154 Antibody-Mediated Inhibition
Authors: Gorbachev Anton V. , DiIulio Nancy A. , Fairchild Robert L.
Published: April 2014

During sensitization with dinitrofluorobenzene for contact hypersensitivity (CHS) responses, hapten-specific CD8+ T cells develop into IFN-γ-producing cells, and CD4+ T cells develop into IL-4/IL-5-producing cells. Administration of IL-12 during sensitization skews CD4+ T cell development to IFN-γ-producing cells, resulting in exaggerated CHS responses. In the current report we tested the role of IL-12 on CD8+ T cell development during sensitization and elicitation of CHS to dinitrofluorobenzene. Administration of IL-12 during hapten sensitization induced the expression of IL-12Rβ2 on both CD4+ and CD8+ T cells, augmented IFN-γ production by these T cell populations, and increased the magnitude and duration of the CHS response to hapten challenge. CHS responses were virtually identical in wild-type and IL-12 p40−/− mice. Since engagement of CD40 on APC may stimulate IL-12 production, we also tested the role of CD40-CD154 interactions on the development of IFN-γ-producing CD4+ and CD8+ T cells following hapten sensitization. Development of IFN-γ-producing CD4+ T cells during hapten sensitization was absent in wild-type mice treated with anti-CD154 mAb or in CD154−/− mice. In contrast, the absence of CD40-CD154 signaling had little or no impact on the development of IFN-γ-producing CD8+ T cells. These results demonstrate that the development of hapten-specific Th1 effector CD4+ T cells in CHS requires both CD40-CD154 interactions and IL-12, whereas the development of IFN-γ-producing effector CD8+ T cells can occur independently of these pathways.
The Role of the CD19/CD21 Complex in B Cell Processing and Presentation of Complement-Tagged Antigens
Authors: Cherukuri Anu , Cheng Paul C. , Pierce Susan K.
Published: April 2014

The CD19/CD21 complex is an essential B cell coreceptor that functions synergistically to enhance signaling through the B cell Ag receptor in response to T cell-dependent, complement-tagged Ags. In this study, we use a recombinant protein containing three tandemly arranged copies of C3d and the Ag hen egg lysozyme, shown to be a highly effective immunogen in vivo, to evaluate the role of the CD19/CD21 complex in Ag processing in B cells. Evidence is provided that coengagement of the CD19/CD21 complex results in more rapid and efficient production of antigenic peptide/class II complexes as compared with B cell Ag receptor-mediated processing alone. The CD19/CD21 complex does not itself target complement-tagged Ags for processing, but rather appears to influence B cell Ag processing through its signaling function. The ability of the CD19/CD21 complex to augment processing may be an important element of the mechanism by which the CD19/CD21 complex functions to promote B cell responses to T cell-dependent complement-tagged Ags in vivo.