bluuebunny/crossref_metadata_2025 huggingface dataset:
Antibodies to C-C Chemokine Receptor 5 in Normal Human IgG Block Infection of Macrophages and Lymphocytes with Primary R5-Tropic Strains of HIV-1
Authors: Bouhlal Hicham , Hocini Hakim , Quillent-Grégoire Caroline , Donkova Vladimira , Rose Stephanie , Amara Ali , Longhi Renato , Haeffner-Cavaillon Nicole , Beretta Alberto , Kaveri Srini V. , Kazatchkine Michel D.
Published: April 2014

In the present study, we demonstrate that normal human IgG for therapeutic use (i.v. Ig) contains natural Abs directed against the CCR5 coreceptor for HIV-1. Abs to CCR5 were isolated from i.v. Ig using an affinity matrix consisting of a synthetic peptide corresponding to the N-terminus of CCR5 coupled to Sepharose. Natural anti-CCR5 Abs inhibited the binding of RANTES to macrophages, demonstrating their interaction with the coreceptor of R5-tropic HIV-1. Affinity-purified anti-CCR5 Ig further inhibited infection of lymphocytes and monocytes/macrophages with primary and laboratory-adapted strains of HIV-1, but did not inhibit infection with X4-tropic HIV. Our results suggest that anti-CCR5 Abs from healthy immunocompetent donors may be suitable for development of novel passive immunotherapy regimens in specific clinical settings in HIV infection.
Molecular Basis of a Selective C1s Deficiency Associated with Early Onset Multiple Autoimmune Diseases
Authors: Dragon-Durey Marie-Agnès , Quartier Pierre , Frémeaux-Bacchi Véronique , Blouin Jacques , de Barace Claire , Prieur Anne-Marie , Weiss Laurence , Fridman Wolf-Herman
Published: April 2014

We have investigated the molecular basis of selective and complete C1s deficiency in 2-year-old girl with complex autoimmune diseases including lupus-like syndrome, Hashimoto’s thyroiditis, and autoimmune hepatitis. This patient’s complement profile was characterized by the absence of CH50 activity, C1 functional activity <10%, and undetectable levels of C1s Ag associated with normal levels of C1r and C1q Ags. Exon-specific amplification of genomic DNA by PCR followed by direct sequence analysis revealed a homozygous nonsense mutation in the C1s gene exon XII at codon 534, caused by a nucleotide substitution from C (CGA for arginine) to T (TGA for stop codon). Both parents were heterozygous for this mutation. We used the new restriction site for endonuclease Fok-1 created by the mutation to detect this mutation in the genomic DNA of seven healthy family members. Four additional heterozygotes for the mutation were identified in two generations. Our data characterize for the first time the genetic defect of a selective and complete C1s deficiency in a Caucasian patient.
CXCL10 (IFN-γ-Inducible Protein-10) Control of Encephalitogenic CD4+ T Cell Accumulation in the Central Nervous System During Experimental Autoimmune Encephalomyelitis
Authors: Fife Brian T. , Kennedy Kevin J. , Paniagua Mary C. , Lukacs Nicholas W. , Kunkel Steven L. , Luster Andrew D. , Karpus William J.
Published: April 2014

Experimental autoimmune encephalomyelitis (EAE) is a CD4+ Th1-mediated demyelinating disease of the CNS that serves as a model for multiple sclerosis. A critical event in the pathogenesis of EAE is the entry of both Ag-specific and Ag-nonspecific T lymphocytes into the CNS. In the present report, we investigated the role of the CXC chemokine CXCL10 (IFN-γ-inducible protein-10) in the pathogenesis of EAE. Production of CXCL10 in the CNS correlated with the development of clinical disease. Administration of anti-CXCL10 decreased clinical and histological disease incidence, severity, as well as infiltration of mononuclear cells into the CNS. Anti-CXCL10 specifically decreased the accumulation of encephalitogenic PLP139–151 Ag-specific CD4+ T cells in the CNS compared with control-treated animals. Anti-CXCL10 administration did not affect the activation of encephalitogenic T cells as measured by Ag-specific proliferation and the ability to adoptively transfer EAE. These results demonstrate an important role for the CXC chemokine CXCL10 in the recruitment and accumulation of inflammatory mononuclear cells during the pathogenesis of EAE.
The Prevention and Treatment of Murine Colitis Using Gene Therapy with Adenoviral Vectors Encoding IL-10
Authors: Lindsay James O. , Ciesielski Cathleen J. , Scheinin Tom , Hodgson Humphrey J. , Brennan Fionula M.
Published: April 2014

IL-10-deficient (IL-10−/−) mice develop colitis with many similarities to Crohn’s disease. Daily IL-10 injections have a short systemic half-life and are unable to induce complete remission in IL-10−/− mice with established disease. In this paper, we investigate the duration, potency, and immunogenicity of gene therapy using an adenoviral vector encoding murine IL-10 (AdvmuIL-10). A single systemic injection of AdvmuIL-10 was sufficient not only to prevent the onset of colitis for at least 10 wk but also to induce clinical and histological remission in mice with established disease. In addition, AdvmuIL-10 diminished the systemic manifestations of disease, including elevated acute-phase proteins, as well as the local consequences of inflammation such as raised stool IL-1β concentrations. Both IL-10 protein and the effects of secreted IL-10 were detectable for 10 wk after AdvmuIL-10 injection. Furthermore, the immunoregulatory effect of a single AdvmuIL-10 injection was manifest both by a reduction in TNF-α, IFN-γ, and RANTES release from stimulated splenocyte cultures, and also by a change in the proportion of CD45RBhigh/low lymphocytes in the spleen compared with control mice. The delivery of AdvmuIL-10 resulted in a significantly diminished host antiadenoviral response compared with control adenoviral vectors. Thus, gene therapy strategies using adenoviral vectors encoding immunoregulatory and antiinflammatory cytokines may prove to be a potent approach for the treatment of chronic inflammatory disease. Antiinflammatory cytokine expression protects against immune responses directed at gene vectors.
Ex Vivo IFN-γ Secretion by Circulating CD8 T Lymphocytes: Implications of a Novel Approach for T Cell Monitoring in Infectious and Malignant Diseases
Authors: Pittet Mikaël J. , Zippelius Alfred , Speiser Daniel E. , Assenmacher Mario , Guillaume Philippe , Valmori Danila , Liénard Danielle , Lejeune Ferdy , Cerottini Jean-Charles , Romero Pedro
Published: April 2014

To elucidate the functional heterogeneity of Ag-specific T lymphocyte populations, we combined labeling of lymphocytes with MHC/peptide tetramers and a cell surface affinity matrix for IFN-γ. Magnetic cell sorting of IFN-γ-positive lymphocytes allowed the selective enrichment and identification of live Ag-specific cytokine-secreting cells by flow cytometry. Naive, memory, and effector Ag-specific populations were evaluated in healthy HLA-A2 individuals. Significant fractions of influenza- and CMV-specific cells secreted IFN-γ upon challenge with cognate peptide, consistent with an effector/memory status. The sensitivity of the approach allowed the detection of significant numbers of CMV-specific IFN-γ-secreting cells ex vivo (i.e., without Ag stimulation). This was not apparent when using previously described assays, namely, ELISPOT or intracellular IFN-γ staining (cytospot). CD8+ T cells specific for the melamoma-associated Ag Melan-A/MART-1 did not produce IFN-γ upon challenge with cognate peptide, reminiscent with their naive functional state in healthy individuals. In contrast, CD45RAlow Melan-A/MART-1 tumor-specific cells from three of three melanoma patients presented levels of activity similar to those found for influenza- or CMV virus-specific lymphocytes, compatible with a functional differentiation into competent effector/memory T lymphocytes in vivo. Notably, a sizable fraction of Melan-A/MART-1-specific cells from a patient secreted IFN-γ ex vivo following peptide-based vaccination. Thus, the high sensitivity of the assay provides a valuable tool to monitor effector T cell responses in different clinical situations.
Cutting Edge: CD43 Functions as a T Cell Counterreceptor for the Macrophage Adhesion Receptor Sialoadhesin (Siglec-1)
Authors: van den Berg Timo K. , Nath Deepa , Ziltener Hermann J. , Vestweber Dietmar , Fukuda Minoru , van Die Irma , Crocker Paul R.
Published: April 2014

Sialoadhesin (Siglec-1) is a macrophage-restricted sialic acid-binding receptor that mediates interactions with hemopoietic cells, including lymphocytes. In this study, we identify sialoadhesin counterreceptors on T lymphocytes. Several major glycoproteins (85, 130, 240 kDa) were precipitated by sialoadhesin-Fc fusion proteins from a murine T cell line (TK-1). Binding of sialoadhesin to these glycoproteins was sialic acid dependent and was abolished by mutation of a critical residue (R97A) of the sialic acid binding site in the membrane distal Ig-like domain of sialoadhesin. The 130- and 240-kDa sialoadhesin-binding glycoproteins were identified as the sialomucins CD43 and P-selectin glycoprotein ligand 1 (CD162), respectively. CD43 expressed in COS cells supported increased binding to immobilized sialoadhesin. Finally, sialoadhesin bound different glycoforms of CD43 expressed in Chinese hamster ovary cells, including unbranched (core 1) and branched (core 2) O-linked glycans, that are normally found on CD43 in resting and activated T cells, respectively. These results identify CD43 as a T cell counterreceptor for sialoadhesin and suggest that in addition to its anti-adhesive role CD43 may promote cell-cell interactions.
Cutting Edge: Regulation of Uterine NKT Cells by a Fetal Class I Molecule Other Than CD1
Authors: Dang Yushe , Heyborne Kent D.
Published: April 2014

The peri-implantation uterus contains an expanded population of NK1.1+ Vα14+ TCRint (NKT) lymphocytes. Although these cells bear the above features in common with other NKT cells populations in thymus, bone marrow, liver, and spleen, they differ from these other populations in terms of an altered Vβ repertoire and absence of a CD4+ component. In this study, we demonstrate that the uterine population also differs from other NKT cell populations because they recognize a class I/class I-like molecule other than CD1, whereas most previously described Vα14+ NKT cells are CD1-restricted. Moreover, the class I/class I-like molecule leading to the uterine NKT cell expansion may be supplied by the fetus. These data demonstrate a novel mechanism whereby the fetus is capable of modulating the maternal immune system.
Cutting Edge: CTLs Rapidly Capture Membrane Fragments from Target Cells in a TCR Signaling-Dependent Manner
Authors: Hudrisier Denis , Riond Joelle , Mazarguil Honoré , Gairin Jean Edouard , Joly Etienne
Published: April 2014

Upon encounter of a CTL with a target cell carrying foreign Ags, the TCR internalizes with its ligand, the peptide-MHC class I complex. However, it is unclear how this can happen mechanistically because MHC molecules are anchored to the target cell’s surface via a transmembrane domain. By using antigenic peptides and lipids that were fluorescently labeled, we found that CTLs promptly capture target cell membranes together with the antigenic peptide as well as various other surface proteins. This efficient and specific capture process requires sustained TCR signaling. Our observations indicate that this process allows efficient acquisition of the Ag by CTL, which may in turn regulate lymphocyte activation or elimination.
Cutting Edge: Nociceptin Stimulates Neutrophil Chemotaxis and Recruitment: Inhibition by Aspirin-Triggered-15-Epi-Lipoxin A4
Authors: Serhan Charles N. , Fierro Iolanda M. , Chiang Nan , Pouliot Marc
Published: April 2014

The nociceptin receptor (Noci-R) is a G protein-coupled receptor present in neural tissues and its activation by nociceptin is involved in the processing of pain signals. Here, we report that Noci-R is present and functional on peripheral blood polymorphonuclear leukocytes (PMN). Human PMN express mRNA for Noci-R, its nucleotide sequence determined, and specific binding with [125I]-labeled nociceptin gave an apparent Kd ∼1.5 nM for this PMN opioid receptor. Nociceptin evoked PMN chemotaxis with maximal activity at 100 pM, without intracellular Ca2+ mobilization. When injected in murine air pouches, nociceptin elicited leukocyte infiltration in a concentration-dependent fashion. Nociceptin-stimulated PMN infiltration was inhibited by treating mice with a synthetic analog of the aspirin-triggered lipid mediator 15-epi-lipoxin A4. The present results identify nociceptin as a potent chemoattractant and provide a novel link between the neural and immune systems that are blocked by aspirin-triggered lipid mediators and may be relevant in neurogenic inflammation.
Cutting Edge: Human γδ T Cells Are Activated by Intermediates of the 2-C-methyl-d-erythritol 4-phosphate Pathway of Isoprenoid Biosynthesis
Authors: Altincicek Boran , Moll Jens , Campos Narciso , Foerster Gesine , Beck Ewald , Hoeffler Jean-François , Grosdemange-Billiard Catherine , Rodríguez-Concepción Manuel , Rohmer Michel , Boronat Albert , Eberl Matthias , Jomaa Hassan
Published: April 2014

Activation of Vγ9/Vδ2 T cells by small nonprotein Ags is frequently observed after infection with various viruses, bacteria, and eukaryotic parasites. We suggested earlier that compounds synthesized by the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway of isopentenyl pyrophosphate synthesis are responsible for the Vγ9/Vδ2 T cell reactivity of many pathogens. Using genetically engineered Escherichia coli knockout strains, we now demonstrate that the ability of E. coli extracts to stimulate γδ T cell proliferation is abrogated when genes coding for essential enzymes of the MEP pathway, dxr or gcpE, are disrupted or deleted from the bacterial genome.
Cutting Edge: Critical Role of Inducible Costimulator in Germinal Center Reactions
Authors: Dong Chen , Temann Ulla-Angela , Flavell Richard A.
Published: April 2014

Inducible costimulator (ICOS) is a new member of the CD28/CTLA-4 family that is expressed on activated and germinal center (GC) T cells. Recently, we reported that ICOS-deficient mice exhibited profound defects in T cell activation and effector function. Ab responses in a T-dependent primary reaction and in a murine asthma model were also diminished. In the current study, we investigate the mechanism by which ICOS regulates humoral immunity and examine B cell GC reactions in the absence of ICOS. We found that ICOS−/− mice, when immunized with SRBC, had smaller GCs. Furthermore, IgG1 class switching in the GCs was impaired. Remarkably, GC formation in response to a secondary recall challenge was completely absent in ICOS knockout mice. These data establish a critical role of ICOS in regulation of humoral immunity.
Testing Time-, Ignorance-, and Danger-Based Models of Tolerance
Authors: Anderson Colin C. , Carroll Joseph M. , Gallucci Stefania , Ridge John P. , Cheever Allen W. , Matzinger Polly
Published: April 2014

In this study, we present data showing that tolerance to Ags in the periphery is not determined by the time at which the Ag appears, or by special properties of tissues in newborn mice or newly developing immune systems. We placed male grafts onto immunoincompetent female mice, allowed the grafts to heal for up to 5 mo, and then repopulated the recipients with fetal liver stem cells. We found that the newly arising T cells were neither tolerant nor ignorant of the grafts, but promptly rejected them, though they did not reject female grafts, nor show any signs of autoimmunity. We also found that the H-Y Ag was continuously cross-presented on host APCs, that this presentation was immunogenic, not tolerogenic, and that it depended on the continuous presence of the graft. In searching for the stimulus that might activate the host APCs, we analyzed mRNA expression with a highly sensitive real-time quantitative PCR assay. By using two different “housekeeping” molecules for comparison, we analyzed the message levels for several stress and/or inflammatory molecules in the healed grafts. We found that the long-healed grafts were not equivalent to “normal” skin because the healed grafts expressed lower levels of GAPDH. Altogether, these data suggest that acceptance vs rejection of peripheral tissues is not attributable to ignorance, timing-based tolerance, or special circulation properties of naive T cells in neonatal tissues. It is more likely attributable to an aspect of the context of Ag presentation that remains to be identified.
Functional Caspase-1 Is Required for Langerhans Cell Migration and Optimal Contact Sensitization in Mice
Authors: Antonopoulos Christos , Cumberbatch Marie , Dearman Rebecca J. , Daniel Richard J. , Kimber Ian , Groves Richard W.
Published: April 2014

Langerhans cell (LC) migration from epidermis to draining lymph node is a critical first step in cutaneous immune responses. Both TNF-α and IL-1β are important signals governing this process, but the potential regulatory role of IL-1α processing by caspase-1 is unknown. In wild-type (WT) mice, application of the contact allergens 2,4-dinitrofluorobenzine and oxazolone lead to a marked reduction in epidermal LC numbers, but in caspase-1-deficient mice this reduction was not observed. Moreover, although intradermal injection of TNF-α (50 ng) induced epidermal LC migration in WT mice, this cytokine failed to induce LC migration in caspase-1-deficient mice. Intradermal IL-1β (50 ng) caused a similar reduction in epidermal LC numbers in both WT and caspase-1-deficient mice, indicating that, given an appropriate signal, caspase-1-deficient epidermal LC are capable of migration. Contact hypersensitivity to both 2,4-dinitrofluorobenzine and oxazolone was inhibited in caspase-1-deficient mice, indicating a functional consequence of the LC migration defect. In organ culture the caspase-1 inhibitor Ac-YVAD-cmk, but not control peptide, potently inhibited the epidermal LC migration that occurs in this system, and reduced spontaneous migration of LC was observed in skin derived from caspase-1-deficient mice. Moreover, Ac-YVAD-cmk applied to BALB/c mouse skin before application of contact sensitizers inhibited LC migration and contact hypersensitivity in vivo. Taken together, these data indicate that caspase-1 may play a central role in the regulation of LC migration and suggest that the activity of this enzyme is amenable to control by specific inhibitors both in vivo and in vitro.
Rapid Peptide Turnover and Inefficient Presentation of Exogenous Antigen Critically Limit the Activation of Self-Reactive CTL by Dendritic Cells
Authors: Ludewig Burkhard , McCoy Kathy , Pericin Marcus , Ochsenbein Adrian F. , Dumrese Tilman , Odermatt Bernhard , Toes Rene E. M. , Melief Cornelis J. M. , Hengartner Hans , Zinkernagel Rolf M.
Published: April 2014

This study evaluated to what extent presentation of exogenously acquired self-Ags via MHC class I molecules on DC might contribute to the activation of self-reactive CTL and subsequent development of autoimmune disease. We show here by using the rat insulin promotor lymphocytic choriomeningitis virus glycoprotein model of autoimmune diabetes that the activation of self-reactive CTL by DC after uptake of exogenous Ag is very limited, first by the short half-life of MHC class I-associated peptides on DC in vitro and in vivo, and second by the rather inefficient MHC class I presentation of cell-associated self-Ags by DC. These two mechanisms are probably crucial in establishing high thresholds for the induction of self-reactive CTL that prevent autoimmune sequelae after release of sequestered and previously immunologically ignored tissue Ags.
A Mucosal IgA-Mediated Excretory Immune System In Vivo
Authors: Robinson Janet K. , Blanchard Thomas G. , Levine Alan D. , Emancipator Steven N. , Lamm Michael E.
Published: April 2014

The capacity of mucosal IgA Abs to serve as an excretory immune system in vivo was investigated. Mice expressing a transgenic TCR were immunized intragastrically with the cognate Ag to elicit a vigorous mucosal IgA Ab response. Soon after i.v. challenge, Ag was detected within the epithelial cells of the small intestinal crypts and to a lesser degree within the epithelial cells higher up the villi, paralleling the gradient in expression of the polymeric Ig receptor and the transport of its ligand, oligomeric IgA. Uptake of Ag into the epithelial cells occurred only from the basolateral aspect and only when Ag complexed to IgA Ab could be present in the lamina propria. The results support the concept that local IgA Abs can excrete Ags from the body by transporting them directly through mucosal epithelial cells, using the same mechanism that transports free IgA into the mucosal secretions.
Translocation of the B Cell Antigen Receptor into Lipid Rafts Reveals a Novel Step in Signaling
Authors: Cheng Paul C. , Brown Bruce K. , Song Wenxia , Pierce Susan K.
Published: April 2014

The cross-linking of the B cell Ag receptor (BCR) leads to the initiation of a signal transduction cascade in which the earliest events involve the phosphorylation of the immunoreceptor tyrosine-based activation motifs of Igα and Igβ by the Src family kinase Lyn and association of the BCR with the actin cytoskeleton. However, the mechanism by which BCR cross-linking initiates the cascade remains obscure. In this study, using various A20-transfected cell lines, biochemical and genetic evidence is provided that BCR cross-linking leads to the translocation of the BCR into cholesterol- and sphingolipid-rich lipid rafts in a process that is independent of the initiation of BCR signaling and does not require the actin cytoskeleton. Translocation of the BCR into lipid rafts did not require the Igα/Igβ signaling complex, was not dependent on engagement of the FcR, and was not blocked by the Src family kinase inhibitor PP2 or the actin-depolymerizing agents cytochalasin D or latrunculin. Thus, cross-linking or oligomerization of the BCR induces the BCR translocation into lipid rafts, defining an event in B cell activation that precedes receptor phosphorylation and association with the actin cytoskeleton.
Compartmentalization of Peyer’s Patch Anlagen Before Lymphocyte Entry
Authors: Hashi Hiroyuki , Yoshida Hisahiro , Honda Kenya , Fraser Stuart , Kubo Hajime , Awane Masaaki , Takabayashi Arimichi , Nakano Hideki , Yamaoka Yoshio , Nishikawa Shin-Ichi
Published: April 2014

We have shown that Peyer’s patch (PP) first develops as a simple and even cell aggregation during embryogenesis. To investigate when and how such a simple cell aggregation forms the complex PP architecture, we analyzed the distribution of cells expressing IL-7Rα (PP inducer cells), VCAM-1 (mesenchymal cells), CD11c (dendritic cells), and mature lymphocytes by whole-mount immunostaining of 17.5 days postcoitus to 2 days postpartum mouse gut. Our results show that compartmentalization of PP anlagen commences at day 18.5 of gestation by clustering and subsequent follicle formation of IL-7Rα+, VCAM-1+, and CD11c+ cells. This process adds the primitive architecture of PP anlage with several follicles in which IL-7Rα+ cells localize in the center, while VCAM-1+ and CD11c+ cells localize at the fringe. This follicle formation is accompanied by the establishment of PP-specific vascular network expressing mucosal addressin cellular adhesion molecule-1. Mature B and T lymphocytes entering in the PP anlage are distributed promptly to their own target zones; B cells to the follicle and T cells to nonfollicular zones. Our analysis of scid/scid mouse indicate that the initial processes including formation of PP-specific vascular network occur in the absence of lymphocytes. These observations indicate that the basic architecture of PP is formed by a set of cell lineages assembled during the initial phase of induction of PP anlagen before entry of mature lymphocytes.
Novel Roles for Lyn in B Cell Migration and Lipopolysaccharide Responsiveness Revealed Using Anti-Double-Stranded DNA Ig Transgenic Mice
Authors: Seo Su-jean , Buckler Jodi , Erikson Jan
Published: April 2014

Lyn-deficient mice produce Abs against dsDNA, yet exhibit exaggerated tolerance to the model Ag hen-egg lysozyme. To investigate this apparent contradiction, and to further examine the function of Lyn in Ag-engaged cells, we have used an anti-dsDNA Ig transgenic model. Previously, looking at these anti-dsDNA B cells in Lyn-sufficient BALB/c mice, we showed that they are regulated by functional inactivation (anergy). In the absence of Lyn, these anti-dsDNA B cells remain unable to secrete Ab. This suggests that functional inactivation of anti-dsDNA B cells does not depend on Lyn, and that the anti-dsDNA Abs that are produced in lyn−/− mice arise from a defect in another mechanism of B cell tolerance. Although the anti-dsDNA B cells remain anergic, Lyn deficiency does restore their ability to proliferate to LPS. This reveals a novel role for Lyn in mediating the LPS unresponsiveness that normally follows surface Ig engagement. Furthermore, Lyn deficiency leads to an altered splenic localization and EBV-induced molecule 1 ligand chemokine responsiveness of anti-dsDNA B cells, as well as an absence of marginal zone B cells, suggesting additional roles for Lyn in controlling the migration and development of specific B cell populations.
Dendritic Cells Acquire Antigens from Live Cells for Cross-Presentation to CTL
Authors: Harshyne Larry A. , Watkins Simon C. , Gambotto Andrea , Barratt-Boyes Simon M.
Published: April 2014

Dendritic cells (DC) can readily capture Ag from dead and dying cells for presentation to MHC class I-restricted CTL. We now show by using a primate model that DC also acquire Ag from healthy cells, including other DC. Coculture assays showed that fluorescently labeled plasma membrane was rapidly and efficiently transferred between DC, and transfer of intracellular proteins was observed to a lesser extent. Acquisition of labeled plasma membrane and intracellular protein was cell contact-dependent and was primarily a function of immature DC, whereas both immature and CD40L-matured DC could serve as donors. Moreover, immature DC could acquire labeled plasma membrane and intracellular proteins from a wide range of hemopoietic cells, including macrophages, B cells, and activated T cells. Notably, macrophages, which readily phagocytose apoptotic bodies, were very inefficient at acquiring labeled plasma membrane and intracellular proteins from other live macrophages or DC. With live-cell imaging techniques, we demonstrate that individual DC physically extract plasma membrane from other DC, generating endocytic vesicles of up to 1 μm in diameter. Finally, DC but not macrophages acquired an endogenous melanoma Ag expressed by live DC and cross-presented Ag to MHC class I-restricted CTL, demonstrating the immunological relevance of our finding. These data show for the first time that DC readily acquire Ag from other live cells. We suggest that Ag acquisition from live cells may provide a novel mechanism whereby DC can present Ag in the absence of direct infection, and may serve to expand and regulate the immune response in vivo.
Concomitant Inhibition of Janus Kinase 3 and Calcineurin-Dependent Signaling Pathways Synergistically Prolongs the Survival of Rat Heart Allografts
Authors: Behbod Fariba , Erwin-Cohen Rebecca A. , Wang Mou-Er , Trawick Barton W. , Qu Xienui , Verani Regina , Kahan Barry D. , Stepkowski Stanislaw M. , Kirken Robert A.
Published: April 2014

The cytoplasmic localized Janus tyrosine kinase 3 (Jak3) is activated by multiple cytokines, including IL-2, IL-4, and IL-7, through engagement of the IL-2R common γ-chain. Genetic inactivation of Jak3 is manifested as SCID in humans and mice. These findings have suggested that Jak3 represents a pharmacological target to control certain lymphoid-derived diseases. Using the rat T cell line Nb2-11c, we document that tyrphostin AG-490 blocked in vitro IL-2-induced cell proliferation (IC50 ∼20 μM), Jak3 autophosphorylation, and activation of its key substrates, Stat5a and Stat5b, as measured by tyrosine/serine phosphorylation analysis and DNA-binding experiments. To test the notion that inhibition of Jak3 provides immunosuppressive potential, a 7-day course of i.v. therapy with 5–20 mg/kg AG-490 was used to inhibit rejection of heterotopically transplanted Lewis (RT1l) heart allografts in ACI (RT1a) recipients. In this study, we report that AG-490 significantly prolonged allograft survival, but also acted synergistically when used in combination with the signal 1 inhibitor cyclosporin A, but not the signal 3 inhibitor, rapamycin. Finally, AG-490 treatment reduced graft infiltration of mononuclear cells and Stat5a/b DNA binding of ex vivo IL-2-stimulated graft infiltrating of mononuclear cells, but failed to affect IL2Rα expression, as judged by RNase protection assays. Thus, inhibition of Jak3 prolongs allograft survival and also potentiates the immunosuppressive effects of cyclosporin A, but not rapamycin.
Normal Cellular Prior Protein Is Preferentially Expressed on Subpopulations of Murine Hemopoietic Cells
Authors: Liu Tong , Li Ruliang , Wong Boon-Seng , Liu Dacai , Pan Tao , Petersen Robert B. , Gambetti Pierluigi , Sy Man-Sun
Published: April 2014

We studied the expression of normal cellular prion protein (PrPC) in mouse lymphoid tissues with newly developed mAbs to PrPC. Most of the mature T and B cells in the peripheral lymphoid organs do not express PrPC. In contrast, most thymocytes are PrPC+. In the bone marrow, erythroid cells and maturing granulocytes are PrPC+. Approximately 50% of the cells in the region of small lymphocytes and progenitor cells also express PrPC. Most of these PrPC+ cells are CD43+, but B220−, surface IgM− (sIgM−), and IL-7R−, a phenotype that belongs to cells not yet committed to the B cell lineage. Another small group of the PrPC+ cell are B220+, and some of these are also sIgM+. The majority of the B220+ cells, however, are PrPC−. Therefore, PrPC is preferentially expressed in early bone marrow progenitor cells and subsets of maturing B cells. Supporting this interpretation is our observation that stimulation of bone marrow cells in vitro with PMA results in a decrease in the number of PrPC+B220− cells with a corresponding increase of sIgM+B220high mature B cells. This result suggests that the PrPC+B220− cells are potential progenitors. Furthermore, in the bone marrow of Rag-1−/− mice, there are an increased number of PrPC+B220− cells, and most of the developmentally arrested pro-B cells in these mice are PrPC+. Collectively, these results suggest that PrPC is expressed preferentially in immature T cells in the thymus and early progenitor cells in the bone marrow, and the expression of PrPC is regulated during hemopoietic differentiation.
Oligodeoxynucleotides Containing CpG Motifs Induce Low Levels of TNF-α in Human B Lymphocytes: Possible Adjuvants for Th1 Responses
Authors: Bohle Barbara , Orel Lukas , Kraft Dietrich , Ebner Christof
Published: April 2014

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) represent potential adjuvants for specific immunotherapy of type I allergies because they foster Th1-like immune responses. However, previous work has shown that CpG-ODN induce systemically active levels of TNF-α in murine macrophages. The goal of the present study was to evaluate the release of TNF-α in human cells by a CpG-ODN proven to induce Th1 immune responses in cells from atopic individuals and in mice. CpG-ODN induced TNF-α in cells from atopic and healthy individuals. However, the amounts were low, as determined by comparison with commonly used Ags. Intracellular cytokine staining of PBMC revealed that CpG-ODN-induced TNF-α derived exclusively from B lymphocytes. TNF-α contributed to the CpG-ODN-augmented proliferation and Ig synthesis in PBMC, but was not involved in IFN-γ synthesis. In conclusion, our findings indicate that certain CpG-ODN induce low amounts of TNF-α in human B lymphocytes and may therefore be used to modulate Th2-biased immune responses in allergic patients.
NK T Cell-Induced Protection Against Diabetes in Vα14-Jα281 Transgenic Nonobese Diabetic Mice Is Associated with a Th2 Shift Circumscribed Regionally to the Islets and Functionally to Islet Autoantigen
Authors: Laloux Véronique , Beaudoin Lucie , Jeske Dirk , Carnaud Claude , Lehuen Agnès
Published: April 2014

The onset of autoimmune diabetes is related to defective immune regulation. Recent studies have shown that NK T cells are deficient in number and function in both diabetic patients and nonobese diabetic (NOD) mice. NK T cells, which are CD1d restricted, express a TCR with an invariant Vα14-Jα281 chain and rapidly produce large amounts of cytokines. Vα14-Jα281 transgenic NOD mice have increased numbers of NK T cells and are protected against diabetes onset. In this study we analyzed where and how NK T cells interfere with the development of the anti-islet autoimmune response. NK T cells, which are usually rare in lymph nodes, are abundant in pancreatic lymph nodes and are also present in islets. IL-4 mRNA levels are increased and IFN-γ mRNA levels decreased in islets from diabetes-free Vα14-Jα281 transgenic NOD mice; the IgG1/IgG2c ratio of autoantibodies against glutamic acid decarboxylase is also increased in these mice. Treatment with IL-12 (a pro-Th1 cytokine) or anti-IL-4 Ab abolishes the diabetes protection in Vα14-Jα281 NOD mice. The protection from diabetes conferred by NK T cells is thus associated with a Th2 shift within islets directed against autoantigen such as glutamic acid decarboxylase. Our findings also demonstrate the key role of IL-4.
Lymphocytes from Autoimmune MRLlpr/lprMice Are Hyperresponsive to IL-18 and Overexpress the IL-18 Receptor Accessory Chain
Authors: Neumann Detlef , Del Giudice Elda , Ciaramella Antonio , Boraschi Diana , Bossù Paola
Published: April 2014

MRL lpr/lpr mice spontaneously develop a severe autoimmune lupus syndrome characterized by strong autoantibody production and massive lymphoproliferation, in which IFN-γ plays a major pathogenic effect. The role of the IFN-γ-inducing cytokine IL-18 in the autoimmune syndrome of lpr/lpr mice has been investigated. In response to IL-18, lymph node cells of lpr/lpr mice produce significant amounts of IFN-γ and proliferate more potently as compared with cells from +/+ mice. Cells likely responsible for such hyperresponsiveness to IL-18 include NK cells and the CD4+/CD8+ self-reactive T lymphocytes characteristically present in lymph nodes of lpr/lpr mice. Analysis of the expression of IL-18R complex revealed that mRNA for the IL-18Rα-chain is constitutively expressed at similar level both in +/+ and lpr/lpr lymphocytes. In contrast, the expression of the accessory receptor chain IL-18Rβ is low in unstimulated +/+ cells but significantly high in lpr/lpr cells. Thus, the abnormally high expression of the IL-18R chain IL-18Rβ could be one of the causes of the hyperresponsiveness of lpr/lpr cells to IL-18 at the basis of consequent enhancement of IFN-γ production and development of IFN-γ-dependent autoimmune pathology.
Primary Hepatocytes from Mice Treated with IL-2/IL-12 Produce T Cell Chemoattractant Activity that Is Dependent on Monokine Induced by IFN-γ (Mig) and Chemokine Responsive to γ-2 (Crg-2)
Authors: Park Jong-Wook , Gruys M. Eilene , McCormick Kathy , Lee Jong-Keuk , Subleski Jeffrey , Wigginton Jon M. , Fenton Robert G. , Wang Ji-Ming , Wiltrout Robert H.
Published: April 2014

The IFN-γ-inducible proteins monokine induced by IFN-γ (Mig) and chemokine responsive to γ-2 (Crg-2) can contribute to IL-12-induced antiangiogenic and leukocyte-recruiting activities, but the extent to which leukocytes vs parenchymal cells in different organs contribute to the production of these molecules remains unclear. The results presented herein show that IFN-γ-dependent induction of Mig and Crg-2 gene expression can occur in many nonlymphoid organs, and these genes are rapidly induced in purified hepatocytes isolated from mice treated with IL-2 plus IL-12, or from Hepa 1-6 hepatoma cells treated in vitro with IFN-γ. In addition to depending on IFN-γ, the ability of IL-12 or IL-2/IL-12 to induce Mig and Crg-2 gene expression in purified hepatocytes also is accompanied by the coordinate up-regulation of the IFN-γ R α and β-chains, in the absence of IL-12R components. Supernatants of primary hepatocytes obtained from mice treated in vivo with IL-2/IL-12 or from hepatocytes treated in vitro with IFN-γ contain increased chemotactic activity for enriched human and mouse CD3+ T cells, as well as mouse DX5+ NK cells. The hepatocyte-derived chemotactic activity for mouse T cells but not NK cells was ablated by Abs specific for Mig and Crg-2. These results suggest that parenchymal cells in some organs may contribute substantially to initiation and/or amplification of inflammatory or antitumor responses.
Mouse CD94 Participates in Qa-1-Mediated Self Recognition by NK Cells and Delivers Inhibitory Signals Independent of Ly-49
Authors: Toyama-Sorimachi Noriko , Taguchi Yuriko , Yagita Hideo , Kitamura Fujiko , Kawasaki Akemi , Koyasu Shigeo , Karasuyama Hajime
Published: April 2014

Inhibitory receptors expressed on NK cells recognize MHC class I molecules and transduce negative signals to prevent the lysis of healthy autologous cells. The lectin-like CD94/NKG2 heterodimer has been studied extensively as a human inhibitory receptor. In contrast, in mice, another lectin-like receptor, Ly-49, was the only known inhibitory receptor until the recent discovery of CD94/NKG2 homologues in mice. Here we describe the expression and function of mouse CD94 analyzed by a newly established mAb. CD94 was detected on essentially all NK and NK T cells as well as small fractions of T cells in all mouse strains tested. Two distinct populations were identified among NK and NK T cells, CD94bright and CD94dull cells, independent of Ly-49 expression. The anti-CD94 mAb completely abrogated the inhibition of target killing mediated by NK recognition of Qa-1/Qdm peptide on target cells. Importantly, CD94bright but not CD94dull cells were found to be functional in the Qa-1/Qdm-mediated inhibition. In the presence of the mAb, activated NK cells showed substantial cytotoxicity against autologous target cells as well as enhanced cytotoxicity against allogeneic and “missing self” target cells. These results suggest that mouse CD94 participates in the protection of self cells from NK cytotoxicity through the Qa-1 recognition, independent of inhibitory receptors for classical MHC class I such as Ly-49.
The Maturation of Dendritic Cells Results in Postintegration Inhibition of HIV-1 Replication
Authors: Bakri Youssef , Schiffer Cécile , Zennou Véronique , Charneau Pierre , Kahn Edmond , Benjouad Abdelaziz , Gluckman Jean Claude , Canque Bruno
Published: April 2014

Maturation of dendritic cells (DC) is known to result in decreased capacity to produce HIV due to postentry block of its replicative cycle. In this study, we compared the early phases of this cycle in immature DC (iDC) and mature DC (mDC) generated from monocytes cultured with GM-CSF and IL-4, trimeric CD40 ligand (DCCD40LT), or monocyte-conditioned medium (DCMCM) being added or not from day 5. Culture day 8 cells exposed to X4 HIV-1LAI or R5 HIV-1Ba-L were analyzed by semiquantitative R-U5 PCR, which detects total HIV DNA. CXC chemokine receptor 4low (CXCR4low) CCR5+ iDC harbored similar viral DNA amounts when exposed to either strain. HIV-1LAI entered more efficiently into DCCD40LT or DCMCM with up-regulated CXCR4. CCR5low DCCD40LT still allowed entry of HIV-1Ba-L, whereas CCR5− DCMCM displayed reduced permissivity to this virus. Comparing amounts of late (long terminal repeat (LTR)-gag PCR) and total (R-U5 PCR) viral DNA products showed that HIV-1Ba-L reverse transcription was more efficient than that of HIV-1LAI, but was not affected by DC maturation. Southern blot detection of linear, circular, and integrated HIV DNA showed that maturation affected neither HIV-1 nuclear import nor integration. When assessing virus transcription by exposing iDC to pNL4-3.GFP or pNL4-3.Luc viruses pseudotyped with the G protein of vesicular stomatitis virus (VSV-G), followed by culture with or without CD40LT or MCM, GFP and luciferase activities decreased by 60–75% in mDC vs iDC. Thus, reduced HIV replication in mDC is primarily due to a postintegration block occurring mainly at the transcriptional level. We could not relate this block to altered expression and nuclear localization of NF-κB proteins and SP1 and SP3 transcription factors.
IL-10 Is Required for Regulatory T Cells to Mediate Tolerance to Alloantigens In Vivo
Authors: Hara Masaki , Kingsley Cherry I. , Niimi Masanori , Read Simon , Turvey Stuart E. , Bushell Andrew R. , Morris Peter J. , Powrie Fiona , Wood Kathryn J.
Published: April 2014

We present evidence that donor-reactive CD4+ T cells present in mice tolerant to donor alloantigens are phenotypically and functionally heterogeneous. CD4+ T cells contained within the CD45RBhigh fraction remained capable of mediating graft rejection when transferred to donor alloantigen-grafted T cell-depleted mice. In contrast, the CD45RBlow CD4+ and CD25+CD4+ populations failed to induce rejection, but rather, were able to inhibit rejection initiated by naive CD45RBhigh CD4+ T cells. Analysis of the mechanism of immunoregulation transferred by CD45RBlow CD4+ T cells in vivo revealed that it was donor Ag specific and could be inhibited by neutralizing Abs reactive with IL-10, but not IL-4. CD45RBlow CD4+ T cells from tolerant mice were also immune suppressive in vitro, as coculture of these cells with naive CD45RBhigh CD4+ T cells inhibited proliferation and Th1 cytokine production in response to donor alloantigens presented via the indirect pathway. These results demonstrate that alloantigen-specific regulatory T cells contained within the CD45RBlow CD4+ T cell population are responsible for the maintenance of tolerance to donor alloantigens in vivo and require IL-10 for functional activity.
Critical Requirement for the Membrane-Proximal Cytosolic Tyrosine Residue for CD28-Mediated Costimulation In Vivo
Authors: Harada Yohsuke , Tokushima Miyoko , Matsumoto Yasuyo , Ogawa Shuhei , Otsuka Masataka , Hayashi Katsuhiko , Weiss Bonnie D. , June Carl H. , Abe Ryo
Published: April 2014

The YMNM motif that exists in the CD28 cytoplasmic domain is known as a binding site for phosphatidylinositol 3-kinase and Grb-2 and is considered to be important for CD28-mediated costimulation. To address the role of the YMNM motif in CD28 cosignaling in primary T cells, we generated transgenic mice on a CD28 null background that express a CD28 mutant lacking binding ability to phosphatidylinositol 3-kinase and Grb-2. After anti-CD3 and anti-CD28 Ab stimulation in vitro, the initial proliferative response and IL-2 secretion in CD28 Y189F transgenic T cells were severely compromised, while later responses were intact. In contrast to anti-CD3 and anti-CD28 Ab stimulation, PMA and anti-CD28 Ab stimulation failed to induce IL-2 production from CD28 Y189F transgenic T cells at any time point. Using the graft-vs-host reaction system, we assessed the role of the YMNM motif for CD28-mediated costimulation in vivo and found that CD28 Y189F transgenic spleen cells failed to engraft and could not induce acute graft-vs-host reaction. Together, these results suggest that the membrane-proximal tyrosine of CD28 is required for costimulation in vivo. Furthermore, these results indicate that the results from in vitro assays of CD28-mediated costimulation may not always correlate with T cell activation in vivo.
MHC-II-Independent CD4+ T Cells Induce Colitis in Immunodeficient RAG−/− Hosts
Authors: Trobonjaca Zlatko , Leithäuser Frank , Möller Peter , Bluethmann Horst , Koezuka Yasuhiko , MacDonald H. Robson , Reimann Jörg
Published: April 2014

CD4+ αβ T cells from either normal C57BL/6 (B6) or MHC-II-deficient (Aα−/− or Aβ−/−) B6 donor mice engrafted into congenic immunodeficient RAG1−/− B6 hosts induced an aggressive inflammatory bowel disease (IBD). Furthermore, CD4+ T cells from CD1d−/− knockout (KO) B6 donor mice but not those from MHC-I−/− (homozygous transgenic mice deficient for β2-microglobulin) KO B6 mice induced a colitis in RAG−/− hosts. Abundant numbers of in vivo activated (CD69highCD44highCD28high) NK1+ and NK1− CD4+ T cells were isolated from the inflamed colonic lamina propria (cLP) of transplanted mice with IBD that produced large amounts of TNF-α and IFN-γ but low amounts of IL-4 and IL-10. IBD-associated cLP Th1 CD4+ T cell populations were polyclonal and MHC-II-restricted when derived from normal B6 donor mice, but oligoclonal and apparently MHC-I-restricted when derived from MHC-II-deficient (Aα−/− or Aβ−/−) B6 donor mice. cLP CD4+ T cell populations from homozygous transgenic mice deficient for β2-microglobulin KO B6 donor mice engrafted into RAG−/− hosts were Th2 and MHC-II restricted. These data indicate that MHC-II-dependent as well as MHC-II-independent CD4+ T cells can induce a severe and lethal IBD in congenic, immunodeficient hosts, but that the former need the latter to express its IBD-inducing potential.
Endogenous Glucocorticoids Play a Positive Regulatory Role in the Anti-Keyhole Limpet Hemocyanin In Vivo Antibody Response
Authors: Fleshner Monika , Deak Terrence , Nguyen Kien T. , Watkins Linda R. , Maier Steven F.
Published: April 2014

Glucocorticoids (GCs) are commonly reported to be immunosuppressive. Studies that support this involve the administration of synthetic GCs such as dexamethasone at high pharmacological doses and using in vitro assay systems that may have limited relevance to the role of GCs during normal in vivo immune responses. Therefore, the following experiments tested the conclusion that GCs are generally immunosuppressive. Adult male Sprague Dawley rats received adrenalectomy (ADX) or sham surgery. ADX rats were given either basal corticosterone (CORT) replacement in their drinking water (25 μg/ml) or no CORT. Rats were immunized with keyhole limpet hemocyanin (KLH), and blood samples were taken. ADX rats with no CORT replacement had reduced anti-KLH IgM and IgG responses compared with sham-operated controls. ADX rats that received basal CORT replacement had partially restored anti-KLH IgM, but still had suppressed anti-KLH IgG. Administration of GC receptor type I (RU28318) and type II (RU40555) receptor antagonists also reduced the anti-KLH IgM and IgG responses. ADX rats that received both basal CORT replacement and low dose injections of CORT on days 5 and 7 after KLH had anti-KLH IgG levels equal to those of sham-operated controls. Finally, the GC elevation 4–7 days after immunization may play a role in stimulating the IgM to IgG2a switch. GC receptor blockade reduced the anti-KLH IgG2a and splenic IFN-γ, but not the anti-KLH IgG1, response. Given that IFN-γ is an important regulator of the IgM to IgG2a switch, it is possible that the small rise in GC found 4–7 days after KLH facilitates IgG2a isotype switching.
Antiviral Cytotoxic T Cells Cross-Reactively Recognize Disparate Peptide Determinants from Related Viruses but Ignore More Similar Self- and Foreign Determinants
Authors: Regner Matthias , Lobigs Mario , Blanden Robert V. , Milburn Peter , Müllbacher Arno
Published: April 2014

We have investigated the reactivities of cytotoxic T (Tc) cells against the two immunodominant, H-2Kk-restricted determinants from the Flavivirus Murray Valley encephalitis virus (MVE), MVE1785 (REHSGNEI) and MVE1971 (DEGEGRVI). The respective Tc cell populations cross-reactively lysed target cells pulsed with determinants from the MVE1785- and MVE1971-corresponding positions of six other flaviviruses, despite low sequence homology in some cases. Notably, anti-MVE1785 Tc cells recognized a determinant (TDGEERVI) that shares with the determinant used for stimulation only the carboxyl-terminal amino acid residue, one of two H-2Kk anchor residues. These reactivity patterns were also observed in peptide-dependent IFN-γ production and the requirements for in vitro restimulation of memory Tc cells. However, the broad cross-reactivity appeared to be limited to flavivirus-derived determinants, as none of a range of determinants from endogenous mouse-derived sequences, similar to the MVE-determinants, were recognized. Neither were cells infected with a number of unrelated viruses recognized. These results raise the paradox that virus-immune Tc cell responses, which are mostly directed against only a few “immunodominant” viral determinants, are remarkably peptide cross-reactive.