bluuebunny/crossref_metadata_2025 huggingface dataset:
Colocalization of Expansion of the Splenic Marginal Zone Population with Abnormal B Cell Activation and Autoantibody Production in B6 Mice with an Introgressed New Zealand Black Chromosome 13 Interval
Authors: Wither Joan E. , Loh Christina , Lajoie Ginette , Heinrichs Svinda , Cai Yong-Chun , Bonventi Gabriel , MacLeod Ralph
Published: April 2014

Polyclonal B cell activation is a prominent feature of the lupus-prone New Zealand Black (NZB) mouse strain. We have previously demonstrated linkage between a region on NZB chromosome 13 and increased costimulatory molecule expression on B cells. In this study we have produced C57BL/6 congenic mice with an introgressed homozygous NZB interval extending from ∼24 to 73 cM on chromosome 13 (denoted B6.NZBc13). We show that B6.NZBc13 female mice not only have enhanced B cell activation but also share many other B cell phenotypic characteristics with NZB mice, including expansion of marginal zone and CD5+ B cell populations, increased numbers of IgM ELISPOTs, and increased serum levels of total IgM and IgM autoantibodies. In addition these mice have increased T cell activation, increased numbers of germinal centers, mild glomerulonephritis, and produce high-titer IgM and IgG anti-chromatin Abs. Male B6.NZBc13 mice have a less pronounced cellular phenotype, lacking expansion of the marginal zone B cell population and IgG anti-chromatin Ab production, indicating the presence of gender dimorphism for this locus. Thus, we have identified a genetic locus that recapitulates with fidelity the B cell phenotypic abnormalities in NZB mice, and we demonstrate that this locus is sufficient to induce an autoimmune phenotype. The data provide further support to the contention that immune abnormalities leading to altered B cell activation and selection contribute to the development of autoimmunity in NZB mice.
TLR Signaling Tailors Innate Immune Responses in Human Microglia and Astrocytes
Authors: Jack Carolyn S. , Arbour Nathalie , Manusow Joshua , Montgrain Vivianne , Blain Manon , McCrea Ellie , Shapiro Aaron , Antel Jack P.
Published: April 2014

The specific signals mediating the activation of microglia and astrocytes as a prelude to, or consequence of, CNS inflammation continue to be defined. We investigated TLRs as novel receptors mediating innate immune responses in human glial cells. We find that microglia express mRNA for TLRs 1–9, whereas astrocytes express robust TLR3, low-level TLR 1, 4, 5, and 9, and rare-to-undetectable TLR 2, 6, 7, 8, and 10 mRNA (quantitative real-time PCR). We focused on TLRs 3 and 4, which can signal through both the MyD88-dependent and -independent pathways, and on the MyD88-restricted TLR2. By flow cytometry, we established that microglia strongly express cell surface TLR2; TLR3 is expressed at higher levels intracellularly. Astrocytes express both cell surface and intracellular TLR3. All three TLRs trigger microglial activation upon ligation. TLR3 signaling induces the strongest proinflammatory polarizing response, characterized by secretion of high levels of IL-12, TNF-α, IL-6, CXCL-10, and IL-10, and the expression of IFN-β. CXCL-10 and IL-10 secretion following TLR4 ligation are comparable to that of TLR3; however, other responses were lower or absent. TLR2-mediated responses are dominated by IL-6 and IL-10 secretion. Astrocytes respond to TLR3 ligation, producing IL-6, CXCL-10, and IFN-β, implicating these cells as contributors to proinflammatory responses. Initial TLR-mediated glial activation also regulates consequent TLR expression; while TLR2 and TLR3 are subject to positive feedback, TLR4 is down-regulated in microglia. Astrocytes up-regulate all three TLRs following TLR3 ligation. Our data indicate that activation of innate immune responses in the CNS is not homogeneous but rather tailored according to cell type and environmental signal.
Activation of Dendritic Cells via Inhibition of Jak2/STAT3 Signaling
Authors: Nefedova Yulia , Cheng Pingyan , Gilkes Daniele , Blaskovich Michelle , Beg Amer A. , Sebti Said M. , Gabrilovich Dmitry I.
Published: April 2014

Signaling via Jak2/STAT3 is critically important for normal dendritic cell (DC) differentiation. In addition, we have previously demonstrated that hyperactivation of the Jak2/STAT3 pathway induced by tumor-derived factors (TDF) may be responsible for abnormal DC differentiation in cancer. In this study, using a novel selective inhibitor of Jak2/STAT3, JSI-124, we investigated the mechanism of the Jak2/STAT3 effect on DCs and the possibility of pharmacological regulation of DC differentiation in cancer. Our experiments have demonstrated that JSI-124 overcomes the differentiation block induced by TDF and promotes the differentiation of mature DCs and macrophages. Surprisingly, inhibition of Jak2/STAT3 signaling resulted in dramatic activation of immature DCs generated in the presence of TDF as well as in control medium. This activation manifested in up-regulation of MHC class II, costimulatory molecules, and a dramatic increase in the ability to stimulate allogeneic or Ag-specific T cells. Inhibition of Jak2/STAT3 signaling resulted in activation of the transcription factor NF-κB. This up-regulation was not due to a conventional pathway involving IκBα, but was probably due to a block of the dominant negative effect of STAT3. This indicates that Jak2/STAT3 play an important role in negative regulation of DC activation, and pharmacological inhibition of the Jak2/STAT3 pathway can be used to enhance DC function.
The Death Receptor Fas (CD95/APO-1) Mediates the Deletion of T Lymphocytes Undergoing Homeostatic Proliferation
Authors: Fortner Karen A. , Budd Ralph C.
Published: April 2014

Murine T cells adoptively transferred into syngeneic lymphopenic recipients undergo proliferation. Despite continued cell division, this lymphopenia-induced or homeostatic proliferation of a limited number of transferred T cells does not fill the T cell compartment. The continued expansion of the transferred T cells, even after stable T cell numbers have been reached, suggests that active cell death prevents further increase in T cell number. In this study, we show that wild-type T cells undergoing homeostatic proliferation are sensitive to Fas-mediated cell death. In the absence of Fas, T cells accumulate to significantly higher levels after transfer into lymphopenic recipients. Fas is, thus, a principal regulator of the expansion of peripheral T cells in response to self-peptide/MHC during T cell homeostasis. As Fas-deficient lpr mice manifest no significant abnormalities in thymic negative selection or in foreign Ag-induced peripheral T cell deletion, their lymphadenopathy may result from unrestrained homeostatic proliferation.
Murine CD160, Ig-Like Receptor on NK Cells and NKT Cells, Recognizes Classical and Nonclassical MHC Class I and Regulates NK Cell Activation
Authors: Maeda Motoi , Carpenito Carmine , Russell Ryan C. , Dasanjh Jyoti , Veinotte Linnea L. , Ohta Hideaki , Yamamura Takashi , Tan Rusung , Takei Fumio
Published: April 2014

Human and mouse NK cells use different families of receptors to recognize MHC class I (MHC I) on target cells. Although human NK cells express both Ig-like receptors and lectin-like receptors specific for MHC I, all the MHC I-specific receptors identified on mouse NK cells to date are lectin-like receptors, and no Ig-like receptors recognizing MHC I have been identified on mouse NK cells. In this study we report the first MHC I-specific Ig-like receptor on mouse NK cells, namely, murine CD160 (mCD160). The expression of mCD160 is restricted to a subset of NK cells, NK1.1+ T cells, and activated CD8+ T cells. The mCD160-Ig fusion protein binds to rat cell lines transfected with classical and nonclassical mouse MHC I, including CD1d. Furthermore, the level of mCD160 on NK1.1+ T cells is modulated by MHC I of the host. Overexpression of mCD160 in the mouse NK cell line KY-2 inhibits IFN-γ production induced by phorbol ester plus ionomycin, whereas it enhances IFN-γ production induced by NK1.1 cross-linking or incubation with dendritic cells. Cross-linking of mCD160 also inhibits anti-NK1.1-mediated stimulation of KY-2 cells. Anti-mCD160 mAb alone has no effect. Thus, mCD160, the first MHC I-specific Ig-like receptor on mouse NK cells, regulates NK cell activation both positively and negatively, depending on the stimulus.
Loss of IL-4 Secretion from Human Type 1a Diabetic Pancreatic Draining Lymph Node NKT Cells
Authors: Kent Sally C. , Chen Yahua , Clemmings Sue M. , Viglietta Vissia , Kenyon Norma Sue , Ricordi Camillo , Hering Bernhard , Hafler David A.
Published: April 2014

Altered frequency and function of peripheral invariant NKT (iNKT) cells have been implicated in the regulation of murine and human type 1a diabetes. To examine regulatory cells from the site of drainage of autoinflammatory tissue and autoantigenic T cell priming in diabetes, we directly cloned iNKT cells from human pancreatic draining lymph nodes (PLN). From 451 T cell clones from control and diabetic PLN, we derived 55 iNKT cells by two methods and analyzed function by cytokine secretion. iNKT cell clones isolated from control PLN secreted IL-4 and IFN-γ upon TCR stimulation. For type 1a diabetic subjects, PLN iNKT cell clones from three samples secreted IFN-γ and no IL-4. In a rare recent onset diabetic sample with islet-infiltrating CD4+ T cells, the phenotype of PLN iNKT cell clones was mixed. From normal and diabetic PLN, one-third of CD1d tetramer+-sorted T cell clones were reactive with CD1d transfectants or proliferated/secreted cytokine in response to α-galactosylceramide-pulsed PBMCs; tetramer-staining T cell clones from diabetic PLN did not secrete IL-4. This is the first report directly examining iNKT cells from lymph nodes draining the site of autoimmunological attack in humans; iNKT cells were altered in cytokine secretion as previously reported for circulating iNKT cells in human type 1a diabetes.
Phosphotyrosine Binding-Mediated Oligomerization of Downstream of Tyrosine Kinase (Dok)-1 and Dok-2 Is Involved in CD2-Induced Dok Phosphorylation
Authors: Boulay Iohann , Némorin Jean-Guy , Duplay Pascale
Published: April 2014

To date, five members of the downstream of tyrosine kinase (Dok) family have been characterized. In T cells, two members, Dok-1 and Dok-2, are expressed. CD2 or CD28 stimulation, but not CD3/TCR stimulation, induces Dok phosphorylation. Recent evidence suggests that they act as negative regulators of the CD2 and CD28 signaling pathways. To identify the molecular mechanisms involved in Dok-mediated inhibition, we have identified proteins that bind to the phosphotyrosine-binding (PTB) domain of Dok-1 and Dok-2. We showed that the Dok PTB domain mediates phosphotyrosine-dependent homotypic and heterotypic interactions of Dok-1 and Dok-2. Moreover, in CD2-stimulated Jurkat cells, Dok-1 coimmunoprecipitates with tyrosine-phosphorylated Dok-2. To study the involvement of PTB-mediated oligomerization in Dok function, we have generated Jurkat clones overexpressing Dok-1 or Dok-2 with a mutation that prevents oligomerization (in either the PTB domain or Tyr146 of Dok-1 and Tyr139 of Dok-2). These mutations abrogate CD2-induced phosphorylation and the ability of Dok-1 or Dok-2 to inhibit CD2-induced ERK1/2 and NFAT activation. Moreover, overexpression of Dok-1Y146F or Dok-2Y139F interferes with CD2-induced phosphorylation of endogenous Dok, whereas overexpression of PTB mutant or wild-type Dok does not. Taken together, these data indicate that PTB-mediated oligomerization of Dok-1 and Dok-2 represents an essential step for Dok phosphorylation and function.
A Novel NF-κB-Regulated Site within the Human Iγ1 Promoter Requires p300 for Optimal Transcriptional Activity
Authors: Dryer Rebecca L. , Covey Lori R.
Published: April 2014

Transcriptional activation of germline (GL) promoters occurs through binding of NF-κB to three evolutionarily conserved sites within a CD40 response region in the human and mouse GL Iγ and Iε promoters. Here we identify and characterize a novel NF-κB binding site (κB6) within the human GL Iγ1 promoter that plays an essential role in basal- and CD40-induced transcription. This site is adjacent to identified CREB/activating transcription factor (ATF) sites, present in the Iγ1 but not the Iγ3 promoter, which are important for the amplification of transcription. Our data suggest a cohesive protein complex regulating Iγ1 promoter activity because disruption of any individual NF-κB or CREB/ATF site markedly lowers the overall inducible activity of the promoter. In addition, alteration of helical phasing within the promoter indicates spatial orientation of CREB/ATF and NF-κB, proteins contributes directly to promoter activity. We found that CREB and p50 transactivators, as well as coactivator p300, interact in vivo with the Iγ1 promoter in the presence and absence of CD40 signaling in Ramos and primary B cells. However, the level of CREB and p300 binding differs as a consequence of activation in primary B cells. Furthermore, overexpression of p300, and not a mutant lacking acetyltransferase activity, significantly increases Iγ1 construct-specific transcription. Together these data support a model whereby CREB and multiple NF-κB complexes bind to the Iγ1 promoter and recruit p300. CD40 signals induce p300-dependent changes that result in optimal Iγ1 promoter activity.
Two Divergent Isotypes of the Fourth Complement Component from a Bony Fish, the Common Carp (Cyprinus carpio)
Authors: Mutsuro Junichi , Tanaka Noriyuki , Kato Yoko , Dodds Alister W. , Yano Tomoki , Nakao Miki
Published: April 2014

Duplication and diversification of several complement components is a striking feature of bony fish complement systems. It gives an interesting insight into an evolutionary strategy for the possible enhancement of the repertoire of innate immunity. The present study is aimed at examining diversity in bony fish C4, a member of the thioester-containing complement components. Two diverged cDNA sequences sharing only ∼32% identity at the amino acid level were isolated from the common carp and designated C4-1 and C4-2. C4-1 and C4-2 share a number of C4-like structural signatures, such as the thioester site and a disulfide-linked three-chain structure. Interestingly, they differ at the residue corresponding to the thioester-catalytic histidine, as seen in the human C4A and C4B isotypes, suggesting their distinct substrate specificities in the binding reaction of the thioester. Phylogenetic analysis indicates that the divergence of C4-1 and C4-2 predated the separation of the cartilaginous and bony fish lineages. Genomic Southern hybridization suggests the presence of single copy genes each encoding C4-1 and C4-2 in the carp genome. An activation fragment, C4a, was shown to be released from each isotype in carp serum activated via the classical and/or lectin pathways. Synthetic peptides representing a putative C2 binding site on C4-1 and C4-2 inhibited the classical pathway-mediated hemolytic activity of carp serum in a dose-dependent manner. The results suggest that C4-1 and C4-2 represent two major lineages of C4 that are present in carp serum, have distinct binding specificities, and are functional in the classical/lectin pathways of complement activation.
Regulation of Rat Basophilic Leukemia-2H3 Mast Cell Secretion by a Constitutive Lyn Kinase Interaction with the High Affinity IgE Receptor (FcεRI)
Authors: Vonakis Becky M. , Gibbons Scott P. , Rotté Masashi J. , Brothers Elizabeth A. , Kim Seok C. , Chichester Kristin , MacDonald Susan M.
Published: April 2014

Signaling through the high affinity IgE receptor is initiated by noncovalently associated Lyn kinase, resulting in the secretion of inflammatory mediators from mast cells. A fraction of the total cellular Lyn is associated via its N-terminal unique domain with the cytoplasmic domain of the FcεRI β subunit before receptor aggregation. In the current study, we stably transfected the unique domain of Lyn into rat basophilic leukemia-2H3 mast cells and examined the consequences on FcεRI-induced signal transduction and mediator secretion to further define the role of the unique domain of Lyn in mast cell secretion. Tyrosine phosphorylation of FcεRI β and γ subunits was partially inhibited in the Lyn unique domain transfectants after Ag stimulation. Ag stimulation of Lyn unique domain transfectants was accompanied by enhanced phosphorylation of MEK and ERK-2, which are required for leukotriene C4 (LTC4) release, and production of LTC4 was increased 3- to 5-fold, compared with cells transfected with vector alone. Conversely, tyrosine phosphorylation of the adaptor protein Gab2, which is essential for mast cell degranulation, was inhibited after Ag stimulation of Lyn unique domain transfectants, and Ag-induced release of histamine was inhibited up to 48%. In rat basophilic leukemia-2H3 cells, Lyn thus plays a dual role by positively regulating FcεRI phosphorylation and degranulation while negatively regulating LTC4 production. This study provides further evidence that the constitutive interaction between the unique domain of Lyn and the FcεRI β subunit is a crucial step in the initiation of FcεRI signaling and that Lyn is limiting for FcεRI-induced secretion of inflammatory mediators.
Expression of T-bet by CD4 T Cells Is Essential for Resistance toSalmonellaInfection
Authors: Ravindran Rajesh , Foley Joseph , Stoklasek Thomas , Glimcher Laurie H. , McSorley Stephen J.
Published: April 2014

Despite the recognized role of the T-bet transcription factor in the differentiation of Th1 cells, T-bet-deficient mice can develop small numbers of IFN-γ-producing CD4 T cells. Although these are not sufficient to allow normal handling of some pathogens, T-bet-deficient mice do resolve infection with the intracellular pathogen Listeria monocytogenes. In contrast, we report that expression of T-bet is required for resistance to Salmonella infection. T-bet-deficient mice succumbed to infection with attenuated Salmonella and did not generate IFN-γ-producing CD4 T cells or isotype-switched Salmonella-specific Ab responses. Spleen cells from Salmonella-infected T-bet-deficient mice secreted increased levels of IL-10, but not IL-4, upon in vitro restimulation. A Salmonella-specific TCR transgenic adoptive transfer system was used to further define the involvement of T-bet expression in the development of Salmonella-specific Th1 cells. Wild-type Salmonella-specific CD4 T cells activated in T-bet-deficient recipient mice displayed no defect in clonal expansion, contraction, or IFN-γ production. In contrast, T-bet-deficient, Salmonella-specific CD4 T cells activated in wild-type recipient mice produced less IFN-γ and more IL-2 upon in vivo restimulation. Therefore, expression of T-bet by CD4 T cells is required for the development of Salmonella-specific Th1 cells, regulation of IL-10 production, and resistance to Salmonella infection.
CD8+ T Cell Epitope-Flanking Mutations Disrupt Proteasomal Processing of HIV-1 Nef
Authors: Milicic Anita , Price David A. , Zimbwa Peter , Booth Bruce L. , Brown Helen L. , Easterbrook Philippa J. , Olsen Kara , Robinson Nicola , Gileadi Uzi , Sewell Andrew K. , Cerundolo Vincenzo , Phillips Rodney E.
Published: April 2014

CTL play a critical role in the control of HIV and SIV. However, intrinsic genetic instability enables these immunodeficiency viruses to evade detection by CTL through mutation of targeted antigenic sites. These mutations can impair binding of viral epitopes to the presenting MHC class I molecule or disrupt TCR-mediated recognition. In certain regions of the virus, functional constraints are likely to limit the capacity for variation within epitopes. Mutations elsewhere in the protein, however, might still enable immune escape through effects on Ag processing. In this study, we describe the coincident emergence of three mutations in a highly conserved region of Nef during primary HIV-1 infection. These mutations (R69K, A81G, and H87R) flank the HLA B*35-restricted VY8 epitope and persisted to fixation as the early CTL response to this Ag waned. The variant form of Nef showed a reduced capacity to activate VY8-specific CTL, although protein stability and expression levels were unchanged. This effect was associated with altered processing by the proteasome that caused partial destruction of the VY8 epitope. Our data demonstrate that a variant HIV genotype can significantly impair proteasomal epitope processing and substantiate the concept of immune evasion through diminished Ag generation. These observations also indicate that the scale of viral escape may be significantly underestimated if only intraepitope variation is evaluated.
gC1q Receptor Ligation Selectively Down-Regulates Human IL-12 Production through Activation of the Phosphoinositide 3-Kinase Pathway
Authors: Waggoner Stephen N. , Cruise Michael W. , Kassel Rachel , Hahn Young S.
Published: April 2014

gC1qR, a complement receptor for C1q, plays a pivotal role in the regulation of inflammatory and antiviral T cell responses. Several pathogens, including hepatitis C virus, exploit gC1qR-dependent regulatory pathways to manipulate host immunity. However, the molecular mechanism(s) of gC1qR signaling involved in regulating inflammatory responses remains unknown. We report the selective inhibition of TLR4-induced IL-12 production after cross-linking of gC1qR on the surface of macrophages and dendritic cells. Suppression of IL-12 did not result from increased IL-10 or TGF-β, but was dependent on PI3K activation. Activation of PI3K and subsequent phosphorylation of Akt define an intracellular pathway mediating gC1qR signaling and cross-talk with TLR4 signaling. This is the first report to identify signaling pathways used by gC1qR-mediated immune suppression, and it establishes a means of complement-mediated immune suppression to inhibit Th1 immunity crucial for clearing pathogenic infection.
Interactions of the Extracellular Matrix Proteoglycans Decorin and Biglycan with C1q and Collectins
Authors: Groeneveld Tom W. L. , Oroszlán Melinda , Owens Rick T. , Faber-Krol Maria C. , Bakker Astrid C. , Arlaud Gérard J. , McQuillan David J. , Kishore Uday , Daha Mohamed R. , Roos Anja
Published: April 2014

Decorin and biglycan are closely related abundant extracellular matrix proteoglycans that have been shown to bind to C1q. Given the overall structural similarities between C1q and mannose-binding lectin (MBL), the two key recognition molecules of the classical and the lectin complement pathways, respectively, we have examined functional consequences of the interaction of C1q and MBL with decorin and biglycan. Recombinant forms of human decorin and biglycan bound C1q via both collagen and globular domains and inhibited the classical pathway. Decorin also bound C1 without activating complement. Furthermore, decorin and biglycan bound efficiently to MBL, but only biglycan could inhibit activation of the lectin pathway. Other members of the collectin family, including human surfactant protein D, bovine collectin-43, and conglutinin also showed binding to decorin and biglycan. Decorin and biglycan strongly inhibited C1q binding to human endothelial cells and U937 cells, and biglycan suppressed C1q-induced MCP-1 and IL-8 production by human endothelial cells. In conclusion, decorin and biglycan act as inhibitors of activation of the complement cascade, cellular interactions, and proinflammatory cytokine production mediated by C1q. These two proteoglycans are likely to down-regulate proinflammatory effects mediated by C1q, and possibly also the collectins, at the tissue level.
Adoptive Transfer of Tumor-Reactive Melan-A-Specific CTL Clones in Melanoma Patients Is Followed by Increased Frequencies of Additional Melan-A-Specific T Cells
Authors: Vignard Virginie , Lemercier Brigitte , Lim Annick , Pandolfino Marie-Christine , Guilloux Yannick , Khammari Amir , Rabu Catherine , Echasserieau Klara , Lang François , Gougeon Marie-Lise , Dreno Brigitte , Jotereau Francine , Labarriere Nathalie
Published: April 2014

In this study, we report the adoptive transfer of highly tumor-reactive Melan-A-specific T cell clones to patients with metastatic melanoma, and the follow-up of these injected cells. These clones were generated from HLA-A*0201 patients by in vitro stimulations of total PBMC with the HLA-A*0201-binding Melan-A peptide analog ELAGIGILTV. Ten stage IV melanoma patients were treated by infusion of these CTL clones with IL-2 and IFN-α. The generated T cell clones, of effector/memory phenotype were selected on the basis of their ability to produce IL-2 in response to HLA-A*0201 Melan-A-positive melanoma lines. Infused clones were detected, by quantitative PCR, in the blood of three patients for periods ranging from 7 to 60 days. Six patients showed regression of individual metastases or disease stabilization, and one patient experienced a complete response, but no correlation was found between the detection of the infused clones in PBMC or tumor samples and clinical responses. Nonetheless, frequencies of Melan-A/A2-specific lymphocytes, measured by tetramer labeling, increased after treatment in most patients. In one of these patients, who showed a complete response, this increase corresponded to the expansion of new clonotypes of higher avidity than those detected before treatment. Together, our results suggest that infused CTL clones may have initiated an antitumor response that may have resulted in the expansion of a Melan-A-specific CTL repertoire.
α-Melanocyte-Stimulating Hormone Suppresses Antigen-Induced Lymphocyte Proliferation in Humans Independently of Melanocortin 1 Receptor Gene Status
Authors: Cooper Ashley , Robinson Samantha J. , Pickard Chris , Jackson Claire L. , Friedmann Peter S. , Healy Eugene
Published: April 2014

Studies in mice indicate that α-melanocyte-stimulating hormone (αMSH) is immunosuppressive, but it is not known whether αMSH suppresses human immune responses to exogenous Ags. Human PBMCs, including monocytes, express the melanocortin 1 receptor (MC1R), and it is thought that the ability of αMSH to alter monocyte-costimulatory molecule expression and IL-10 release is mediated by this receptor. However, the MC1R gene is polymorphic, and certain MC1R variants compromise receptor signaling via cAMP, resulting in red hair and fair skin. Here, we have investigated whether αMSH can suppress Ag-induced lymphocyte proliferation in humans and whether these effects are dependent on MC1R genotype. αMSH suppressed streptokinase-streptodornase-induced lymphocyte proliferation, with maximal inhibition at 10−13–10−11 M αMSH. Anti-IL-10 Abs failed to prevent suppression by αMSH, indicating that it was not due to MC1R-mediated IL-10 release by monocytes. Despite variability in the degree of suppression between subjects, similar degrees of αMSH-induced immunosuppression were seen in individuals with wild-type, heterozygous variant, and homozygous/compound heterozygous variant MC1R alleles. RT-PCR of streptokinase-streptodornase-stimulated PBMCs for all five melanocortin receptors demonstrated MC1R expression by monocytes/macrophages, MC1R and MC3R expression by B lymphocytes, but no melanocortin receptor expression by T lymphocytes. In addition, αMSH did not significantly inhibit anti-CD3 Ab-induced lymphocyte proliferation, whereas αMSH and related analogs (SHU9119 and MTII) inhibited Ag-induced lymphocyte proliferation in monocyte-depleted and B lymphocyte-depleted assays. These findings demonstrate that αMSH, acting probably via MC1R on monocytes and B lymphocytes, and possibly also via MC3R on B lymphocytes, has immunosuppressive effects in humans but that suppression of Ag-induced lymphocyte proliferation by αMSH is independent of MC1R gene status.
Gene Family Clustering Identifies Functionally Associated Subsets of Human In Vivo Blood and Tonsillar Dendritic Cells
Authors: Lindstedt Malin , Lundberg Kristina , Borrebaeck Carl A. K.
Published: April 2014

Human dendritic cells (DCs) are a distinct but heterogeneous lineage of APCs operating as the link between innate and adaptive immune responses, with the function to either maintain tolerance or trigger immunity. The DC lineage consists of several subpopulations with unique phenotypes; however, their functional characteristics and transcriptional similarities remain largely unknown. To further characterize the phenotypes and transcriptomes of the subsets, we purified myeloid CD16+, blood DC Ag 1+ (BDCA1+), and BDCA3+ DC populations, as well as plasmacytoid CD123+ DCs, from tonsillar tissue and peripheral blood. Transcriptional profiling and hierarchical clustering visualized that BDCA1+ DCs clustered with BDCA3+ DCs, whereas CD16+ DCs and CD123+ DCs clustered as distinct populations in blood. Differential expression levels of chemokines, ILs, and pattern recognition receptors were demonstrated, which emphasize innate DC subset specialization. Even though highly BDCA1+ and BDCA3+ DC-specific gene expression was identified in blood, the BDCA1+ DCs and BDCA3+ DCs from tonsils displayed similar transcriptional activity, most likely due to the pathogenic or inflammatory maturational signals present in tonsillar tissues. Of note, plasmacytoid DCs displayed less plasticity in their transcriptional activity compared with myeloid DCs. The data demonstrated a functionally distinct association of each of the seven subsets based on their signatures, involving regulatory genes in adaptive and innate immunity.
The CD85J/Leukocyte Inhibitory Receptor-1 Distinguishes between Conformed and β2-Microglobulin-Free HLA-G Molecules
Authors: Gonen-Gross Tsufit , Achdout Hagit , Arnon Tal I. , Gazit Roi , Stern Noam , Hořejší Václav , Goldman-Wohl Debra , Yagel Simcha , Mandelboim Ofer
Published: April 2014

For a proper development of the placenta, maternal NK cells should not attack the fetal extravillous cytotrophoblast cells. This inhibition of maternal NK cells is partially mediated via the nonclassical MHC class I molecule HLA-G. Recently, we demonstrated that HLA-G forms disulfide-linked high molecular complexes on the surface of transfected cells. In the present study, we demonstrate that HLA-G must associate with β2m for its interaction with CD85J/leukocyte Ig-like receptor-1 (LIR-1). Although HLA-G free H chain complexes are expressed on the surface, they are not recognized and possibly interfere with CD85J/LIR-1 and HLA-G interaction. The formation of these complexes on the cell surface might represent a novel mechanism developed specifically by the HLA-G protein aimed to control the efficiency of the CD85J/LIR-1-mediated inhibition. We also show that endogenous HLA-G complexes are expressed on the cell surface. These findings provide novel insights into the delicate interaction between extravillous cytotrophoblast cells and NK cells in the decidua.
Engagement of 4-1BB Inhibits the Development of Experimental Allergic Conjunctivitis in Mice
Authors: Fukushima Atsuki , Yamaguchi Tomoko , Ishida Waka , Fukata Kazuyo , Mittler Robert S. , Yagita Hideo , Ueno Hisayuki
Published: April 2014

The 4-1BB receptor acts as a costimulator in CD8+ T cell activation. Agonistic stimulation through this molecule by treatment with anti-4-1BB Abs has been demonstrated to inhibit various experimentally induced diseases in animals. However, the effect of anti-4-1BB Abs on experimental allergic diseases has not been reported. We investigated the effect of anti-4-1BB Abs on the development and progression of experimental allergic conjunctivitis in mice. To examine the effects of Abs during the induction or effector phase, actively immunized mice or passively immunized mice by splenocyte transfer were treated with agonistic anti-4-1BB Abs, blocking anti-4-1BB ligand Abs, or normal rat IgG. Eosinophil infiltration into the conjunctiva was significantly reduced in wild-type mice by the anti-4-1BB Ab treatment during either induction or effector phase. Th2 cytokine production by splenocytes and total serum IgE were significantly reduced by the anti-4-1BB Ab treatment, while IFN-γ production was increased. The anti-4-1BB Ab treatment induced a relative increase of CD8-positive cell numbers in the spleens. Moreover, inhibition of eosinophil infiltration by the treatment with anti-4-1BB Abs was also noted in actively immunized IFN-γ knockout mice. Taken altogether, in vivo treatment with agonistic anti-4-1BB Abs in either induction or effector phase inhibits the development of experimental allergic conjunctivitis, and this inhibition is likely to be mediated by suppression of Th2 immune responses rather than up-regulation of IFN-γ.
Vα24-Invariant NKT Cells from Patients with Allergic Asthma Express CCR9 at High Frequency and Induce Th2 Bias of CD3+ T Cells upon CD226 Engagement
Authors: Sen Yang , Yongyi Bi , Yuling He , Luokun Xie , Li He , Jie Xiong , Tao Deng , Gang Zhou , Junyan Liu , Chunsong Hu , Zhang Xuejun , Youxin Jin , Feili Gong , Boquan Jin , Jinquan Tan
Published: April 2014

We have demonstrated that Vα24+Vβ11+ invariant (Vα24+i) NKT cells from patients with allergic asthma express CCR9 at high frequency. CCR9 ligand CCL25 induces chemotaxis of asthmatic Vα24+i NKT cells but not the normal cells. A large number of CCR9-positive Vα24+i NKT cells are found in asthmatic bronchi mucosa, where high levels of Th2 cytokines are detected. Asthmatic Vα24+i NKT cells, themselves Th1 biased, induce CD3+ T cells into an expression of Th2 cytokines (IL-4 and IL-13) in cell-cell contact manner in vitro. CD226 are overexpressed on asthmatic Vα24+i NKT cells. CCL25/CCR9 ligation causes directly phosphorylation of CD226, indicating that CCL25/CCR9 signals can cross-talk with CD226 signals to activate Vα24+i NKT cells. Prestimulation with immobilized CD226 mAb does not change ability of asthmatic Vα24+i NKT cells to induce Th2-cytokine production, whereas soluble CD226 mAb or short hairpin RNA of CD226 inhibits Vα24+i NKT cells to induce Th2-cytokine production by CD3+ T cells, indicating that CD226 engagement is necessary for Vα24+i NKT cells to induce Th2 bias of CD3+ T cells. Our results are providing with direct evidence that aberration of CCR9 expression on asthmatic Vα24+i NKT cells. CCL25 is first time shown promoting the recruitment of CCR9-expressing Vα24+i NKT cells into the lung to promote other T cells to produce Th2 cytokines to establish and develop allergic asthma. Our findings provide evidence that abnormal asthmatic Vα24+i NKT cells induce systemically and locally a Th2 bias in T cells that is at least partially critical for the pathogenesis of allergic asthma.
Early Presence of Regulatory Cells in Transplanted Rats Rendered Tolerant by Donor-Specific Blood Transfusion
Authors: Kitade Hiroaki , Kawai Masaru , Rutgeerts Omer , Landuyt Willy , Waer Mark , Mathieu Chantal , Pirenne Jacques
Published: April 2014

Mechanisms by which donor-specific blood transfusion (DSBT) promotes organ allograft acceptance are unclear. In a rat fully mismatched cardiac allograft model, we found that DSBT alone (without immunotherapy) induces the development of regulatory T cells (DSBT-Tregs) posttransplant, thereby shedding new light in the mechanisms of the transfusion effect. Compartments and timing of expansion, requirements, and phenotype of DSBT-Tregs are unknown. It is generally assumed that some time is necessary before Tregs develop. However, we show—by adoptive transfer from DSBT—tolerant into naive recipients: 1) the presence of DSBT-Tregs at 5 days posttransplant in spleen and lymph nodes; 2) their gradual expansion in these compartments; and 3) their presence in the graft 14 of 30 days posttransplant. DSBT-Tregs are donor specific and do not protect third-party allografts. Splenocytes from DSBT-treated nontransplanted recipients or from transplanted DSBT-untreated (rejecting) recipients do not transfer tolerance, indicating that both DSBT and graft are required for sufficient numbers of DSBT-Tregs to develop. Thymectomy (or splenectomy) before DSBT (not at transplantation) abrogate DSBT-Tregs generation and tolerance, showing that thymus (and spleen) are required for DSBT-Tregs generation (not for expansion/maintenance). In contrast with other Tregs models, DSBT-Tregs activity is not restricted to CD4+CD25+ but to CD4+CD45RC− cells, whereas CD4+CD45RC+ cells act as effector cells and accelerate rejection. In conclusion, DSBT alone induces—rapidly posttransplant—the development of alloantigen-specific Tregs in lymphoid tissues and in the graft. DSBT, graft, thymus, and spleen are required for DSBT-Tregs generation. DSBT-Tregs in this model are CD4+CD45RC− (identical to Tregs protecting from autoimmunity in rats).
Mechanisms of Hypotonicity-Induced Calcium Signaling and Integrin Activation by Arachidonic Acid-Derived Inflammatory Mediators in B Cells
Authors: Zhu Peimin , Liu Xiaohong , Labelle Edward F. , Freedman Bruce D.
Published: April 2014

We previously characterized the initial steps in the activation of novel (calcium-permeant) nonselective cation channels (NSCCs) and calcium release-activated calcium channels in primary murine B lymphocytes. Phospholipase C products, namely diacylglycerol and d-myo-inositol 1,4,5-trisphosphate, were identified as proximal intracellular agonists of these respective channels following mechanical stimulation of B cells. However, neither the distal steps in NSCC activation nor the contribution of these channels to sustained mechanical signaling were defined in these previous studies. In this study, single cell measurements of intracellular Ca2+ were used to define the mechanisms of NSCC activation and demonstrate a requirement for arachidonic acid liberated from diacylglycerol. Several arachidonic acid-derived derivatives were identified that trigger Ca2+ entry into B cells, including the lipoxygenase product 5-hydroperoxyeicosatetranenoic acid and the cytochrome P450 hydroxylase product 20-hydroxyeicosatetraenoic; however, the cytochrome P450 epoxygenase product 5,6-epoxyeicosatrienoic acid is primarily responsible for hypotonicity-induced responses. In addition to regulating calcium entry, our data suggest that eicosanoid-activated NSCCs have a separate and direct role in regulating the avidity of integrins on B cells for extracellular matrix proteins, including ICAM-1 and VCAM-1. Thus, in addition to defining a novel osmotically activated signal transduction pathway in B cells, our results have broad implications for understanding how inflammatory mediators dynamically and rapidly regulate B cell adhesion and trafficking.
Chronic Morphine Treatment Promotes Specific Th2 Cytokine Production by Murine T Cells In Vitro via a Fas/Fas Ligand-Dependent Mechanism
Authors: Greeneltch Kristy M. , Kelly-Welch Ann E. , Shi Yufang , Keegan Achsah D.
Published: April 2014

Improper homeostasis of Th1 and Th2 cell differentiation can promote pathological immune responses such as autoimmunity and asthma. A number of factors govern the development of these cells including TCR ligation, costimulation, death effector expression, and activation-induced cell death (AICD). Although chronic morphine administration has been shown to selectively promote Th2 development in unpurified T cell populations, the direct effects of chronic morphine on Th cell skewing and cytokine production by CD4+ T cells have not been elucidated. We previously showed that morphine enhances Fas death receptor expression in a T cell hybridoma and human PBL. In addition, we have demonstrated a role for Fas, Fas ligand (FasL), and TRAIL in promoting Th2 development via killing of Th1 cells. Therefore, we analyzed whether the ability of morphine to affect Th2 cytokine production was mediated by regulation of Fas, FasL, and TRAIL expression and AICD directly in purified Th cells. We found that morphine significantly promoted IL-4 and IL-13 production but did not alter IL-5 or IFN-γ. Furthermore, morphine enhanced the mRNA expression of Fas, FasL and TRAIL and promoted Fas-mediated AICD of CD4+ T cells. Additionally, blockade of Fas/FasL interaction by anti-FasL inhibited the morphine-induced production of IL-4 and IL-13 and AICD of CD4+ T cells. These results suggest that morphine preferentially enhances Th2 cell differentiation via killing of Th1 cells in a Fas/FasL-dependent manner.
Paradoxical Anti-Inflammatory Actions of TNF-α: Inhibition of IL-12 and IL-23 via TNF Receptor 1 in Macrophages and Dendritic Cells
Authors: Zakharova Maria , Ziegler H. Kirk
Published: April 2014

IL-12 and TNF-α are central proinflammatory cytokines produced by macrophages and dendritic cells. Disregulation of TNF-α is associated with sepsis and autoimmune diseases such as rheumatoid arthritis. However, new evidence suggests an anti-inflammatory role for TNF-α. TNF-α-treated murine macrophages produced less IL-12p70 and IL-23, after stimulation with IFN-γ and LPS. Frequency of IL-12p40-producing macrophages correspondingly decreased as measured by intracellular cytokine staining. IL-12p40 production was also inhibited in dendritic cells. TNFR1 was established as the main receptor involved in IL-12p40 regulation, because IL-12p40 levels were not affected by TNF-α in TNFR1−/−-derived macrophages. Macrophages activated during Listeria monocytogenes infection were more susceptible to inhibition by TNF-α than cells from naive animals, which suggests a regulatory role for TNF-α in later stages of infection. This nonapoptotic anti-inflammatory regulation of IL-12 and IL-23 is an important addition to the multitude of TNF-α-induced responses determined by cell-specific receptor signaling.
Tight Regulation of IFN-γ Transcription and Secretion in Immature and Mature B cells by the Inhibitory MHC Class I Receptor, Ly49G2
Authors: Hart Gili , Flaishon Liat , Becker-Herman Shirly , Shachar Idit
Published: April 2014

To complete their maturation and to participate in the humoral immune response, immature B cells that leave the bone marrow are targeted to specific areas in the spleen, where they differentiate into mature cells. Previously, we showed that immature B cells actively down-regulate their integrin-mediated migration to lymph nodes or sites of inflammation, enabling their targeting to the spleen to allow their final maturation. This inhibition is mediated by IFN-γ, which is transcribed and secreted at low levels by these immature B cells and is down-regulated at the mature stage. The activating MHC class I receptor, Ly49D, which is expressed at high levels on immature B cells, stimulates this IFN-γ secretion. In this study we show that B cells coexpress the inhibitory MHC class I receptor, Ly49G2. In addition, we demonstrate a tight regulation in the expression of the Ly49 family members on B cells that depends on their cell surface levels. High levels of Ly49G2 have a dominant inhibitory effect on Ly49D expressed at low levels on immature bone marrow and mature B cells, resulting in inhibition of IFN-γ secretion. However, low levels of the inhibitory receptor, Ly49G2, coexpressed with high levels of the activating receptor, Ly49D, on the immigrating immature B cells enable the secretion of specific low levels of IFN-γ. This expression pattern insures the inhibitory control of peripheral immature B cell to prevent premature encounter with an Ag while enabling entry to the lymph nodes during the mature stage.
Estrogen Enhances Susceptibility to Experimental Autoimmune Myasthenia Gravis by Promoting Type 1-Polarized Immune Responses
Authors: Delpy Laurent , Douin-Echinard Victorine , Garidou Lucile , Bruand Corinne , Saoudi Abdelhadi , Guéry Jean-Charles
Published: April 2014

Myasthenia gravis (MG) is an organ-specific autoimmune disease caused in most cases by autoantibodies against the nicotinic acetylcholine receptor (AChR). It is now well documented that many autoimmune diseases, including MG, are more prevalent in women than in men, and that fluctuations in disease severity occur during pregnancy. These observations raise the question of the potential role of sex hormones, such as estrogens, as mediators of sex differences in autoimmunity. In the present study, we have analyzed the effect of 17β-estradiol (E2) on the pathogenesis of experimental autoimmune myasthenia gravis (EAMG), an animal model of MG. We show that treatment with E2 before Ag priming is necessary and sufficient to promote AChR-specific Th1 cell expansion in vivo. This time-limited exposure to E2 enhances the production of anti-AChR IgG2ab (specific for b allotype; e.g., B6) and IgG2b, but not IgG1, and significantly increases the severity of EAMG in mice. Interestingly, the E2-mediated augmentation in AChR-specific Th1 response correlates with an enhanced production of IL-12 by splenic APCs through the recruitment of CD8α+ dendritic cells. These data provide the first evidence that estrogen enhances EAMG, and sheds some light on the role of sex hormones in immune responses and susceptibility to autoimmune disease in women.
Human Embryonic Stem Cell-Derived NK Cells Acquire Functional Receptors and Cytolytic Activity
Authors: Woll Petter S. , Martin Colin H. , Miller Jeffrey S. , Kaufman Dan S.
Published: April 2014

Human embryonic stem cells (hESCs) provide a unique resource to analyze early stages of human hematopoiesis. However, little is known about the ability to use hESCs to evaluate lymphocyte development. In the present study, we use a two-step culture method to demonstrate efficient generation of functional NK cells from hESCs. The CD56+CD45+ hESC-derived lymphocytes express inhibitory and activating receptors typical of mature NK cells, including killer cell Ig-like receptors, natural cytotoxicity receptors, and CD16. Limiting dilution analysis suggests that these cells can be produced from hESC-derived hemopoietic progenitors at a clonal frequency similar to CD34+ cells isolated from cord blood. The hESC-derived NK cells acquire the ability to lyse human tumor cells by both direct cell-mediated cytotoxicity and Ab-dependent cellular cytotoxicity. Additionally, activated hESC-derived NK cells up-regulate cytokine production. hESC-derived lymphoid progenitors provide a novel means to characterize specific cellular and molecular mechanisms that lead to development of specific human lymphocyte populations. These cells may also provide a source for innovative cellular immune therapies.
Critical Role for the Tapasin-Docking Site of TAP2 in the Functional Integrity of the MHC Class I-Peptide-Loading Complex
Authors: Leonhardt Ralf M. , Keusekotten Kirstin , Bekpen Cemalettin , Knittler Michael R.
Published: April 2014

The transporter associated with Ag processing (TAP) translocates antigenic peptides into the endoplasmic reticulum for binding onto MHC class I (MHC I) molecules. Tapasin organizes a peptide-loading complex (PLC) by recruiting MHC I and accessory chaperones to the N-terminal regions (N domains) of the TAP subunits TAP1 and TAP2. To investigate the function of the tapasin-docking sites of TAP in MHC I processing, we expressed N-terminally truncated variants of TAP1 and TAP2 in combination with wild-type chains, as fusion proteins or as single subunits. Strikingly, TAP variants lacking the N domain in TAP2, but not in TAP1, build PLCs that fail to generate stable MHC I-peptide complexes. This correlates with a substantially reduced recruitment of accessory chaperones into the PLC demonstrating their important role in the quality control of MHC I loading. However, stable surface expression of MHC I can be rescued in post-endoplasmic reticulum compartments by a proprotein convertase-dependent mechanism.