bluuebunny/crossref_metadata_2025 huggingface dataset:
Leptin-Induced IL-6 Production Is Mediated by Leptin Receptor, Insulin Receptor Substrate-1, Phosphatidylinositol 3-Kinase, Akt, NF-κB, and p300 Pathway in Microglia
Authors: Tang Chih-Hsin , Lu Da-Yuu , Yang Rong-Sen , Tsai Huei-Yann , Kao Ming-Ching , Fu Wen-Mei , Chen Yuh-Fung
Published: April 2014

Leptin, the adipocyte-secreted hormone that centrally regulates weight control, is known to function as an immunomodulatory regulator. We investigated the signaling pathway involved in IL-6 production caused by leptin in microglia. Microglia expressed the long (OBRl) and short (OBRs) isoforms of the leptin receptor. Leptin caused concentration- and time-dependent increases in IL-6 production. Leptin-mediated IL-6 production was attenuated by OBRl receptor antisense oligonucleotide, PI3K inhibitor (Ly294002 and wortmannin), Akt inhibitor (1L-6-hydroxymethyl-chiro-inositol-2-((R)-2-O-methyl-3-O-octadecylcarbonate)), NF-κB inhibitor (pyrrolidine dithiocarbamate), IκB protease inhibitor (l-1-tosylamido-2-phenylenylethyl chloromethyl ketone), IκBα phosphorylation inhibitor (Bay 117082), or NF-κB inhibitor peptide. Transfection with insulin receptor substrate (IRS)-1 small-interference RNA or the dominant-negative mutant of p85 and Akt also inhibited the potentiating action of leptin. Stimulation of microglia with leptin activated IκB kinase α/IκB kinase β, IκBα phosphorylation, IκBα degradation, p65 phosphorylation at Ser276, p65 and p50 translocation from the cytosol to the nucleus, and κB-luciferase activity. Leptin-mediated an increase of IκB kinase α/IκB kinase β activity, κB-luciferase activity, and p65 and p50 binding to the NF-κB element was inhibited by wortmannin, Akt inhibitor, and IRS-1 small-interference RNA. The binding of p65 and p50 to the NF-κB elements, as well as the recruitment of p300 and the enhancement of histone H3 and H4 acetylation on the IL-6 promoter was enhanced by leptin. Our results suggest that leptin increased IL-6 production in microglia via the leptin receptor/IRS-1/PI3K/Akt/NF-κB and p300 signaling pathway.
Prevalence of Newly Generated Naive Regulatory T Cells (Treg) Is Critical for Treg Suppressive Function and Determines Treg Dysfunction in Multiple Sclerosis
Authors: Haas Jürgen , Fritzsching Benedikt , Trübswetter Petra , Korporal Mirjam , Milkova Linda , Fritz Brigitte , Vobis Diana , Krammer Peter H. , Suri-Payer Elisabeth , Wildemann Brigitte
Published: April 2014

The suppressive function of regulatory T cells (Treg) is impaired in multiple sclerosis (MS) patients. The mechanism underlying the Treg functional defect is unknown. Treg mature in the thymus and the majority of cells circulating in the periphery rapidly adopt a memory phenotype. Because our own previous findings suggest that the thymic output of T cells is impaired in MS, we hypothesized that an altered Treg generation may contribute to the suppressive deficiency. We therefore determined the role of Treg that enter the circulation as recent thymic emigrants (RTE) and, unlike their CD45RO+ memory counterparts, express CD31 as typical surface marker. We show that the numbers of CD31+-coexpressing CD4+CD25+CD45RA+CD45RO−FOXP3+ Treg (RTE-Treg) within peripheral blood decline with age and are significantly reduced in MS patients. The reduced de novo generation of RTE-Treg is compensated by higher proportions of memory Treg, resulting in a stable cell count of the total Treg population. Depletion of CD31+ cells from Treg diminishes the suppressive capacity of donor but not patient Treg and neutralizes the difference in inhibitory potencies between the two groups. Overall, there was a clear correlation between Treg-mediated suppression and the prevalence of RTE-Treg, indicating that CD31-expressing naive Treg contribute to the functional properties of the entire Treg population. Furthermore, patient-derived Treg, but not healthy Treg, exhibit a contracted TCR Vβ repertoire. These observations suggest that a shift in the homeostatic composition of Treg subsets related to a reduced thymic-dependent de novo generation of RTE-Treg with a compensatory expansion of memory Treg may contribute to the Treg defect associated with MS.
Omalizumab Reverses the Phenotypic and Functional Effects of IgE-Enhanced FcεRI on Human Skin Mast Cells
Authors: Gomez Gregorio , Jogie-Brahim Sherryline , Shima Mika , Schwartz Lawrence B.
Published: April 2014

The dramatic effects of the anti-IgE mAb omalizumab to lower free IgE levels and FcεRI levels on basophils contrast with more modest clinical effects. Accordingly, whether IgE modulates FcεRI levels and FcεRI-dependent mediator release in vitro on human skin mast cells (MCTC type) that had matured in vivo is of interest. IgE reversibly enhanced FcεRI levels on MCTC cells in a dose- and time-dependent manner (up-regulation t1/2 of 4–5 days with 1–3 μg/ml IgE), without affecting cell proliferation. A molar ratio of omalizumab to IgE of 0.9 at baseline prevented receptor up-regulation by 50%, whereas adding omalizumab to MCTC cells already with IgE-enhanced FcεRI levels at molar ratios of 5, 12.5, and 31 reduced FcεRI levels to baseline with respective t1/2 values of 8.7, 6.3, and 4.8 days. MCTC cells with IgE-enhanced FcεRI levels were more sensitive to stimulation with a low dose of anti-FcεRI mAb in terms of degranulation and production of PGD2, GM-CSF, IL-6, IL-13, and TNF-α. Reducing up-regulated FcεRI levels with omalizumab also reduced mediator release to a low dose of anti-FcεRI mAb to baseline by 3–4 wk. Thus, reducing free IgE should decrease the hypersensitivity of allergic individuals to low naturally occurring concentrations of allergens.
Tumor-Associated Embryonic Antigen-Expressing Vaccines that Target CCR6 Elicit Potent CD8+ T Cell-Mediated Protective and Therapeutic Antitumor Immunity
Authors: Biragyn Arya , Schiavo Roberta , Olkhanud Purevdorj , Sumitomo Kenya , King Alan , McCain Megan , Indig Fred E. , Almanzar Giovanni , Baatar Dolgor
Published: April 2014

Despite its potency, the wider use of immunotherapy for B cell malignancies is hampered by the lack of well-defined tumor-specific Ags. In this study, we demonstrate that an evolutionarily conserved 37-kDa immature laminin receptor protein (OFA-iLRP), a nonimmunogenic embryonic Ag expressed by a variety of tumors, is rendered immunogenic if targeted to the APCs using the CCR6 ligands MIP3α/CCL20 and mDF2β. The CCR6 targeting facilitated efficient Ag cross-presentation and induction of tumor-neutralizing CTLs. Although the Ag targeting alone, without activation of dendritic cells (DCs), is proposed to induce tolerance, and MIP3α does not directly activate DCs, the MIP3α-based vaccine efficiently induced protective and therapeutic antitumor responses. The responses were as strong as those elicited by the OFA-iLRP fusions with moieties that activated DCs and Th1-type cytokine responses, mDF2β, or mycobacterial Hsp70 Ag. Although the same cDNA encodes the dimerized high-affinity mature 67-kDa mLRP that is expressed in normal tissues to stabilize the binding of laminin to cell surface integrins, the vaccines expressing OFA-iLRP elicited long-term protective CD8+ T cell-mediated memory responses against syngeneic B cell lymphoma, indicating the potential application of these simple vaccines as preventive and therapeutic formulations for human use.
Cutting Edge: Transgenic Expression of Human MUC1 in IL-10−/− Mice Accelerates Inflammatory Bowel Disease and Progression to Colon Cancer
Authors: Beatty Pamela L. , Plevy Scott E. , Sepulveda Antonia R. , Finn Olivera J.
Published: April 2014

Epithelial cell MUC1 is aberrantly expressed on human epithelial adenocarcinomas where it functions as a regulator of immune responses and an oncogene. Normally expressed at low levels in healthy colonic epithelium, MUC1 was reported to be overexpressed in human inflammatory bowel disease (IBD) and thus may be expected to play an important role in regulating chronic inflammation and its progression to colitis-associated colon cancer. Studies in the immunobiology and pathology of IBD and colitis-associated colon cancer have been done in various mouse models but none could properly address the role of MUC1 due to low homology between the mouse and the human molecule. We report that IL-10−/− mice, a widely accepted mouse model of IBD, crossed to human MUC1-transgenic mice, develop MUC1+ IBD characterized by an earlier age of onset, higher inflammation scores, and a much higher incidence and number of colon cancers compared with IL-10−/− mice.
Cutting Edge: Genetic Variation Influences FcεRI-Induced Mast Cell Activation and Allergic Responses
Authors: Yamashita Yumi , Charles Nicolas , Furumoto Yasuko , Odom Sandra , Yamashita Toshiyuki , Gilfillan Alasdair M. , Constant Stephanie , Bower Molly A. , Ryan John J. , Rivera Juan
Published: April 2014

Mast cell responses are influenced by a diverse array of environmental factors, but little is known about the effect of genetic background. In this study, we report that 129/Sv mice had high levels of circulating IgE, increased expression of the high-affinity receptor for IgE (FcεRI), and greater sensitivity to anaphylaxis when compared with C57BL/6 mice. Bone marrow-derived mast cells (BMMCs) from 129/Sv mice showed more robust degranulation upon the engagement of FcεRI. Deficiency of the Src family kinase Lyn enhanced degranulation in 129/Sv BMMCs but inhibited this response in C57BL/6 cells. C57BL/6 lyn−/− BMMCs had reduced expression of the Src family kinase Fyn, and increasing its expression markedly enhanced degranulation. In human mast cells the silencing of Lyn or Fyn expression resulted in hyperdegranulation or hypodegranulation, respectively. The findings demonstrate a genetic influence on the extent of a mast cell’s response and identify Fyn kinase as a contributory determinant.
Cutting Edge: High Molecular Weight Hyaluronan Promotes the Suppressive Effects of CD4+CD25+ Regulatory T Cells
Authors: Bollyky Paul L. , Lord James D. , Masewicz Susan A. , Evanko Stephen P. , Buckner Jane H. , Wight Thomas N. , Nepom Gerald T.
Published: April 2014

Hyaluronan is a glycosaminoglycan present in the extracellular matrix. When hyaluronan is degraded during infection and injury, low m.w. forms are generated whose interactions influence inflammation and angiogenesis. Intact high m.w. hyaluronan, conversely, conveys anti-inflammatory signals. We demonstrate that high m.w. hyaluronan enhances human CD4+CD25+ regulatory T cell functional suppression of responder cell proliferation, whereas low m.w. hyaluronan does not. High m.w. hyaluronan also up-regulates the transcription factor FOXP3 on CD4+CD25+ regulatory T cells. These effects are only seen with activated CD4+CD25+ regulatory T cells and are associated with the expression of CD44 isomers that more highly bind high m.w. hyaluronan. At higher concentrations, high m.w. hyaluronan also has direct suppressive effects on T cells. We propose that the state of HA in the matrix environment provides contextual cues to CD4+CD25+ regulatory T cells and T cells, thereby providing a link between the innate inflammatory network and the regulation of adaptive immune responses.
Cutting Edge: CD8+ T Cell Clones Possess the Potential to Differentiate into both High- and Low-Avidity Effector Cells
Authors: Kroger Charles J. , Alexander-Miller Martha A.
Published: April 2014

The property of functional avidity is recognized to be of critical importance in determining pathogen clearance. An unresolved question with regard to this property is whether distinct naive subsets exist that display inherent differences in their peptide sensitivity requirements for activation, i.e., functional avidity, or whether differences in peptide sensitivity are induced following peptide encounter. In this study, we demonstrate that naive populations that can give rise to both high- and low-avidity cells do not contain subsets that exhibit differences in the amount of peptide required for activation. Furthermore, we show that an individual T cell clone can generate both high- and low-avidity effectors. The work presented here provides the first formal demonstration that an individual cell can give rise to both high- and low-avidity progeny, suggesting that avidity modulation at the level of an individual cell may play an important role in the CD8+ T cell response generated in vivo.
Oral Administration of High Molecular Mass Poly-γ-Glutamate Induces NK Cell-Mediated Antitumor Immunity
Authors: Kim Tae Woo , Lee Tae Young , Bae Hyun Cheol , Hahm Jeong Ho , Kim Yang Hyun , Park Chung , Kang Tae Heung , Kim Chul Joong , Sung Moon Hee , Poo Haryoung
Published: April 2014

We analyzed the in vivo tumor regression activity of high molecular mass poly-γ-glutamate (γ-PGA) from Bacillus subtilis sups. chungkookjang. C57BL/6 mice were orally administered 10-, 100-, or 2000-kDa γ-PGA or β-glucan (positive control), and antitumor immunity was examined. Our results revealed higher levels of NK cell-mediated cytotoxicity and IFN-γ secretion in mice treated with higher molecular mass γ-PGA (2000 kDa) vs those treated with lower molecular mass γ-PGA (10 or 100 kDa) or β-glucan. We then examined the effect of oral administration of 10- or 2000-kDa γ-PGA on protection against B16 tumor challenge in C57BL/6 mice. Mice receiving high molecular mass γ-PGA (2000 kDa) showed significantly smaller tumor sizes following challenge with the MHC class I-down-regulated tumor cell lines, B16 and TC-1 P3 (A15), but not with TC-1 cells, which have normal MHC class I expression. Lastly, we found that γ-PGA-induced antitumor effect was decreased by in vivo depletion of NK cells using mAb PK136 or anti-asialo GM1 Ab, and that was completely blocked in NK cell-deficient B6 beige mice or IFN-γ knockout mice. Taken together, we demonstrated that oral administration of high molecular mass γ-PGA (2000 kDa) generated significant NK cell-mediated antitumor activity in mice bearing MHC class I-deficient tumors.
The CD16−/CD56bright Subset of NK Cells Is Resistant to Oxidant-Induced Cell Death
Authors: Thorén Fredrik B. , Romero Ana I. , Hermodsson Svante , Hellstrand Kristoffer
Published: April 2014

Phagocyte-derived reactive oxygen species (“oxygen radicals”) have been ascribed a suppressive role in immunoregulation by inducing dysfunction and apoptotic cell death in lymphocytes. Earlier studies show that human NK cells are exceptionally sensitive to oxygen radical-induced apoptosis and functional inhibition. Two subsets of human CD56+ NK cells have been identified: the highly cytotoxic CD56dim cells which constitute >90% of NK cells in peripheral blood, and the less cytotoxic but efficiently cytokine-producing CD56bright cells. In this study, we demonstrate that the CD56bright subset of NK cells, in contrast to CD56dim cells, remains viable and functionally intact after exposure to phagocyte-derived or exogenously added oxygen radicals. The resistance of CD56bright cells to oxidative stress was accompanied by a high capacity of neutralizing exogenous hydrogen peroxide, and by a high cell-surface expression of antioxidative thiols. Our results imply that CD56bright NK cells are endowed with an efficient antioxidative defense system that protects them from oxygen radical-induced inactivation.
Apoptosis-Inducing Factor Regulates Death in Peripheral T Cells
Authors: Srivastava Smita , Banerjee Hridesh , Chaudhry Ashutosh , Khare Anupriya , Sarin Apurva , George Anna , Bal Vineeta , Durdik Jeannine M. , Rath Satyajit
Published: April 2014

Apoptosis-inducing factor (Aif) is a mitochondrial flavoprotein with multiple roles in apoptosis as well as in cellular respiration and redox regulation. The harlequin (Hq) mouse strain carries an aif locus modification causing reduced Aif expression. We demonstrate that activated CD4+ and CD8+ peripheral T cells from Hq mice show resistance to neglect-induced death (NID) triggered by growth factor withdrawal, but not to death induced by multiple agents that trigger DNA damage. Aif translocates to the nucleus in cells undergoing NID, and, in Hq T cell blasts, resistance to NID is associated with reduced cytosolic release of mitochondrial cytochrome c, implicating Aif in this event. In contrast, Hq T cell blasts express higher levels of CD95L, demonstrating increased susceptibility to activation-induced cell death (AICD) and apoptosis triggered by hydrogen peroxide. Superoxide scavenging protects from AICD in wild-type, but not Hq, T cell blasts, suggesting that Aif plays a crucial superoxide-scavenging role to regulate T cell AICD. Finally, the altered pattern of death susceptibility is reproduced by siRNA-mediated reduction of Aif expression in normal T cells. Thus, Aif serves nonredundant roles, both proapoptotic and antiapoptotic, in activated peripheral T cells.
Distinct Activities of Stromal Cells Involved in the Organogenesis of Lymph Nodes and Peyer’s Patches
Authors: Okuda Masato , Togawa Atsushi , Wada Hiromi , Nishikawa Shin-ichi
Published: April 2014

It is now well established that the interaction between “inducer” cells of hemopoietic origin and “organizer” cells of mesenchymal lineage is involved in the organogenesis of lymph node (LN) and Peyer’s patch (PP). Organizer cells are defined by the expression of VCAM-1 and ICAM-1 and the production of homeostatic chemokines. However, several studies suggested the presence of a diversity among these cells from different lymphoid tissues. Thus, we attempted to define the difference of organizer cells of LN and PP in terms of gene expression profile. Microarray analyses of organizer cells revealed that these cells isolated from embryonic mesenteric LN expressed higher levels of genes that are related to inflammation, tissue remodeling, and development of mesenchymal lineage compared with those from PP. Several transcription factors related to epithelial-mesenchymal interactions were also up-regulated in organizer cells from LN. These results indicate that organizer cells in LN and PP are indeed distinct and suggest that the organizer cells in LN are at a more activated stage than those in PP.
Dendritic Cells Trigger Tumor Cell Death by a Nitric Oxide-Dependent Mechanism
Authors: Nicolas Alexandra , Cathelin Dominique , Larmonier Nicolas , Fraszczak Jennifer , Puig Pierre-Emmanuel , Bouchot André , Bateman Andrew , Solary Eric , Bonnotte Bernard
Published: April 2014

Dendritic cells (DCs) are well known for their capacity to induce adaptive antitumor immune response through Ag presentation and tumor-specific T cell activation. Recent findings reveal that besides this role, DCs may display additional antitumor effects. In this study, we provide evidence that LPS- or IFN-γ-activated rat bone marrow-derived dendritic cells (BMDCs) display killing properties against tumor cells. These cytotoxic BMDCs exhibit a mature DC phenotype, produce high amounts of IL-12, IL-6, and TNF-α, and retain their phagocytic properties. BMDC-mediated tumor cell killing requires cell-cell contact and depends on NO production, but not on perforin/granzyme or on death receptors. Furthermore, dead tumor cells do not exhibit characteristics of apoptosis. Thus, intratumoral LPS injections induce an increase of inducible NO synthase expression in tumor-infiltrating DCs associated with a significant arrest of tumor growth. Altogether, these results suggest that LPS-activated BMDCs represent powerful tumoricidal cells which enforce their potential as anticancer cellular vaccines.
CTLs Target Th Cells That Acquire Bystander MHC Class I-Peptide Complex from APCs
Authors: Cox Jennifer H. , McMichael Andrew J. , Screaton Gavin R. , Xu Xiao-Ning
Published: April 2014

CTLs can acquire MHC class I-peptide complexes from their target cells, whereas CD4+ T cells obtain MHC class II-peptide complexes from APCs in a TCR-specific manner. As a consequence, Ag-specific CTL can kill each other (fratricide) or CD4+ T cells become APCs themselves. The purpose of the acquisition is not fully understood and may be either inhibition or prolongation of an immunological response. In this study, we demonstrate that human CD4+ Th cells are able to capture membrane fragments from APC during the process of immunological synapse formation. The fragments contain not only MHC class II-peptide complexes but also MHC class I-peptide complexes, rendering these cells susceptible to CTL killing in an Ag-specific manner. The control of CD4+ Th cells by Ag-specific CTL, therefore, maybe another mechanism to regulate CD4+ T cell expansion in normal immune responses or cause immunopathoglogy during the course of viral infections such as HIV.
Brain Microenvironment Promotes the Final Functional Maturation of Tumor-Specific Effector CD8+ T Cells
Authors: Masson Frédérick , Calzascia Thomas , Di Berardino-Besson Wilma , de Tribolet Nicolas , Dietrich Pierre-Yves , Walker Paul R.
Published: April 2014

During the priming phase of an antitumor immune response, CD8+ T cells undergo a program of differentiation driven by professional APCs in secondary lymphoid organs. This leads to clonal expansion and acquisition both of effector functions and a specific adhesion molecule pattern. Whether this program can be reshaped during the effector phase to adapt to the effector site microenvironment is unknown. We investigated this in murine brain tumor models using adoptive transfer of tumor-specific CD8+ T cells, and in spontaneous immune responses of patients with malignant glioma. Our data show proliferation of Ag-experienced tumor-specific T cells within the brain parenchyma. Moreover, CD8+ T cells further differentiated in the brain, exhibiting enhanced IFN-γ and granzyme B expression and induction of αE(CD103)β7 integrin. This unexpected integrin expression identified a subpopulation of CD8+ T cells conditioned by the brain microenvironment and also had functional consequences: αE(CD103)β7-expressing CD8+ T cells had enhanced retention in the brain. These findings were further investigated for CD8+ T cells infiltrating human malignant glioma; CD8+ T cells expressed αE(CD103)β7 integrin and granzyme B as in the murine models. Overall, our data indicate that the effector site plays an active role in shaping the effector phase of tumor immunity. The potential for local expansion and functional reprogramming should be considered when optimizing future immunotherapies for regional tumor control.
KIR2DS1-Positive NK Cells Mediate Alloresponse against the C2 HLA-KIR Ligand Group In Vitro
Authors: Chewning Joseph H. , Gudme Charlotte N. , Hsu Katharine C. , Selvakumar Annamalai , Dupont Bo
Published: April 2014

The inhibitory 2DL1 and activating 2DS1 killer Ig-like receptors (KIR) both have shared ligand specificity for codon sequences in the C2 group HLA-Cw Ags. In this study, we have investigated NK cell activation by allogeneic target cells expressing different combinations of the HLA-KIR ligand groups C1, C2, and Bw4. We demonstrate that fresh NK cells as well as IL-2-propagated NK cells from 2DS1-positive donors that are homozygous for the C1 ligand group are activated in vitro by B lymphoblastoid cell lines expressing the C2 group. This response is, in part, due to the absence of C1 group recognition mediated by the inhibitory receptor 2DL2/3. This “missing self” alloresponse to C2, however, is rarely observed in NK cells from donors lacking 2DS1. Even in presence of 2DS1, the NK alloresponse is dramatically reduced in donors that have C2 group as “self.” Analysis of selected NK clones that express 2DS1 mRNA and lack mRNA for 2DL1 demonstrates that activation by the C2 ligand and mAb cross-linking of 2DS1 in these clones induces IFN-γ. Furthermore, this C2 group-induced activation is inhibited by Abs to both HLA class I and the receptor. Collectively, these studies demonstrate that NK cells from 2DS1-positive donors are activated by target cells that express the C2 group as an alloantigen. This leads to increased IFN-γ-positive fresh NK cells and induces NK allocytotoxicity in IL2-propagated polyclonal NK cells and NK clones. This study also provides support for the concept that incompatibility for the HLA-KIR ligand groups C1, C2, and Bw4 dominates NK alloactivation in vitro.
Resistance to Experimental Autoimmune Encephalomyelitis and Impaired T Cell Priming by Dendritic Cells in Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase Substrate-1 Mutant Mice
Authors: Tomizawa Takeshi , Kaneko Yuka , Kaneko Yoriaki , Saito Yasuyuki , Ohnishi Hiroshi , Okajo Jun , Okuzawa Chie , Ishikawa-Sekigami Tomomi , Murata Yoji , Okazawa Hideki , Okamoto Koichi , Nojima Yoshihisa , Matozaki Takashi
Published: April 2014

Src homology 2 domain-containing protein tyrosine phosphatase (SHP) substrate-1 (SHPS-1) is a transmembrane protein that binds the protein tyrosine phosphatases SHP-1 and SHP-2 through its cytoplasmic region and is expressed on the surface of CD11c+ dendritic cells (DCs) and macrophages. In this study, we show that mice that express a mutant form of SHPS-1 lacking most of the cytoplasmic region are resistant to experimental autoimmune encephalomyelitis (EAE) in response to immunization with a peptide derived from myelin oligodendrocyte glycoprotein (MOG (35–55)). The MOG (35–55)-induced proliferation of, and production of IFN-γ, IL-2, and IL-17, by T cells from immunized SHPS-1 mutant mice were reduced compared with those apparent for wild-type cells. The abilities of splenic DCs from mutant mice to stimulate an allogenic MLR and to prime Ag-specific T cells were reduced. Both IL-12-stimulated and TLR-dependent cytokine production by DCs of mutant mice were also impaired. Finally, SHPS-1 mutant mice were resistant to induction of EAE by adoptive transfer of MOG (35–55)-specific T cells. These results show that SHPS-1 on DCs is essential for priming of naive T cells and the development of EAE. SHPS-1 is thus a potential therapeutic target in inflammatory disorders of the CNS and other autoimmune diseases.
Reciprocal Regulation of SH3 and SH2 Domain Binding via Tyrosine Phosphorylation of a Common Site in CD3ε
Authors: Kesti Tapio , Ruppelt Anja , Wang Jing-Huan , Liss Michael , Wagner Ralf , Taskén Kjetil , Saksela Kalle
Published: April 2014

Recruitment of cellular signaling proteins by the CD3 polypeptides of the TCR complex mediates T cell activation. We have screened a human Src homology 3 (SH3) domain phage display library for proteins that can bind to the proline-rich region of CD3ε. This screening identified Eps8L1 (epidermal growth factor receptor pathway substrate 8-like 1) together with the N-terminal SH3 domain of Nck1 and Nck2 as its preferred SH3 partners. Studies with recombinant proteins confirmed strong binding of CD3ε to Eps8L1 and Nck SH3 domains. CD3ε bound well also to Eps8 and Eps8L3, and modestly to Eps8L2, but not detectably to other SH3 domains tested. Interestingly, binding of Nck and Eps8L1 SH3 domains was mapped to a PxxDY motif that shared its tyrosine residue (Y166) with the ITAM of CD3ε. Phosphorylation of this residue abolished binding of Eps/Nck SH3 domains in peptide spot filter assays, as well as in cells cotransfected with a dominantly active Lck kinase. TCR ligation-induced binding and phosphorylation-dependent loss of binding were also demonstrated between Eps8L1 and endogenous CD3ε in Jurkat T cells. Thus, phosphorylation of Y166 serves as a molecular switch during T cell activation that determines the capacity of CD3ε to interact with either SH3 or SH2 domain-containing proteins.
Early TCR Expression and Aberrant T Cell Development in Mice with Endogenous Prerearranged T Cell Receptor Genes
Authors: Serwold Thomas , Hochedlinger Konrad , Inlay Matthew A. , Jaenisch Rudolf , Weissman Irving L.
Published: April 2014

The factors that regulate the rate of production of T cells by the thymus remain incompletely defined. To test whether generation of functional T cell receptors limits the rate of thymic T cell export, we made use of a line of mice, LN3αβ, that have endogenously prerearranged TCR genes. The prerearranged TCR genes were expressed abnormally early in hemopoietic development, indicating that RAG-mediated recombination, rather than transcription factor expression, is the key determinant of the initiation of robust TCR transcription. Thymic T cell export rates were similar between wild-type (wt) and LN3αβ mice, indicating that T cell maturation rates in these mice are determined by factors other than TCR gene rearrangement. In competitive bone marrow chimeras, however, LN3αβ thymocytes were out-competed by wt cells and failed to develop beyond the double-negative 4 stage. Furthermore, wt progenitors transplanted intrathymically into LN3αβ mice proliferated excessively, suggesting that increased proliferative signals in the LN3αβ thymus compensate for faulty T cell development driven by early TCR expression.
IFN-γ Acts Directly on Activated CD4+ T Cells during Mycobacterial Infection to Promote Apoptosis by Inducing Components of the Intracellular Apoptosis Machinery and by Inducing Extracellular Proapoptotic Signals
Authors: Li Xujian , McKinstry K. Kai , Swain Susan L. , Dalton Dyana K.
Published: April 2014

Despite many studies, the regulation of CD4+ T cell apoptosis during the shutdown of immune responses is not fully understood. We have investigated the molecular mechanisms of IFN-γ in regulating apoptosis of CD4+ T cells during bacillus Calmette-Guérin (BCG) infection of mice. Our data provide new insight into the regulation of CD4+ T cell apoptosis by IFN-γ. As CD4+ T cells responded to BCG infection, there was a coordinated increase in IFN-γ production by effector CD4+ T cells and a coordinated IFN-γ-dependent up-regulation of many diverse apoptosis-pathway genes in effector CD4+ T cells. Unexpectedly, IFN-γ up-regulated transcripts and protein expression of Bcl-2, Bax, Bim, Bid, Apaf-1, and caspase-9 in activated CD4+ T cells—components of the apoptosis machinery that are involved in promoting mitochondrial damage-mediated apoptosis. Wild-type, but not IFN-γ knockout, CD4+ T cells underwent apoptosis that was associated with damaged mitochondrial membranes. IFN-γ also up-regulated expression of cell-extrinsic signals of apoptosis, including TRAIL, DR5, and TNFR1. Cell-extrinsic apoptosis signals from TNF-α, TRAIL, and NO were capable of damaging the mitochondrial membranes in activated CD4+ T cells. Moreover, activated CD4+ T cells from BCG-infected DR5, TNFR1, and inducible NO synthase knockout mice had impaired caspase-9 activity, suggesting impaired mitochondria-pathway apoptosis. We propose that IFN-γ promotes apoptosis of CD4+ T cells during BCG infection as follows: 1) by sensitizing CD4+ T cells to apoptosis by inducing intracellular apoptosis molecules and 2) by inducing cell-extrinsic apoptosis signals that kill CD4+ effector T cells.
IL-2 Regulates Expansion of CD4+ T Cell Populations by Affecting Cell Death: Insights from Modeling CFSE Data
Authors: Ganusov Vitaly V. , Milutinović Dejan , De Boer Rob J.
Published: April 2014

It is generally accepted that IL-2 influences the dynamics of populations of T cells in vitro and in vivo. However, which parameters for cell division and/or death are affected by IL-2 is not well understood. To get better insights into the potential ways of how IL-2 may influence the population dynamics of T cells, we analyze data on the dynamics of CFSE-labeled polyclonal CD4+ T lymphocytes in vitro after anti-CD3 stimulation at different concentrations of exogenous IL-2. Inferring cell division and death rates from CFSE-delabeling experiments is not straightforward and requires the use of mathematical models. We find that to adequately describe the dynamics of T cells at low concentrations of exogenous IL-2, the death rate of divided cells has to increase with the number of divisions cells have undergone. IL-2 hardly affects the average interdivision time. At low IL-2 concentrations 1) fewer cells are recruited into the response and successfully complete their first division; 2) the stochasticity of cell division is increased; and 3) the rate, at which the death rate increases with the division number, increases. Summarizing, our mathematical reinterpretation suggests that the main effect of IL-2 on the in vitro dynamics of naive CD4+ T cells occurs by affecting the rate of cell death and not by changing the rate of cell division.
The Inflamed Central Nervous System Drives the Activation and Rapid Proliferation of Foxp3+ Regulatory T Cells
Authors: O’Connor Richard A. , Malpass Katy H. , Anderton Stephen M.
Published: April 2014

Resolution of experimental autoimmune encephalomyelitis requires a large cohort of Foxp3+ regulatory T cells (Tregs) within the CNS. In this study, we have used the passive transfer of murine experimental autoimmune encephalomyelitis using myelin-reactive T cells to study the development of this Treg response. Rapid proliferation of Tregs within the CNS (which is not seen in lymphoid organs) drives a switch in the balance of CNS proliferation from T effectors to Tregs, correlating with recovery. This proliferative burst drives a local over-representation of Vβ8+ Tregs in the CNS, indicative of an oligoclonal expansion. There is also evidence for a small, but detectable, myelin oligodendrocyte glycoprotein-reactive Treg component expanded without prior immunization. Furthermore, CNS-derived Tregs, taken during recovery, suppressed the proliferation of CNS-derived effectors in response to myelin oligodendrocyte glycoprotein. Under these conditions, Tregs could also limit the level of IFN-γ production, but not IL-17 production, by CNS-derived effectors. These data establish the CNS as an environment that permits extensive Treg proliferation and are the first to demonstrate Treg expansion specifically within the tissues during the natural resolution of autoimmune inflammation.
Induction of Regulatory T Cells and Dominant Tolerance by Dendritic Cells Incapable of Full Activation
Authors: Yates Stephen F. , Paterson Alison M. , Nolan Kathleen F. , Cobbold Stephen P. , Saunders Nigel J. , Waldmann Herman , Fairchild Paul J.
Published: April 2014

Transplants tolerated through a process known as infectious tolerance evoke continuous recruitment of regulatory T (Treg) cells that are necessary to maintain the unresponsive state. This state is maintained long-term and requires continuous Ag exposure. It is not known, however, whether infectious tolerance operates through sustained recruitment of pre-existing regulatory cells, induction of regulatory cells, or both. Using mice deficient in natural Treg cells, we show here that quiescent donor dendritic cells (DC) laden with histocompatibility Ag can induce Treg cells de novo that mediate transplantation tolerance. In contrast, fully activated DC fail to do so. These findings suggest that DC incapable of delivering full activation signals to naive T cells may favor their polarization toward a regulatory phenotype. Furthermore, they suggest a role for quiescent endogenous DC in the maintenance of the tolerant state.
Cross-Talk between Myeloid-Derived Suppressor Cells and Macrophages Subverts Tumor Immunity toward a Type 2 Response
Authors: Sinha Pratima , Clements Virginia K. , Bunt Stephanie K. , Albelda Steven M. , Ostrand-Rosenberg Suzanne
Published: April 2014

Although the immune system has the potential to protect against malignancies, many individuals with cancer are immunosuppressed. Myeloid-derived suppressor cells (MDSC) are elevated in many patients and animals with tumors, and contribute to immune suppression by blocking CD4+ and CD8+ T cell activation. Using the spontaneously metastatic 4T1 mouse mammary carcinoma, we now demonstrate that cross-talk between MDSC and macrophages further subverts tumor immunity by increasing MDSC production of IL-10, and by decreasing macrophage production of IL-12. Cross-talk between MDSC and macrophages requires cell-cell contact, and the IL-12 decrease is dependent on MDSC production of IL-10. Treatment with the chemotherapeutic drug gemcitabine, which reduces MDSC, promotes rejection of established metastatic disease in IL-4Rα−/− mice that produce M1 macrophages by allowing T cell activation, by maintaining macrophage production of IL-12, and by preventing increased production of IL-10. Therefore, MDSC impair tumor immunity by suppressing T cell activation and by interacting with macrophages to increase IL-10 and decrease IL-12 production, thereby promoting a tumor-promoting type 2 response, a process that can be partially reversed by gemcitabine.
IL-4 and IL-13 Negatively Regulate TNF-α- and IFN-γ-Induced β-Defensin Expression through STAT-6, Suppressor of Cytokine Signaling (SOCS)-1, and SOCS-3
Authors: Albanesi Cristina , Fairchild Heather R. , Madonna Stefania , Scarponi Claudia , De Pità Ornella , Leung Donald Y. M. , Howell Michael D.
Published: April 2014

Human β-defensins (HBDs) are a major class of antimicrobial peptides that play an important role in the innate immune response, however, the induction and regulation of these antimicrobial peptides is not well understood. We demonstrate here that stimulation of keratinocytes with TNF-α/IFN-γ induces HBD-2 and HBD-3 by activating STAT-1 and NF-κB signaling. We further demonstrate that IL-4 and IL-13 activate STAT-6 and induce the suppressors of cytokine signaling (SOCS)-1 and -3. This interferes with STAT-1 and NF-κB signaling, thereby inhibiting TNF-α/IFN-γ-mediated induction of HBD-2 and HBD-3. These data suggest that targeting the STAT-1-signaling pathway or suppressor of cytokine signaling expression enhances β-defensin expression and represents a new therapeutic strategy for reduction of infection in human diseases associated with β-defensin deficiency.
Deletional Self-Tolerance to a Melanocyte/Melanoma Antigen Derived from Tyrosinase Is Mediated by a Radio-Resistant Cell in Peripheral and Mesenteric Lymph Nodes
Authors: Nichols Lisa A. , Chen Yiming , Colella Teresa A. , Bennett Clare L. , Clausen Björn E. , Engelhard Victor H.
Published: April 2014

Self-tolerance to melanocyte differentiation Ags limits the ability to generate therapeutic antimelanoma responses. However, the mechanisms responsible for CD8 T cell tolerance to these Ags are unknown. We have used a newly generated TCR-transgenic mouse to establish the basis of tolerance to one such Ag from tyrosinase. Despite expression of tyrosinase transcripts in the thymus, central deletion does not shape the tyrosinase-specific CD8 T cell repertoire. We demonstrate that this endogenously expressed melanocyte Ag is constitutively presented in both peripheral and mesenteric lymph nodes, leading to abortive activation and deletion of tyrosinase-specific CD8 T cells. Importantly, this Ag is not presented by either radio-sensitive dendritic cells, or by radio-resistant Langerhans cells. Thus, for this endogenous Ag, cross-tolerization does not appear to be an operative mechanism. Instead, we find radioresistant tyrosinase mRNA expression in lymphoid compartments where CD8 T cell deletion occurs. This suggests that direct presentation of tyrosinase by radio-resistant lymph node resident cells is entirely responsible for tolerance to this endogenous melanocyte differentiation Ag.
Cutting Edge: Opposite Effects of IL-1 and IL-2 on the Regulation of IL-17+ T Cell Pool IL-1 Subverts IL-2-Mediated Suppression
Authors: Kryczek Ilona , Wei Shuang , Vatan Linhua , Escara-Wilke June , Szeliga Wojciech , Keller Evan T. , Zou Weiping
Published: April 2014

In this report, we show that IL-17+CD4+ and IL-17+CD8+ T cells are largely found in lung and digestive mucosa compartments in normal mice. Endogenous and exogenous IL-1 dramatically contribute to IL-17+ T cell differentiation mediated by TGFβ and IL-6. IL-1 is capable of stimulating IL-17+ T cell differentiation in the absence of IL-6. Furthermore, although IL-2 reduces IL-17+ T cell differentiation, IL-1 completely disables this effect. Mechanistically, IL-1 and IL-2 play opposite roles in regulating the expression of several molecules regulating Th17 cell differentiation, including the orphan nuclear receptor RORγt, the IL-1 receptor, and the IL-23 receptor. IL-1 subverts the effects of IL-2 on the expression of these gene transcripts. Altogether, our work demonstrates that IL-6 is important but not indispensable for IL-17+ T cell differentiation and that IL-1plays a predominant role in promoting IL-17+ T cell induction. Thus, the IL-17+ T cell pool may be controlled by the local cytokine profile in the microenvironment.
TNF-α Drives Human CD14+ Monocytes to Differentiate into CD70+ Dendritic Cells Evoking Th1 and Th17 Responses
Authors: Iwamoto Sanju , Iwai Shin-ichi , Tsujiyama Kazuko , Kurahashi Chika , Takeshita Kumiko , Naoe Michio , Masunaga Atsuko , Ogawa Yoshio , Oguchi Katsuji , Miyazaki Akira
Published: April 2014

Many mechanisms involving TNF-α, Th1 responses, and Th17 responses are implicated in chronic inflammatory autoimmune disease. Recently, the clinical impact of anti-TNF therapy on disease progression has resulted in re-evaluation of the central role of this cytokine and engendered novel concept of TNF-dependent immunity. However, the overall relationship of TNF-α to pathogenesis is unclear. Here, we demonstrate a TNF-dependent differentiation pathway of dendritic cells (DC) evoking Th1 and Th17 responses. CD14+ monocytes cultured in the presence of TNF-α and GM-CSF converted to CD14+ CD1alow adherent cells with little capacity to stimulate T cells. On stimulation by LPS, however, they produced high levels of TNF-α, matrix metalloproteinase (MMP)-9, and IL-23 and differentiated either into mature DC or activated macrophages (Mφ). The mature DC (CD83+ CD70+ HLA-DR high CD14low) expressed high levels of mRNA for IL-6, IL-15, and IL-23, induced naive CD4 T cells to produce IFN-γ and TNF-α, and stimulated resting CD4 T cells to secret IL-17. Intriguingly, TNF-α added to the monocyte culture medium determined the magnitude of LPS-induced maturation and the functions of the derived DC. In contrast, the Mφ (CD14highCD70+CD83−HLA-DR−) produced large amounts of MMP-9 and TNF-α without exogenous TNF stimulation. These results suggest that the TNF priming of monocytes controls Th1 and Th17 responses induced by mature DC, but not inflammation induced by activated Mφ. Therefore, additional stimulation of monocytes with TNF-α may facilitate TNF-dependent adaptive immunity together with GM-CSF-stimulated Mφ-mediated innate immunity.
Xenogeneic β2-Microglobulin Substitution Alters NK Cell Function
Authors: Benoît Loralyn A. , Tan Rusung
Published: April 2014

Recently, it has been shown that human β2-microglobulin (h-β2m) blocks the association between the NK cell inhibitory receptor Ly49C and H-2Kb. Given this finding, we therefore sought to assess the immunobiology of NK cells derived from C57BL/6 (H-2b) mice expressing exclusively h-β2m. Initial analysis revealed that the Ly49C expression profile of NK cells from h-β2m+ mice was modified, despite the fact that H-2Kb expression was normal in these mice. Moreover, the NK cells were not anergic in that IL-2 treatment of h-β2m+ NK cells in vitro enabled efficient lysis of prototypic tumor cell lines as well as of syngeneic h-β2m+ lymphoblasts. This loss of self-tolerance appeared to correlate with the activation status of h-β2m+ NK cells because quiescent h-β2m+ transplant recipients maintained h-β2m+ grafts but polyinosine:polycytidylic acid-treated recipients acutely rejected h-β2m+ grafts. NK cell reactivity toward h-β2m+ targets was attributed to defective Ly49C interactions with h-β2m:H-2Kb molecules. With regard to NK cell regulatory mechanisms, we observed that h-β2m:H-2Kb complexes in the cis-configuration were inefficient at regulating Ly49C and, furthermore, that receptor-mediated uptake of h-β2m:H-2Kb by Ly49C was impaired compared with uptake of mouse β2m:H-2Kb. Thus, we conclude that transgenic expression of h-β2m alters self-MHC class I in such a way that it modulates the NK cell phenotype and interferes with regulatory mechanisms, which in turn causes in vitro-expanded and polyinosine:polycytidylic acid-activated NK cells to be partially self-reactive similar to what is seen with NK cells derived from MHC class I-deficient mice.
Molecular and Functional Characterization of IL-15 in Rainbow TroutOncorhynchus mykiss:A Potent Inducer of IFN-γ Expression in Spleen Leukocytes
Authors: Wang Tiehui , Holland Jason W. , Carrington Allison , Zou Jun , Secombes Christopher J.
Published: April 2014

IL-15 is a member of the common γ-chain family of cytokines that possess a heterogeneous repertoire of activities on various cells of the immune system. We report here the first functional characterization of a fish IL-15 in rainbow trout. The trout IL-15 gene is 6-kb long and contains six exons and five introns that transcribe into a 1.2-kb mRNA containing seven out-of-frame AUG initiation codons and translate into a 193-aa peptide. Potential sites for transcriptional activators and repressors have been identified in the trout IL-15 gene. Like IL-15 from other species, trout IL-15 is closely linked to an INPP4B gene, but there is also a BCL10 gene located between the IL-15 and INPP4B genes. Three alternative splicing variants of the trout IL-15 gene have also been identified and their expression in vivo was studied. Trout IL-15 expression is present in all the tissues and cell lines studied. Recombinant trout IFN-γ selectively increased IL-15 expression but had little effect on other cytokines such as IL-1β and IL-11. Recombinant trout IL-15 preferentially stimulated splenic leukocytes from healthy fish, where it induced a large increase in IFN-γ expression, with little, if any, effect on IL-1β expression. This effect was quite long-lived, and was still apparent 24 h poststimulation. Although the exact cell types being affected have still to be determined, it is clear that once produced IL-15 will have a profound affect on the ability of the fish immune system to activate antimicrobial defenses and genes induced themselves by IFN-γ.
CD8+ Dendritic Cells Use LFA-1 to Capture MHC-Peptide Complexes from Exosomes In Vivo
Authors: Segura Elodie , Guérin Coralie , Hogg Nancy , Amigorena Sebastian , Théry Clotilde
Published: April 2014

Exosomes are secreted vesicles formed in late endocytic compartments. Mature dendritic cells (DCs) secrete exosomes bearing functional MHC-peptide complexes and high levels of ICAM-1. Such exosomes can activate Ag-specific naive T cells but only after recapture by recipient APCs. In this study, we addressed the molecular mechanisms of interaction between exosomes and recipient DCs. We show that exosomes can be presented by mouse DCs without the need for internalization and processing. Exosomes interact with DCs through a specific saturable receptor. Although the two major ligands of ICAM-1, LFA-1 and Mac-1, are expressed by lymphoid organ DCs, only LFA-1 is required for exosome capture by these cells. Accordingly, we show that CD8+ DCs express higher levels of LFA-1 than CD8− DCs, and that they are the main recipients of exosomes in vivo. We propose a new role for LFA-1 on DCs, as a receptor for exosomes to favor Ag transfer between DCs in vivo.
CD86 Regulates IgG1 Production via a CD19-Dependent Mechanism
Authors: Kin Nicholas W. , Sanders Virginia M.
Published: April 2014

CD86 signals directly in a B cell to activate PI3K and increase the rate of IgG1 production, without affecting germline transcription. However, the mechanism by which CD86 activates PI3K in a B cell and the relevance of CD86 stimulation in vivo remains unknown. We show that the addition of CD28/Ig to CD40 ligand/IL-4-activated wild-type, but not CD86- or CD19-deficient, B cells increased the level of phosphorylation for Lyn and CD19, as well as the amount of Lyn, Vav, and PI3K that immunoprecipitated with CD19. Adoptive transfer of CD86-deficient B cells and wild-type CD4+ T cells into RAG2-deficient mice and immunization with trinitrophenylated keyhole limpet hemocyanin resulted in an IL-4 and germline IgG1 response equivalent to control mice, but a decrease in serum IgG1. Thus, our findings suggest that CD86 plays a key role in regulating the level of IgG1 produced in vitro and in vivo, and that Lyn and CD19 may be the signaling intermediates activated by CD86 proximal to PI3K.