bluuebunny/crossref_metadata_2025 huggingface dataset:
CCR7 Is Critically Important for Migration of Dendritic Cells in Intestinal Lamina Propria to Mesenteric Lymph Nodes
Authors: Jang Myoung Ho , Sougawa Nagako , Tanaka Toshiyuki , Hirata Takako , Hiroi Takachika , Tohya Kazuo , Guo Zijin , Umemoto Eiji , Ebisuno Yukihiko , Yang Bo-Gie , Seoh Ju-Young , Lipp Martin , Kiyono Hiroshi , Miyasaka Masayuki
Published: April 2014

Although dendritic cells (DCs) located in the small intestinal lamina propria (LP-DCs) migrate to mesenteric lymph nodes (MLNs) constitutively, it is unclear which chemokines regulate their trafficking to MLNs. In this study we report that LP-DCs in unperturbed mice require CCR7 to migrate to MLNs. In vitro, LP-DCs expressing CCR7 migrated toward CCL21, although the LP-DCs appeared morphologically and phenotypically immature. In MLNs, DCs bearing the unique LP-DC phenotype (CD11chighCD8αintCD11blowαLlowβ7high and CD11chighCD8α−CD11bhighαLlowβ7high) were abundant in wild-type mice, but were markedly fewer in CCL19-, CCL21-Ser-deficient plt/plt mice and were almost absent in CCR7-deficient mice, indicating the critical importance of CCR7 in LP-DC trafficking to MLNs. Interestingly, CCR7+ DCs in MLNs with the unique LP-DC phenotype had numerous vacuoles containing cellular debris in the cytoplasm, although MLN-DCs themselves were poorly phagocytic, suggesting that the debris was derived from the LP, where the LP-DCs ingested apoptotic intestinal epithelial cells (IECs). Consistent with this, LP-DCs ingested IECs vigorously in vitro. By presenting IEC-associated Ag, the LP-DCs also induce T cells to produce IL-4 and IL-10. Collectively, these results strongly suggest that LP-DCs with unique immunomodulatory activities migrate to MLNs in a CCR7-dependent manner to engage in the presentation of IEC-associated Ags acquired in the LP.
Early Growth Response-1 Is Required for CD154 Transcription
Authors: Cron Randy Q. , Bandyopadhyay Rupa , Genin Anna , Brunner Michael , Kersh Gilbert J. , Yin Jiyi , Finkel Terri H. , Crow Mary K.
Published: April 2014

CD154 (CD40 ligand) expression on CD4 T cells is normally tightly controlled, but abnormal or dysregulated expression of CD154 has been well documented in autoimmune diseases, such as systemic lupus erythematosus. Beyond regulation by NFAT proteins, little is known about the transcriptional activation of the CD154 promoter. We identified a species-conserved purine-rich sequence located adjacent to the CD154 transcriptional promoter proximal NFAT site, which binds early growth response (Egr) transcription factors. Gel shift assays and chromatin immunoprecipitation assays reveal that Egr-1, Egr-3, and NFAT1 present in primary human CD4 T cells are capable of binding this combinatorial site in vitro and in vivo, respectively. Multimerization of this NFAT/Egr sequence in the context of a reporter gene demonstrates this sequence is transcriptionally active upon T cell activation in primary human CD4 T cells. Overexpression of Egr-1, but not Egr-3, is capable of augmenting transcription of this reporter gene as well as that of an intact CD154 promoter. Conversely, overexpression of small interfering RNA specific for Egr-1 in primary human CD4 T cells inhibits CD154 expression. Similarly, upon activation, CD154 message is notably decreased in splenic CD4 T cells from Egr-1-deficient mice compared with wild-type controls. Our data demonstrate that Egr-1 is required for CD154 transcription in primary CD4 T cells. This has implications for selective targeting of Egr family members to control abnormal expression of CD154 in autoimmune diseases such as systemic lupus erythematosus.
Progesterone-Induced Blocking Factor Activates STAT6 via Binding to a Novel IL-4 Receptor
Authors: Kozma Noemi , Halasz Melinda , Polgar Beata , Poehlmann Tobias G. , Markert Udo R. , Palkovics Tamas , Keszei Marton , Par Gabriella , Kiss Katalin , Szeberenyi Jozsef , Grama Laszlo , Szekeres-Bartho Julia
Published: April 2014

Progesterone-induced blocking factor (PIBF) induces Th2-dominant cytokine production. Western blotting and EMSA revealed phosphorylation as well as nuclear translocation of STAT6 and inhibition of STAT4 phosphorylation in PIBF-treated cells. The silencing of STAT6 by small interfering RNA reduced the cytokine effects. Because the activation of the STAT6 pathway depends on the ligation of IL-4R, we tested the involvement of IL-4R in PIBF-induced STAT6 activation. Although PIBF does not bind to IL-4R, the blocking of the latter with an Ab abolished PIBF-induced STAT6 activation, whereas the blocking of the IL-13R had no effect. PIBF activated suppressor of cytokine signaling-3 and inhibited IL-12-induced suppressor of cytokine signaling-1 activation. The blocking of IL-4R counteracted all the described effects, suggesting that the PIBF receptor interacts with IL-4R α-chain, allowing PIBF to activate the STAT6 pathway. PIBF did not phosphorylate Jak3, suggesting that the γ-chain is not needed for PIBF signaling. Confocal microscopic analysis revealed a colocalization and at 37°C a cocapping of the FITC PIBF-activated PIBF receptor and PE anti-IL-4R-labeled IL-4R. After the digestion of the cells with phosphatidylinositol-specific phospholipase C, the STAT6-activating effect of PIBF was lost, whereas that of IL-4 remained unaltered. These data suggest the existence of a novel type of IL-4R composed of the IL-4R α-chain and the GPI-anchored PIBF receptor.
Dynamics of MHC Class II-Activating Signals in Murine Resting B Cells
Authors: Nashar Toufic O. , Drake James R.
Published: April 2014

MHC class II (MHC II) proteins are competent signaling molecules on APC. However, little is known about the mechanisms that control generation of their activating signals. Previous reports highlighted a number of factors that could affect the nature and outcome of MHC II signals, including the inability of MHC II ligation on resting vs activated murine B cells to induce mobilization of Ca2+. In the present study, we report that ligation of MHC II on resting murine B cells reproducibly induces mobilization of intracellular Ca2+ using both mAbs and cognate T cells as ligands. Mobilization of Ca2+ was independent of MHC II haplotype, isotype, or mouse genetic background. MHC II-mediated mobilization of Ca2+ is completely inhibited by inhibitors of src-like kinases and syk, and MHC II ligation increases overall tyrosine phosphorylation level. Moreover, MHC II ligation results in specific up-regulation of CD86. However, induction of these responses is dependent on the type of anti-MHC II Ab used, suggesting that epitope specificity and/or the nature of ligation is important. Moreover, we demonstrate that MHC II-derived signals are strictly regulated by the order and timing of BCR and CD40 signals, suggesting coordination of these signals preserves the integrity of early B cell priming events. Thus, the mode and the context of MHC II ligation influence generation of MHC II-derived activating signals in resting B cells. Based on these results, a new model that highlights the role of MHC II-activating signals in regulation of Ag presentation by B cells is proposed.
LF 15-0195 Treatment Protects against Central Nervous System Autoimmunity by Favoring the Development of Foxp3-Expressing Regulatory CD4 T Cells
Authors: Duplan Valérie , Beriou Gaëlle , Heslan Jean-Marie , Bruand Corinne , Dutartre Patrick , Mars Lennart T. , Liblau Roland S. , Cuturi Maria-Cristina , Saoudi Abdelhadi
Published: April 2014

Experimental autoimmune encephalomyelitis (EAE) is an instructive model for the human demyelinating disease multiple sclerosis. Lewis (LEW) rats immunized with myelin-basic protein (MBP) develop EAE characterized by a single episode of paralysis, from which they recover spontaneously and become refractory to a second induction of disease. LF 15-0195 is a novel molecule that has potent immunosuppressive effects in several immune-mediated pathological manifestations, including EAE. In the present study, we show that a 30-day course of LF 15-0195 treatment not only prevents MBP-immunized LEW rats from developing EAE but also preserves their refractory phase to reinduction of disease. This effect is Ag driven since it requires priming by the autoantigen during the drug administration. In contrast to other immunosuppressive drugs, short-term treatment with this drug induces a persistent tolerance with no rebound of EAE up to 4 mo after treatment withdrawal. This beneficial effect of LF 15-0195 on EAE does not result from the deletion of MBP-specific Vβ8.2 encephalitogenic T cells. In contrast, this drug favors the differentiation of MBP-specific CD4 T cells into Foxp3-expressing regulatory T cells that, upon adoptive transfer in syngeneic recipients, prevent the development of actively induced EAE. Finally, we demonstrate that the tolerance induced by LF 15-0195 treatment is not dependent on the presence of TGF-β. Together, these data demonstrate that short-term treatment with LF 15-0195 prevents MBP-immunized LEW rats from EAE by favoring the development of Foxp-3-expressing regulatory CD4 T cells.
Dopamine Selectively Induces Migration and Homing of Naive CD8+ T Cells via Dopamine Receptor D3
Authors: Watanabe Yoshiko , Nakayama Takashi , Nagakubo Daisuke , Hieshima Kunio , Jin Zhe , Katou Fuminori , Hashimoto Kenji , Yoshie Osamu
Published: April 2014

The nervous systems affect immune functions by releasing neurohormones and neurotransmitters. A neurotransmitter dopamine signals via five different seven-transmembrane G protein-coupled receptors termed D1 to D5. The secondary lymphoid tissues are highly innervated by sympathetic nerve fibers that store dopamine at high contents. Lymphocytes also produce dopamine. In this study, we examined expression and function of dopamine receptors in lymphocytes. We found that D3 was the predominant subtype of dopamine receptors in the secondary lymphoid tissues and selectively expressed by naive CD8+ T cells of both humans and mice. Dopamine induced calcium flux and chemotaxis in mouse L1.2 cells stably expressing human D3. These responses were almost completely inhibited by pertussis toxin, indicating that D3 was coupled with the Gαi class of G proteins. Consistently, dopamine selectively induced chemotactic responses in naive CD8+ T cells of both humans and mice in a manner sensitive to pertussis toxin and D3 antagonists. Dopamine was highly synergistic with CCL19, CCL21, and CXCL12 in induction of chemotaxis in naive CD8+ T cells. Dopamine selectively induced adhesion of naive CD8+ T cells to fibronectin and ICAM-1 through activation of integrins. Intraperitoneal injection of mice with dopamine selectively attracted naive CD8+ T cells into the peritoneal cavity. Treatment of mice with a D3 antagonist U-99194A selectively reduced homing of naive CD8+ T cells into lymph nodes. Collectively, naive CD8+ T cells selectively express D3 in both humans and mice, and dopamine plays a significant role in migration and homing of naive CD8+ T cells via D3.
Nonmutated Self-Antigen-Derived Cancer Vaccine Peptides Elicit an IgE-Independent but Mast Cell-Dependent Immediate-Type Skin Reaction without Systemic Anaphylaxis
Authors: Yamada Akira , Yano Hirohisa , Takao Yukari , Ono Takeharu , Matsumoto Tomoko , Itoh Kyogo
Published: April 2014

We previously reported an unexpected phenomenon, i.e., several cancer vaccine peptides, including a cyclophilin B-derived peptide (CypB-84), elicited an immediate-type skin reaction in prevaccination skin tests. These peptides were prohibited in the subsequent vaccinations because of a possible induction of systemic anaphylaxis. In this study, we investigated mechanisms involved in the peptide-elicited inflammatory reactions in BALB/c mice whose MHC class I molecule (Kd) shared similar binding motifs with the human HLA-A24 molecule. Among 11 peptides tested, all of which had been scheduled for use in clinical trials with HLA-A24+ cancer patients, three peptides (CypB-84, ART1-170, and ART4-13) elicited immediate footpad reactions in BALB/c mice similar to the skin reactions in humans. The footpad reaction was also observed in C57BL/6, athymic nu/nu, and CB17-SCID mice, but not in mast cell-deficient WBB6F1w/wv mice, indicating the reaction was not mediated by specific immunity, but was mast cell-dependent. Furthermore, the reactions were not correlated to in vivo antitumor effects of the peptides. An anaphylaxis was not elicited when the peptides were systemically injected due to a very rapid clearance of the peptides from the plasma by in vivo degradation. These results suggest that certain peptides of cancer vaccine candidates exhibit an IgE-independent but mast cell-dependent inflammatory response with no elicitation of systemic anaphylaxis, and may provide new insights for further development of peptide-based vaccinations for cancer patients.
The Decreased Susceptibility of Bcr/Abl Targets to NK Cell-Mediated Lysis in Response to Imatinib Mesylate Involves Modulation of NKG2D Ligands, GM1 Expression, and Synapse Formation
Authors: Cebo Christelle , Da Rocha Sylvie , Wittnebel Sebastian , Turhan Ali G. , Abdelali Jalil , Caillat-Zucman Sophie , Bourhis Jean Henri , Chouaib Salem , Caignard Anne
Published: April 2014

Chronic myeloid leukemia is a clonal multilineage myeloproliferative disease of stem cell origin characterized by the presence of the Bcr/Abl oncoprotein, a constitutively active tyrosine kinase. In previous studies, we have provided evidence that Bcr/Abl overexpression in leukemic cells increased their susceptibility to NK-mediated lysis by different mechanisms. In the present study, using UT-7/9 cells, a high level Bcr/Abl transfectant of UT-7 cells, we show that the treatment of Bcr/Abl target by imatinib mesylate (IM), a specific Abl tyrosine kinase inhibitor, hampers the formation of the NK/target immunological synapse. The main effect of IM involves an induction of surface GM1 ganglioside on Bcr/Abl transfectants that prevents the redistribution of MHC-related Ag molecules in lipid rafts upon interaction with NK cells. IM also affects cell surface glycosylation of targets, as assessed by binding of specific lectins resulting in the subsequent modulation of their binding to lectin type NK receptor, particularly NKG2D. In addition, we demonstrate that the tyrosine kinase activity repression results in a decrease of MHC-related Ags-A/B and UL-16-binding protein expression on Bcr/Abl transfectants UT-7/9. We show that NKG2D controls the NK-mediated lysis of UT-7/9 cells, and IM treatment inhibits this activating pathway. Taken together, our results show that the high expression of Bcr/Abl in leukemic cells controls the expression of NKG2D receptor ligands and membrane GM1 via a tyrosine kinase-dependent mechanism and that the modulation of these molecules by IM interferes with NK cell recognition and cytolysis of the transfectants.
B Cell Antigen Receptor and CD40 Differentially Regulate CD22 Tyrosine Phosphorylation
Authors: Fujimoto Manabu , Kuwano Yoshihiro , Watanabe Rei , Asashima Nobuko , Nakashima Hiroko , Yoshitake Satoko , Okochi Hitoshi , Tamaki Kunihiko , Poe Jonathan C. , Tedder Thomas F. , Sato Shinichi
Published: April 2014

Cell surface molecules on lymphocytes positively or negatively modulate the Ag receptor signaling, and thus regulate the fate of the cell. CD22 is a B cell-specific cell surface protein that contains multiple ITIMs in the cytoplasmic tail, and critically regulates B cell activation and survival. CD22 regulation on B cell signaling is complex because CD22 can have both positive and negative roles in various contexts. We generated phosphospecific polyclonal Abs reacting four major CD22 tyrosine motifs (Y762, Y807, Y822, and Y842) and analyzed the pattern and intensity of phosphorylation of these tyrosine residues. The tyrosine motifs, Y762, Y822, and Y842, are considered as ITIM, whereas the other, Y807, is suggested to be important for Grb2 recruitment. Approximately 10% of the four tyrosine residues were constitutively phosphorylated. Upon anti-IgM ligation, CD22 Y762 underwent most rapid phosphorylation, whereas all four tyrosine residues were eventually phosphorylated equally at ∼35% of all CD22 molecules in the cell. By contrast, anti-CD40 stimulation specifically up-regulated anti-IgM-induced phosphorylation of tyrosines within two ITIM motifs, Y762 and Y842, which was consistent with in vivo finding of the negative role of CD22 in CD40 signaling. Thus, CD22 phosphorylation is not only quantitatively but also qualitatively regulated by different stimulations, which may determine the outcome of B cell signaling.
Regulation of Th2 Cytokine Expression in NKT Cells: Unconventional Use of Stat6, GATA-3, and NFAT2
Authors: Wang Zheng-Yu , Kusam Saritha , Munugalavadla Veerendra , Kapur Reuben , Brutkiewicz Randy R. , Dent Alexander L.
Published: April 2014

NKT cells are unique in that they can produce high levels of both Th1 and Th2 cytokines, yet little is known about how NKT cells control the transcription of Th2 cytokines. The expression of IL-4 by NKT cells is independent of the Th2-associated transcription factor Stat6. We have found that Stat6 is critical for the expression of IL-5, IL-10, and IL-13 by NKT cells. However, the Th2 cell-associated transcription factor GATA-3, normally induced by Stat6 activation, is expressed at low levels in NKT cells. CD4+ NKT cells are highly enriched for Th2 cytokine expression compared with CD4− NKT cells, and we searched for transcription factors that are up-regulated in CD4+ NKT cells that could control Th2 cytokine expression. We found that the NFAT family member NFAT2 is selectively increased in CD4+ NKT cells. We tested the roles of NFAT2 and also GATA-3 in Th2 cytokine expression by retrovirus-mediated gene transduction into NKT cells and nonpolarized conventional T cells. Expression of NFAT2 increased the expression of IL-4 in both NKT cells and conventional T cells, and NFAT2 activated IL-10 in conventional T cells but not in NKT cells. GATA-3 strongly activated IL-4, IL-5, and IL-13 expression in conventional T cells but had comparatively weak effects on these cytokines in NKT cells. Thus, NFAT2, GATA-3, and Stat6 have surprisingly different roles in NKT cells than in conventional T cells. We propose that one mechanism by which CD4+ NKT cells express IL-4 independent of Stat6 is via increased NFAT2 activity.
Development of Functional B Cells in a Line of SCID Mice with Transgenes Coding for Anti-Double-Stranded DNA Antibody
Authors: Bosma Gayle C. , Oshinsky Jennifer , Kiefer Kerstin , Nakajima Pamela B. , Charan Deepshika , Congelton Cecil , Radic Marko , Bosma Melvin J.
Published: April 2014

Deletion or inactivation of anti-self (DNA) B cells has been reported in non-autoimmune mice bearing Ig transgenes that code for Abs with specificity for dsDNA or ssDNA. However, we report a case in which anti-dsDNA B cells appear to escape both deletion and inactivation. We show that B cells (B220+IgM+) can develop in non-autoimmune SCID mice bearing two site-directed transgenes, 3H9(56R) and Vκ8, that together code for an anti-dsDNA Ab. The B cells appear inactive, because the mice (56RVκ8 SCID mice) generally lack serum Ig. However, 56RVκ8 SCID mice are able to produce IgG Ab with specificity for dsDNA when they become “leaky” for T cells or are reconstituted with exogenous T cells from B cell-deficient JH−/− donors. Thus, anti-dsDNA B cells that escape deletion in 56RVκ8 SCID mice appear fully functional and can differentiate, class switch, and give rise to IgG-producing cells in the presence of T cells and self-Ag.
Anaphylactic Release of Mucosal Mast Cell Granule Proteases: Role of Serpins in the Differential Clearance of Mouse Mast Cell Proteases-1 and -2
Authors: Pemberton Alan D. , Wright Steven H. , Knight Pamela A. , Miller Hugh R. P.
Published: April 2014

The granule-derived mouse mast cell proteases-1 and -2 (mMCP-1 and -2) colocalize in similar quantities in mucosal mast cells but micrograms of mMCP-1 compared with nanograms of mMCP-2 are detected in peripheral blood during intestinal nematode infection. This differential systemic response was investigated both in vitro and in vivo. Bone marrow-derived mucosal mast cell homologs released similar quantities of mMCP-1 and-2 concomitantly with β-hexosaminidase in response to calcium ionophore (∼60% release) or IgE/DNP (25% release). In contrast, serum from mice sensitized by infection with Nippostrongylus brasiliensis 10 days earlier contained >1500-fold more mMCP-1 (10,130 ± 1,609 ng/ml) than mMCP-2 (6.4 ± 1 ng/ml), but, in gut lumen, the difference was ∼8-fold. After OVA sensitization, >600-fold more mMCP-1 (7,861 ± 2,209 ng/ml) than mMCP-2 (12.8 ± 4.7 ng/ml) was present in blood 1 h after challenge, but, in gut lumen, there were relatively comparable levels of mMCP-1 and -2. To estimate the rates of systemic accumulation and clearance, 10 μg of mMCP-1 or -2 was injected i.p. Plasma levels of injected mMCP-2 peaked (1%) at 15 min then declined, whereas levels of mMCP-1 were maximal (∼25%) at 3 h. Inactivation of mMCP-1 with PMSF before injection resulted in mMCP-2-like kinetics, but inhibition of mMCP-1 by serum gave kinetics similar to that of native mMCP-1. mMCP-1 isolated from serum is complexed with serpins and we conclude that both the accumulation and the longevity of mMCP-1 in blood is due to complex formation, protecting it from a pathway that rapidly clears mMCP-2, which is unable to form complexes with serpins.
IFN-Dependent Down-Regulation of the NKG2D Ligand H60 on Tumors
Authors: Bui Jack D. , Carayannopoulos Leonidas N. , Lanier Lewis L. , Yokoyama Wayne M. , Schreiber Robert D.
Published: April 2014

In this study, we show that IFN-γ or IFN-α reduce expression of H60 on 3′-methylcholanthrene (MCA) sarcomas from 129/Sv mice. As determined by flow cytometry using either NKG2D tetramers or NKG2D ligand-specific mAb, H60 was identified as the NKG2D ligand most frequently expressed on these sarcomas, and its expression was selectively down-regulated by either IFN-γ or IFN-α in a manner that was dose- and time-dependent and reversible. Down-regulation occurred at the transcript level and was STAT1-dependent. It also had functional consequences. IFN-γ-treated MCA sarcomas with high levels of H60 were resistant to killing by IL-2-activated NK cells. Resistance was not solely dependent on enhanced MHC class I expression but rather also required H60 down-regulation. IFN-γ-treated tumor cells also displayed diminished capacity to down-regulate NKG2D on freshly isolated NK cells. Transplanted tumor cells reisolated from immunocompetent mice displayed reduced H60 expression and increased MHC class I expression compared with tumor cells that were either left unmanipulated or reisolated from mice treated with neutralizing IFN-γ-specific mAb. This report thus represents the first demonstration that certain cytokines and specifically the IFNs regulate expression of specific NKG2D ligands on murine tumors. This process most likely helps to specify the type of immune effector cell populations that participate in host-protective antitumor responses.
IL-7 Induces Immunological Improvement in SIV-Infected Rhesus Macaques under Antiviral Therapy
Authors: Beq Stéphanie , Nugeyre Marie-Thérèse , Fang Raphaël Ho Tsong , Gautier David , Legrand Roger , Schmitt Nathalie , Estaquier Jérôme , Barré-Sinoussi Françoise , Hurtrel Bruno , Cheynier Rémi , Israël Nicole
Published: April 2014

Despite efficient antiretroviral therapy (ART), CD4+ T cell counts often remain low in HIV-1-infected patients. This has led to IL-7, a crucial cytokine involved in both thymopoiesis and peripheral T cell homeostasis, being suggested as an additional therapeutic strategy. We investigated whether recombinant simian IL-7-treatment enhanced the T cell renewal initiated by ART in rhesus macaques chronically infected with SIVmac251. Six macaques in the early chronic phase of SIV infection received antiretroviral treatment. Four macaques also received a 3-wk course of IL-7 injections. Viral load was unaffected by IL-7 treatment. IL-7 treatment increased the number of circulating CD4+ and CD8+ memory T cells expressing activation (HLA-DR+, CD25+) and proliferation (Ki-67+) markers. It also increased naive (CD45RAbrightCD62L+) T cell counts by peripheral proliferation and enhanced de novo thymic production. The studied parameters returned to pretreatment values by day 29 after the initiation of treatment, concomitantly to the appearance of anti-IL-7 neutralizing Abs, supporting the need for a nonimmunogenic molecule for human treatment. Thus, IL-7, which increases T cell memory and de novo renewal of naive T cells may have additional benefits in HIV-infected patients receiving ART.
Identification of CD8+ T Cell Epitopes within Lytic Antigens of Human Herpes Virus 8
Authors: Ribechini Eliana , Fortini Cinzia , Marastoni Mauro , Traniello Serena , Spisani Susanna , Monini Paolo , Gavioli Riccardo
Published: April 2014

The human herpesvirus 8 (HHV-8) is a γ herpesvirus with oncogenic potential which establishes a chronic infection that is normally controlled by the immune system of healthy individuals. In particular, CTL responses seem to play a key role in control of the infection. In this study, we characterized epitope-specific CTL responses in healthy HHV-8-seropositive individuals against four HHV-8 lytic Ags: open reading frames (ORF) 26, 70, K3, and K5. We found that the majority of subjects responded to at least one HHV-8 lytic Ag-derived epitope, and some of these epitopes represented dominant targets, suggesting that they could be relevant targets of CTL-mediated immunity in vivo, and may be involved in host control of HHV-8. Specifically, we identified three CTL epitopes from ORF 26, which are presented by HLA-A2, six CTL epitopes from ORF 70 presented by HLA-A2 (three epitopes), -A24 (two epitopes), and -B7 (one epitope), three CTL epitopes from ORF K3 presented by HLA-A2 (two epitopes) and -B7 (one epitope), and one HLA-A2 presented epitope derived from ORF K5. The identified epitopes may be regarded as useful tools for understanding the role of CTL responses to lytic Ags in individuals affected by HHV-8-associated disorders, and for the development of immunotherapies for the treatment/prevention of HHV-8-associated malignancies.
The 77C→G Mutation in the Human CD45 (PTPRC) Gene Leads to Increased Intensity of TCR Signaling in T Cell Lines from Healthy Individuals and Patients with Multiple Sclerosis
Authors: Do Hue-Tran , Baars Wiebke , Borns Katja , Windhagen Anja , Schwinzer Reinhard
Published: April 2014

The 77C→G mutation in exon A of the human CD45 gene occurs with low frequency in healthy individuals. An enhanced frequency of 77C→G individuals has been reported in cohorts of patients suffering from multiple sclerosis, systemic sclerosis, autoimmune hepatitis, and HIV-1. To investigate the mechanisms by which the variant allele may contribute to disease susceptibility, we compared T cell reactivity in heterozygous carriers of the mutation (healthy individuals and multiple sclerosis patients) and wild-type controls. In vitro-generated T cell lines and freshly isolated CD4+CD45R0+ primed/memory T cells from 77C→G individuals aberrantly expressed CD45RA isoforms and showed enhanced proliferation and IL-2 production when stimulated with anti-TCR/CD3 mAb or Ag. Mutant T cell lines contained a more active pool of p56lck tyrosine kinase and responded with increased phosphorylation of Zap70 and TCR-ζ and an enhanced Ca2+ flux to TCR/CD3 stimulation. These data suggest that 77C→G may act as a risk factor for certain diseases by increasing the intensity of TCR signaling.
Split Tolerance in Peripheral B Cell Subsets in Mice Expressing a Low Level of Igκ-Reactive Ligand
Authors: Aït-Azzouzene Djemel , Verkoczy Laurent , Duong Bao , Skog Patrick , Gavin Amanda L. , Nemazee David
Published: April 2014

Peripheral B cell tolerance differs from central tolerance in anatomic location, in the stage of B cell development, and in the diversity of Ag-responsive cells. B cells in secondary lymphoid organs are heterogeneous, including numerous subtypes such as B-1, marginal zone, transitional, and follicular B cells, which likely respond differently from one another to ligand encounter. We showed recently that central B cell tolerance mediated by receptor editing was induced in mice carrying high levels of a ubiquitously expressed κ-macroself Ag, a synthetic superantigen reactive to Igκ. In this study, we characterize a new transgenic line that has a distinctly lower expression pattern from those described previously; the B cell tolerance phenotype of these mice is characterized by the presence of significant numbers of immature κ+ B cells in the spleen, the loss of mature follicular and marginal zone B cells, the persistence of κ+ B-1 cells in the peritoneal cavity, and significant levels of serum IgM,κ. These findings suggest distinct signaling thresholds for tolerance among peripheral B cell subsets reactive with an identical ligand.
DNA Microarray Gene Expression Profile of T Cells with the Splice Variants of TCRζ mRNA Observed in Systemic Lupus Erythematosus
Authors: Tsuzaka Kensei , Nozaki Kyoko , Kumazawa Chika , Shiraishi Kiyono , Setoyama Yumiko , Yoshimoto Keiko , Suzuki Katsuya , Abe Tohru , Takeuchi Tsutomu
Published: April 2014

We have reported that the TCRζ mRNA with alternatively spliced 3′ UTR (ζ mRNA/as-3′-untranslated region (UTR)) and ζ mRNA lacking exon 7 (ζ mRNA/exon 7−) observed in systemic lupus erythematosus patient T cells can lead to down-regulation of both ζ and TCR/CD3 complexes. To determine whether these T cells expressing decreased ζ exhibit differential transcription patterns, we transfected retrovirus vectors containing wild-type ζ cDNA, ζ cDNA/as-3′ UTR, and ζ cDNA/exon 7− into murine T cell hybridoma MA5.8 cells which lack ζ expression to construct the MA5.8 mutants WT, AS3′ UTR, and EX7−, respectively. FACS analyses demonstrated reduced cell surface expression of ζ and TCR/CD3 complexes on the AS3′ UTR mutant and the EX7− mutant in comparison to that on the WT mutant. Total RNA was collected after stimulating the MA5.8 mutants with anti-CD3 Ab. Reverse-transcribed cDNA was applied to the mouse cDNA microarray containing 8691 genes, and the results were confirmed by real-time PCR. The results showed that 36 genes encoding cytokines and chemokines, including IL-2, IL-15, IL-18, and TGF-β2, were down-regulated in both the AS3′ UTR mutant and the EX7− mutant. Another 16 genes were up-regulated in both, and included genes associated with membranous proteins and cell damage granules, including the genes encoding poliovirus receptor-related 2, syndecan-1, and granzyme A. Increased protein expression of these genes was confirmed by Western blot and FACS analyses. Identification of these responsive genes in T cells in which the ζ and TCR/CD3 complexes were down-regulated may help to better understand the pathogenesis of systemic lupus erythematosus.
Sodium Salicylate Promotes Neutrophil Apoptosis by Stimulating Caspase-Dependent Turnover of Mcl-1
Authors: Derouet Mathieu , Thomas Luke , Moulding Dale A. , Akgul Cahit , Cross Andrew , Moots Robert J. , Edwards Steven W.
Published: April 2014

Mcl-1 is an antiapoptotic member of the Bcl-2 family of proteins that plays a central role in cell survival of neutrophils and other cells. The protein is unusual among family members in that it has a very short half-life of 2–3 h. In this report, we show that sodium salicylate (at 10 mM) greatly enhances the rate at which neutrophils undergo apoptosis and, in parallel, greatly accelerates the turnover rate of Mcl-1, decreasing its half-life to only 90 min. Whereas constitutive and GM-CSF-modified Mcl-1 turnover is regulated by the proteasome, the accelerated sodium salicylate-induced Mcl-1 turnover is mediated largely via caspases. Sodium salicylate resulted in rapid activation of caspase-3, -8, -9, and -10, and salicylate-accelerated Mcl-1 turnover was partly blocked by caspase inhibitors. Sodium salicylate also induced dramatic changes in the activities of members of the MAPK family implicated in Mcl-1 turnover and apoptosis. For example, sodium salicylate blocked GM-CSF-stimulated Erk and Akt activation, but resulted in rapid and sustained activation of p38-MAPK, an event mimicked by okadaic acid that also accelerates Mcl-1 turnover and neutrophil apoptosis. These data thus shed important new insights into the dynamic and highly regulated control of neutrophil apoptosis that is effected by modification in the rate of Mcl-1 turnover.
Prostaglandin E2 Is Generally Required for Human Dendritic Cell Migration and Exerts Its Effect via EP2 and EP4 Receptors
Authors: Legler Daniel F. , Krause Petra , Scandella Elke , Singer Eva , Groettrup Marcus
Published: April 2014

The control of dendritic cell (DC) migration is pivotal for the initiation of cellular immune responses. In this study, we demonstrate that the migration of human monocyte-derived (Mo)DCs as well as of ex vivo peripheral blood DCs toward CCL21, CXCL12, and C5a is stringently dependent on the presence of the proinflammatory mediator PGE2, although DCs expressed CXCR4 and C5aR on their surface and DC maturation was accompanied by CCR7 up-regulation independently of PGE2. The necessity of exogenous PGE2 for DC migration is not due to the suppression of PGE2 synthesis by IL-4, which is used for MoDC differentiation, because maturation-induced endogenous production of PGE2 cannot promote DC migration. Surprisingly, PGE2 was absolutely required at early time points of maturation to enable MoDC chemotaxis, whereas PGE2 addition during terminal maturation events was ineffective. In contrast to mouse DCs, which exclusively rely on EP4 receptor triggering for migration, human MoDCs require a signal mediated by EP2 or EP4 either alone or in combination. Our results provide clear evidence that PGE2 is a general and mandatory factor for the development of a migratory phenotype of human MoDCs as well as for peripheral blood myeloid DCs.
OX40 Costimulation Synergizes with GM-CSF Whole-Cell Vaccination to Overcome Established CD8+ T Cell Tolerance to an Endogenous Tumor Antigen
Authors: Murata Satoshi , Ladle Brian H. , Kim Peter S. , Lutz Eric R. , Wolpoe Matthew E. , Ivie Susan E. , Smith Holly M. , Armstrong Todd D. , Emens Leisha A. , Jaffee Elizabeth M. , Reilly R. Todd
Published: April 2014

T cell costimulation via OX40 is known to increase CD4+ T cell expansion and effector function and enhances the development of T cell memory. OX40 costimulation can also prevent, and even reverse, CD4+ T cell anergy. However, the role of OX40 in CD8+ T cell function is less well defined, particularly in the setting of immune tolerance. To determine the effects of OX40 costimulation on the induction of the host CD8+ T cell repertoire to an endogenous tumor Ag, we examined the fate of CD8+ T cells specific for the immunodominant rat HER-2/neu epitope, RNEU420–429, in FVB MMTV-neu (neu-N) mice, which express rat HER-2/neu protein in a predominantly mammary-restricted fashion. We show that the RNEU420–429-specific T cell repertoire in neu-N mice expands transiently after vaccination with a neu-targeted GM-CSF-secreting whole-cell vaccine, but quickly declines to an undetectable level. However, OX40 costimulation, when combined with GM-CSF-secreting tumor-targeted vaccination, can break established CD8+ T cell tolerance in vivo by enhancing the expansion, and prolonging the survival and effector function of CD8+ T cells specific for RNEU420–429. Moreover, we demonstrate that OX40 expression is up-regulated on both CD4+ and CD8+ T cells shortly after administration of a GM-CSF expressing vaccine. These studies highlight the increased efficacy of OX40 costimulation when combined with a GM-CSF-secreting vaccine, and define a new role for OX40 costimulation of CD8+ T cells in overcoming tolerance and boosting antitumor immunity.
Effect of CD4+CD25+ and CD4+CD25− T Regulatory Cells on the Generation of Cytolytic T Cell Response to a Self but Human Tumor-Associated Epitope In Vitro
Authors: Chattopadhyay Subhasis , Mehrotra Shikhar , Chhabra Arvind , Hegde Upendra , Mukherji Bijay , Chakraborty Nitya G.
Published: April 2014

CD4+ T cells naturally expressing CD25 molecules (natural T regulatory cells (Tregs)) have a role in maintaining self tolerance and in regulating responses to infectious agents, transplantation Ags, and tumor Ags. CD4+ Tregs induced from CD4+CD25− precursors (induced Tregs) also regulate immune responses in the periphery. However, which of these Tregs is a major impediment in generating antitumor CTL responses is not clear. We show that although the CD4+CD25+ subsets isolated from peripheral blood-derived lymphocytes do suppress the proliferation of CD4+CD25− effector T cells, they do not suppress the activation and expansion of the self but melanoma-associated, melanoma Ag-reactive T cell 1 (MART-1)27–35-specific CD8+ T cells stimulated by the respective peptide-loaded matured dendritic cells in vitro. The CD4+CD25− counterparts, in contrast, lead to the generation of CD25+ glucocorticoid-inducible TNFR+-Forkhead/winged helix transcription factor+ populations and efficiently suppress the activation and expansion of the MART-127–35 epitope-specific CTLs. Our data suggest that when CTL precursors are optimally stimulated, natural Tregs are not a formidable constraint toward generating a robust antitumor CTL response, but induced Tregs could be.
HIV-1 Selectively Infects a Subset of Nonmaturing BDCA1-Positive Dendritic Cells in Human Blood
Authors: Granelli-Piperno Angela , Shimeliovich Irina , Pack Maggi , Trumpfheller Christine , Steinman Ralph M.
Published: April 2014

The infection of cultured monocyte-derived dendritic cells (DCs) with HIV-1 involves CD4 and CCR5 receptors, while transmission to T cells is enhanced at least in part by the lectin DC-SIGN/CD209. In the present study, we studied BDCA-1+ myeloid DCs isolated directly from human blood. These cells express CD4 and low levels of CCR5 and CXCR4 coreceptors, but not DC-SIGN. The myeloid DCs replicate two R5 viruses, BaL and YU2, and transfer infection to activated T cells. The virus productively infects a small fraction of the blood DCs that fail to mature in culture, as indicated by the maturation markers CD83 and DC-LAMP/CD208, and the expression of high CD86 and MHC class II, in contrast to many noninfected DCs. A greater proportion of BDCA-1+ DCs are infected when the virus is pseudotyped with the vesicular stomatitis envelope VSV-G (5–15%), as compared with the R5 virus (0.3–3.5%), indicating that HIV-1 coreceptors may limit the susceptibility of DCs to become infected, or the endocytic route of viral entry used by HIV/vesicular stomatitis virus enhances infectivity. When infected and noninfected cells are purified by cell sorting, the former uniformly express HIV p24 gag and are virtually inactive as stimulators of the allogeneic MLR, in contrast to potent stimulation by noninfected DCs from the same cultures. These results point to two roles for a small fraction of blood DCs in HIV-1 pathogenesis: to support productive infection and to evade the direct induction of T cell-mediated immunity.
Pertussis Toxin B-Oligomer Suppresses IL-6 Induced HIV-1 and Chemokine Expression in Chronically Infected U1 Cells via Inhibition of Activator Protein 1
Authors: Rizzi Chiara , Crippa Massimo P. , Jeeninga Rienk E. , Berkhout Ben , Blasi Francesco , Poli Guido , Alfano Massimo
Published: April 2014

Pertussis toxin B-oligomer (PTX-B) inhibits HIV replication in T lymphocytes and monocyte-derived macrophages by interfering with multiple steps of the HIV life cycle. PTX-B prevents CCR5-dependent (R5) virus entry in a noncompetitive manner, and it also exerts suppressive effects on both R5- and CXCR4-dependent HIV expression at a less-characterized postentry level. We demonstrate in this study that PTX-B profoundly inhibits HIV expression in chronically infected promonocytic U1 cells stimulated with several cytokines and, particularly, the IL-6-mediated effect, a cytokine that triggers viral production in these cells independently of NF-κB activation. From U1 cells we have subcloned a cell line, named U1-CR1, with increased responsiveness to IL-6. In these cells, PTX-B neither down-regulated the IL-6R nor prevented IL-6 induced signaling in terms of STAT3 phosphorylation and DNA binding. In contrast, PTX-B inhibited AP-1 binding to target DNA and modified its composition with a proportional increases in FosB, Fra2, and ATF2. PTX-B inhibited IL-6-induced HIV-1 long-terminal repeat-driven transcription from A, C, E, and F viral subtypes, which contain functional AP-1 binding sites, but failed to inhibit transcription from subtypes B and D LTR devoid of these sites. In addition, PTX-B inhibited the secretion of IL-6-induced, AP-1-dependent genes, including urokinase-type plasminogen activator, CXCL8/IL-8, and CCL2/monocyte chemotactic protein-1. Thus, PTX-B suppression of IL-6 induced expression of HIV and cellular genes in chronically infected promonocytic cells is strongly correlated to inhibition of AP-1.
The Central Role of the Alternative Complement Pathway in Human Disease
Authors: Thurman Joshua M. , Holers V. Michael
Published: April 2014

The complement system is increasingly recognized as important in the pathogenesis of tissue injury in vivo following immune, ischemic, or infectious insults. Within the complement system, three pathways are capable of initiating the processes that result in C3 activation: classical, alternative, and lectin. Although the roles that proinflammatory peptides and complexes generated during complement activation play in mediating disease processes have been studied extensively, the relative contributions of the three activating pathways is less well understood. Herein we examine recent evidence that the alternative complement pathway plays a key and, in most instances, obligate role in generating proinflammatory complement activation products in vivo. In addition, we discuss new concepts regarding the mechanisms by which the alternative pathway is activated in vivo, as recent clinical findings and experimental results have provided evidence that continuous active control of this pathway is necessary to prevent unintended targeting and injury to self tissues.
Cutting Edge: Identification of E-Cadherin as a Ligand for the Murine Killer Cell Lectin-Like Receptor G1
Authors: Gründemann Carsten , Bauer Monika , Schweier Oliver , von Oppen Nanette , Lässing Ute , Saudan Philippe , Becker Karl-Friedrich , Karp Klaus , Hanke Thomas , Bachmann Martin F. , Pircher Hanspeter
Published: April 2014

The killer cell lectin-like receptor G1 (KLRG1) is expressed by NK cells and by T cells. In both humans and mice, KLRG1 identifies Ag-experienced T cells that are impaired in their proliferative capacity but are capable of performing effector functions. In this study, we identified E-cadherin as a ligand for murine KLRG1 by using fluorescently labeled, soluble tetrameric complexes of the extracellular domain of the murine KLRG1 molecule as staining reagents in expression cloning. Ectopic expression of E-cadherin in B16.BL6 target cells did not affect cell-mediated lysis by lymphokine-activated NK cells and by CD8 T cells but inhibited Ag-induced proliferation and induction of cytolytic activity of CD8 T cells. E-cadherin is expressed by normal epithelial cells, Langerhans cells, and keratinocytes and is usually down-regulated on metastatic cancer cells. KLRG1 ligation by E-cadherin in healthy tissue may thus exert an inhibitory effect on primed T cells.
Cutting Edge: Deficiency in the E3 Ubiquitin Ligase Cbl-b Results in a Multifunctional Defect in T Cell TGF-β Sensitivity In Vitro and In Vivo
Authors: Wohlfert Elizabeth A. , Gorelik Leonid , Mittler Robert , Flavell Richard A. , Clark Robert B.
Published: April 2014

Mice deficient in the E3 ubiquitin ligase Cbl-b have CD28-independent T cells and develop autoimmunity. We previously reported that Cbl-b−/− CD4+CD25− T effector cells are resistant in vitro to the antiproliferative effects of CD4+CD25+ regulatory T cells and TGF-β. We have now asked whether the resistance noted in Cbl-b−/− T cells is restricted solely to TGF-β’s antiproliferative effects, whether the TGF-β resistance has in vivo relevance, and whether a defect can be identified in the TGF-β signaling pathway. We now demonstrate the following: 1) in vitro, Cbl-b deficiency prevents the TGF-β-mediated induction of Foxp3+ functional regulatory T cells; 2) in vivo, Cbl-b−/− mice show a significantly enhanced response to a tumor that is strictly TGF-β regulated; and 3) Cbl-b−/− T effector cells have defective TGF-β-mediated Smad2 phosphorylation. These studies are the first to document that the E3 ubiquitin ligase Cbl-b plays an integral role in T cell TGF-β signaling, and that its absence results in multifunctional TGF-β-related defects that have important disease-related implications.
Cutting Edge: A Novel Nonoxidative Phagosomal Mechanism Exerted by Cathepsin-D Controls Listeria monocytogenes Intracellular Growth
Authors: Cerro-Vadillo Elida del , Madrazo-Toca Fidel , Carrasco-Marín Eugenio , Fernandez-Prieto Lorena , Beck Christian , Leyva-Cobián Francisco , Saftig Paul , Alvarez-Dominguez Carmen
Published: April 2014

Deciphering how Listeria monocytogenes exploits the host cell machinery to invade mammalian cells is a key issue in understanding the pathogenesis of this food-borne pathogen, which can cause diseases ranging from gastroenteritis to meningitis and abortion. In this study, we show that the lysosomal aspartyl-protease cathepsin-D (Ctsd) is of considerable importance for nonoxidative listericidal defense mechanisms. We observed enhanced susceptibility to L. monocytogenes infection of fibroblasts and bone-marrow macrophages and increased intraphagosomal viability of bacteria in fibroblasts isolated from Ctsd-deficient mice compared with wild type. These findings are further supported by prolonged survival of L. monocytogenes in Ctsd-deficient mice after infection. Transient transfection of Ctsd in wild-type cells was sufficient to revert these wild-type phagosomes back to microbicidal compartments. Based on infection experiments with mutant bacteria, in vitro degradation, and immunoprecipitation experiments, we suggest that a major target of cathepsin D is the main virulence factor listeriolysin O.
Cutting Edge: Prostaglandin D2 Enhances Leukotriene C4 Synthesis by Eosinophils during Allergic Inflammation: Synergistic In Vivo Role of Endogenous Eotaxin
Authors: Mesquita-Santos Fabio P. , Vieira-de-Abreu Adriana , Calheiros Andrea S. , Figueiredo Isabela H. , Castro-Faria-Neto Hugo C. , Weller Peter F. , Bozza Patrícia T. , Diaz Bruno L. , Bandeira-Melo Christianne
Published: April 2014

In addition to the well-recognized ability of prostaglandin D2 (PGD2) to regulate eosinophil trafficking, we asked whether PGD2 was also able to activate eosinophils and control their leukotriene C4 (LTC4)-synthesizing machinery. PGD2 administration to presensitized mice enhanced in vivo LTC4 production and formation of eosinophil lipid bodies–potential LTC4-synthesizing organelles. Immunolocalization of newly formed LTC4 demonstrated that eosinophil lipid bodies were the sites of LTC4 synthesis during PGD2-induced eosinophilic inflammation. Pretreatment with HQL-79, an inhibitor of PGD synthase, abolished LTC4 synthesis and eosinophil lipid body formation triggered by allergic challenge. Although PGD2 was able to directly activate eosinophils in vitro, in vivo PGD2-induced lipid body-driven LTC4 synthesis within eosinophils was dependent on the synergistic activity of endogenous eotaxin acting via CCR3. Our findings, that PGD2 activated eosinophils and enhanced LTC4 synthesis in vivo in addition to the established PGD2 roles in eosinophil recruitment, heighten the interest in PGD2 as a target for antiallergic therapies.