bluuebunny/crossref_metadata_2025 huggingface dataset:
TCR-Mediated Up-Regulation of c-FLIPshort Correlates with Resistance Toward CD95-Mediated Apoptosis by Blocking Death-Inducing Signaling Complex Activity
Authors: Kirchhoff Sabine , Müller Wolfgang W. , Krueger Andreas , Schmitz Ingo , Krammer Peter H.
Published: April 2014

To investigate apoptosis resistance upon restimulation in human peripheral blood T lymphocytes, we used the following in vitro model. This model represents the main features of T cell reactivity: freshly isolated PHA-activated T cells cultured in IL-2 for a prolonged period of time develop a CD95 (APO-1/Fas) apoptosis-sensitive phenotype. These T cells represent activation-induced cell death-sensitive T cells during the down phase of an immune response. A fraction of apoptosis-sensitive activated T cells becomes apoptosis resistant upon TCR/CD3 restimulation. CD95 apoptosis sensitivity requires formation of a functional receptor associated death-inducing signaling complex (DISC), i.e., a protein complex of CD95 receptors, the adaptor Fas-associated death domain protein (FADD)/MORT1 and caspase-8 (FADD-like IL-1β-converting enzyme (FLICE), MACH, Mch5). We identified activation of procaspase-8 at the DISC as the main target for the protective activity of TCR/CD3 restimulation. We found that procaspase-8 cleavage is reduced in T cells after TCR/CD3 restimulation. In addition, we detected up-regulation of c-FLIPS (the short splice variant of the cellular FLICE inhibitory protein) and strongly enhanced recruitment of c-FLIPS into the DISC. These data suggest that the recruitment of c-FLIPS into the DISC results in reduced DISC and caspase-8 activity.
Ku86 Variant Expression and Function in Multiple Myeloma Cells Is Associated with Increased Sensitivity to DNA Damage
Authors: Tai Yu-Tzu , Teoh Gerrard , Lin Boris , Davies Faith E. , Chauhan Dharminder , Treon Steven P. , Raje Noopur , Hideshima Teru , Shima Yoshihito , Podar Klaus , Anderson Kenneth C.
Published: April 2014

Ku is a heterodimer of Ku70 and Ku86 that binds to double-stranded DNA breaks (DSBs), activates the catalytic subunit (DNA-PKcs) when DNA is bound, and is essential in DSB repair and V(D)J recombination. Given that abnormalities in Ig gene rearrangement and DNA damage repair are hallmarks of multiple myeloma (MM) cells, we have characterized Ku expression and function in human MM cells. Tumor cells (CD38+CD45RA−) from 12 of 14 (86%) patients preferentially express a 69-kDa variant of Ku86 (Ku86v). Immunoblotting of whole cell extracts (WCE) from MM patients shows reactivity with Abs targeting Ku86 N terminus (S10B1) but no reactivity with Abs targeting Ku86 C terminus (111), suggesting that Ku86v has a truncated C terminus. EMSA confirmed a truncated C terminus in Ku86v and further demonstrated that Ku86v in MM cells had decreased Ku-DNA end binding activity. Ku86 forms complexes with DNA-PKcs and activates kinase activity, but Ku86v neither binds DNA-PKcs nor activates kinase activity. Furthermore, MM cells with Ku86v have increased sensitivity to irradiation, mitomycin C, and bleomycin compared with patient MM cells or normal bone marrow donor cells with Ku86. Therefore, this study suggests that Ku86v in MM cells may account for decreased DNA repair and increased sensitivity to radiation and chemotherapeutic agents, whereas Ku86 in MM cells confers resistance to DNA damaging agents. Coupled with a recent report that Ku86 activity correlates with resistance to radiation and chemotherapy, these results have implications for the potential role of Ku86 as a novel therapeutic target.
The Expression of MHC Class II Genes in Macrophages Is Cell Cycle Dependent
Authors: Xaus Jordi , Comalada Mònica , Barrachina Marta , Herrero Carmen , Goñalons Eduard , Soler Concepció , Lloberas Jorge , Celada Antonio
Published: April 2014

Using different drugs, we stopped the cell cycle of bone marrow-derived macrophages at different points. After IFN-γ stimulation, macrophages arrested at the G1 phase of the cell cycle did not increase cell surface expression of the MHC class II IA. This inhibition is specific, because, under the same conditions, IFN-γ induces the expression of Fcγ receptors and the inducible NO synthase mRNA. Treatments that inhibit macrophage proliferation by blocking the cell cycle at the G1 phase, such as adenosine, forskolin, or LPS, blocked the IFN-γ induction of IA. Under IFN-γ treatment, the steady-state levels of IAα and IAβ mRNA did not increase in cells arrested at the G1 phase and the half-life of the MHC mRNA was not modified. These data suggest that the cell cycle modulation of IFN-γ-induced MHC II gene expression occurs at the transcriptional level. The expression of the class II transactivator mRNA induced by IFN-γ was also blocked when macrophages were arrested at the G1 phase of the cell cycle, suggesting that the lack of IFN-γ response occurs at the early steps of MHC class II expression. Finally, macrophages arrested at the G1 phase showed increased basal levels of cell surface IA due to an increase of the translational efficiency. These data show that the expression of MHC class II genes is regulated by the cell cycle.
Cloning of a Novel Scavenger Receptor Cysteine-Rich Type I Transmembrane Molecule (M160) Expressed by Human Macrophages
Authors: Grønlund Jørn , Vitved Lars , Lausen Mette , Skjødt Karsten , Holmskov Uffe
Published: April 2014

We report the cloning of a novel human type I cell surface Ag mainly expressed by macrophages. The primary structure was established by molecular cloning, which yielded a 4579-bp cDNA sequence encoding a polypeptide chain of 1453 amino acid residues with 16 potential N-glycosylation sites. We designated this molecule M160. The domain organization features 12 scavenger receptor cysteine-rich domains followed by a transmembrane region and a cytoplasmic domain that occurs in two forms, a predominant form (M160-α) of 71 residues and an alternatively spliced form (M160-β) of 39 residues. M160-α contains three possible phosphorylation sites, which are lost in the alternatively spliced form. RT-PCR analyses showed M160 to be expressed by alveolar macrophages and by the monocyte cell lines HL60, U937, and THP1, but not by Jurkat or Raji cells. Stimulation of U937 cells with phorbol ester resulted in an increased expression of M160 from day 5 onward. RT-PCR analysis of 19 different human tissues showed signals for M160-α of varying intensity in all tissues, whereas M160-β was confined to the spleen. We conclude that M160 is a new member of the scavenger receptor cysteine-rich superfamily expressed by the monocyte/macrophage cell lineage.
The Role of Macrophage Inflammatory Protein-1α/CCL3 in Regulation of T Cell-Mediated Immunity toCryptococcus neoformansInfection
Authors: Olszewski Michal A. , Huffnagle Gary B. , McDonald Roderick A. , Lindell Dennis M. , Moore Bethany B. , Cook Donald N. , Toews Galen B.
Published: April 2014

Macrophage inflammatory protein-1α (MIP-1α/CCL3) is a CC chemokine required for optimal recruitment of leukocytes in response to cryptococcal Ags. MIP-1α is expressed in the lungs by day 6 post Cryptococcus neoformans infection and could play a role in the development of cell-mediated immunity. To address this possibility, wild-type (MIP-1α+/+) mice and MIP-1α knockout (MIP-1α−/−) mice were infected intratracheally with a highly virulent strain of C. neoformans (145A). MIP-1α message was detected in the lungs on days 3, 7, and 14 in MIP-1α+/+ mice, but it was undetectable in MIP-1α−/− mice. On day 16, MIP-1α−/− mice had a 7-fold increase in C. neoformans burden in the lungs, but no decrease in pulmonary leukocyte recruitment. MIP-1α+/+ and MIP-1α−/− mice had similar numbers of recruited lymphocytes and monocytes/macrophages. Notably, MIP-1α−/− mice had a significantly greater number of eosinophils. MIP-1α−/− mice had extremely high levels of serum IgE. This switch of immune response to a T2 phenotype was associated with enhanced IL-4 and IL-13 expression in the lungs of MIP-1α−/− mice compared with MIP-1α +/+ mice. Progression of pulmonary cryptococcosis in the presence of nonprotective T2 immunity resulted in profound lung damage in MIP-1α−/− mice (eosinophilic crystal deposition, destruction of lung parenchyma, and pulmonary hemorrhage). Twelve-week survival was dramatically decreased in MIP-1α−/− mice. These studies, together with our previous studies, demonstrate that MIP-1α plays a role in both the afferent (T1/T2 development) and efferent (T1-mediated leukocyte recruitment) phases of cell-mediated immunity to C. neoformans.
The Attachment (G) Glycoprotein of Respiratory Syncytial Virus Contains a Single Immunodominant Epitope That Elicits Both Th1 and Th2 CD4+ T Cell Responses
Authors: Varga Steven M. , Wissinger Erika L. , Braciale Thomas J.
Published: April 2014

BALB/c mice immunized with a vaccinia virus expressing the attachment (G) glycoprotein of respiratory syncytial virus (RSV) develop a virus-specific CD4+ T cell response that consists of a mixture of Th1 and Th2 CD4+ T cells following intranasal infection with live RSV. Recent work has shown that both Th1 and Th2 CD4+ T cells are elicited to a single region comprising aa 183–197 of the G protein. To more precisely define the CD4+ T cell epitope(s) contained within this region, we created a panel of amino- and carboxyl-terminal truncated as well as single alanine-substituted peptides spanning aa 183–197. These peptides were used to examine the ex vivo cytokine response of memory effector CD4+ T cells infiltrating the lungs of G-primed RSV-infected mice. Analysis of lung-derived memory effector CD4+ T cells using intracellular cytokine staining and/or ELISA of effector T cell culture supernatants revealed a single I-Ed-restricted CD4+ T cell epitope with a core sequence mapping to aa 185–193. In addition, we examined the T cell repertoire of the RSV G peptide-specific CD4+ T cells and show that the CD4+ T cells directed to this single immunodominant G epitope use a restricted range of TCR Vβ genes and predominantly express Vβ14 TCR.
Stromal Cell-Derived Factor-1-CXC Chemokine Receptor 4 Interactions Play a Central Role in CD4+ T Cell Accumulation in Rheumatoid Arthritis Synovium
Authors: Nanki Toshihiro , Hayashida Kenji , El-Gabalawy Hani S. , Suson Sharon , Shi Kenrin , Girschick Hermann J. , Yavuz Sule , Lipsky Peter E.
Published: April 2014

Rheumatoid arthritis (RA) is characterized by the accumulation of CD4+ memory T cells in the inflamed synovium. To address the mechanism, we analyzed chemokine receptor expression and found that the frequency of CXC chemokine receptor (CXCR)4 expressing synovial tissue CD4+ memory T cells was significantly elevated. CXCR4 expression could be enhanced by IL-15, whereas stromal cell-derived factor (SDF)-1, the ligand of CXCR4, was expressed in the RA synovium and could be increased by CD40 stimulation. SDF-1 stimulated migration of rheumatoid synovial T cells and also inhibited activation-induced apoptosis of T cells. These results indicate that SDF-1-CXCR4 interactions play important roles in CD4+ memory T cell accumulation in the RA synovium, and emphasize the role of stromal cells in regulating rheumatoid inflammation.
Activated Human T Lymphocytes Express a Functional C3a Receptor
Authors: Werfel Thomas , Kirchhoff Konstanze , Wittmann Miriam , Begemann Gabriele , Kapp Alexander , Heidenreich Feodor , Götze Otto , Zwirner Jörg
Published: April 2014

The C3a molecule is an anaphylatoxin of the C system with a wide spectrum of proinflammatory effects predominantly on cells of myeloid origin. In this study we investigated the expression of the high affinity receptor for C3a (C3aR) in human T lymphocytes using receptor-specific mAb. C3aR expression was detected in CD4+ and CD8+ blood- or skin-derived T cell clones (TCC) from birch pollen-sensitized patients with atopic dermatitis. No significant difference in C3aR expression in CD4+ or CD8+ TCCs could be observed. In contrast to C3a(desArg), C3a led to a transient calcium flux in TCCs expressing the C3aR, whereas C3aR-negative TCCs were unreactive. Circulating T cells from patients suffering from severe inflammatory skin diseases expressed the C3aR, whereas no expression of C3aR could be found in unstimulated T lymphocytes from patients with mild inflammatory skin diseases or from healthy individuals. Type I IFNs, which are potent stimulators of cellular immunity, were identified as up-regulators of C3aR expression in vitro in freshly isolated or cloned T lymphocytes. Moreover, C3aR+ T cells were found at the sites of injection in IFN-β-treated patients with multiple sclerosis. These data provide direct evidence for the expression of C3aR on activated human T lymphocytes; this may point to a biological function of C3a in T cell-dependent diseases.
Cutting Edge: C-C Chemokine Receptor 6 Is Essential for Arrest of a Subset of Memory T Cells on Activated Dermal Microvascular Endothelial Cells Under Physiologic Flow Conditions In Vitro
Authors: Fitzhugh David J. , Naik Shubhada , Caughman S. Wright , Hwang Sam T.
Published: April 2014

Memory T cells (mTC) express multiple chemokine receptors (including CCR4 and CCR6) that may potentially be involved in their arrest on inflamed endothelia. Herein, we specifically addressed whether CCR6 is required for mTC to arrest on TNF-α-activated human dermal microvascular endothelial cells (HDMEC) in vitro under shear stress conditions. Recombinant liver and activation-regulated chemokine (LARC)/CCL20 (a CCR6 ligand) induced firm arrest of cutaneous lymphocyte Ag+ mTC in a flow chamber system using purified substrates. Strikingly, desensitization of CCR6 with LARC, but not thymus and activation-regulated chemokine/CCL17 or secondary lymphoid tissue chemokine/CCL21, caused a 50–75% decrease (p < 0.001) in arrest of mTC on HDMEC, which was indistinguishable from the reduction observed when total mTC were treated with pertussis toxin (p > 0.5). CCR6-depleted mTC also had a markedly reduced ability to arrest on HDMEC. Our results suggest that LARC production by activated endothelial cells and CCR6 expression by mTC may be critical components in the pertussis toxin-sensitive arrest of mTC on activated HDMEC.
Cutting Edge: Naturally Occurring Soluble Form of Mouse Toll-Like Receptor 4 Inhibits Lipopolysaccharide Signaling
Authors: Iwami Ken-ichiro , Matsuguchi Tetsuya , Masuda Akio , Kikuchi Takeshi , Musikacharoen Tipayaratn , Yoshikai Yasunobu
Published: April 2014

Toll-like receptors (TLRs) are a family of proteins playing important roles in host defense. Mice defective of functional TLR4 are hyporesponsive to LPS, suggesting that TLR4 is essential for LPS signaling. Here we report the cloning of an alternatively spliced mouse TLR4 (mTLR4) mRNA. The additional exon exists between the second and third exon of the reported mTLR4 gene and contains an in-frame stop codon. The alternatively spliced mRNA encodes 86 aa of the reported mTLR4 and an additional 36 aa. This alternatively spliced mTLR4 mRNA expressed a partially secretary 20-kDa protein, which we named soluble mTLR4 (smTLR4). In a mouse macrophage cell line, the exogenously expressed smTLR4 significantly inhibited LPS-mediated TNF-α production and NF-κB activation. Additionally, in mouse macrophages, LPS increased the mRNA for smTLR4. Taken together, our results indicate that smTLR4 may function as a feedback mechanism to inhibit the excessive LPS responses in mouse macrophages.
Cutting Edge: Chromatin Remodeling at the IL-4/IL-13 Intergenic Regulatory Region for Th2-Specific Cytokine Gene Cluster
Authors: Takemoto Naofumi , Kamogawa Yumiko , Jun Lee Hyun , Kurata Hirokazu , Arai Ken-ichi , O’Garra Anne , Arai Naoko , Miyatake Shoichiro
Published: April 2014

During the differentiation of naive Th cells into Th2 effector cells, the entire IL-4/IL-13 locus is remodeled into an accessible chromatin conformation. Here we show that ectopic expression and activation of Stat6 or GATA-3 in Th cells developing under Th1-polarizing conditions lead to the induction of chromatin remodeling not only at the flanking regions of the IL-4 and IL-13 genes but also at the IL-4/IL-13 intergenic regulatory region for the IL-4/IL-13/IL-5 gene cluster. Furthermore, we demonstrate that GATA-3 and another Th2-specific, inducible protein complex interact with the IL-4/IL-13 intergenic DNase I hypersensitive region specifically in Th2 cells.
CDK4 Expression and Activity Are Required for Cytokine Responsiveness in T Cells
Authors: Modiano Jaime F. , Mayor Jocelyne , Ball Carrie , Fuentes Maren K. , Linthicum D. Scott
Published: April 2014

Stimulation of lymphocytes through the Ag receptor can lead to cytokine responsiveness or unresponsiveness. We examined the importance of cyclin-dependent kinase (CDK)4 to establish and maintain IL-2 responsiveness in human T cells. Our results show that a herbimycin A- and staurosporine-sensitive phase of CDK4 expression and activity preceded the acquisition of IL-2-responsiveness in mitogen-stimulated peripheral blood T cells. Intriguingly, CDK4 expression and activity were demonstrable in purified unstimulated peripheral blood T cells from ∼30% (5/16) of healthy individuals examined for this study. These T cells proliferated in response to IL-2 without additional mitogens, and both the expression and activity of CDK4 and the ability to respond to cytokines were resistant to herbimycin A and staurosporine. The pattern of CDK4 expression and response to IL-2 in this subset of individuals resembled that seen in the human IL-2-dependent Kit-225 T cell line. However, in contrast to normal T cells, Kit-225 cells were rendered unresponsive to IL-2 by stimulation through the Ag receptor. In these cells, PHA, anti-CD3, or PMA induced marked reductions of CDK4 expression and activity that paralleled IL-2 unresponsiveness, and these effects were not reversible by IL-2. Furthermore, IL-2-dependent proliferation could be similarly inhibited in Kit-225 cells by overexpression of the CDK inhibitors p16/Ink4-a or p21/Waf-1a or by overexpression of a kinase-inactive CDK4 mutant. The data indicate that CDK4 expression and activity are necessary to induce and maintain cytokine responsiveness in T cells, suggesting that CDK4 is important to link T cell signaling pathways to the machinery that controls cell cycle progression.
CD30 Shedding from Karpas 299 Lymphoma Cells Is Mediated by TNF-α-Converting Enzyme
Authors: Hansen Hinrich P. , Dietrich Sebastian , Kisseleva Tatiana , Mokros Thilo , Mentlein Rolf , Lange Hans H. , Murphy Gillian , Lemke Hilmar
Published: April 2014

CD30 is a costimulatory receptor on activated lymphocytes and a number of human lymphoma cells. Specific ligation of membrane-bound CD30 or cellular stimulation by PMA results in a metalloproteinase-mediated down-regulation of CD30 and release of its soluble ectodomain (sCD30). In this report, it is demonstrated that PMA-induced CD30 cleavage from Karpas 299 cells was mediated by a membrane-anchored metalloproteinase which was active on intact cells following 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate extraction of membrane preparations. Moreover, CD30 shedding was blocked by the synthetic hydroxamic acid-based metalloproteinase inhibitor BB-2116 (IC50, 230 nM) and the natural tissue inhibitor of metalloproteinases (TIMP)-3 (IC50, 30 nM), but not by the matrix metalloproteinase inhibitors TIMP-1 and TIMP-2. This inhibition profile is similar to that of the TNF-α- converting enzyme (TACE) and, indeed, mRNA transcripts of the membrane-bound metalloproteinase-disintegrin TACE could be detected in Karpas 299 cells. The ectodomain of TACE was expressed in bacteria as a GST fusion protein (GST-TACE) which cleaved CD30 from the surface of Karpas 299 cells and concomitantly increased the level of sCD30 in the cell supernatants. Hence, TACE does not only control the release of TNF-α, but also that of sCD30.
MHC Recognition in Thymic Development: Distinct, Parallel Pathways for Survival and Lineage Commitment
Authors: Chang David , Valdez Patricia , Ho Thomas , Robey Ellen
Published: April 2014

The molecular events triggered by MHC recognition and how they lead to the emergence of mature CD4 and CD8 lineage thymocytes are not yet understood. To address these questions, we have examined what signals are necessary to drive the development of CD8 lineage thymocytes in TCRα− mice in which TCR/MHC engagement cannot occur. We find that the combination of constitutive Notch activity and constitutive Bcl-2 expression are necessary and sufficient to allow the appearance of mature CD8 lineage thymocytes in TCRα− mice. In addition, Notch activity alone in TCRα− mice can induce the up-regulation of HES1, suggesting that thymocytes are competent to respond to Notch signaling in the absence of MHC recognition. These data indicate that survival and lineage commitment represent distinct, parallel pathways that occur as a consequence of MHC recognition, both of which are necessary for the development of mature CD8 lineage T cells.
Intestinal CD8αα and CD8αβ Intraepithelial Lymphocytes Are Thymus Derived and Exhibit Subtle Differences in TCRβ Repertoires
Authors: Imhof Beat A. , Dunon Dominique , Courtois David , Luhtala Marko , Vainio Olli
Published: April 2014

Intraepithelial lymphocytes (IEL) of the small intestine are anatomically positioned to be in the first line of cellular defense against enteric pathogens. Therefore, determining the origin of these cells has important implications for the mechanisms of T cell maturation and repertoire selection. Recent evidence suggests that murine CD8αα intestinal IELs (iIELs) can mature and undergo selection in the absence of a thymus. We analyzed IEL origin by cell transfer, using two congenic chicken strains. Embryonic day 14 and adult thymocytes did not contain any detectable CD8αα T cells. However, when TCR+ thymocytes were injected into congenic animals, they migrated to the gut and developed into CD8αα iIELs, while TCR− T cell progenitors did not. The TCR Vβ1 repertoire of CD8αα+ TCR Vβ1+ iIELs contained only part of the TCR Vβ1 repertoire of total iIELs, and it exhibited no new members compared with CD8+ T cells in the thymus. This indicated that these T cells emigrated from the thymus at an early stage in their developmental process. In conclusion, we show that while CD8αα iIELs originate in the thymus, T cells acquire the expression of CD8αα homodimers in the gut microenvironment.
Immortalized Myeloid Suppressor Cells Trigger Apoptosis in Antigen-Activated T Lymphocytes
Authors: Apolloni Elisa , Bronte Vincenzo , Mazzoni Alessandra , Serafini Paolo , Cabrelle Anna , Segal David M. , Young Howard A. , Zanovello Paola
Published: April 2014

We described a generalized suppression of CTL anamnestic responses that occurred in mice bearing large tumor nodules or immunized with powerful recombinant viral immunogens. Immune suppression entirely depended on GM-CSF-driven accumulation of CD11b+/Gr-1+ myeloid suppressor cells (MSC) in secondary lymphoid organs. To further investigate the nature and properties of MSC, we immortalized CD11b+/Gr-1+ cells isolated from the spleens of immunosuppressed mice, using a retrovirus encoding the v-myc and v-raf oncogenes. Immortalized cells expressed monocyte/macrophage markers (CD11b, F4/80, CD86, CD11c), but they differed from previously characterized macrophage lines in their capacities to inhibit T lymphocyte activation. Two MSC lines, MSC-1 and MSC-2, were selected based upon their abilities to inhibit Ag-specific proliferative and functional CTL responses. MSC-1 line was constitutively inhibitory, while suppressive functions of MSC-2 line were stimulated by exposure to the cytokine IL-4. Both MSC lines triggered the apoptotic cascade in Ag-activated T lymphocytes by a mechanism requiring cell-cell contact. Some well-known membrane molecules involved in the activation of apoptotic pathways (e.g., TNF-related apoptosis-inducing ligand, Fas ligand, TNF-α) were ruled out as candidate effectors for the suppression mechanism. The immortalized myeloid lines represent a novel, useful tool to shed light on the molecules involved in the differentiation of myeloid-related suppressors as well as in the inhibitory pathway they use to control T lymphocyte activation.
Reversal of CD8+ T Cell Ignorance and Induction of Anti-Tumor Immunity by Peptide-Pulsed APC
Authors: Dalyot-Herman Nava , Bathe Oliver F. , Malek Thomas R.
Published: April 2014

In the present report, we have studied the potential of naive and activated effector CD8+ T cells to function as anti-tumor T cells to a solid tumor using OVA-specific T cells from TCR-transgenic OT-I mice. Adoptive transfer of naive OT-I T cells into tumor-bearing syngeneic mice did not inhibit tumor cell growth. The adoptively transferred OT-I T cells did not proliferate in lymphoid tissue of tumor-bearing mice and were not anergized by the tumor. In contrast, adoptive transfer of preactivated OT-I CTL inhibited tumor growth in a dose-dependent manner, indicating that E.G7 was susceptible to immune effector cells. Importantly, naive OT-I T cells proliferated and elicited an anti-tumor response if they were adoptively transferred into normal or CD4-deficient mice that were then vaccinated with GM-CSF-induced bone marrow-derived OVA-pulsed APC. Collectively, these data indicate that even though naive tumor-specific T cells are present at a relatively high fraction they remain ignorant of the tumor and demonstrate that a CD8-mediated anti-tumor response can be induced by Ag-pulsed APC without CD4 T cell help.
Targeting Weak Antigens to CD64 Elicits Potent Humoral Responses in Human CD64 Transgenic Mice
Authors: Keler Tibor , Guyre Paul M. , Vitale Laura A. , Sundarapandiyan Karuna , van de Winkel Jan G. J. , Deo Yashwant M. , Graziano Robert F.
Published: April 2014

Previous studies have documented that targeting foreign Ags to IgG FcγR leads to enhanced Ag-specific responses in vitro and in vivo. However, the ability to overcome immunologic nonresponsiveness by targeting poorly immunogenic Ags to FcγR has not been investigated. To address this question in a simple model, we immunized transgenic mice expressing human CD64 (FcγRI) and their nontransgenic littermates with Fab′ derived from the murine anti-human CD64 mAb m22. The m22 Fab′ served as both the targeting molecule and the Ag. We found that only CD64-expressing mice developed anti-Id titers to m22. Furthermore, chemically linked multimers of m22 Fab′, which mediated efficient internalization of the human CD64, were significantly more potent than monomeric m22 F(ab′)2 at inducing anti-Id responses. In all cases, the humoral responses were specific for m22 Id and did not react with other murine IgG1 Fab′ fragments. Chemical addition of a second murine Fab′ (520C9 anti-human HER2/neu) to m22 Fab′ multimers demonstrated that IgG1 and IgG2a anti-Id titers could be generated to 520C9 only in the CD64-expressing mice. These results show that targeting to CD64 can overcome immunological nonresponsiveness to a weak immunogen. Therefore, targeting to CD64 may be an effective method to enhance the activity of nonimmunogenic tumor vaccines.
Dendritic Cell Migration Controlled by α1b-Adrenergic Receptors
Authors: Maestroni Georges J. M.
Published: April 2014

Dendritic cells (DC) bring Ags into lymphoid organs via lymphatic vessels. In this study, we investigated the possibility that the sympathetic neurotransmitter norepinephrine (NE) influences DC migration. Murine epidermal Langerhans cells mobilization is enhanced by systemic treatment with the α2-adrenergic antagonist yohimbine and inhibited by local treatment with the specific α1-adrenergic antagonist prazosin (PRA). Consistently, NE enhances spontaneous emigration of DC from ear skin explants, and PRA inhibits this effect. In addition, local treatment with PRA during sensitization with FITC inhibits the contact hypersensitivity response 6 days later. In vitro, bone marrow-derived immature, but not CD40-stimulated mature DC migrate in response to NE, and this effect is neutralized by PRA. NE seems to exert both a chemotactic and chemokinetic activity on immature DC. Coherently, immature, but not mature DC, express mRNA coding for the α1b-adrenergic receptor subtype. Inactivation of this adrenergic receptor by the specific and irreversible antagonist chloroethylclonidine hinders the migration of injected DC from the footpad to regional lymph nodes. Thus, besides regulating lymph flow, the sympathetic innervation of lymphatic vessels may participate in directing DC migration from the site of inflammation to regional lymph nodes. Alternatively, the chemokinetic activity of NE may enhance the ability of DC to sample local Ags, and hence increase the number of DC migrating to the draining lymph nodes. This finding might improve our understanding of the biological basis of skin diseases and allergic reactions, and opens new pharmacological possibilities to modulate the immune response.
Early Th1 Response in Unprimed Nonobese Diabetic Mice to the Tyrosine Phosphatase-Like Insulinoma-Associated Protein 2, an Autoantigen in Type 1 Diabetes
Authors: Trembleau Sylvie , Penna Giuseppe , Gregori Silvia , Magistrelli Giovanni , Isacchi Antonella , Adorini Luciano
Published: April 2014

The insulinoma-associated protein 2 (IA-2) is a phosphatase-like autoantigen inducing T and B cell responses associated with human insulin-dependent diabetes mellitus (IDDM). We now report that T cell responses to IA-2 can also be detected in the nonobese diabetic (NOD) mouse, a model of human IDDM. Cytokine secretion in response to purified mouse rIA-2, characterized by high IFN-γ and relatively low IL-10 and IL-6 secretion, was elicited in spleen cells from unprimed NOD mice. Conversely, no response to IA-2 was induced in spleen cells from BALB/c, C57BL/6, or Biozzi AB/H mice that express, like NOD, the I-Ag7 class II molecule, but are not susceptible to spontaneous IDDM. The IA-2-induced IFN-γ response in NOD spleen cells could already be detected at 3 wk and peaked at 8 wk of age, whereas the IL-10 secretion was maximal at 4 wk of age and then waned. IA-2-dependent IFN-γ secretion was induced in CD4+ cells from spleen as well as pancreatic and mesenteric lymph nodes. It required Ag presentation by I-Ag7 molecules and engagement of the CD4 coreceptor. Interestingly, cytokines were produced in the absence of cell proliferation and IL-2 secretion. The biological relevance of the response to IA-2 is indicated by the enhanced IDDM following a single injection of the recombinant protein emulsified in IFA into 18-day-old NOD mice. In addition, IFN-γ production in response to IA-2 and IDDM acceleration could be induced by IL-12 administration to 12-day-old NOD mice. These results identify IA-2 as an early T cell-inducing autoantigen in the NOD mouse and indicate a role for the IA-2-induced Th1 cell response in IDDM pathogenesis.
Resistance to TNF-Induced Cytotoxicity Correlates with an Abnormal Cleavage of Cytosolic Phospholipase A2
Authors: El Mahdani Nour-Eddine , Ameyar Maya , Cai Zhenzi , Colard Odile , Masliah Joëlle , Chouaib Salem
Published: April 2014

To investigate the mechanism underlying the absence of arachidonic acid (AA) release by TNF in TNF-resistant cells, we first performed comparative analysis of phospholipid pools in both TNF-sensitive (MCF7) and their equivalent resistant cells (C1001). Quantification and incorporation studies of [3H]AA indicated that TNF-resistant cells were not depleted in AA. Furthermore, distribution of this fatty acid in different phospholipid pools was similar in both sensitive cells and their resistant counterparts, ruling out a defect in phospholipid pools. Since phospholipase A2 (PLA2) are the main enzymes releasing free AA, we investigated their relative contribution in the acquisition of cell resistance to TNF-induced cell death and AA release. For this purpose, we used two PLA2 inhibitors, methylarachidonyl fluorophosphate (MAFP) and bromoenol lactone (BEL), which selectively and irreversibly inhibit the cytosolic PLA2 (cPLA2) and the Ca2+-independent PLA2, respectively. Although a significant inhibitory effect of MAFP on both TNF-induced AA release and PLA2 activity in MCF7 was observed, BEL had no effect. The inhibitory effect of MAFP on cPLA2 activity correlated with an inhibition of TNF-induced cell death. Western blot analysis revealed that TNF induced a differential cleavage of cPLA2 in TNF-sensitive vs TNF-resistant cells. Although the p70 (70-kDa) form of cPLA2 was specifically increased in TNF-sensitive cells, a cleaved form, p50 (50 kDa), was selectively observed in TNF-resistant C1001 cells in the presence or absence of TNF. These findings suggest that the acquisition of cell resistance to this cytokine may involve an abnormal cPLA2 cleavage.
The Development, Maturation, and Turnover Rate of Mouse Spleen Dendritic Cell Populations
Authors: Kamath Arun T. , Pooley Joanne , O’Keeffe Meredith A. , Vremec David , Zhan Yifan , Lew Andrew M. , D’Amico Angela , Wu Li , Tough David F. , Shortman Ken
Published: April 2014

Three distinct subtypes of dendritic cells (DC) are present in mouse spleen, separable as CD4−8α−, CD4+8α−, and CD4−8α+ DC. We have tested whether these represent stages of development or activation within one DC lineage, or whether they represent separate DC lineages. All three DC subtypes appear relatively mature by many criteria, but all retain a capacity to phagocytose particulate material in vivo. Although further maturation or activation could be induced by bacterially derived stimuli, phagocytic capacity was retained, and no DC subtype was converted to the other. Continuous elimination of CD4+8− DC by Ab depletion had no effect on the levels of the other DC subtypes. Bromodeoxyuridine labeling experiments indicated that all three DC subtypes have a rapid turnover (half-life, 1.5–2.9 days) in the spleen, with none being the precursor of another. The three DC subtypes showed different kinetics of development from bone marrow precursors. The CD8α+ spleen DC, apparently the most mature, displayed an extremely rapid turnover based on bromodeoxyuridine uptake and the fastest generation from bone marrow precursors. In conclusion, the three splenic DC subtypes behave as rapidly turning over products of three independent developmental streams.
Exposure to Cigarette Tar Inhibits Ribonucleotide Reductase and Blocks Lymphocyte Proliferation
Authors: McCue Jesica M. , Link Karen L. , Eaton Sandra S. , Freed Brian M.
Published: April 2014

Cigarette smoking causes profound suppression of pulmonary T cell responses, which has been associated with increased susceptibility to respiratory tract infections and decreased tumor surveillance. Exposure of human T cells to cigarette tar or its major phenolic components, hydroquinone and catechol, causes an immediate cessation of DNA synthesis without cytotoxicity. However, little is known of the mechanisms by which this phenomenon occurs. In this report we demonstrate that hydroquinone and catechol inhibit lymphocyte proliferation by quenching the essential tyrosyl radical in the M2 subunit of ribonucleotide reductase.
HLA-B2702 (77–83/83–77) Peptide Binds to β-Tubulin on Human NK Cells and Blocks Their Cytotoxic Capacity
Authors: Tarazona Raquel , López-Lluch Guillermo , Galiani Maria-Dolores , Aguado Enrique , Barahona Fernando , Solana Rafael , Peña José
Published: April 2014

It has been described that peptides derived from a highly conserved region of the α1 helix of the first domain of HLA class I Ags exhibit immunomodulatory capacity blocking both T and NK cell cytotoxicity. In vivo treatment with these peptides prolongs survival of MHC-mismatched allografts. However, the molecular bases of these effects are still unclear. In this study, we further analyze the mechanisms by which the dimeric peptide HLA-B2702 (77–83/83–77) induces suppression of NK cell cytotoxicity. This peptide inhibits natural and redirected lysis mediated by NK cells without significantly affecting effector-target cell binding. We have also isolated and sequenced a protein that binds this inhibitory peptide, which structurally corresponds to β-tubulin. Tubulin is the major protein of microtubules and is involved in target cell killing. Furthermore, B2702 peptide promotes GTP-independent tubulin assembly, producing aggregates that cannot be depolymerized by cold. Treatment of NK cells with Taxol or demecolcine, which interfere with microtubule organization, also prevents NK cell cytotoxicity. Taken together, these results support the hypothesis that the peptide B2702 (77–83/83–77) exerts its inhibitory effect on NK cell cytotoxicity by inducing polymerization of microtubules and interfering with their normal assembly/disassembly dynamics.
CD4+ Th1 and CD8+ Type 1 Cytotoxic T Cells Both Play a Crucial Role in the Full Development of Contact Hypersensitivity
Authors: Wang Binghe , Fujisawa Hiroshi , Zhuang Lihua , Freed Irwin , Howell Brandon G. , Shahid Shabana , Shivji Gulnar M. , Mak Tak W. , Sauder Daniel N.
Published: April 2014

The role of CD4+ vs CD8+ T cells in contact hypersensitivity (CHS) remains controversial. In this study, we used gene knockout (KO) mice deficient in CD4+ or CD8+ T cells to directly address this issue. Mice lacking either CD4+ or CD8+ T cells demonstrated depressed CHS responses to dinitrofluorobenzene and oxazolone compared with wild-type C57BL/6 mice. The depression of CHS was more significant in CD8 KO mice than in CD4 KO mice. Furthermore, in vivo depletion of either CD8+ T cells from CD4 KO mice or CD4+ T cells from CD8 KO mice virtually abolished CHS responses. Lymph node cells (LNCs) from hapten-sensitized CD4 and CD8 KO mice showed a decreased capacity for transferring CHS. In vitro depletion of either CD4+ T cells from CD8 KO LNCs or CD8+ T cells from CD4 KO LNCs resulted in a complete loss of CHS transfer. LNCs from CD4 and CD8 KO mice produced significant amounts of IFN-γ, indicating that both CD4+ and CD8+ T cells are able to secrete IFN-γ. LNCs from CD8, but not CD4, KO mice were able to produce IL-4 and IL-10, suggesting that IL-4 and IL-10 are mainly derived from CD4+ T cells. Intracellular cytokine staining of LNCs confirmed that IFN-γ-positive cells consisted of CD4+ (Th1) and CD8+ (type 1 cytotoxic T) T cells, whereas IL-10-positive cells were exclusively CD4+ (Th2) T cells. Collectively, these results suggest that both CD4+ Th1 and CD8+ type 1 cytotoxic T cells are crucial effector cells in CHS responses to dinitrofluorobenzene and oxazolone in C57BL/6 mice.
Functional Equivalency of B7-1 and B7-2 for Costimulating Plasmid DNA Vaccine-Elicited CTL Responses
Authors: Santra Sampa , Barouch Dan H. , Jackson Shawn S. , Kuroda Marcelo J. , Schmitz Joern E. , Lifton Michelle A. , Sharpe Arlene H. , Letvin Norman L.
Published: April 2014

A costimulatory signal in addition to an Ag-specific stimulus is required for optimal activation of T lymphocytes. CD28, the primary positive costimulatory receptor on T cells, has two identified ligands, B7-1 and B7-2. Whether B7-1 and B7-2 have identical, overlapping, or distinct functions remains unresolved. In this study, we show that mice lacking B7-2 were unable to generate CTL responses following immunization with a plasmid DNA vaccine. The ability of these B7-2-deficient mice to generate CTL responses following plasmid gp120 DNA vaccination was fully reconstituted by coadministering either a plasmid expressing B7-2 or B7-1. Moreover, the ability to generate CTL responses following plasmid DNA vaccination in mice lacking both B7-1 and B7-2 could be reconstituted by administering either plasmid B7-1 or plasmid B7-2 with the vaccine construct. These data demonstrate that either B7-1 or B7-2 administered concurrently with a plasmid DNA vaccine can fully costimulate vaccine-elicited CTL responses. Functional differences between B7-1 and B7-2 observed in vivo therefore may not reflect inherent differences in the interactions of CD28 with these ligands.
Ig Light Chain Receptor Editing in Anergic B Cells
Authors: Tze Lina E. , Baness Elizabeth A. , Hippen Keli L. , Behrens Timothy W.
Published: April 2014

Receptor editing in the bone marrow (BM) serves to modify the Ag receptor specificity of immature self-reactive B cells, while anergy functionally silences self-reactive clones. Here, we demonstrate that anergic B cells in hen egg lysozyme Ig (HEL-Ig)/soluble HEL double transgenic mice show evidence of having undergone receptor editing in vivo, as demonstrated by the presence of elevated levels of endogenous κ light chain rearrangements in the BM and spleen. In an in vitro IL-7-driven BM culture system, HEL-Ig BM B cells grown in the presence of soluble HEL down-regulated surface IgM expression and also showed induction of new endogenous κ light chain rearrangements. Using a panel of soluble protein ligands with reduced affinity for the HEL-Ig receptor, the editing response was shown to correlate in a dose-dependent fashion with the strength of signaling through the B cell receptor. The finding that the level of B cell receptor cross-linking sufficient to induce anergy in B cells is also capable of engaging the machinery required for receptor editing suggests an intimate relationship between these two mechanisms in maintaining B cell tolerance.
Stat4 Regulates Multiple Components of IFN-γ-Inducing Signaling Pathways
Authors: Lawless Victoria A. , Zhang Shangming , Ozes Osman N. , Bruns Heather A. , Oldham India , Hoey Timothy , Grusby Michael J. , Kaplan Mark H.
Published: April 2014

Stat4 is activated in response to IL-12. Most functions of IL-12, including the induction of IFN-γ, are compromised in the absence of Stat4. Since the precise role of Stat4 in IFN-γ induction has not been established, experiments were conducted to examine Stat4 activation of IFN-γ and other genes required for cytokine-induced expression of IFN-γ. We first examined IL-12 signaling components. Basal expression of IL-12Rβ1 and IL-12Rβ2 is decreased in Stat4-deficient cells compared with that in control cells. However, IL-12 was still capable of inducing equivalent phosphorylation of Jak2 and Tyk2 in wild-type and Stat4-deficient activated T cells. We have further determined that other cytokine signaling pathways that induce IFN-γ production are defective in the absence of Stat4. IL-18 induces minimal IFN-γ production from Stat4-deficient activated T cells compared with control cells. This is due to defective IL-18 signaling, which results from the lack of IL-12-induced, and Stat4-dependent, expression of the IL-18R. Following IL-12 pretreatment to induce IL-18R, wild-type, but not Stat4-deficient, activated T cells demonstrated IL-18-induced NF-κB DNA-binding activity. In addition, IL-12-pretreated Stat4-deficient activated T cells have minimal IFN-γ production followed by stimulation with IL-18 alone or in combination with IL-12 compared with control cells. Thus, Stat4 activation by IL-12 is required for the function of multiple cytokine pathways that result in induction of IFN-γ.
Ligation of CD27 on Murine B Cells Responding to T-Dependent and T-Independent Stimuli Inhibits the Generation of Plasma Cells
Authors: Raman Vanitha S. , Bal Vineeta , Rath Satyajit , George Anna
Published: April 2014

B cells can be stimulated either allogenically with the Th cell clone D10G4.1 and bone marrow-derived dendritic cells or polyclonally with LPS to proliferate and undergo terminal differentiation to Ig-secreting plasma cells in vitro. The addition of anti-CD27 to such cultures inhibits Ig secretion, and inhibition is more marked in T-dependent cultures than in T-independent cultures. Both IgM and secondary isotypes are affected, and addition of anti-CD27 even 4 days after culture initiation inhibits Ig secretion. Anti-CD27 does not affect B cell proliferation or the acquisition of activation markers by B cells, and no marked loss of B cell viability is detected in cells cultured in the presence of anti-CD27, suggesting that the inhibition of Ig secretion is not due to inhibition of early activation events or to death of activated cells in vitro. However, the presence of anti-CD27 significantly inhibits the induction of Blimp-1 and J chain transcripts, which are turned on in cells committed to plasma cell differentiation. Furthermore, mice immunized under cover of anti-CD27 make less Ag-specific IgM and IgG, but have equivalent T cell responses when compared with control mice. These data suggest that ligation of CD27, a member of the TNFR family, on the B cell surface may prevent terminal differentiation of activated B cells into Ig-secreting plasma cells.
Diminution of the AML1 Transcription Factor Function Causes Differential Effects on the Fates of CD4 and CD8 Single-Positive T Cells
Authors: Hayashi Keitaro , Natsume Waka , Watanabe Toshio , Abe Natsumi , Iwai Naomi , Okada Hitoshi , Ito Yoshiaki , Asano Masahide , Iwakura Yoichiro , Habu Sonoko , Takahama Yousuke , Satake Masanobu
Published: April 2014

In the thymic cortex, T lymphocytes are positively selected to survive and committed either to the CD4 single-positive (SP) or the CD8 SP lineage. The SP cells then pass through a step of maturation in the medulla and are delivered to peripheral lymphoid tissues. We examined the role of AML1, the gene encoding a transcription factor, in the above processes by using the transgenic mice expressing a dominant interfering form of AML1 as well as mice targeted heterozygously for AML1. One phenotypic change seen in the AML1-diminished mice was the reduction in the numbers of both CD4 SP and CD8 SP thymocytes, reflecting the partial impairment of the transition from the double-positive to SP stage. In addition, distinct from the above abnormality, perturbed were several aspects of SP cells, including the maturation of SP thymocytes, the recent thymic emigration, and the proliferative responsiveness of peripheral T cells to TCR stimulation. Interestingly, the AML1 diminution caused inhibitory and enhancing effects on the CD4 SP and CD8 SP cells, respectively. These differential effects are most likely related to the reduction in the peripheral CD4 SP/CD8 SP ratio observed in the AML1-diminished mice. The AML1 transcription factor thus maintains the homeostasis of each SP subset by functioning at the later stages of T lymphocyte differentiation.
Genetic Analyses of NFKB1 and OCA-B Function: Defects in B Cells, Serum IgM Level, and Antibody Responses in Nfkb1−/−Oca-b−/− Mice
Authors: Kim Unkyu , Gunther Cary S. , Roeder Robert G.
Published: April 2014

Defined patterns of gene expression during cell differentiation are likely to be ensured by multiple factors playing redundant roles. By generating mice deficient in both NFKB1 and OCA-B, we show here that the two transcription factors are required for B-1 cell differentiation and serum IgM production. In addition, relative to Nfkb1−/− or Oca-b−/− mice, the Nfkb1−/−Oca-b−/− mice show a decrease in conventional B cell frequencies in the spleen and augmented reductions in T-independent and T-dependent Ab responses. These results suggest that NFKB1 and OCA-B play compensatory roles in multiple aspects of B cell differentiation.
Early Programming of T Cell Populations Responding to Bacterial Infection
Authors: Mercado Roberto , Vijh Sujata , Allen S. Elise , Kerksiek Kristen , Pilip Ingrid M. , Pamer Eric G.
Published: April 2014

The duration of infection and the quantity of Ag presented in vivo are commonly assumed to influence, if not determine, the magnitude of T cell responses. Although the cessation of in vivo T cell expansion coincides with bacterial clearance in mice infected with Listeria monocytogenes, closer analysis suggests that control of T cell expansion and contraction is more complex. In this report, we show that the magnitude and kinetics of Ag-specific T cell responses are determined during the first day of bacterial infection. Expansion of Ag-specific T lymphocyte populations and generation of T cell memory are independent of the duration and severity of in vivo bacterial infection. Our studies indicate that the Ag-specific T cell response to L. monocytogenes is programmed before the peak of the innate inflammatory response and in vivo bacterial replication.