bluuebunny/crossref_metadata_2025 huggingface dataset:
Diverse Transcriptional Response of CD4+ T Cells to Stromal Cell-Derived Factor (SDF)-1: Cell Survival Promotion and Priming Effects of SDF-1 on CD4+ T Cells
Authors: Suzuki Yasuhiro , Rahman Monzur , Mitsuya Hiroaki
Published: April 2014

Stromal cell-derived factor (SDF)-1 is a ligand for the chemokine receptor CXCR4 which is broadly expressed in lymphocytes, but the effects of SDF-1 on T cells are largely unknown. When examined using complementary DNA microarray, up-regulation of genes which are associated with DNA repair, detoxification, apoptosis, cell morphology, cell adhesion, and signal transduction was seen in CD4+ T cells upon SDF-1 exposure. SDF-1 was shown to promote CD4+ T cell survival through the phosphatidylinositol 3-kinase (PI3K)- and mitogen-activated protein kinase (MAPK)-cascades without cell cycle progression. The proapoptotic Bcl-2 antagonistic of cell death protein was also seen inactivated by the SDF-1-mediated activation of MAPK-extracellular signal-regulated kinases (MEK)-extracellular signal-regulated kinase-ribosomal S6 kinases- and PI3K-pathways. Moreover, the genes known to be associated with cell survival were up-regulated upon SDF-1 exposure and were linked to the MAPK-MEK and PI3K-pathways. Thus, SDF-1 promotes cell survival by two mechanisms: posttranslational inactivation of the cell death machinery and an increased transcription of cell survival-related genes. SDF-1 also primed resting CD4+ T cells for cytokine- and TCR-mediated stimuli. These data suggest that the SDF-1-mediated cell survival combined with its priming function would set T cells to respond to immunologic challenges.
The Role of IFN-γ in the Production of Th2 Subpopulations: Implications for Variable Th2-Mediated Pathologies in Autoimmunity
Authors: Wensky Allen , Garibaldi Marcondes Maria Cecília , Lafaille Juan J.
Published: April 2014

It has become increasingly apparent in studies of mutant mice and observations of disease that cytokine production by fully committed effector T cells within the Th1 and Th2 phenotype can vary within each group. This can potentially influence the type and effectiveness of a given immune response. The factors responsible for inducing variable Th1 and Th2 subtype responses have not been well established. Using transgenic mice expressing the myelin basic protein-specific TCR, we demonstrate here that two distinct populations of Th2 cells that are characterized primarily by differential IL-4 and IL-5 expression levels can be generated depending upon the levels of IFN-γ present at the time of priming. We also demonstrate that populations expressing high levels of IL-4 relative to IL-5 vs those with intermediate levels of IL-4 relative to IL-5 are stable and possess distinct effector functions in an experimental autoimmune encephalomyelitis model.
Direct Recognition of Foreign MHC Determinants by Naive T Cells Mobilizes Specific Vβ Families Without Skewing of the Complementarity-Determining Region 3 Length Distribution
Authors: Sebille Fabien , Gagne Katia , Guillet Marina , Degauque Nicolas , Pallier Annaïck , Brouard Sophie , Vanhove Bernard , Delsuc Marc-André , Soulillou Jean-Paul
Published: April 2014

The capacity of T cells to interact with nonself-APC, also referred to as direct allorecognition, is an essential feature of the cellular response involved in graft rejection. However, there is no study on TCR repertoire biases associated with direct restricted T cell activation. In this paper, we have addressed the impact of direct recognition on the whole naive T cell repertoire, using a new approach that provides, for the first time, an integrated depiction of the quantitative and qualitative alterations in the TCR Vβ transcriptome. This method can differentiate resting patterns from polyclonally activated ones, as evidenced by superantigen usage. According to this new readout, we show that direct recognition of nonself-MHC molecules triggers mRNA accumulation of several TCR Vβ families, specific to the combination studied. Moreover, in marked contrast to the situation that prevails in indirect allorecognition, T cell activation through the direct presentation pathway was not associated with skewing of the complementarity determining region (CDR) 3 length distribution. Altogether, these data argue for the significance of TCR contacts with the MHC framework in direct allorecognition. In addition, the TCR diversity mobilized by this interaction and the massive TCRβ mRNA accumulation observed after a few days of culture suggest that a significant proportion of naive T cells receive a signal leading to TCRβ transcriptional activation even though only a few of them engage in mitosis.
Tumor-Specific Responses in Lymph Nodes Draining Murine Sarcomas Are Concentrated in Cells Expressing P-Selectin Binding Sites
Authors: Tanigawa Keishi , Takeshita Nobuhiro , Craig Ronald A. , Phillips Katie , Knibbs Randall N. , Chang Alfred E. , Stoolman Lloyd M.
Published: April 2014

Tumor-draining lymph node (TDLN) cells develop substantial antitumor activity after activation on immobilized αCD3 and culture in low-dose IL-2. This study found that the minor subset of TDLN T cells expressing binding sites for the adhesion receptor P-selectin (Plighigh T cells) produced T lymphoblasts with the most tumor-specific IFN-γ synthesis in vitro and antitumor activity following adoptive transfer in vivo. The Plighigh T cells constituted <25% of the cells with the phenotype of recently activated cells including high levels of CD69, CD44, or CD25, and low levels of CD62L. The cultured Plighigh TDLN were 10- to 20-fold more active against established pulmonary micrometastases than cultured unfractionated TDLN, and >30-fold more active than cultured TDLN cells depleted of the Plighigh fraction before expansion (Pliglow cells). Tumor-specific IFN-γ synthesis in vitro paralleled the antitumor activities of the cultured fractions in vivo, implying that increased Tc1 and Th1 effector functions contributed to the tumor suppression. Neither nonspecific interaction with the P-selectin chimera used for sorting nor endogenous costimulatory activity in the Plighigh fraction accounted for the marked increase in antitumor activities after culture. The cultured Plighigh fraction contained a variety of potential effector cells; however, the CD8 and CD4 subsets of αβ T cells accounted for 95–97% of its antitumor activity. The authors propose that P-selectin sorting increased antitumor activities by concentrating Tc1 and Th1 pre-effector/effector cells before culture.
Human Endothelial-Cell Specific Molecule-1 Binds Directly to the Integrin CD11a/CD18 (LFA-1) and Blocks Binding to Intercellular Adhesion Molecule-1
Authors: Béchard David , Scherpereel Arnaud , Hammad Hamida , Gentina Thibaut , Tsicopoulos Anne , Aumercier Marc , Pestel Jöel , Dessaint Jean-Paul , Tonnel André-Bernard , Lassalle Philippe
Published: April 2014

ICAMs are ligands for LFA-1, a major integrin of mononuclear cells involved in the immune and inflammatory processes. We previously showed that endothelial cell specific molecule-1 (ESM-1) is a proteoglycan secreted by endothelial cells under the control of inflammatory cytokines. Here, we demonstrate that ESM-1 binds directly to LFA-1 onto the cell surface of human blood lymphocytes, monocytes, and Jurkat cells. The binding of ESM-1 was equally dependent on Ca2+, Mg2+, or Mn2+ divalent ions, which are specific, saturable, and sensitive to temperature. An anti-CD11a mAb or PMA induced a transient increase in binding, peaking 5 min after activation. Direct binding of ESM-1 to LFA-1 integrin was demonstrated by specific coimmunoprecipitation by CD11a and CD18 mAbs. A cell-free system using a Biacore biosensor confirmed that ESM-1 and LFA-1 dynamically interacted in real time with high affinity (Kd = 18.7 nM). ESM-1 consistently inhibited the specific binding of soluble ICAM-1 to Jurkat cells in a dose-dependent manner. These results suggest that ESM-1 and ICAM-1 interact with LFA-1 on binding sites very close to but distinct from the I domain of CD11a. Through this mechanism, ESM-1 could be implicated in the regulation of the LFA-1/ICAM-1 pathway and may therefore influence both the recruitment of circulating lymphocytes to inflammatory sites and LFA-1-dependent leukocyte adhesion and activation.
Differentiation of Regulatory T Cells 1 Is Induced by CD2 Costimulation
Authors: Wakkach Abdelilah , Cottrez Françoise , Groux Hervé
Published: April 2014

Induction and maintenance of peripheral tolerance is an important phenomenon for the control of homeostasis in the immune system. There is now compelling evidence for CD4+ T cells that prevent immune pathology, both in autoimmunity and in transplantation. However, the mechanisms involved in the specific differentiation of these T cells are unknown. We had previously shown that repetitive stimulations of naive T cells in the presence of IL-10 induce the differentiation of T regulatory cells 1. We further dissected the mechanism of IL-10 function and demonstrated that IL-10 acts by the down-regulation of most costimulatory molecules without modifying the expression of CD58. Using artificial APCs expressing various costimulatory molecules, we demonstrated that, in contrast to other costimulation patterns, costimulation via CD2 alone, in the absence of costimulations through CD28- or LFA-1, induced T cell anergy in an IL-10-independent pathway along with the differentiation of Ag-specific regulatory T cells. T regulatory cell-1 differentiation via CD2 was very efficient as both high IL-10 secretion and regulatory function were observed after the first stimulation of naive T cells with CD32-CD58 L cells. The possibility to rapidly induce the differentiation of Ag-specific regulatory T cells will certainly accelerate their characterization and their potential use as regulators of T cell-mediated diseases.
Human NKT Cells Mediate Antitumor Cytotoxicity Directly by Recognizing Target Cell CD1d with Bound Ligand or Indirectly by Producing IL-2 to Activate NK Cells
Authors: Metelitsa Leonid S. , Naidenko Olga V. , Kant Anita , Wu Hong-Wei , Loza Matthew J. , Perussia Bice , Kronenberg Mitchell , Seeger Robert C.
Published: April 2014

α-Galactosylceramide (αGalCer) stimulates NKT cells and has antitumor activity in mice. Murine NKT cells may directly kill tumor cells and induce NK cell cytotoxicity, but the mechanisms are not well defined. Newly developed human CD1d/αGalCer tetrameric complexes were used to obtain highly purified human αGalCer-reactive NKT cell lines (>99%), and the mechanisms of NKT cell cytotoxicity and activation of NK cells were investigated. Human NKT cells were cytotoxic against CD1d− neuroblastoma cells only when they were rendered CD1d+ by transfection and pulsed with αGalCer. Four other CD1d− tumor cell lines of diverse origin were resistant to NKT cells, whereas Jurkat and U937 leukemia cell lines, which are constitutively CD1d+, were killed. Killing of the latter was greatly augmented in the presence of αGalCer. Upon human CD1d/αGalCer recognition, NKT cells induced potent cytotoxicity of NK cells against CD1d− neuroblastoma cell lines that were not killed directly by NKT cells. NK cell activation depended upon NKT cell production of IL-2, and was enhanced by secretion of IFN-γ. These data demonstrate that cytotoxicity of human NKT cells can be CD1d and ligand dependent, and that TCR-stimulated NKT cells produce IL-2 that is required to induce NK cell cytotoxicity. Thus, NKT cells can mediate potent antitumor activity both directly by targeting CD1d and indirectly by activating NK cells.
CD28 Is Not Required for c-Jun N-Terminal Kinase Activation in T Cells
Authors: Rivas Fabiola V. , O’Herrin Sean , Gajewski Thomas F.
Published: April 2014

Studies in Jurkat cells have shown that combined stimulation through the TCR and CD28 is required for activation of c-Jun N-terminal kinase (JNK), suggesting that JNK activity may mediate the costimulatory function of CD28. To examine the role of JNK signaling in CD28 costimulation in normal T cells, murine T cell clones and CD28+/+ or CD28−/− TCR transgenic T cells were used. Although ligation with anti-CD28 mAb augmented JNK activation in Th1 and Th2 clones stimulated with low concentrations of anti-CD3 mAb, higher concentrations of anti-CD3 mAb alone were sufficient for JNK activation even in the absence of anti-CD28. JNK activity was comparably induced in both CD28+/+ and CD28−/− 2C/recombinase-activating gene 2(RAG2)−/− T cells stimulated with anti-CD3 mAb alone, and with Ld/peptide dimers, a direct αβ TCR ligand. Moreover, JNK activation was also detected in 2C/RAG2−/− T cells stimulated with P815 cells that express the relevant alloantigen Ld whether or not B7-1 was coexpressed. However, IL-2 production by both Th1 clones and CD28+/+ 2C/RAG2−/− T cells was detected only upon TCR and CD28 coengagement. Thus, CD28 coligation is not necessary, and stimulation through the TCR is sufficient, for JNK activation in normal murine T cells. The concept that JNK mediates the costimulatory function of CD28 needs to be reconsidered.
Selective Expansion and Partial Activation of Human NK Cells and NK Receptor-Positive T Cells by IL-2 and IL-15
Authors: Dunne Jean , Lynch Sara , O’Farrelly Cliona , Todryk Stephen , Hegarty John E. , Feighery Conleth , Doherty Derek G.
Published: April 2014

IL-2 and IL-15 are lymphocyte growth factors produced by different cell types with overlapping functions in immune responses. Both cytokines costimulate lymphocyte proliferation and activation, while IL-15 additionally promotes the development and survival of NK cells, NKT cells, and intraepithelial lymphocytes. We have investigated the effects of IL-2 and IL-15 on proliferation, cytotoxicity, and cytokine secretion by human PBMC subpopulations in vitro. Both cytokines selectively induced the proliferation of NK cells and CD56+ T cells, but not CD56− lymphocytes. All NK and CD56+ T cell subpopulations tested (CD4+, CD8+, CD4−CD8−, αβTCR+, γδTCR+, CD16+, CD161+, CD158a+, CD158b+, KIR3DL1+, and CD94+) expanded in response to both cytokines, whereas all CD56− cell subpopulations did not. Therefore, previously reported IL-15-induced γδ and CD8+ T cell expansions reflect proliferations of NK and CD56+ T cells that most frequently express these phenotypes. IL-15 also expanded CD8α+β− and Vα24Vβ11 TCR+ T cells. Both cytokines stimulated cytotoxicity by NK and CD56+ T cells against K562 targets, but not the production of IFN-γ, TNF-α, IL-2, or IL-4. However, they augmented cytokine production in response to phorbol ester stimulation or CD3 cross-linking by inducing the proliferation of NK cells and CD56+ T cells that produce these cytokines at greater frequencies than other T cells. These results indicate that IL-2 and IL-15 act at different stages of the immune response by expanding and partially activating NK receptor-positive lymphocytes, but, on their own, do not influence the Th1/Th2 balance of adaptive immune responses.
Regulation of IgE Production Requires Oligomerization of CD23
Authors: Kilmon Michelle A. , Ghirlando Rodolfo , Strub Marie-Paule , Beavil Rebecca L. , Gould Hannah J. , Conrad Daniel H.
Published: April 2014

Here we describe the production of a rabbit polyclonal Ab (RAS1) raised against the stalk of murine CD23. RAS1 inhibits release of CD23 from the surface of both M12 and B cells resulting in an increase of CD23 on the cell surface. Despite this increase, these cells are unable to bind IgE as determined by FACS. CD23 has previously been shown to bind IgE with both a high (4–10 × 107 M−1) and low (4–10 × 106 M−1) affinity. Closer examination by direct binding of 125I-IgE revealed that RAS1 blocks high affinity binding while having no effect on low affinity binding. These data support the model proposing that oligomers of CD23 mediate high affinity IgE binding. These experiments suggest that RAS1 binding to cell surface CD23 results in a shift from oligomers to monomers, which, according to the model, only bind IgE with low affinity. These experiments also suggest that high affinity binding of IgE is required for IgE regulation by CD23 and is demonstrated by the fact that treatment of Ag/Alum-immunized mice treated with RAS1 results in a significant increase in IgE production similar to the levels seen in CD23-deficient mice. These mice also had significantly decreased levels of serum soluble CD23 and Ag-specific IgG1. RAS1 had no effect on IgE or Ag-specific IgG1 production in CD23-deficient mice.
Eosinophils Promote Allergic Disease of the Lung by Regulating CD4+ Th2 Lymphocyte Function
Authors: MacKenzie Jason R. , Mattes Joerg , Dent Lindsay A. , Foster Paul S.
Published: April 2014

Eosinophils are primarily thought of as terminal effectors of allergic responses and of parasite elimination. However, limited studies suggest a more discrete immunomodulatory role for this leukocyte during these inflammatory responses. In this investigation, we highlight the potential of eosinophils to act as APCs and thus modulators of allergic responses by influencing Th2 cell function. In response to Ag provocation of the allergic lung, eosinophils rapidly trafficked to sites of Ag deposition (airways lumen) and presentation (lung-associated lymph nodes and T cell-rich paracortical zones). Eosinophils from the allergic lung expressed class II MHC peptides, T cell costimulatory molecules (CD80 and CD86), and rapidly internalized and processed Ag that was sampled from within the airway lumen. Ag-loaded eosinophils promoted the production of IL-4, IL-5, and IL-13 in cocultures with in vitro-polarized Th2 cells and induced IL-5 production in a dose-dependent manner from Ag-specific CD4+ T cells isolated from allergic mice. In addition, Ag-loaded eosinophils primed for Th2 cell-driven allergic disease of the lung when transferred to naive mice. Thus, eosinophils have the potential to not only activate Th2 cells to release disease-modulating cytokines but also to assist in priming the immune system for allergic responses. This investigation highlights the potential of eosinophils to not only act as terminal effector cells but also to actively modulate allergic inflammation by amplifying Th2 cell responses.
A New IFN-Like Cytokine, Limitin Modulates the Immune Response Without Influencing Thymocyte Development
Authors: Takahashi Isao , Kosaka Hiroshi , Oritani Kenji , Heath William R. , Ishikawa Jun , Okajima Yu , Ogawa Megumu , Kawamoto Sin-ichiro , Yamada Masahide , Azukizawa Hiroaki , Itami Satoshi , Yoshikawa Kunihiko , Tomiyama Yoshiaki , Matsuzawa Yuji
Published: April 2014

A novel IFN-like molecule, limitin, was recently identified and revealed to suppress B lymphopoiesis through the IFN-αβ receptor, although it lacked growth suppression on myeloid and erythroid progenitors. Here we have studied diverse effects of limitin on T lymphocytes and compared limitin with previously known IFNs. Like IFN–α and -β, limitin modified immunity in the following responses. It suppressed mitogen- and Ag-induced T cell proliferation through inhibiting the responsiveness to exogenous IL-2 rather than suppressing the production of IL-2. In contrast, limitin enhanced cytotoxic T lymphocyte activity associated with the perforin-granzyme pathway. To evaluate the effect of limitin in vivo, a lethal graft-versus-host disease assay was established. Limitin-treatment of host mice resulted in the enhancement of graft-versus-host disease. Limitin did not influence thymocyte development either in fetal thymus organ cultures or in newborn mice injected with limitin-Ig, suggesting that limitin is distinguishable from IFN-α and -β. From these findings, it can be speculated that the human homolog of limitin may be applicable for clinical usage because of its IFN-like activities with low adverse effects on, for example, T lymphopoiesis, erythropoiesis, and myelopoiesis.
Disruption of NF-κB Signaling Reveals a Novel Role for NF-κB in the Regulation of TNF-Related Apoptosis-Inducing Ligand Expression
Authors: Baetu Tudor M. , Kwon Hakju , Sharma Sonia , Grandvaux Nathalie , Hiscott John
Published: April 2014

The NF-κB family of transcription factors functions broadly in the host control of immunoregulatory gene expression, inflammation, and apoptosis. Using Jurkat T cells engineered to inducibly express a transdominant repressor of IκBα, we examined the role of NF-κB in the regulation of cytokine and apoptotic gene expression. In this T cell model, as well as in primary T lymphocytes, expression of TNF-related apoptosis-inducing ligand (TRAIL) apoptotic signaling protein was dramatically down-regulated by inhibition of NF-κB binding activity. TRAIL acts through membrane death receptors to induce apoptosis of activated T lymphocytes and can be up-regulated by a variety of physiological and pharmacological inducers. However, regulation of TRAIL gene expression has not been defined. Treatment with TCR mimetics (PMA/ionomycin, PHA, and anti-CD3/CD28 Abs) resulted in a rapid increase in the expression of TRAIL mRNA and cell surface TRAIL protein. Induction of the transdominant repressor of IκBα dramatically down-regulated surface expression of TRAIL, indicating an essential role for NF-κB in the regulation of TRAIL. The induced expression of TRAIL was linked to a c-Rel binding site in the proximal TRAIL promoter at position −256 to −265; mutation of this site or an adjacent κB site resulted in a complete loss of the inducibility of the TRAIL promoter. The regulation of TRAIL expression by NF-κB may represent a general mechanism that contributes to the control of TRAIL-mediated apoptosis in T lymphocytes.
Signaling Lymphocytic Activation Molecule Is Expressed on CD40 Ligand-Activated Dendritic Cells and Directly Augments Production of Inflammatory Cytokines
Authors: Bleharski Joshua R. , Niazi Kayvan R. , Sieling Peter A. , Cheng Genhong , Modlin Robert L.
Published: April 2014

Dendritic cells (DC) comprise a key part of the innate immune system that, upon activation, profoundly influences the nature of the adaptive T cell response. In this study, we present evidence that signaling lymphocytic activation molecule (SLAM), a molecule first identified in activated T and B cells, is strongly up-regulated in DC activated through CD40, as well as in response to inflammatory stimuli, including polyinosinic polycytidylic acid and LPS. mRNA encoding both membrane-bound and soluble secreted isoforms of SLAM was detected in CD40 ligand-activated DC, comprising two of the four known SLAM isoforms. Expression of membrane-bound SLAM protein peaked at 12 h poststimulation with CD40 ligand, gradually returning to baseline levels after 6 days. SLAM up-regulation appears to be a direct result of the induction of DC maturation, as inflammatory cytokines released during this process do not affect SLAM expression. Functionally, engagement of SLAM enhances DC production of IL-12 and IL-8, while having no effect on production of IL-10. Because SLAM is involved in the activation of T cells, the expression of SLAM on DC may provide a bidirectional signaling mechanism in which interacting DC and T cells are simultaneously and synergistically activated to mount proinflammatory Th1 responses.
Pituitary Adenylate Cyclase-Activating Polypeptide Inhibits Collagen-Induced Arthritis: An Experimental Immunomodulatory Therapy
Authors: Abad Catalina , Martinez Carmen , Leceta Javier , Gomariz Rosa P. , Delgado Mario
Published: April 2014

Rheumatoid arthritis is a chronic, systemic, autoimmune, and inflammatory disorder that affects the synovial lining of the joints. We describe the beneficial effects of the pituitary adenylate cyclase-activating polypeptide (PACAP) in the collagen-induced arthritis experimental murine model being proposed as a novel therapeutic approach in the treatment of rheumatoid arthritis. PACAP greatly decreases arthritis frequency and severity in the studied mice by improving clinical symptoms, ameliorating joint damage, and blocking both the inflammatory and autoimmune mediators which are the main keys of the pathogenesis of this disease. With this study, PACAP emerges as a promising candidate for the treatment of a pathology with a high world incidence but currently no effective treatment.
CD19 Can Regulate B Lymphocyte Signal Transduction Independent of Complement Activation
Authors: Hasegawa Minoru , Fujimoto Manabu , Poe Jonathan C. , Steeber Douglas A. , Tedder Thomas F.
Published: April 2014

B lymphocytes are critically regulated by signals transduced through the CD19-CD21 cell surface receptor complex, where complement C3d binding to CD21 supplies an already characterized ligand. To determine the extent that CD19 function is controlled by complement activation, CD19-deficient mice (that are hyporesponsive to transmembrane signals) and mice overexpressing CD19 (that are hyperresponsive) were crossed with CD21- and C3-deficient mice. Cell surface CD19 and CD21 expression were significantly affected by the loss of CD21 and C3 expression, respectively. Mature B cells from CD21-deficient littermates had ∼36% higher cell surface CD19 expression, whereas CD21/35 expression was increased by ∼45% on B cells from C3-deficient mice. Negative regulation of CD19 and CD21 expression by CD21 and C3, respectively, may be functionally significant because small increases in cell surface CD19 overexpression can predispose to autoimmunity. Otherwise, B cell development and function in CD19-deficient and -overexpressing mice were not significantly affected by a simultaneous loss of CD21 expression. Although CD21-deficient mice were found to express a hypomorphic cell surface CD21 protein at low levels that associated with mouse CD19, C3 deficiency did not significantly affect B cell development and function in CD19-deficient or -overexpressing mice. These results, and the severe phenotype exhibited by CD19-deficient mice compared with CD21- or C3-deficient mice, collectively demonstrate that CD19 can regulate B cell signaling thresholds independent of CD21 engagement and complement activation.
Vaccination with Cytoplasmic ErbB-2 DNA Protects Mice from Mammary Tumor Growth Without Anti-ErbB-2 Antibody
Authors: Pilon Shari A. , Piechocki Marie P. , Wei Wei-Zen
Published: April 2014

Wild-type ErbB-2 (E2) positive D2F2/E2 tumors are rejected by active vaccination with ErbB-2 DNA. However, anti-ErbB-2 Ab response can cause cardiac toxicity or interfere with cellular immunity. It will be advantageous to induce only cellular immunity by active vaccination. A panel of E2 DNA vaccines were constructed, and their vaccination efficacy was ranked as E2 > tyrosine kinase-deficient ErbB-2 (E2A) > full-length ErbB-2 targeted to the cytoplasm (cytE2) > tyrosine kinase-deficient cytE2 (cytE2A). E2A is a tyrosine kinase-deficient mutant containing a single residue substitution. CytE2 or cytE2A encodes a full-length protein that is targeted to and rapidly degraded in the cytosol by the proteasomes. Covaccination with cytE2A and GM-CSF or IL-2 DNA resulted in equivalent anti-tumor activity as E2. However, anti-ErbB-2 Ab was induced by E2 or E2A, but not cytE2 or cytE2A. Therefore, cytE2A appears to induce anti-tumor immunity without an Ab response. ErbB-2-specific CTL were detected in mice immunized with cytE2A and GM-CSF and have rejected tumor challenge. Depletion of CD8, but not CD4 T cells reduced anti-tumor immunity, indicating CTL as the effector cells. Covaccination with E2A and cytE2A induced synergistic anti-tumor activity, supporting enhanced peptide presentation from cytE2A, which was further evidenced by superior CTL activation using APCs expressing cytE2 vs E2. Taken together, cytoplasmic ErbB-2 DNA induced anti-tumor CTL, but not humoral response, demonstrating the feasibility of eliciting individual effector mechanism by targeted DNA vaccine.
Modulation of Human T Cell Responses and Macrophage Functions by Onchocystatin, a Secreted Protein of the Filarial NematodeOnchocerca volvulus
Authors: Schönemeyer Annett , Lucius Richard , Sonnenburg Bettina , Brattig Norbert , Sabat Robert , Schilling Klaus , Bradley Janette , Hartmann Susanne
Published: April 2014

Immune responses of individuals infected with filarial nematodes are characterized by a marked cellular hyporesponsiveness and a shift of the cytokine balance toward a Th2/Th3 response. This modulation of cellular immune responses is considered as an important mechanism to avoid inflammatory immune responses that could eliminate the parasites. We investigated the immunomodulatory potential of a secreted cysteine protease inhibitor (onchocystatin) of the human pathogenic filaria Onchocerca volvulus. Recombinant onchocystatin (rOv17), a biologically active cysteine protease inhibitor that inhibited among others the human cysteine proteases cathepsins L and S, suppressed the polyclonally stimulated and the Ag-driven proliferation of human PBMC. Stimulated as well as unstimulated PBMC in the presence of rOv17 produced significantly more IL-10, which was paralleled in some situations by a decrease of IL-12p40 and preceded by an increase of TNF-α. At the same time, rOv17 reduced the expression of HLA-DR proteins and of the costimulatory molecule CD86 on human monocytes. Neutralization of IL-10 by specific Abs restored the expression of HLA-DR and CD86, whereas the proliferative block remained unaffected. Depletion of monocytes from the PBMC reversed the rOv17-induced cellular hyporeactivity, indicating monocytes to be the target cells of immunomodulation. Therefore, onchocystatin has the potential to contribute to a state of cellular hyporesponsiveness and is a possible pathogenicity factor essential for the persistence of O. volvulus within its human host.
Activation of Eotaxin-3/CCL26 Gene Expression in Human Dermal Fibroblasts Is Mediated by STAT6
Authors: Hoeck Jutta , Woisetschläger Maximilian
Published: April 2014

Allergic inflammatory conditions such as asthma are characterized by an accumulation of eosinophils at sites of inflammation. Eotaxin-3/CCL26 is a member of the family of CC chemokines, which are known to be potent chemoattractants for eosinophils. This chemokine was shown to be up-regulated by IL-4 and IL-13 in endothelial cells. This study demonstrates that eotaxin-3 transcription and eotaxin-3 protein expression are stimulated by IL-4 and IL-13 in a time- and dose-dependent fashion in human dermal fibroblasts. In contrast to eotaxin-1/CCL11, TNF-α could not act as inducer on its own nor did it synergize with IL-4. The activities of eotaxin-3 promoter luciferase constructs were significantly increased by IL-4 and IL-13 in human dermal fibroblasts. This effect was mediated by a binding site for the transcription factor STAT6 in the eotaxin-3 promoter sequence. Mutations in the STAT6 binding site abrogated up-regulation of eotaxin-3 promoter activity. In STAT6-defective human embryonic kidney 293 cells, the wild-type luciferase construct, but not the STAT6 binding mutant, was inducible by IL-4 only upon cotransfection of STAT6 expression vector. In addition, eotaxin-3 protein was detectable in the supernatants of STAT6-transfected human embryonic kidney 293 cells upon IL-4 or IL-13 stimulation. In the same experiments, TNF-α induced activation of the monocyte chemoattractant protein-1/CCL2 gene was independent of STAT6 transfection. These results indicate that IL-4 and IL-13 activate eotaxin-3 gene expression in a STAT6-dependent fashion. Although both eotaxin-1 and -3 are regulated by this transcription factor, the response of the eotaxin-3 gene to TNF-α stimulation appears to be different.
The HA-2 Minor Histocompatibility Antigen Is Derived from a Diallelic Gene Encoding a Novel Human Class I Myosin Protein
Authors: Pierce Richard A. , Field Erin D. , Mutis Tuna , Golovina Tatiana N. , Kap-Herr Chris Von , Wilke. Martina , Pool Jos , Shabanowitz Jeffrey , Pettenati Mark J. , Eisenlohr Laurence C. , Hunt Donald F. , Goulmy Els , Engelhard Victor H.
Published: April 2014

Human minor histocompatibility Ags (mHag) present significant barriers to successful bone marrow transplantation. However, the structure of human mHag and the basis for antigenic disparities are still largely unknown. Here we report the identification of the gene encoding the human mHag HA-2 as a previously unknown member of the class I myosin family, which we have designated MYO1G. The gene is located on the short arm of chromosome 7. Expression of this gene is limited to cells of hemopoietic origin, in keeping with the previously defined tissue expression of the HA-2 Ag. RT-PCR amplification of MYO1G from different individuals led to the identification of two genetic variants, designated MYO1GV and MYO1GM. The former encodes the peptide sequence previously shown to be the HA-2 epitope (YIGEVLVSV), whereas the latter shows a single amino acid change in this peptide (YIGEVLVSM). This change has only a modest effect on peptide binding to the class I MHC-restricted element HLA-A*0201, and a minimal impact on recognition by T cells when added exogenously to target cells. Nonetheless, as detected using either T cells or mass spectrometry, this amino acid change results in a failure of the latter peptide to be presented at the surface of cells that express MYO1GM endogenously. These studies have thus identified a new mHag-encoding gene, and thereby provide additional information about both the genetic origins of human mHag as well as the underlying basis of an Ag-positive vs Ag-negative state.
Down-Regulation of CD28 Expression by TNF-α
Authors: Bryl Ewa , Vallejo Abbe N. , Weyand Cornelia M. , Goronzy Jörg J.
Published: April 2014

Aging and chronic inflammatory syndromes, such as rheumatoid arthritis, are associated with high frequencies of CD4+CD28null T cells, which are rarely seen in healthy individuals younger than 40 years. Inasmuch as rheumatoid arthritis and aging are also associated with elevated levels of TNF-α, we examined whether this proinflammatory cytokine influences CD28 expression. Incubation of T cell lines and clones as well as Jurkat cells with TNF-α induced a reduction in the levels of cell surface expression of CD28. This effect of TNF-α was reversible; however, continuous culture of CD4+CD28+ T cell clones in TNF-α resulted in the appearance of a CD28null subset. In reporter gene bioassays, TNF-α was found to inhibit the activity of the CD28 minimal promoter. Inactivation of the promoter was accompanied by a marked reduction in DNA-protein complex formation by two DNA sequence motifs corresponding to the transcriptional initiator of the CD28 gene. Indeed, in vitro transcription assays showed that nuclear extracts from TNF-α-treated cells failed to activate transcription of DNA templates under the control of a consensus TATA box and the CD28 initiator sequences. In contrast, similar extracts from unstimulated T cells supported transcription. These results demonstrate that TNF-α directly influences CD28 gene transcription. We propose that the emergence of CD4+CD28null T cells in vivo is facilitated by increased production of TNF-α.
Antibody Repertoire Development in Fetal And Neonatal Piglets. IV. Switch Recombination, Primarily in Fetal Thymus, Occurs Independent of Environmental Antigen and Is Only Weakly Associated with Repertoire Diversification
Authors: Butler John E. , Sun Jishan , Weber Patrick , Ford Steven P. , Rehakova Zuzana , Sinkora Jiri , Lager Kelly
Published: April 2014

The epitheliochorial placenta of swine is considered a barrier to Ag and selective transport of IgG, so this species should be an excellent model with which to determine whether switch recombination is Ag dependent. Analysis of Ig levels and Ig isotype profiles in >150 normal and virus-infected fetuses from 38–110 days of gestation (DG) suggested that IgG, IgA, and IgM were most likely the result of de novo fetal synthesis. Although transcripts for IgM could be recovered at DG 50 (114 DG is full gestation) in all major fetal lymphoid tissues, those for IgG and IgA first became prominent at 60 DG in thymus, and transcription and spontaneous secretion became especially pronounced in this organ in older fetuses. Data on transcription, secretion, and serum isotype profiles suggest that although all fetal IgA and IgM may result from de novo synthesis, some IgG may result from low-level selective transport. The complementarity-determining region 3 spectratypes of thymic IgA and IgG transcripts at 70 and 90 days, respectively, were as polyclonal as that of IgM, indicating a broad repertoire of switched B cells although the VDJs transcribed with these switched isotypes in normal fetuses were not diversified in comparison to those from animals exposed to environmental Ags such as age-matched, virus-infected fetuses, colonized isolator piglets, and conventional adults. However, VDJs expressed with switched isotypes were more diversified than those expressed with IgM. Thus, switch recombination in fetal life does not appear to be driven by environmental Ag and is only weakly coupled to VDJ diversification. These findings, and the fact that the oligoclonal IgA and IgM repertoires in a noninductive site of the mucosal immune system (parotid gland) become polyclonal in piglets reared germfree, suggest that initial expansion of the switched cells in the B cell compartment of fetal and neonatal piglets is not driven by environmental Ag.
T Cell Selection and Differential Activation on Structurally Related HLA-DR4 Ligands
Authors: Gebe John A. , Novak Erik J. , Kwok William W. , Farr Andy G. , Nepom Gerald T. , Buckner Jane H.
Published: April 2014

Plasticity of TCR interactions during CD4+ T cell activation by an MHC-peptide complex accommodates variation in the peptide or MHC contact sites in which recognition of an altered ligand by the T cell can modify the T cell response. To explore the contribution of this form of TCR cross-recognition in the context of T cell selection on disease-associated HLA molecules, we have analyzed the relationship between TCR recognition of the DRB1*0401- and DRB1*0404-encoded HLA class II molecules associated with rheumatoid arthritis. Thymic reaggregation cultures demonstrated that CD4+ T cells selected on either DRB1*0401 or DRB1*0404 could be subsequently activated by the other MHC molecule. Using HLA tetramer technology we identify hemagglutinin residue 307–319-specific T cells restricted by DRB1*0401, but activated by hemagglutinin residues 307–319, in the context of DRB1*0404. One such clone exhibits an altered cytokine profile upon activation with the alternative MHC ligand. This altered phenotype persists when both class II molecules are present. These findings directly demonstrate that T cells selected on an MHC class II molecule carry the potential for activation on altered self ligands when encountering Ags presented on a related class II molecule. In individuals heterozygous for these alleles the possibility of TCR cross-recognition could lead to an aberrant immune response.
HS1,2 Enhancer Regulation of Germline ε and γ2b Promoters in Murine B Lymphocytes: Evidence for Specific Promoter-Enhancer Interactions
Authors: Laurencikiene Jurga , Deveikaite Vilma , Severinson Eva
Published: April 2014

During an immune response, activated B cells develop into high rate Ig-secreting plasma cells. They also switch from production of IgM to IgG, IgA, or IgE. This process requires a DNA recombination event, which is regulated at the transcriptional level by the production of isotype-specific, sterile germline (GL) transcripts. Induction of these transcripts is controlled by GL promoters and, possibly, by IgH 3′ enhancers. We investigated the interaction of the GL ε and γ2b promoters with the HS1,2 enhancer using transiently transfected mouse primary B cells and cell lines. The constructs used for the transfections contained a GL promoter upstream and HS1,2 downstream of a luciferase reporter gene. Both GL ε and γ2b promoters synergized strongly with the HS1,2 enhancer in activated primary B cells, a mature B cell line, and a plasma cell line. We show that the major activity of HS1,2 in activated primary B cells occurs within a 310-bp fragment that includes NF-κB, OCT, and NF of activated B cells (Ets/AP-1) sites. By mutating the consensus sequences for various transcription factors, we have determined which sites in HS1,2 are important for synergy with the GL ε and γ2b promoters. Our findings indicate that different sites in HS1,2 might selectively interact with the GL ε and γ2b promoters. We also provide evidence that B cell-specific activator protein is not an absolute suppressor of HS1,2 activity.
Humanization and Epitope Mapping of Neutralizing Anti-Human Fas Ligand Monoclonal Antibodies: Structural Insights into Fas/Fas Ligand Interaction
Authors: Nisihara Tsukasa , Ushio Yoshitaka , Higuchi Hirohumi , Kayagaki Nobuhiko , Yamaguchi Noriko , Soejima Kenji , Matsuo Seishi , Maeda Hiroaki , Eda Yasuyuki , Okumura Ko , Yagita Hideo
Published: April 2014

Fas ligand (L)/CD95L, a proapoptotic member of the TNF family, is a potential target for clinical intervention in various diseases. In the present study, we generated a humanized anti-human FasL mAb and characterized the epitopes of neutralizing mAbs by extensive alanine-scanning mutagenesis of human FasL. The predicted molecular model of FasL trimer revealed that the mAbs recognize largely overlapped conformational epitopes that are composed of two clusters, one around the outer tip-forming D-E loop and another near the top of FasL. Both of these sites on FasL are critically involved in the direct interaction with the corresponding receptor, Fas. These results suggest that the mAbs efficiently neutralize FasL cytotoxicity by masking both of these FasL/Fas contact sites.
Crystal Structures of Two Closely Related but Antigenically Distinct HLA-A2/Melanocyte-Melanoma Tumor-Antigen Peptide Complexes
Authors: Sliz Piotr , Michielin Olivier , Cerottini Jean-Charles , Luescher Immanuel , Romero Pedro , Karplus Martin , Wiley Don C.
Published: April 2014

We have determined high-resolution crystal structures of the complexes of HLA-A2 molecules with two modified immunodominant peptides from the melanoma tumor-associated protein Melan-A/Melanoma Ag recognized by T cells-1. The two peptides, a decamer and nonamer with overlapping sequences (ELAGIGILTV and ALGIGILTV), are modified in the second residue to increase their affinity for HLA-A2. The modified decamer is more immunogenic than the natural peptide and a candidate for peptide-based melanoma immunotherapy. The crystal structures at 1.8 and 2.15 Å resolution define the differences in binding modes of the modified peptides, including different clusters of water molecules that appear to stabilize the peptide-HLA interaction. The structures suggest both how the wild-type peptides would bind and how three categories of cytotoxic T lymphocytes with differing fine specificity might recognize the two peptides.
An Important Role of CDK Inhibitor p18INK4c in Modulating Antigen Receptor-Mediated T Cell Proliferation
Authors: Kovalev Grigoriy I. , Franklin David S. , Coffield V. McNeil , Xiong Yue , Su Lishan
Published: April 2014

The inhibitors of cyclin-dependent kinase (CDK) 4 (INK4) bind CDK4/6 to prevent their association with D-cyclins and G1 cell cycle initiation and progression. We report here that among the seven CDK inhibitors, p18INK4c played an important role in modulating TCR-mediated T cell proliferation. Loss of p18INK4c in T cells led to hyperproliferation in response to CD3 stimulation. p18INK4c-null mice developed lymphoproliferative disorder and T cell lymphomas. Expression of IL-2, IL-2R-α, and the major G1 cell cycle regulatory proteins was not altered in p18-null T cells. Both FK506 and rapamycin efficiently inhibited proliferation of p18-null T cells. In activated T cells, p18INK4c remained constant, and preferentially associated with and inhibited CDK6 but not CDK4. We propose that p18INK4c sets an inhibitory threshold in T cells and one function of CD28 costimulation is to counteract the p18INK4c inhibitory activity on CDK6-cyclin D complexes. The p18INK4c protein may provide a novel target to modulate T cell immunity.
Antigen-Specific CD8+ T Cells Persist in the Upper Respiratory Tract Following Influenza Virus Infection
Authors: Wiley James A. , Hogan Robert J. , Woodland David L. , Harmsen Allen G.
Published: April 2014

Because little is known about lymphocyte responses in the nasal mucosa, lymphocyte accumulation in the nasal mucosa, nasal-associated lymphoid tissue (NALT), and cervical lymph nodes (CLN) were determined after primary and heterosubtypic intranasal influenza challenge of mice. T cell accumulation peaked in the nasal mucosa on day 7, but peaked slightly earlier in the CLN (day 5) and later (day 10) in the NALT. Tetrameric staining of nasal mucosal cells revealed a peak accumulation of CD8 T cells specific for either the H-2Db influenza nucleoprotein epitope 366–374 (DbNP366) or the H-2Db polymerase 2 protein epitope 224–233 (DbPA224) at 7 days. By day 13, DbPA224-specific CD8 T cells were undetectable in the mucosa, whereas DbNP366-specific CD8 T cells persisted for at least 35 days in the mucosa and spleen. After heterosubtypic virus challenge, the accumulation of CD8 T cells in the nasal mucosa was quicker, more intense, and predominantly DbNP366 specific relative to the primary inoculation. The kinetics and specificity of the CD8 T cell response were similar to those in the CLN, but the responses in the NALT and spleen were again slower and more protracted. These results indicate that similar to what was reported in the lung, DbNP366-specific CD8 T cells persist in the nasal mucosa after primary influenza infection and predominate in an intensified nasal mucosal response to heterosubtypic challenge. In addition, differences in the kinetics of the CD8 T cell responses in the CLN, NALT, and spleen suggest different roles of these lymphoid tissues in the mucosal response.
ATP-Mediated Killing of Intracellular Mycobacteria by Macrophages Is a P2X7-Dependent Process Inducing Bacterial Death by Phagosome-Lysosome Fusion
Authors: Fairbairn Ian P. , Stober Carmel B. , Kumararatne Dinakantha S. , Lammas David A.
Published: April 2014

Mycobacterium tuberculosis survives within host macrophages by actively inhibiting phagosome fusion with lysosomes. Treatment of infected macrophages with ATP induces both cell apoptosis and rapid killing of intracellular mycobacteria. The following studies were undertaken to characterize the effector pathway(s) involved. Macrophages were obtained from p47phox and inducible NO synthase gene-disrupted mice (which are unable to produce reactive oxygen and nitrogen radicals, respectively) and P2X7 gene-disrupted mice. RAW murine macrophages transfected with either the natural resistance-associated macrophage protein gene 1 (Nramp1)-resistant or Nramp1-susceptible gene were also used. The cells were infected with bacille Calmette-Guérin (BCG), and intracellular mycobacterial trafficking was analyzed using confocal and electron microscopy. P2X7 receptor activation was essential for effective ATP-induced mycobacterial killing, as its bactericidal activity was radically diminished in P2X7−/− macrophages. ATP-mediated killing of BCG within p47phox−/−, inducible NO synthase−/−, and Nramps cells was unaffected, demonstrating that none of these mechanisms have a role in the ATP/P2X7 effector pathway. Following ATP stimulation, BCG-containing phagosomes rapidly coalesce and fuse with lysosomes. Blocking of macrophage phospholipase D activity with butan-1-ol blocked BCG killing, but not macrophage death. ATP stimulates phagosome-lysosome fusion with concomitant mycobacterial death via P2X7 receptor activation. Macrophage death and mycobacterial killing induced by the ATP/P2X7 signaling pathway can be uncoupled, and diverge proximal to phospholipase D activation.