bluuebunny/crossref_metadata_2025 huggingface dataset:
Altered Peptide Ligand-Mediated TCR Antagonism Can Be Modulated by a Change in a Single Amino Acid Residue Within the CDR3β of an MHC Class I-Restricted TCR
Authors: Kalergis Alexis M. , Nathenson Stanley G.
Published: April 2014

The Ag receptor of cytotoxic CD8+ T lymphocytes recognizes peptides of 8–10 aa bound to MHC class I molecules. This Ag recognition event leads to the activation of the CD8+ lymphocyte and subsequent lysis of the target cell. Altered peptide ligands are analogues derived from the original antigenic peptide that commonly carry amino acid substitutions at TCR contact residues. TCR engagement by these altered peptide ligands usually impairs normal T cell function. Some of these altered peptide ligands (antagonists) are able to specifically antagonize and inhibit T cell activation induced by the wild-type antigenic peptide. Despite significant advances made in understanding TCR antagonism, the molecular interactions between the TCR and the MHC/peptide complex responsible for the inhibitory activity of antagonist peptides remain elusive. To approach this question, we have identified altered peptide ligands derived from the vesicular stomatitis virus peptide (RGYVYQGL) that specifically antagonize an H-2Kb/vesicular stomatitis virus-specific TCR. Furthermore, by site-directed mutagenesis, we altered single amino acid residues of the complementarity-determining region 3 of the β-chain of this TCR and tested the effect of these point mutations on Ag recognition and TCR antagonism. Here we show that a single amino acid change on the TCR CDR3β loop can modulate the TCR-antagonistic properties of an altered peptide ligand. Our results highlight the role of the TCR complementarity-determining region 3 loops for controlling the nature of the T cell response to TCR/altered peptide ligand interactions, including those leading to TCR antagonism.
IL-10 Gene Expression Is Controlled by the Transcription Factors Sp1 and Sp3
Authors: Tone Masahide , Powell Mark J. , Tone Yukiko , Thompson Sara A. J. , Waldmann Herman
Published: April 2014

IL-10 is an 18-kDa cytokine with a key role in homeostatic control of inflammatory and immune responses. We have investigated how transcription of the IL-10 gene is regulated, so as to be able to understand the circumstances of IL-10 expression in both health and disease. In the mouse, IL-10 gene expression is regulated by a TATA-type promoter with a critical cis-acting element containing GGA repeats located at −89 to −77. Its complementary sequence is similar to the cis-acting elements (TCC repeats) in the promoters of genes encoding epidermal growth factor receptor and CD58. All these elements comprise a common CCTCCT sequence with less conserved C + T-rich sequences. Eliminating this CCTCCT sequence results in a marked reduction in promoter activity, suggesting a necessary role in IL-10 gene expression. Despite its dissimilarity to the G + C-rich Sp1 consensus sequence (GC box), Sp1 and Sp3 transcription factors could be shown to bind to this motif. The requirement for Sp1 and Sp3 in transcription of IL-10 was confirmed using Drosophila SL2 cells, which lack endogenous Sp factors. These results suggest that the transcription of IL-10 is positively regulated by both Sp1 and Sp3.
Posttranscriptional Regulation of IL-10 Gene Expression Through Sequences in the 3′-Untranslated Region
Authors: Powell Mark J. , Thompson Sara A. J. , Tone Yukiko , Waldmann Herman , Tone Masahide
Published: April 2014

IL-10 is an 18-kDa immunoregulatory cytokine the transcription of which is controlled by the ubiquitously expressed transcription factors Sp1 and Sp3. Although many cell types express IL-10 mRNA, not all make detectable amounts of protein, and levels of protein expression vary enormously. We show here that much of this variation can be accounted for by posttranscriptional mechanisms. Multiple copies of potential mRNA destabilizing motifs AUUUA and related sequences can be found to the 3′-untranslated region (UTR) of IL-10 mRNA distributed through three potential regulatory regions. Evidence of RNA-destabilizing activities in all three regions was deduced from luciferase reporter assays. The half-life of RNA containing the 3′-UTR of IL-10 mRNA was quite short in both nonstimulated (t1/2 = 1 h), and PMA-stimulated EL-4 cell (t1/2 = 3 h). In contrast, the half-life of RNA lacking the 3′-UTR was much longer (t1/2 = >12 h) whether cells were stimulated or not. This suggests that many cells are poised to secrete IL-10 and will do so if they receive appropriate posttranscriptional signals.
Dominant Epitopes and Allergic Cross-Reactivity: Complex Formation Between a Fab Fragment of a Monoclonal Murine IgG Antibody and the Major Allergen from Birch Pollen Bet v 1
Authors: Mirza Osman , Henriksen Anette , Ipsen Henrik , Larsen Jørgen N. , Wissenbach Margit , Spangfort Michael D. , Gajhede Michael
Published: April 2014

The symptoms characteristic of allergic hypersensitivity are caused by the release of mediators, i.e., histamine, from effector cells such as basophils and mast cells. Allergens with more than one B cell epitope cross-link IgE Abs bound to high affinity FcεRI receptors on mast cell surfaces leading to aggregation and subsequent mediator release. Thus, allergen-Ab complexes play a crucial role in the cascade leading to the allergic response. We here report the structure of a 1:1 complex between the major birch pollen allergen Bet v 1 and the Fab fragment from a murine monoclonal IgG1 Ab, BV16, that has been solved to 2.9 Å resolution by x-ray diffraction. The mAb is shown to inhibit the binding of allergic patients’ IgE to Bet v 1, and the allergen-IgG complex may therefore serve as a model for the study of allergen-IgE interactions relevant in allergy. The size of the BV16 epitope is 931 Å2 as defined by the Bet v 1 Ab interaction surface. Molecular interactions predicted to occur in the interface are likewise in agreement with earlier observations on Ag-Ab complexes. The epitope is formed by amino acids that are conserved among major allergens from related species within the Fagales order. In combination with a surprisingly high inhibitory capacity of BV16 with respect to allergic patients’ serum IgE binding to Bet v 1, these observations provide experimental support for the proposal of dominant IgE epitopes located in the conserved surface areas. This model will facilitate the development of new and safer vaccines for allergen immunotherapy in the form of mutated allergens.
Senescent Jejunal Mast Cells and Eosinophils in the Mouse Preferentially Translocate to the Spleen and Draining Lymph Node, Respectively, During the Recovery Phase of Helminth Infection
Authors: Friend Daniel S. , Gurish Michael F. , Austen K. Frank , Hunt John , Stevens Richard L.
Published: April 2014

Because mice infected with Trichinella spiralis experience a pronounced, but transient, mastocytosis and eosinophilia in their intestine, this disease model was used to follow the fate of senescent T cell-dependent mast cells (MCs) and eosinophils. Very few MCs or eosinophils undergoing apoptosis were found in the jejunum during the resolution phase of the infection, even though apoptotic MCs were common in the large intestine. Although the mesenteric draining lymph nodes contained large numbers of apoptotic eosinophils, MCs were rarely found at this location. During the recovery phase, large numbers of MCs were present in the spleen, and many of these cells possessed segmented nuclei. These splenic MCs were not proliferating. Although MCs from the jejunum and spleen of noninfected mice failed to express mouse MC protease (mMCP) 9, essentially all of the MCs in the jejunal submucosa and spleen of T. spiralis-infected mice expressed this serine protease during the recovery phase. The MCs in the jejunum expressed mMCP-9 before any mMCP-9-containing cells could be detected in the spleen. The fact that mMCP-9-containing MCs were detected in splenic blood vessels as these cells began to disappear from the jejunum supports the view that many jejunal MCs translocate to the spleen during the recovery phase of the infection. During this translocation process, some senescent jejunal MCs undergo nuclear segmentation. These studies reveal for the first time different exit and disposal pathways for T cell-dependent eosinophils and MCs after their expansion in the jejunum during a helminth infection.
CD8+ CTL from Lungs ofMycobacterium tuberculosis-Infected Mice Express Perforin In Vivo and Lyse Infected Macrophages
Authors: Serbina Natalya V. , Liu Chau-Ching , Scanga Charles A. , Flynn JoAnne L.
Published: April 2014

CD8+ T lymphocytes have been implicated in the protective immune response against human and murine tuberculosis. However, the functional role that this cell subset plays during the resolution of infection remains controversial. In this study, we demonstrate the presence of Mycobacterium tuberculosis-specific CD8+ CTL in the lungs and lung-draining lymph nodes of mice infected with M. tuberculosis via the aerosol or i.v. route. These cells expressed perforin in vivo and specifically recognized and lysed M. tuberculosis-infected macrophages in a perforin-dependent manner after a short period of in vitro restimulation. The efficiency of lysis of infected macrophages was dependent upon the time allowed for interaction between macrophage and M. tuberculosis bacilli. Recognition of infected targets by CD8+ CTL was β2-microglobulin and MHC class I dependent and was not CD1d restricted. The presented data indicate that CD8+ T cells contribute to the protective immune response during M. tuberculosis infection by exerting cytotoxic function and lysing infected macrophages.
Expression of P-Selectin at Low Site Density Promotes Selective Attachment of Eosinophils Over Neutrophils
Authors: Edwards Bruce S. , Curry Mark S. , Tsuji Hisashi , Brown David , Larson Richard S. , Sklar Larry A.
Published: April 2014

The selective interaction of neutrophils with E-selectin and eosinophils with P-selectin has been previously reported, but the relevance of selectin site density and fluid shear has not been studied in detail. We have developed a new approach to examine these interactions in cell suspensions that integrates an on-line cone-plate viscometer with a flow cytometer. We find that eosinophils and neutrophils both use P-selectin glycoprotein ligand-1 to form stable conjugates with P-selectin Chinese hamster ovary cell transfectants, with a preferential adhesion of eosinophils. Further, the difference in cell adhesion between neutrophils and eosinophils is magnified at P-selectin expression levels below ∼20 sites/μm2, a range likely to be relevant to endothelial cell expression levels in conditions associated with eosinophilia. The unique behavior is retained over shear rates ranging from 100 to 1500/s but is magnified at low shear. Results from parallel-plate flow chamber assays suggest that preferential eosinophil adhesion reflects an enhanced efficiency of initial PSGL-1 bond formation with P-selectin rather than a unique ability of eosinophils to mediate rolling interactions of longer duration on low-density P-selectin substrates. These differences may account in part for the increase in eosinophil accumulation in allergic diseases.
Cyclic Tensile Strain Acts as an Antagonist of IL-1β Actions in Chondrocytes
Authors: Xu Zhongfa , Buckley Michael J. , Evans Christopher H. , Agarwal Sudha
Published: April 2014

Inflammatory cytokines play a major role in cartilage destruction in diseases such as osteoarthritis and rheumatoid arthritis. Because physical therapies such as continuous passive motion yield beneficial effects on inflamed joints, we examined the intracellular mechanisms of mechanical strain-mediated actions in chondrocytes. By simulating the effects of continuous passive motion with cyclic tensile strain (CTS) on chondrocytes in vitro, we show that CTS is a potent antagonist of IL-1β actions and acts as both an anti-inflammatory and a reparative signal. Low magnitude CTS suppresses IL-1β-induced mRNA expression of multiple proteins involved in catabolic responses, such as inducible NO synthase, cyclo-oxygenase II, and collagenase. CTS also counteracts cartilage degradation by augmenting mRNA expression for tissue inhibitor of metalloproteases and collagen type II that are inhibited by IL-1β. Additionally, CTS augments the reparative process via hyperinduction of aggrecan mRNA expression and abrogation of IL-1β-induced suppression of proteoglycan synthesis. Nonetheless, the presence of an inflammatory signal is a prerequisite for the observed CTS actions, as exposure of chondrocytes to CTS alone has little effect on these parameters. Functional analysis suggests that CTS-mediated anti-inflammatory actions are not mediated by IL-1R down-regulation. Moreover, as an effective antagonist of IL-1β, the actions of CTS may involve disruption/regulation of signal transduction cascade of IL-1β upstream of mRNA transcription. These observations are the first to show that CTS directly acts as an anti-inflammatory signal on chondrocytes and provide a molecular basis for its actions.
The Role of the CC Chemokine, RANTES, in Acute Lung Allograft Rejection
Authors: Belperio John A. , Burdick Marie D. , Keane Michael P. , Xue Ying Ying , Lynch Joseph P. , Daugherty Bruce L. , Kunkel Steven L. , Strieter Robert M.
Published: April 2014

Lung transplantation is a therapeutic option for patients with end-stage lung disease. Acute allograft rejection is a major complication of lung transplantation and is characterized by the infiltration of activated mononuclear cells. The specific mechanisms that recruit these leukocytes have not been fully elucidated. The CC chemokine, RANTES, is a potent mononuclear cell chemoattractant. In this study we investigated RANTES involvement during acute lung allograft rejection in humans and in a rat model system. Patients with allograft rejection had a 2.3-fold increase in RANTES in their bronchoalveolar lavages compared with healthy allograft recipients. Rat lung allografts demonstrated a marked time-dependent increase in levels of RANTES compared with syngeneic control lungs. RANTES levels correlated with the temporal recruitment of mononuclear cells and the expression of RANTES receptors CCR1 and CCR5. To determine RANTES involvement in lung allograft rejection, lung allograft recipients were passively immunized with either anti-RANTES or control Abs. In vivo neutralization of RANTES attenuated acute lung allograft rejection and reduced allospecific responsiveness by markedly decreasing mononuclear cell recruitment. These experiments support the idea that RANTES, and the expression of its receptors have an important role in the pathogenesis of acute lung allograft rejection.
Differential Involvement of Src Family Kinases in Fcγ Receptor-Mediated Phagocytosis
Authors: Suzuki Takeshi , Kono Hajime , Hirose Naoto , Okada Masato , Yamamoto Tadashi , Yamamoto Kazuhiko , Honda Zen-ichiro
Published: April 2014

The tyrosine phosphorylation cascade originated from Fcγ receptors (FcγRs) is essential for macrophage functions including phagocytosis. Although the initial step is ascribed to Src family tyrosine kinases, the role of individual kinases in phagocytosis signaling is still to be determined. In reconstitution experiments, we first showed that expression in the RAW 264.7 cell line of C-terminal Src kinase (Csk) inhibited and that of a membrane-anchored, gain-of-function Csk abolished the FcγR-mediated signaling that leads to phagocytosis in a kinase-dependent manner. We next tested reconstruction of the signaling in the membrane-anchored, gain-of-function Csk-expressing cells by introducing Src family kinases the C-terminal negative regulatory sequence of which was replaced with a c-myc epitope. Those constructs derived from Lyn and Hck (a-Lyn and a-Hck) that associated with detergent-resistant membranes successfully reconstructed FcγR-mediated Syk activation, filamentous actin rearrangement, and phagocytosis. In contrast, c-Src-derived construct (a-Src), that was excluded from detergent-resistant membranes, could not restore the series of phagocytosis signaling. Tyrosine phosphorylation of Vav and c-Cbl was restored in common by a-Lyn, a-Hck, and a-Src, but FcγRIIB tyrosine phosphorylation, which is implicated in negative signaling, was reconstituted solely by a-Lyn and a-Hck. These findings suggest that Src family kinases are differentially involved in FcγR-signaling and that selective kinases including Lyn and Hck are able to fully transduce phagocytotic signaling.
Ribozymes as Tools for Therapeutic Target Validation in Arthritis
Authors: Jarvis Thale C. , Bouhana Karyn S. , Lesch Mark E. , Brown Suzy A. , Parry Tom J. , Schrier Denis J. , Hunt Stephen W. , Pavco Pamela A. , Flory Craig M.
Published: April 2014

In this paper we describe a method for validating therapeutic gene targets in arthritic disease. Ribozymes are catalytic oligonucleotides capable of highly sequence-specific cleavage of RNA. We designed ribozymes that cleave the mRNA encoding stromelysin, a matrix metalloproteinase implicated in cartilage catabolism. Ribozymes were initially screened in cultured fibroblasts to identify sites in the mRNA that were accessible for binding and cleavage. Accessible sites for ribozyme binding were found in various regions of the mRNA, including the 5′ untranslated region, the coding region, and the 3′ untranslated region. Several ribozymes that mediated sequence-specific and dose-dependent inhibition of stromelysin expression were characterized. Site selection in cell culture was predictive of in vivo bioactivity. An assay for measuring cartilage catabolism in rabbit articular cartilage explants was developed. Ribozymes inhibited IL-1-stimulated stromelysin mRNA expression in articular cartilage explants, yet failed to inhibit proteoglycan degradation. This indicated that up-regulation of stromelysin was not essential for IL-1-induced cartilage catabolism. Broad applications of this approach in therapeutic target validation are discussed.
Critical Involvement of the Chemotactic Axis CXCR4/Stromal Cell-Derived Factor-1α in the Inflammatory Component of Allergic Airway Disease
Authors: Gonzalo Jose-Angel , Lloyd Clare M. , Peled Amnon , Delaney Tracy , Coyle Anthony J. , Gutierrez-Ramos Jose-Carlos
Published: April 2014

Stromal cell-derived factor-1α/β (SDF-1α/β) is phylogenetically a primitive chemokine widely expressed in a variety of tissues and cell types. This expression is detectable in the absence of stimuli provided by bacterial or viral infections and allergic or autoimmune disorders. Based on these and other findings, SDF-1α has not been considered an inflammatory chemokine, but, rather, has been believed to be involved in certain homeostatic processes, such as leukocyte recirculation. SDF-1α is a potent chemoattractant for lymphocytes and monocytes that mediates its activity via the chemokine receptor CXCR4. Study of the role of SDF-1α/CXCR4 in vivo during inflammation has been limited by the fact that transgenic mice that have been made deficient in either molecule die early in life due to developmental defects. The present study was aimed at evaluating the functional relevance of the SDF-1α/CXCR4 axis during an inflammatory process. Neutralizing Abs to CXCR4 reduced lung eosinophilia (bronchoalveolar lavage fluid and interstitium) by half, indicating that CXCR4-mediated signals contribute to lung inflammation in a mouse model of allergic airway disease (AAD). This reduction in inflammation was accompanied by a significant decrease in airway hyper-responsiveness. SDF-1α neutralization resulted in similar reduction in both lung allergic inflammation and airway hyper-responsiveness. Retroviral delivery of a CXCR4 cDNA to leukocytes resulted in greater inflammation when transduced mice were subjected to a mouse model of AAD. These results highlight that, although considered a noninflammatory axis, the involvement of CXCR4 and SDF-1α is critical during AAD, and this receptor and its ligand are potentially relevant in other inflammatory processes.
Mechanism of Paclitaxel Activity in Kaposi’s Sarcoma
Authors: Sgadari Cecilia , Toschi Elena , Palladino Clelia , Barillari Giovanni , Carlei Davide , Cereseto Anna , Ciccolella Cristiana , Yarchoan Robert , Monini Paolo , Stürzl Michael , Ensoli Barbara
Published: April 2014

Kaposi’s sarcoma (KS) is an angioproliferative disease characterized by proliferation of spindle-shaped cells predominantly of endothelial cell origin, neoangiogenesis, inflammatory cell infiltration, and edema. At least in early stage, KS behaves as a reactive lesion sustained by the action of inflammatory cytokines and growth factors, has a polyclonal nature, and can regress. However, in time it can become monoclonal, especially in the nodular stage, evolving into a true sarcoma, likely in association with the increased expression of antiapoptotic oncogenes. We have recently demonstrated by immunohistochemical analysis that Bcl-2, a proto-oncogene known to prolong cellular viability and to antagonize apoptosis, is highly expressed in spindle cells and vessels of both AIDS-KS and classical KS lesions and that its expression increases with lesion stage. Paclitaxel, a microtubule-stabilizing drug known to inhibit Bcl-2 antiapoptotic activity and to be highly effective in the treatment of certain neoplasms, has recently been found to be active also in patients with advanced HIV-associated KS. In this report we investigated the mechanism(s) of paclitaxel activity in KS. By using a model of experimental KS induced by the inoculation of KS-derived spindle cells in nude mice and primary cultures of KS spindle cells, we found that paclitaxel promotes regression of KS lesions in vivo and that it blocks the growth, migration, and invasion of KS cells in vitro. Furthermore, paclitaxel treatment promoted apoptosis and down-regulated Bcl-2 protein expression in KS cells in vitro and in KS-like lesions in mice. Our results suggest that paclitaxel interferes with KS by down-regulating Bcl-2 antiapoptotic effect.
Repeated Administration of Cytosine-Phosphorothiolated Guanine-Containing Oligonucleotides Together with Peptide/Protein Immunization Results in Enhanced CTL Responses with Anti-Tumor Activity
Authors: Davila Eduardo , Celis Esteban
Published: April 2014

The development of therapeutic anti-cancer vaccines designed to elicit CTL responses with anti-tumor activity has become a reality thanks to the identification of several tumor-associated Ags and their corresponding peptide T cell epitopes. However, peptide-based vaccines, in general, fail to elicit sufficiently strong CTL responses capable of producing therapeutic anti-tumor effects (i.e., prolongation of survival, tumor reduction). Here we report that repeated administration of synthetic oligonucleotides containing foreign cytosine-phosphorothiolated guanine (CpG) motifs increased 10- to 100-fold the CTL response to immunization with various synthetic peptides corresponding to well-known T cell epitopes. Moreover, repeated CpG administration allowed the induction of CTL to soluble protein even in the absence of additional adjuvant. Our results indicate that the potentiating effect of CpG in CTL responses required the participation of Th lymphocytes. Repeated CpG administration resulted in overt splenomegaly and lymphadenopathy with a significant increase in the numbers of CTL precursors and dendritic cells. Protein vaccination in combination with repeated CpG therapy was effective in delaying tumor cell growth and extending survival in mice bearing melanoma tumors. These findings support the contention that repeated administration of CpG-oligonucleotides enhances the effect of peptide and protein vaccines leading to potent anti-tumor responses, presumably through the induction of Th1 and dendritic cells, which are essential for optimal CTL responses. The immunostimulatory properties of CpG motifs may be key in inducing a consistent long term immunity to tumor-associated Ags when using peptides or proteins as T cell-inducing vaccines.
Flt3-Ligand and Granulocyte Colony-Stimulating Factor Mobilize Distinct Human Dendritic Cell Subsets In Vivo
Authors: Pulendran Bali , Banchereau Jacques , Burkeholder Susan , Kraus Elizabeth , Guinet Elisabeth , Chalouni Cecile , Caron Dania , Maliszewski Charles , Davoust Jean , Fay Joseph , Palucka Karolina
Published: April 2014

Dendritic cells (DCs) have a unique ability to stimulate naive T cells. Recent evidence suggests that distinct DC subsets direct different classes of immune responses in vitro and in vivo. In humans, the monocyte-derived CD11c+ DCs induce T cells to produce Th1 cytokines in vitro, whereas the CD11c− plasmacytoid T cell-derived DCs elicit the production of Th2 cytokines. In this paper we report that administration of either Flt3-ligand (FL) or G-CSF to healthy human volunteers dramatically increases distinct DC subsets, or DC precursors, in the blood. FL increases both the CD11c+ DC subset (48-fold) and the CD11c− IL-3R+ DC precursors (13-fold). In contrast, G-CSF only increases the CD11c− precursors (>7-fold). Freshly sorted CD11c+ but not CD11c− cells stimulate CD4+ T cells in an allogeneic MLR, whereas only the CD11c− cells can be induced to secrete high levels of IFN-α, in response to influenza virus. CD11c+ and CD11c− cells can mature in vitro with GM-CSF + TNF-α or with IL-3 + CD40 ligand, respectively. These two subsets up-regulate MHC class II costimulatory molecules as well as the DC maturation marker DC-lysosome-associated membrane protein, and they stimulate naive, allogeneic CD4+ T cells efficiently. These two DC subsets elicit distinct cytokine profiles in CD4+ T cells, with the CD11c− subset inducing higher levels of the Th2 cytokine IL-10. The differential mobilization of distinct DC subsets or DC precursors by in vivo administration of FL and G-CSF offers a novel strategy to manipulate immune responses in humans.
CTL Control of EBV in Nasopharyngeal Carcinoma (NPC): EBV-Specific CTL Responses in the Blood and Tumors of NPC Patients and the Antigen-Processing Function of the Tumor Cells
Authors: Lee Steven P. , Chan Anthony T. C. , Cheung Siu-Tim , Thomas Wendy A. , CroomCarter Debbie , Dawson Chris W. , Tsai Ching-Hwa , Leung Sing-Fai , Johnson Philip J. , Huang Dolly P.
Published: April 2014

Undifferentiated nasopharyngeal carcinoma (NPC) is latently infected with EBV and expresses a restricted number of viral proteins. Studies in healthy virus carriers have demonstrated that at least some of these proteins can act as targets for HLA class I-restricted CTLs. Therefore we have explored the possibility of a CTL-based therapy for NPC by characterizing EBV-specific CTL responses in 10 newly diagnosed NPC cases and 21 healthy virus carriers from Southeast Asia. Using the autologous EBV-transformed lymphoblastoid cell line, virus-specific CTL were reactivated in vitro from PBMC, cloned, and screened for cytotoxicity against target cells expressing individual EBV proteins from recombinant vaccinia vectors. EBV-specific CTLs were identified in 6 of 10 patients and 14 of 21 controls and mainly targeted the EBV nuclear Ag 3 (EBNA3) family of viral latent proteins. However, in 3 of 10 patients and 11 of 21 controls, CTLs specific for the NPC-associated protein LMP2 were also detected, albeit at low frequency. EBV-specific CTLs were detected in tumor biopsy material obtained from 3 of 6 of the patients, indicating that functional CTL are present at the tumor site, but none was specific for tumor-associated viral proteins. To assess the Ag-presenting function in NPC we studied two NPC-derived cell lines (C15 and c666.1) and demonstrated that both were capable of processing and presenting endogenously synthesized protein to HLA class I-restricted CTL clones. Overall, our data provide a sound theoretical basis for therapeutic strategies that aim to boost or elicit LMP2-specific CTL responses in NPC patients.
Rapid, B Lymphoid-Restricted Engraftment Mediated by a Primitive Bone Marrow Subpopulation
Authors: Searles A. Elena , Pohlmann Suzanne J. , Pierce L. Jeanne , Perry S. Scott , Slayton William B. , Mojica Mariluz P. , Spangrude Gerald J.
Published: April 2014

Utilizing multiparameter flow cytometry, we have defined a subset of bone marrow cells containing lymphoid-restricted differentiation potential after i.v. transplantation. Bone marrow cells characterized by expression of the Sca-1 and c-kit Ags and lacking Ags of differentiating lineages were segregated into subsets based on allele-specific Thy-1.1 Ag expression. Although hematopoietic stem cells were recovered in the Thy-1.1low subset as previously described, the Thy-1.1neg subset consisted of progenitor cells that preferentially reconstituted the B lymphocyte lineage after i.v. transplantation. Recipients of Thy-1.1neg cells did not survive beyond 30 days, presumably due to the failure of erythroid and platelet lineages to recover after transplants. Thy-1.1neg cells predominantly reconstituted the bone marrow and peripheral blood of lethally irradiated recipients with B lineage cells within 2 weeks, although a low frequency of myeloid lineage cells was also detected. In contrast, myeloid progenitors outnumbered lymphoid progenitors when the Thy-1.1neg population was assayed in culture. When Thy-1.1low stem cells were rigorously excluded from the Thy-1.1neg subset, reconstitution of T lymphocytes was rarely observed in peripheral blood after i.v. transplantation. Competitive repopulation studies showed that the B lymphoid reconstitution derived from Thy-1.1neg cells was not sustained over a 20-wk period. Therefore, the Thy-1.1neg population defined in these studies includes transplantable, non-self-renewing B lymphocyte progenitor cells.
Anti-TCR-Specific DNA Vaccination Demonstrates a Role for a CD8+ T Cell Clone in the Induction of Allograft Tolerance by Donor-Specific Blood Transfusion
Authors: Vignes Caroline , Chiffoleau Elise , Douillard Patrice , Josien Régis , Pêche Hélène , Heslan Jean-Marie , Usal Claire , Soulillou Jean-Paul , Cuturi Maria Cristina
Published: April 2014

Donor-specific allograft tolerance can be induced in the adult rat by pregraft donor-specific blood transfusion (DST). This tolerance appeared to be mediated by regulatory cells and to the production of the suppressive cytokine TGF-β1. A potential immunoregulatory CD8+ clone bearing a Vβ18-Dβ1-Jβ2.7 TCR gene rearrangement was previously identified in DST-treated recipients. To assess the functional role of this T cell clone in the induction of tolerance by DST, we have vaccinated DST-treated recipients with a plasmid construct encoding for the Vβ18-Dβ1-Jβ2.7 TCR β-chain. DST-induced allograft tolerance was abolished by anti-TCR Vβ18-Dβ1-Jβ2.7 DNA vaccination in six of seven recipients, whereas vaccination with the vector alone, or with the construct encoding a TCR Vβ13 β-chain, had no effect. However, the transcript number of the Vβ18-Dβ1-Jβ2.7 chain was unchanged in allografts from vaccinated DST-treated rats, suggesting that this clone was not depleted by vaccination, but rather was altered in its function. Moreover, TCR Vβ18-Dβ1-Jβ2.7 DNA vaccination restored the anti-donor alloantibody production, partially restore the capacity of spleen cells from tolerized recipients to proliferate in vitro against donor cells, and decreased the inhibitory effect of TGF-β1, seen in DST-treated recipients, in spleen cells from vaccinated DST-treated ones. This study strongly suggests that this CD8+ TCR Vβ18-Dβ1-Jβ2.7 T cell clone has an effective immunoregulatory function in allograft tolerance induced by DST.
Cutting Edge: Naive T Cells Masquerading as Memory Cells
Authors: Murali-Krishna Kaja , Ahmed Rafi
Published: April 2014

This study shows that naive CD8 T cells can acquire characteristics of memory T cells in the absence of stimulation with specific Ag simply by the process of homeostatic proliferation under lymphopenic conditions. This Ag-independent T cell differentiation pathway did not result in up-regulation of early activation markers (CD69, CD25, CD71), but expression of several memory markers (CD44, CD122, Ly6C) increased progressively with successive divisions. These markers were then stably expressed, and these cells also became more responsive functionally to specific Ag. Thus, all “memory” phenotype T cells in an individual may not be true Ag-experienced cells and may include naive cells masquerading as memory cells. These findings are specially relevant in cases of disease or treatment-induced lymphopenia such as in HIV-infected individuals or transplant recipients. In addition, this study may have implications for autoimmunity because homeostatic proliferation of naive T cells requires interaction with self peptide plus MHC molecules.
The NF-κB Cascade Is Important in Bcl-xL Expression and for the Anti-Apoptotic Effects of the CD28 Receptor in Primary Human CD4+ Lymphocytes
Authors: Khoshnan Ali , Tindell Charles , Laux Isett , Bae David , Bennett Brydon , Nel Andre E.
Published: April 2014

We explored the role of the NF-κB pathway in the survival of primary human CD4+ T lymphocytes during CD28 costimulation. Transduction of proliferating CD4+ T cells with a tetracycline-regulated retrovirus encoding for a dominant-interfering, degradation-resistant I-κBα (inhibitor of κBα factor) mutant induced apoptosis. Using DNA arrays, we show that Bcl-xL features as a prominent anti-apoptotic member among a number of early CD28-inducible genes. A 1.2-kb segment of the proximal Bcl-xL promoter, linked to a luciferase reporter, responded to CD3/CD28 stimulation in Jurkat cells. Mutation of an NF-κB site around −840 decreased, while ectopic expression of I-κB kinase-β (IKKβ) enhanced reporter gene activity. Na+-salicylate and cyclopentenone PGs, direct inhibitors of IKKβ, interfered in the activation of the Bcl-xL promoter and induced apoptosis in CD28-costimulated CD4+ T cells. Moreover, salicylate blocked nuclear localization of NF-κB factors that bind to the NF-κB binding site in the Bcl-xL promoter, as well as the expression of Bcl-xL protein. HuT-78, a lymphoblastoid T cell line with constitutive NF-κB activity, contained elevated levels of Bcl-xL protein and, similar to proliferating CD4+ T cells, was resistant to apoptotic stimuli such as anti-Fas and TNF-α. In contrast, the same stimuli readily induced apoptosis in a Jurkat T cell clone with no detectable Bcl-xL expression. Jurkat BMS2 cells also differed from HuT-78 in collapse of mitochondrial membrane potential and superoxide generation in the mitochondrium. Taken together, these data demonstrate that CD3/CD28-induced activation of IKKβ and expression of Bcl-xL promote the survival of primary human CD4+ T lymphocytes.
Infection of CD4+ Memory T Cells by HIV-1 Requires Expression of Phosphodiesterase 4
Authors: Sun Yu , Li Linsong , Lau Fion , Beavo Joseph A. , Clark Edward A.
Published: April 2014

Using PCR to monitor HIV-1 RNA genome reverse transcription and nuclear import of preintegration complexes, we found that memory, but not naive, CD4+ T cells could support transport of HIV-1 DNA to nuclei upon TCR/CD3 and IL-2 stimulation. Moreover, memory CD4+ T cells, unlike naive CD4+ T cells, express high levels of phosphodiesterase 4 (PDE4) constitutively. Selective blocking of PDE4 activity inhibited IL-2R expression and thereby led to abolishing HIV-1 DNA nuclear import in memory T cells; however, full-length viral DNA synthesis was not affected. Thus, blocking PDE4 prevents initiation of HIV-1 DNA circle formation in T cells. The fact that PDE4 is expressed constitutively at higher levels in memory vs naive CD4+ T cells may help HIV-1 readily infect memory T cells.
Defective Th Function Induced by a Dominant-Negative cAMP Response Element Binding Protein Mutation Is Reversed by Bcl-2
Authors: Zhang Feng , Rincon Mercedes , Flavell Richard A. , Aune Thomas M.
Published: April 2014

cAMP response element binding protein (CREB) is a critical regulator of diverse stimulus-dependent transcriptional events. Following TCR stimulation, CREB is rapidly induced in CD4+ Th cell precursors, but not in effector Th cells. However, its role in mature T cell function is incompletely defined. Transgenic mice expressing a CREB dominant-negative (dn) mutation in the T cell lineage exhibited normal T cell development in the thymus, normal T cell homeostasis in the periphery, and normal T cell clonal expansion following Ag challenge. However, this mutation caused selective inhibition of Th cell function in vitro and in vivo, and increased susceptibility of Th cells to activation-induced cell death. Th cells expressing the CREB-dn mutation contained reduced levels of the inhibitor of programmed cell death, BCL-2; overexpression of BCL-2 in transgenic mice reversed both susceptibility to activation-induced cell death in CREB-dn T cells and the defect in effector cytokine production. Thus, CREB plays a critical role in Th cell function and development of Th cell-mediated adaptive immune responses, at least in part, by inhibiting stimulus-dependent cell death.
Ly-49P Activates NK-Mediated Lysis by Recognizing H-2Dd 1
Authors: Silver Elizabeth T. , Gong Dong-Er , Chang Chew Shun , Amrani Abdelaziz , Santamaria Pere , Kane Kevin P.
Published: April 2014

Little is known regarding the ligand specificity of Ly-49 activating receptor subfamily members expressed by NK cells. A new Ly-49 activating receptor related to Ly-49A in its extracellular domain, designated Ly-49P, was recently cloned from 129 strain mice. We independently cloned an apparent allele of Ly-49P expressed by nonobese diabetic and nonobese diabetes-resistant mouse strain NK cells. We found it to be reactive with the A1 Ab thought to recognize a polymorphic epitope expressed only by the Ly-49A inhibitory receptor of the C57BL/6 strain. Rat RNK-16 cells transfected with Ly-49P mediated reverse Ab-dependent cellular cytotoxicity of FcR-positive target cells, indicating that Ly-49P can activate NK-mediated lysis. We determined that RNK-16 lysis of Con A blasts induced by Ly-49P was MHC dependent, resulting in efficient lysis of H-2Dd-bearing targets. We found that the Dd α1/α2 domain is required for Ly-49P-mediated RNK-16 activation, as determined by exon shuffling and transfection. Thus, Ly-49P is the second activating Ly-49 receptor demonstrated to induce NK cytotoxicity by recognizing a class I MHC molecule.
Defective Thymocyte Development and Perturbed Homeostasis of T cells in STAT-Induced STAT Inhibitor-1/Suppressors of Cytokine Signaling-1 Transgenic Mice
Authors: Fujimoto Minoru , Naka Tetsuji , Nakagawa Reiko , Kawazoe Yoshinori , Morita Yoshiaki , Tateishi Akihiro , Okumura Koichi , Narazaki Masashi , Kishimoto Tadamitsu
Published: April 2014

Previous experiments have shown that STAT-induced STAT inhibitor-1 (SSI-1; also named suppressors of cytokine signaling-1 (SOCS-1) or Janus kinase binding protein) is predominantly expressed in lymphoid organs and functions in vitro as a negative regulator of cytokine signaling. To determine the function of SOCS-1 in vivo, we generated SSI-1 transgenic mice using the lck proximal promoter that drives transgene expression in T cell lineage. In thymocytes expressing SSI-1 transgene, tyrosine phosphorylation of STATs in response to cytokines such as IFN-γ, IL-6, and IL-7 was inhibited, suggesting that SSI-1 suppresses cytokine signaling in primary lymphocytes. In addition, lck-SSI-1 transgenic mice showed a reduction in the number of thymocytes as a result of the developmental blocking during triple-negative stage. They also exhibited a relative increase in the percentage of CD4+ T cells, a reduction in the number of γδ T cells, as well as the spontaneous activation and increased apoptosis of peripheral T cells. Thus, enforced expression of SSI-1 disturbs the development of thymocytes and the homeostasis of peripheral T cells. All these features of lck-SSI-1 transgenic mice strikingly resemble the phenotype of mice lacking common γ-chain or Janus kinase-3, suggesting that transgene-derived SSI-1 inhibits the functions of common γ-chain-using cytokines. Taken together, these results suggest that SSI-1 can also inhibit a wide variety of cytokines in vivo.
T Cell Reactivity with Allergoids: Influence of the Type of APC
Authors: Kahlert Helga , Grage-Griebenow Evelin , Stüwe Hans-Thomas , Cromwell Oliver , Fiebig Helmut
Published: April 2014

The use of allergoids for allergen-specific immunotherapy has been established for many years. The characteristic features of these chemically modified allergens are their strongly reduced IgE binding activity compared with the native form and the retained immunogenicity. T cell reactivity of chemically modified allergens is documented in animals, but in humans indirect evidence of reactivity has been concluded from the induction of allergen-specific IgG during immunotherapy. Direct evidence of T cell reactivity was obtained recently using isolated human T cells. To obtain further insight into the mechanism of action of allergoids, we compared the Ag-presenting capacity of different APC types, including DC and macrophages, generated from CD14+ precursor cells from the blood of grass pollen allergic subjects, autologous PBMC, and B cells. These APC were used in experiments together with Phl p 5-specific T cell clones under stimulation with grass pollen allergen extract, rPhl p 5b, and the respective allergoids. Using DC and macrophages, allergoids exhibited a pronounced and reproducible T cell-stimulating capacity. Responses were superior to those with PBMC, and isolated B cells failed to present allergoids. Considerable IL-12 production was observed only when using the DC for Ag presentation of both allergens and allergoids. The amount of IL-10 in supernatants was dependent on the phenotype of the respective T cell clone. High IL-10 production was associated with suppressed IL-12 production from the DC in most cases. In conclusion, the reactivity of Th cells with allergoids is dependent on the type of the APC.
Recirculatory and Sessile CD4+ T Lymphocytes Differ on CD45RC Expression
Authors: Ramírez Francisco , Mason Don
Published: April 2014

CD4+ T cell subsets are unequally distributed in rat secondary lymphoid organs. Those with the memory phenotype CD45RClow Thy-1− L-selectin− are present at a higher frequency in Peyer’s patches (PP) than in lymph nodes and spleen, and increase in numbers with age in all three tissues, particularly in the PP. Homing experiments revealed that CD4+ T cells that recirculate through secondary lymphoid organs are mainly CD45RChigh. It was also apparent that the ability of recirculating cells to enter different lymphoid organs varies; less cells enter PP than the spleen or lymph nodes. Our results also reveal the existence of a nonrecirculating population of CD4+ T cells in secondary lymphoid organs, which are predominantly, if not exclusively, CD45RClow. Our results show that secondary lymphoid organs differ in their CD4+ T cell subset composition as a consequence of having different ratios of recirculatory:nonrecirculatory CD4+ T cells, and these cells display a different CD45RC phenotype.
Differential Splicing of Antigen-Encoding RNA Reduces Endogenous Epitope Presentation That Regulates the Expansion and Cytotoxicity of T Cells
Authors: Kienzle Norbert , Buck Marion , Silins Sharon L. , Burrows Scott R. , Moss Denis J. , Winterhalter Adam , Brooks Andrew , Khanna Rajiv
Published: April 2014

The activation of CTLs is dependent on the recognition of MHC-bound peptide present on the surface of APCs. We give evidence in this study that differential splicing of Ag-encoding RNA can decrease the antigenic dose in APCs and regulate the recall of human memory CTLs. Differential splicing of RNA that encoded an immunodominant HLA-B8-restricted CTL epitope of EBV reduced the functional presentation of this epitope, and consequently the in vitro expansion and activity of CTLs, as measured by MHC/peptide-tetramer staining and cytotoxicity assays. The reduced activity of the stimulated CTLs was not only due to lower numbers of Ag-specific CTLs but, surprisingly, was also characterized by decreased cytotoxicity of the CTLs to target cells presenting limiting amounts of the peptide epitope. As indicated by TCR repertoire analysis, the reduction in CTL activity was not caused by stimulation of distinct populations of TCR clonotypes. This study demonstrates how a common eukaryotic posttranscriptional mechanism of gene regulation can modulate the endogenous presentation of Ag and ultimately contribute to the fine tuning of immunological memory cells, which are important in the fight against pathogens and tumors and in autoimmunity.
Oligosaccharides of Hyaluronan Are Potent Activators of Dendritic Cells
Authors: Termeer Christian C. , Hennies Jörn , Voith Ursula , Ahrens Thomas , M. Weiss Johannes , Prehm Peter , Simon Jan C.
Published: April 2014

The extracellular matrix component hyaluronan (HA) exists physiologically as a high m.w. polymer but is cleaved at sites of inflammation, where it will be contacted by dendritic cells (DC). To determine the effects of HA on DC, HA fragments of different size were established. Only small HA fragments of tetra- and hexasaccharide size (sHA), but not of intermediate size (m.w. 80,000–200,000) or high m.w. HA (m.w. 1,000,000–600,000) induced immunophenotypic maturation of human monocyte-derived DC (up-regulation of HLA-DR, B7-1/2, CD83, down-regulation of CD115). Likewise, only sHA increased DC production of the cytokines IL-1β, TNF-α, and IL-12 as well as their allostimulatory capacity. These effects were highly specific for sHA, because they were not induced by other glycosaminoglycans such as chondroitin sulfate or heparan sulfate or their fragmentation products. Interestingly, sHA-induced DC maturation does not involve the HA receptors CD44 or the receptor for hyaluronan-mediated motility, because DC from CD44-deficient mice and wild-type mice both responded similarly to sHA stimulation, whereas the receptor for hyaluronan-mediated motility is not detectable in DC. However, TNF-α is an essential mediator of sHA-induced DC maturation as shown by blocking studies with a soluble TNFR1. These findings suggest that during inflammation, interaction of DC with small HA fragments induce DC maturation.
Transgenic Expression of Ly49A on T Cells Impairs a Specific Antitumor Response
Authors: Brawand Pierre , Lemonnier François A. , MacDonald H. Robson , Cerottini Jean-Charles , Held Werner
Published: April 2014

Inhibitory MHC receptors determine the reactivity and specificity of NK cells. These receptors can also regulate T cells by modulating TCR-induced effector functions such as cytotoxicity, cytokine production, and proliferation. Here we have assessed the capacity of mouse T cells expressing the inhibitory MHC class I receptor Ly49A to respond to a well-defined tumor Ag in vivo using Ly49A transgenic mice. We find that the presence of Ly49A on the vast majority of lymphocytes prevents the development of a significant Ag-specific CD8+ T cell response and, consequently, the rejection of the tumor. Despite minor alterations in the TCR repertoire of CD8+ T cells in the transgenic lines, precursors of functional tumor-specific CD8+ T cells exist but could not be activated most likely due to a lack of appropriate CD4+ T cell help. Surprisingly, all of these effects are observed in the absence of a known ligand for the Ly49A receptor as defined by its ability to regulate NK cell function. Indeed, we found that the above effects on T cells may be based on a weak interaction of Ly49A with Kb or Db class I molecules. Thus, our data demonstrate that enforced expression of a Ly49A receptor on conventional T cells prevents a specific immune response in vivo and suggest that the functions of T and NK cells are differentially sensitive to the presence of inhibitory MHC class I receptors.