bluuebunny/crossref_metadata_2025 huggingface dataset:
Cre Recombinase-Mediated Inactivation of H-2Dd Transgene Expression: Evidence for Partial Missing Self-Recognition by Ly49A NK Cells
Authors: Ioannidis Vassilios , Zimmer Jacques , Beermann Friedrich , Held Werner
Published: April 2014

We have established H-2Dd-transgenic (Tg) mice, in which H-2Dd expression can be extinguished by Cre recombinase-mediated deletion of an essential portion of the transgene (Tg). NK cells adapted to the expression of the H-2Dd Tg in H-2b mice and acquired reactivity to cells lacking H-2Dd, both in vivo and in vitro. H-2Dd-Tg mice crossed to mice harboring an Mx-Cre Tg resulted in mosaic H-2Dd expression. That abrogated NK cell reactivity to cells lacking Dd. In Dd single Tg mice it is the Ly49A+ NK cell subset that reacts to cells lacking Dd, because the inhibitory Ly49A receptor is no longer engaged by its Dd ligand. In contrast, Ly49A+ NK cells from Dd × MxCre double Tg mice were unable to react to Dd-negative cells. These Ly49A+ NK cells retained reactivity to target cells that were completely devoid of MHC class I molecules, suggesting that they were not anergic. Variegated Dd expression thus impacts specifically missing Dd but not globally missing class I reactivity by Ly49A+ NK cells. We propose that the absence of Dd from some host cells results in the acquisition of only partial missing self-reactivity.
Requirement for Ca2+/Calmodulin-Dependent Kinase Type IV/Gr in Setting the Thymocyte Selection Threshold
Authors: Raman Vidya , Blaeser Frank , Ho Nga , Engle Deborah L. , Williams Calvin B. , Chatila Talal A.
Published: April 2014

The outcome of thymocyte selection is influenced by the nature of Ca2+ signals transduced by the TCR. Robust Ca2+ responses characterize high-affinity, negatively selecting peptide/TCR interactions, while modest responses typify lower-affinity, positively selecting interactions. To elucidate mechanisms by which thymocytes decode distinct Ca2+ signals, we examined selection events in mice lacking Ca2+/calmodulin-dependent protein kinase type IV/Gr (CaMKIV/Gr), which is enriched in thymocytes. CaMKIV/Gr-deficient thymocytes exhibited impaired positive selection and defective Ca2+-dependent gene transcription. Significantly, CaMKIV/Gr deficiency raised the selection threshold of peptide/TCR interactions such that a peptide that normally induced weak negative selection instead promoted positive selection. These results demonstrate an important role for CaMKIV/Gr in sensitizing thymocytes to selection by low-affinity peptides.
Without CD4 Help, CD8 Rejection of Pig Xenografts Requires CD28 Costimulation But Not Perforin Killing
Authors: Zhan Yifan , Brady Jamie L. , Sutherland Robyn M. , Lew Andrew M.
Published: April 2014

Although CD4 cells are major mediators in cellular rejection of fetal pig pancreas (FPP) in the mouse, rejection still occurs in the absence of CD4 cells, albeit with delayed kinetics. CD4 cell-independent mechanisms of cellular rejection are poorly understood. To investigate the involvement of CD8 T cells in FPP rejection and their activation requirements, we used mice transgenic for anti-CD4 Ab; this is the most complete model of CD4 cell deficiency. We showed that in such mice FPP was infiltrated with CD8 cells starting from 2 wk posttransplantation and FPP was eventually rejected 8 wk posttransplantation. Ab depletion of CD8 cells greatly improved the survival of FPP and reduced cell infiltration at the graft site. This suggests that CD8 cells can mediate the rejection of porcine xenografts in the absence of CD4 cells. This CD8-mediated rejection of FPP is independent of their perforin-mediated lytic function, as graft survival was not affected in mice deficient in perforin. The production of IFN-γ and IL-5 by the graft infiltrates indicates that CD8 cells may act through cytokine-mediated mechanisms. Remarkably, in the absence of CD4 cells, lymphocyte infiltration at the graft site was absent in mice transgenic for CTLA4Ig such that the islet grafts flourished beyond 24 wk. In contrast, rejection was little affected by CD40 ligand deficiency. Therefore, we show that CD8 cells are activated to mediate FPP rejection independent of perforin and that this CD4-independent activation of CD8 cells critically depends on B7/CD28 costimulation.
Kinetics of Antigen-Induced Phenotypic and Functional Maturation of Human Monocyte-Derived Dendritic Cells
Authors: Hsieh Szu-Min , Pan Sung-Ching , Hung Chien-Ching , Tsai Hsing-Chun , Chen Mao-Yuan , Lee Chun-Nan , Chang Shan-Chwen
Published: April 2014

Dendritic cells (DCs), a critical component of innate immunity, are the most potent APCs. When DCs mature, they can elicit strong T cell responses. We studied the kinetics of Ag-induced phenotypic and functional maturation of human monocyte-derived DCs using an in vitro T cell-independent culture system. With this model, we herein show that an Ag that has recently or repetitively been exposed (“exposed Ag”) rapidly induces a high level of maturation; however, an Ag that has never or only remotely been exposed (“unexposed Ag”) slowly induces a low level of maturation. The kinetics of Ag-induced maturation of DCs possibly implies a novel mechanism for immunological memory that would provide maximal host protection from repetitively invading pathogens in the environment.
Constitutive Expression of LIGHT on T Cells Leads to Lymphocyte Activation, Inflammation, and Tissue Destruction
Authors: Shaikh Raziya B. , Santee Sybil , Granger Steven W. , Butrovich Kristine , Cheung Tim , Kronenberg Mitchell , Cheroutre Hilde , Ware Carl F.
Published: April 2014

LIGHT, a member of the TNF family of cytokines (homologous to lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for herpesvirus entry mediator, a receptor expressed on T cells), is induced on activated T cells and mediates costimulatory and antitumor activity in vitro. Relatively little information is available on the in vivo effects of LIGHT expression, particularly within the T cell compartment. In this work, we describe transgenic mice that express human LIGHT under the control of the CD2 promoter, resulting in constitutive transgene expression in cells of the T lymphocyte lineage. LIGHT-transgenic animals exhibit abnormalities in both lymphoid tissue architecture and the distribution of lymphocyte subsets. They also show signs of inflammation that are most severe in the intestine, along with tissue destruction of the reproductive organs. These LIGHT-mediated effects were recapitulated when immune-deficient mice were reconstituted with bone marrow from LIGHT-transgenic donor mice. T cells in the LIGHT-transgenic mice have an activated phenotype and mucosal T cells exhibit enhanced Th1 cytokine activity. The results indicate that LIGHT may function as an important regulator of T cell activation, and implicate LIGHT signaling pathways in inflammation focused on mucosal tissues.
Transduction of an IL-2 Gene into Human Melanoma-Reactive Lymphocytes Results in Their Continued Growth in the Absence of Exogenous IL-2 and Maintenance of Specific Antitumor Activity
Authors: Liu Ke , Rosenberg Steven A.
Published: April 2014

IL-2-dependent activated cells undergo apoptotic death when IL-2 is withdrawn either in vitro or after in vivo cell transfer. To attempt to sustain their survival after IL-2 withdrawal, melanoma-reactive human T lymphocytes were retrovirally transduced with an exogenous human IL-2 gene. Transduced PBMC and cloned CD8+ T cells produced IL-2 and maintained viability after IL-2 withdrawal. Upon restimulation, IL-2 transductants proliferated in the absence of exogenous IL-2 and could be actively grown, and their survival could be maintained without added IL-2 for over 8 wk. PBMCs similarly transduced with a control vector did not produce IL-2 and failed to proliferate in the absence of IL-2. A CD8+ T cell clone, when transduced with an IL-2 gene, manifested the same phenotypes as PBMCs in the absence of exogenous IL-2. Furthermore, an Ab reactive with the α-chain of IL-2R complex reduced the viability mediated by IL-2 secretion of the IL-2 transductants. Moreover, transduction of an IL-2 gene did not affect the high degree of recognition and specificity of transductants against melanoma targets. These tumor-reactive IL-2 transductants may be valuable for in vitro studies and for improved adoptive transfer therapies for patients with metastatic melanoma.
Immunoreceptor Tyrosine-Based Inhibitory Motif of the IL-4 Receptor Associates with SH2-Containing Phosphatases and Regulates IL-4-Induced Proliferation
Authors: Kashiwada Masaki , Giallourakis Cosmas C. , Pan Ping-Ying , Rothman Paul B.
Published: April 2014

Immunoreceptor tyrosine-based inhibitory motifs (ITIM) have been implicated in the negative modulation of immunoreceptor signaling pathways. The IL-4R α-chain (IL-4Rα) contains a putative ITIM in the carboxyl terminal. To determine the role of ITIM in the IL-4 signaling pathway, we ablated the ITIM of IL-4Rα by deletion and site-directed mutagenesis and stably expressed the wild-type (WT) and mutant hIL-4Rα in 32D/insulin receptor substrate-2 (IRS-2) cells. Strikingly, 32D/IRS-2 cells expressing mutant human (h)IL-4Rα were hyperproliferative in response to IL-4 compared with cells expressing WT hIL-4Rα. Enhanced tyrosine phosphorylation of Stat6, but not IRS-2, induced by hIL-4 was observed in cells expressing mutant Y713F. Using peptides corresponding to the ITIM of hIL-4Rα, we demonstrate that tyrosine-phosphorylated peptides, but not their nonphosphorylated counterparts, coprecipitate SH2-containing tyrosine phosphatase-1, SH2-containing tyrosine phosphatase-2, and SH2-containing inositol 5′-phosphatase. The in vivo association of SH2-containing inositol 5′-phosphatase with IL-4Rα was verified by coimmunoprecipitation with anti-IL-4Rα Abs. These results demonstrate a functional role for ITIM in the regulation of IL-4-induced proliferation.
SH2 Domain-Containing Inositol Polyphosphate 5′-Phosphatase Is the Main Mediator of the Inhibitory Action of the Mast Cell Function-Associated Antigen
Authors: Xu Rong , Abramson Jakub , Fridkin Mati , Pecht Israel
Published: April 2014

The mast cell function-associated Ag (MAFA) is a type II membrane glycoprotein originally found on the plasma membrane of rat mucosal-type mast cells (RBL-2H3 line). A C-type lectin domain and an immunoreceptor tyrosine-based inhibitory motif (ITIM) are located in the extracellular and intracellular domains of MAFA, respectively. MAFA clustering has previously been shown to suppress the secretory response of these cells to the FcεRI stimulus. Here we show that the tyrosine of the ITIM undergoes phosphorylation, on MAFA clustering, that is markedly enhanced on pervanadate treatment of the cells. Furthermore, the Src homology 3 domain of the protein tyrosine kinase Lyn binds directly to a peptide containing nonphosphorylated MAFA ITIM and PAAP motif. Results of both in vitro and in vivo experiments suggest that Lyn is probably responsible for this ITIM phosphorylation, which increases the Src homology domain 2 (SH2) affinity of Lyn for the peptide. In vitro measurements established that tyrosine-phosphorylated MAFA ITIM peptides also bind the SH2 domains of inositol 5′-phosphatase (SHIP) as well as protein tyrosine phosphatase-2. However, the former single domain is bound 8-fold stronger than both of the latter. Further support for the role of SHIP in the action of MAFA stems from in vivo experiments in which tyrosine-phosphorylated MAFA was found to bind primarily SHIP. In RBL-2H3 cells overexpressing wild-type SHIP, MAFA clustering causes markedly stronger inhibition of the secretory response than in control cells expressing normal SHIP levels or cells overexpressing either wild-type protein tyrosine phosphatase-2 or its dominant negative form. In contrast, on overexpression of the SH2 domain of SHIP, the inhibitory action of MAFA is essentially abolished. Taken together, these results suggest that SHIP is the primary enzyme responsible for mediating the inhibition by MAFA of RBL-2H3 cell response to the FcεRI stimulus.
Functional and Structural Similarity of Vγ9Vδ2 T Cells in Humans and Aotus Monkeys, a Primate Infection Model for Plasmodium falciparum Malaria
Authors: Daubenberger Claudia A. , Salomon Maxence , Vecino William , Hübner Beatrice , Troll Heike , Rodriques Raul , Patarroyo Manuel E. , Pluschke Gerd
Published: April 2014

γδ T cells are implicated to play crucial roles during early immune responses to pathogens. A subset of human γδ T cells carrying the Vγ9Vδ2 TCR recognize small, phosphorylated nonpeptidic Ags. However, the precise role of these cells and the ligands recognized in human immune responses against pathogens remains unclear because of the lack of suitable animal models. We have analyzed the reactivity of spleen cells of the New World monkey Aotus nancymaae against isopentenyl pyrophosphate (IPP), a phosphorylated microbial metabolite selectively activating Vγ9Vδ2 T cells. Spleen cells were stimulated by IPP and the expanding cell population expressed the Vγ9 TCR. TRGV-J and TRDV-D-J rearrangements expressed by IPP-stimulated cells of Aotus were analyzed by RT-PCR and DNA sequencing. The TRGV-J and TRDV-D-J rearrangements expressed by IPP-stimulated Aotus and human γδ T cells were similar with respect to 1) TCR gene segment usage, 2) a high degree of germline sequence homology of the TCR gene segments used, and 3) the diversity of the CDR3 regions. Phylogenetic analysis of human, Pan troglodytes, and A. nancymaae TRGV gene segments showed that the interspecies differences are smaller than the intraspecies differences with TRGV9 gene segments located on a distinct clade of the phylogenetic tree. The structural and functional conservation of Vγ9Vδ2 T cells in A. nancymaae and humans implicates a functionally important and evolutionary conserved mechanism of recognition of phosphorylated microbial metabolites.
IFN-αβ-Dependent, IFN-γ Secretion by Bone Marrow-Derived Macrophages Controls an Intracellular Bacterial Infection
Authors: Gigliotti Rothfuchs Antonio , Gigliotti Dulceaydee , Palmblad Karin , Andersson Ulf , Wigzell Hans , Rottenberg Martín E.
Published: April 2014

Several reports have indicated that cell lineages apart from NK and T cells can also express IFN-γ. However, the biological relevance of this finding is uncertain. We show in this study that bone marrow-derived macrophages (BMMs) express IFN-γ at the mRNA and protein level early after infection with Chlamydia pneumoniae. Increased IFN-γ mRNA accumulation by infected BMMs is early, transient, and requires both bacterial and host protein synthesis. The induction of IFN-γ mRNA levels is independent of IL-12 and was dramatically enhanced in IL-10−/− BMMs. Such IL-10−/− BMMs contained less bacteria than the wild-type controls, whereas IFN-γR−/− BMMs showed increased C. pneumoniae load. Inducible NO synthase (iNOS) also participates in the control of bacterial load, as shown by the enhanced numbers of C. pneumoniae in iNOS−/− BMMs. However, the increased accumulation of iNOS mRNA and NO in C. pneumoniae-infected BMMs depended on the presence of IFN-αβ, but was independent of IFN-γ. Interestingly, IFN-αβ are also required for increased IFN-γ mRNA accumulation in C. pneumoniae-infected BMMs. Accordingly, IFN-αβR−/− BMMs showed higher levels of C. pneumoniae than wild-type BMMs. Our findings unravel an autocrine/paracrine macrophage activation pathway by showing an IFN-αβ-dependent IFN-γ and iNOS induction in response to infection, which protects macrophages against intracellular bacterial growth.
Immunization with gp96 from Listeria monocytogenes-Infected Mice Is Due to N-Formylated Listerial Peptides
Authors: Sponaas Anne-Marit , Zuegel Ulrich , Weber Stephan , Hurwitz Robert , Winter Ralf , Lamer Stephanie , Jungblut Peter R. , Kaufmann Stefan H. E.
Published: April 2014

N-Formylated (N-f-met) peptides derived from proteins of the intracellular bacterium Listeria monocytogenes generate a protective, H2-M3-restricted CD8 T cell response in C57BL/6 mice. N-f-met peptide-specific CTL were generated in vitro when mice previously immunized with gp96 isolated from donor mice infected with L. monocytogenes were stimulated with these peptides. No significant peptide-specific CTL activity was observed in mice immunized with gp96 from uninfected animals. Masses corresponding to one N-f-met peptide were found by matrix-assisted laser desorption/ionization-mass spectrometry on gp96 isolated from C57BL/6 mice infected with L. monocytogenes, but not on gp96 from noninfected mice. Therefore, bacterial N-f-met peptides from intracellular bacteria can bind to gp96 in the infected host, and gp96 loaded with these peptides can generate N-f-met-peptide-specific CTL. We assume a unique role of gp96 in Ag processing through the H2-M3 pathway.
A Novel Mimetic Antigen Eliciting Protective Antibody toNeisseria meningitidis
Authors: Granoff Dan M. , Moe Gregory R. , Giuliani Marzia M. , Adu-Bobie Jeannette , Santini Laura , Brunelli Brunella , Piccinetti Francesca , Zuno-Mitchell Patricia , Lee Sharon S. , Neri Paolo , Bracci Luisa , Lozzi Luisa , Rappuoli Rino
Published: April 2014

Molecular mimetic Ags are of considerable interest as vaccine candidates. Yet there are few examples of mimetic Ags that elicit protective Ab against a pathogen, and the functional activity of anti-mimetic Abs has not been studied in detail. As part of the Neisseria meningitidis serogroup B genome sequencing project, a large number of novel proteins were identified. Herein, we provide evidence that genome-derived Ag 33 (GNA33), a lipoprotein with homology to Escherichia coli murein transglycosylase, elicits protective Ab to meningococci as a result of mimicking an epitope on loop 4 of porin A (PorA) in strains with serosubtype P1.2. Epitope mapping of a bactericidal anti-GNA33 mAb using overlapping peptides shows that the mAb recognizes peptides from GNA33 and PorA that share a QTP sequence that is necessary but not sufficient for binding. By flow cytometry, mouse antisera prepared against rGNA33 and the anti-GNA33 mAb bind as well as an anti-PorA P1.2 mAb to the surface of eight of nine N. meningitidis serogroup B strains tested with the P1.2 serosubtype. Anti-GNA33 Abs also are bactericidal for most P1.2 strains and, for susceptible strains, the activity of an anti-GNA33 mAb is similar to that of an anticapsular mAb but less active than an anti-P1.2 mAb. Anti-GNA Abs also confer passive protection against bacteremia in infant rats challenged with P1.2 strains. Thus, GNA33 represents one of the most effective immunogenic mimetics yet described. These results demonstrate that molecular mimetics have potential as meningococcal vaccine candidates.
CXCR2 Deficiency Confers Impaired Neutrophil Recruitment and Increased Susceptibility DuringToxoplasma gondiiInfection
Authors: Del Rio Laura , Bennouna Soumaya , Salinas Jesus , Denkers Eric Y.
Published: April 2014

Neutrophil migration to the site of infection is a critical early step in host immunity to microbial pathogens, in which chemokines and their receptors play an important role. In this work, mice deficient in expression of the chemokine receptor CXCR2 were infected with Toxoplasma gondii and the outcome was monitored. Gene-deleted animals displayed completely defective neutrophil recruitment, which was apparent at 4 h and sustained for at least 36 h. KitW/KitW-v animals also displayed defective polymorphonuclear leukocyte migration, suggesting mast cells as one source of chemokines driving the response. Tachyzoite infection and replication were accelerated in CXCR2−/− animals, resulting in establishment of higher cyst numbers in the brain relative to wild-type controls. Furthermore, serum and spleen cell IFN-γ levels in infected, gene-deleted mice were reduced 60–75% relative to infected normal animals, and spleen cell TNF-α was likewise reduced by ∼50%. These results highlight an important role for CXCR2 in neutrophil migration, which may be important for early control of infection and induction of immunity during Toxoplasma infection.
Cytoadherence of Plasmodium falciparum-Infected Erythrocytes Is Mediated by a Redox-Dependent Conformational Fraction of CD36
Authors: Gruarin Paola , Primo Luca , Ferrandi Chiara , Bussolino Federico , Tandon Narendra N. , Arese Paolo , Ulliers Daniela , Alessio Massimo
Published: April 2014

The adherence of Plasmodium falciparum-infected RBC (IRBC) to postcapillary venular endothelium is an important determinant of the pathogenesis of severe malaria complications. Cytoadherence of IRBC to endothelial cells involves specific receptor/ligand interactions. The glycoprotein CD36 expressed on endothelial cells is the major receptor involved in this interaction. Treatment of CD36-expressing cells with reducing agents, such as DTT and N-acetylcysteine, was followed by CD36 conformational change monitorable by the appearance of the Mo91 mAb epitope. Only a fraction of the surface expressed CD36 molecules became Mo91 positive, suggesting the presence of two subpopulations of molecules with different sensitivities to reduction. The Mo91 epitope has been localized on a peptide (residues 260–279) of the C-terminal, cysteine-rich region of CD36. Treatment with reducing agents inhibited the CD36-dependent cytoadherence of IRBC to CD36-expressing cells and dissolved pre-existent CD36-mediated IRBC/CD36-expressing cell aggregates. CD36 reduction did not impair the functionality of CD36, since the reactivity of other anti-CD36 mAbs as well as the binding of oxidized low density lipoprotein, a CD36 ligand, were maintained. The modifications induced by reduction were reversible. After 14 h CD36 was reoxidized, the cells did not express the Mo91 epitope, and cytoadherence to IRBC was restored. The results indicate that IRBCs bind only to a redox-modulated fraction of CD36 molecules expressed on the cell surface. The present data indicate the therapeutic potential of reducing agents, such as the nontoxic drug N-acetylcysteine, to prevent or treat malaria complications due to IRBC cytoadhesion.
Fc Receptor-Mediated Immunity Against Bordetella pertussis
Authors: Rodriguez Maria Eugenia , Hellwig Sandra M. M. , Hozbor Daniela F. , Leusen Jeanette , van der Pol W. -Ludo , van de Winkel Jan G. J.
Published: April 2014

The relevance of specific Abs for the induction of cellular effector functions against Bordetella pertussis was studied. IgG-opsonized B. pertussis was efficiently phagocytosed by human polymorphonuclear leukocytes (PMN). This process was mediated by the PMN IgG receptors, FcγRIIa (CD32) and FcγRIIIb (CD16), working synergistically. Furthermore, these FcγR triggered efficient PMN respiratory burst activity and mediated transfer of B. pertussis to lysosomal compartments, ultimately resulting in reduced bacterial viability. Bacteria opsonized with IgA triggered similar PMN activation via FcαR (CD89). Simultaneous engagement of FcαRI and FcγR by B. pertussis resulted in increased phagocytosis rates, compared with responses induced by either isotype alone. These data provide new insights into host immune mechanisms against B. pertussis and document a crucial role for Ig-FcR interactions in immunity to this human pathogen.
Role of p38 Mitogen-Activated Protein Kinase in Chemokine-Induced Emigration and Chemotaxis In Vivo
Authors: Cara Denise C. , Kaur Jaswinder , Forster Melanie , McCafferty Donna-Marie , Kubes Paul
Published: April 2014

It has been proposed that L-selectin engagement with ligand activates p38 mitogen-activated protein kinase (MAPK) and can impact on downstream events of leukocyte rolling, including adhesion, and emigration. Using a novel chemotactic assay in vivo, we visualized slow release of chemokine from an agarose gel positioned 350 μm from a postcapillary venule, which induced directed migration (chemotaxis) of neutrophils. In this system, keratinocyte-derived cytokine induced phosphorylation of p38 MAPK, which phosphorylated a downstream protein (ATF-2). This latter event was blocked by the concentration of p38 inhibitors used in this study. Mice were treated with two different p38 inhibitors: SKF86002 and SB203580. Neither inhibitor affected rolling or adhesion in microvessels. Intravenous treatment with SFK86002 (5, 10, and 20 mg/kg) 30 min before the inflammatory stimulus inhibited the total number of emigrated cells at a dose of 20 mg/kg (62%, p < 0.05), despite the presence of many adherent cells within the vessels. A similar inhibition was observed with 20 mg/kg of a second p38 inhibitor SB203580 (67%, p < 0.05). In addition to emigration, both p38 inhibitors impaired the ability of emigrated cells to migrate through the tissue toward the chemotactic stimulus. In fact, the majority of emigrated leukocytes in p38 inhibitor-treated animals remained within 50 μm of the venule. Superfusion of the tissue with SKF86002 (0.7 mM) to impact only on emigrated and not vascular leukocytes resulted in no impairment in emigration, but in a significant reduction in chemotaxis away from the vessel wall. Again, the majority of emigrated leukocytes remained within 50 μm of the blood vessel. Our results suggest that p38 does not affect rolling or adhesion, but that it is involved in leukocyte emigration and chemotaxis through interstitium in response to keratinocyte-derived cytokine in vivo.
Neutrophil Proteinase 3-Mediated Induction of Bioactive IL-18 Secretion by Human Oral Epithelial Cells
Authors: Sugawara Shunji , Uehara Akiko , Nochi Tomonori , Yamaguchi Takahiro , Ueda Haruyasu , Sugiyama Akiko , Hanzawa Kazuo , Kumagai Katsuo , Okamura Haruki , Takada Haruhiko
Published: April 2014

IL-18, a potent IFN-γ-inducing cytokine, is expressed by various nonimmune cells as well as macrophages, suggesting that it has important physiological and immunological roles. The present study focused on the mechanism of active IL-18 induction from human oral epithelial cells. The epithelial cells and the cell lines constitutively express IL-18 mRNA and the 24-kDa precursor form of IL-18. Bioactive IL-18 exhibiting IFN-γ-inducing activity was detected in the supernatant of the cells on costimulation with neutrophil proteinase 3 (PR3) and LPS for 24 h after IFN-γ-priming for 3 days. An active 18-kDa form of IL-18 was detected in lysate and supernatant of the cells only after the above treatment and the induction was inhibited by cycloheximide and by serine proteinase inhibitors. After the treatment, lactate dehydrogenase activity was not detected in the cell culture supernatant, and PR3 was detected only in the membrane and not in cytoplasm fractions of the cells. Caspase-1 was not detected in the cells even after the treatment and the IL-18 induction was not inhibited by a caspase-1 inhibitor. These results suggest that the PR3-mediated induction of bioactive IL-18 secretion from oral epithelial cells in combination with LPS after IFN-γ-priming occurred via a caspase-1-independent pathway, and provide new insight into the possible involvement of a neutrophil proteinase in the induction of bioactive IL-18 in oral inflammation such as periodontitis.
A Functional IFN-γ-Inducible Protein-10/CXCL10-Specific Receptor Expressed by Epithelial and Endothelial Cells That Is Neither CXCR3 Nor Glycosaminoglycan
Authors: Soejima Kenzo , Rollins Barrett J.
Published: April 2014

Interferon-γ-inducible protein-10 (IP-10)/CXCL10 is a CXC chemokine that attracts T lymphocytes and NK cells through activation of CXCR3, the only chemokine receptor identified to date that binds IP-10/CXCL10. We have found that several nonhemopoietic cell types, including epithelial and endothelial cells, have abundant levels of a receptor that binds IP-10/CXCL10 with a Kd of 1–6 nM. Surprisingly, these cells expressed no detectable CXCR3 mRNA. Furthermore, no cell surface expression of CXCR3 was detectable by flow cytometry, and the binding of 125I-labeled IP-10/CXCL10 to these cells was not competed by the other high affinity ligands for CXCR3, monokine induced by IFN-γ/CXCL9, and I-TAC/CXCL11. Although IP-10/CXCL10 binds to cell surface heparan sulfate glycosaminoglycan (GAG), the receptor expressed by these cells is not GAG, since the affinity of IP-10/CXCL10 for this receptor is much higher than it is for GAG, its binding is not competed by platelet factor 4/CXCL4, and it is present on cells that are genetically incapable of synthesizing GAG. Furthermore, in contrast to IP-10/CXCL10 binding to GAG, IP-10/CXCL10 binding to these cells induces new gene expression and chemotaxis, indicating the ability of this receptor to transduce a signal. These high affinity IP-10/CXCL10-specific receptors on epithelial cells may be involved in cell migration and, perhaps, in the spread of metastatic cells as they exit from the vasculature. (All of the lung cancer cells we examined also expressed CXCR4, which has been shown to play a role in breast cancer metastasis.) CXCR3-negative endothelial cells may also use this receptor to mediate the angiostatic activity of IP-10/CXCL10, which is also expressed by these cells in an autocrine manner.
IL-13 Fusion Cytotoxin Ameliorates Chronic Fungal-Induced Allergic Airway Disease in Mice
Authors: Blease Kate , Jakubzick Claudia , Schuh Jane M. , Joshi Bharat H. , Puri Raj K. , Hogaboam Cory M.
Published: April 2014

IL-13 has emerged as a major contributor to allergic and asthmatic responses, and as such it represents an attractive target in these diseases. In this study, IL-13-responsive cells in the lung were targeted via the intranasal administration of IL-13-PE38QQR (IL-13-PE), comprised of human IL-13 and a derivative of Pseudomonas exotoxin, to Aspergillus fumigatus-sensitized mice challenged with A. fumigatus spores, or conidia. Mice received 50, 100, or 200 ng of IL-13-PE or diluent alone (i.e., control group) on alternate days from day 14 to day 28 after the conidia challenge. The control group of mice exhibited significant airway hyperreactivity, goblet cell hyperplasia, and peribronchial fibrosis at day 28 after conidia. Although the two lower doses of IL-13-PE had limited therapeutic effects in mice with fungal-induced allergic airway disease, the highest dose of IL-13-PE tested significantly reduced all features of airway disease compared with the control group. Whole lung mRNA expression of IL-4Rα and IL-13Rα1 was markedly reduced, whereas bronchoalveolar lavage and whole lung levels of IFN-γ were significantly elevated in mice treated with 200 ng of IL-13-PE compared with the control group. This study demonstrates that a therapy designed to target IL-13-responsive cells in the lung ameliorates established fungal-induced allergic airway disease in mice.
Involvement of Phosphoinositide 3-Kinases in Neutrophil Activation and the Development of Acute Lung Injury
Authors: Yum Ho-Kee , Arcaroli John , Kupfner John , Shenkar Robert , Penninger Josef M. , Sasaki Takehiko , Yang Kuang-Yao , Park Jong Sung , Abraham Edward
Published: April 2014

Activated neutrophils contribute to the development and severity of acute lung injury (ALI). Phosphoinositide 3-kinases (PI3-K) and the downstream serine/threonine kinase Akt/protein kinase B have a central role in modulating neutrophil function, including respiratory burst, chemotaxis, and apoptosis. In the present study, we found that exposure of neutrophils to endotoxin resulted in phosphorylation of Akt, activation of NF-κB, and expression of the proinflammatory cytokines IL-1β and TNF-α through PI3-K-dependent pathways. In vivo, endotoxin administration to mice resulted in activation of PI3-K and Akt in neutrophils that accumulated in the lungs. The severity of endotoxemia-induced ALI was significantly diminished in mice lacking the p110γ catalytic subunit of PI3-K. In PI3-Kγ−/− mice, lung edema, neutrophil recruitment, nuclear translocation of NF-κB, and pulmonary levels of IL-1β and TNF-α were significantly lower after endotoxemia as compared with PI3-Kγ+/+ controls. Among neutrophils that did accumulate in the lungs of the PI3-Kγ−/− mice after endotoxin administration, activation of NF-κB and expression of proinflammatory cytokines was diminished compared with levels present in lung neutrophils from PI3-Kγ+/+ mice. These results show that PI3-K, and particularly PI3-Kγ, occupies a central position in regulating endotoxin-induced neutrophil activation, including that involved in ALI.
The Endogenous Opioid Spinorphin Blocks fMet-Leu-Phe-Induced Neutrophil Chemotaxis by Acting as a Specific Antagonist at the N-Formylpeptide Receptor Subtype FPR
Authors: Liang Thomas S. , Gao Ji-Liang , Fatemi Omid , Lavigne Mark , Leto Thomas L. , Murphy Philip M.
Published: April 2014

Spinorphin is an endogenous heptapeptide (leucylvalylvalyltyrosylprolyltryptophylthreonine), first isolated from bovine spinal cord, whose sequence matches a conserved region of β-hemoglobin. Also referred to as LVV-hemorphin-4 and a member of the nonclassical opioid hemorphin family, spinorphin inhibits enkephalin-degrading enzymes and is analgesic. Recently, spinorphin was reported to block neutrophil activation induced by the chemotactic N-formylpeptide N-formylmethionylleucylphenylalanine (fMLF), suggesting a potential role as an endogenous negative regulator of inflammation. Here we use both gain- and loss-of-function genetic tests to identify the specific mechanism of spinorphin action on neutrophils. Spinorphin induced calcium flux in normal mouse neutrophils, but was inactive in neutrophils from mice genetically deficient in the fMLF receptor subtype FPR (N-formylpeptide receptor). Consistent with this, spinorphin induced calcium flux in human embryonic kidney 293 cells transfected with mouse FPR, but had no effect on cells expressing the closely related fMLF receptor subtype FPR2. Despite acting as a calcium-mobilizing agonist at FPR, spinorphin was a weak chemotactic agonist and effectively blocked neutrophil chemotaxis induced by fMLF at concentrations selective for FPR. Spinorphin did not affect mouse neutrophil chemotaxis induced by concentrations of fMLF that selectively activate FPR2. Thus, spinorphin blocks fMLF-induced neutrophil chemotaxis by acting as a specific antagonist at the fMLF receptor subtype FPR.
Skin Allograft Maintenance in a New Synchimeric Model System of Tolerance
Authors: Iwakoshi Neal N. , Markees Thomas G. , Turgeon Nicole , Thornley Thomas , Cuthbert Amy , Leif Jean , Phillips Nancy E. , Mordes John P. , Greiner Dale L. , Rossini Aldo A.
Published: April 2014

Treatment of mice with a single donor-specific transfusion plus a brief course of anti-CD154 mAb uniformly induces donor-specific transplantation tolerance characterized by the deletion of alloreactive CD8+ T cells. Survival of islet allografts in treated mice is permanent, but skin grafts eventually fail unless recipients are thymectomized. To analyze the mechanisms underlying tolerance induction, maintenance, and failure in euthymic mice we created a new analytical system based on allo-TCR-transgenic hemopoietic chimeric graft recipients. Chimeras were CBA (H-2k) mice engrafted with small numbers of syngeneic TCR-transgenic KB5 bone marrow cells. These mice subsequently circulated a self-renewing trace population of anti-H-2b-alloreactive CD8+ T cells maturing in a normal microenvironment. With this system, we studied the maintenance of H-2b allografts in tolerized mice. We documented that alloreactive CD8+ T cells deleted during tolerance induction slowly returned toward pretreatment levels. Skin allograft rejection in this system occurred in the context of 1) increasing numbers of alloreactive CD8+ cells; 2) a decline in anti-CD154 mAb concentration to levels too low to inhibit costimulatory functions; and 3) activation of the alloreactive CD8+ T cells during graft rejection following deliberate depletion of regulatory CD4+ T cells. Rejection of healed-in allografts in tolerized mice appears to be a dynamic process dependent on the level of residual costimulation blockade, CD4+ regulatory cells, and activated alloreactive CD8+ thymic emigrants that have repopulated the periphery after tolerization.
Deficiency of 5-Lipoxygenase Accelerates Renal Allograft Rejection in Mice
Authors: Goulet Jennifer L. , Griffiths Robert C. , Ruiz Phillip , Mannon Roslyn B. , Flannery Pat , Platt Jeffrey L. , Koller Beverly H. , Coffman Thomas M.
Published: April 2014

Acute renal allograft rejection is associated with alterations in renal arachidonic acid metabolism, including enhanced synthesis of leukotrienes (LTs). LTs, the products of the 5-lipoxygenase (5-LO) pathway, are potent lipid mediators with a broad range of biologic activities. Previous studies, using pharmacological agents to inhibit LT synthesis or activity, have implicated these eicosanoids in transplant rejection. To further investigate the role of LTs in acute graft rejection, we transplanted kidneys from CByD2F1 mice into fully allogeneic 129 mice that carry a targeted mutation in the 5lo gene. Unexpectedly, allograft rejection was significantly accelerated in 5-LO-deficient mice compared with wild-type animals. Despite the marked reduction in graft survival, the 5lo mutation had no effect on the hemodynamics or morphology of the allografts. Although LTB4 levels were reduced, renal thromboxane B2 production and cytokine expression were not altered in 5-LO-deficient allograft recipients. These findings suggest that, along with their proinflammatory actions, metabolites of 5-LO can act to enhance allograft survival.
Mediators of Innate Immunity That Target Immature, But Not Mature, Dendritic Cells Induce Antitumor Immunity When Genetically Fused with Nonimmunogenic Tumor Antigens
Authors: Biragyn Arya , Surenhu Munhsuren , Yang De , Ruffini Pier Adelchi , Haines Bryan Allen , Klyushnenkova Elena , Oppenheim Joost J. , Kwak Larry W.
Published: April 2014

Chemokine receptors are differentially expressed on immature and mature dendritic cells (DC). Herein, we demonstrate for the first time that murine antimicrobial peptides β-defensins 2 and 3 bind murine CCR6, similarly to inflammatory chemokine macrophage-inflammatory protein 3α, and they chemoattract bone marrow-derived immature, but not mature DC. Using various chemokines or defensins fused with nonimmunogenic tumor Ags, we studied their capacity to delivery Ags to subsets of immune cells to elicit antitumor immunity. We demonstrate that DNA immunizations with fusion constructs with β-defensin 2 or inflammatory chemokines that target immature DC, but not homeostatic chemokines secondary lymphoid tissue chemokine, CCL21, or stromal cell-derived factor 1, CXCL12, which chemoattract mature DC, elicit humoral, protective, and therapeutic immunity against two different syngeneic lymphomas.
Evidence for Increased T Cell Turnover and Decreased Thymic Output in HIV Infection
Authors: Douek Daniel C. , Betts Michael R. , Hill Brenna J. , Little Susan J. , Lempicki Richard , Metcalf Julia A. , Casazza Joseph , Yoder Christian , Adelsberger Joseph W. , Stevens Randy A. , Baseler Michael W. , Keiser Philip , Richman Douglas D. , Davey Richard T. , Koup Richard A.
Published: April 2014

The effects of HIV infection upon the thymus and peripheral T cell turnover have been implicated in the pathogenesis of AIDS. In this study, we investigated whether decreased thymic output, increased T cell proliferation, or both can occur in HIV infection. We measured peripheral blood levels of TCR rearrangement excision circles (TREC) and parameters of cell proliferation, including Ki67 expression and ex vivo bromodeoxyuridine incorporation in 22 individuals with early untreated HIV disease and in 15 HIV-infected individuals undergoing temporary interruption of therapy. We found an inverse association between increased T cell proliferation with rapid viral recrudescence and a decrease in TREC levels. However, during early HIV infection, we found that CD45RO−CD27high (naive) CD4+ T cell proliferation did not increase, despite a loss of TREC within naive CD4+ T cells. A possible explanation for this is that decreased thymic output occurs in HIV-infected humans. This suggests that the loss of TREC during HIV infection can arise from a combination of increased T cell proliferation and decreased thymic output, and that both mechanisms can contribute to the perturbations in T cell homeostasis that underlie the pathogenesis of AIDS.
Augmentation Versus Inhibition: Effects of Conjunctional OX-40 Receptor Monoclonal Antibody and IL-2 Treatment on Adoptive Immunotherapy of Advanced Tumor
Authors: Kjaergaard Jørgen , Peng Liaomin , Cohen Peter A. , Drazba Judith A. , Weinberg Andrew D. , Shu Suyu
Published: April 2014

Therapeutic efficacy of adoptive immunotherapy of malignancies is proportional to the number of effector T cells transferred. Traditionally, exogenous IL-2 treatment has been used to promote the survival and function of transferred cells. Recently, we described the therapeutic effects of in vivo ligation of the costimulatory receptor, OX-40R, on activated T cells during early tumor growth. In this study, we examined the effects of IL-2 and OX-40R mAb on adoptive immunotherapy of advanced tumors. For treatment of 10-day 3-methylcholanthrene 205 pulmonary metastases, systemic transfer of 50 × 106 activated tumor-draining lymph node T cells resulted in >99% reduction of metastatic nodules. With either IL-2 or OX-40R mAb conjunctional treatment, only 20 × 106 cells were required. Advanced 10-day 3-methylcholanthrene 205 intracranial tumors could be cured by the transfer of 15 × 106 L-selectinlow T cells derived from draining lymph nodes. In this situation, IL-2 administration inhibited therapeutic effects of the transferred cells. By contrast, 5 × 106 T cells were sufficient to cure all mice if OX-40R mAb was administrated. Studies on trafficking of systemically transferred T cells revealed that IL-2, but not OX-40R mAb, impeded tumor infiltration by T cells. Tumor regression required participation of both CD4 and CD8 T cells. Because only CD4 T cells expressed OX-40R at cell transfer, direct CD4 T cell activation is possible. Alternatively, OX-40R might be up-regulated on transferred T cells at the tumor site, rendering them reactive to the mAb. Our study suggests OX-40R mAb to be a reagent of choice to augment T cell adoptive immunotherapy in clinical trials.
Cutting Edge: Regulation of CD8+ T Cell Proliferation by 2B4/CD48 Interactions
Authors: Kambayashi Taku , Assarsson Erika , Chambers Benedict J. , Ljunggren Hans-Gustaf
Published: April 2014

The biological function of 2B4, a CD48-binding molecule expressed on T cells with an activation/memory phenotype, is not clear. In this report, we demonstrate that proliferation of CD8+ T cells is regulated by 2B4. Proliferative responses of CD8+ T cells were significantly reduced by anti-2B4 Ab. The effects were not potentiated by anti-CD48 Ab, suggesting that the observed responses were driven by 2B4/CD48 interactions. Surprisingly, the 2B4/CD48-dependent proliferative responses were also observed in the absence of APCs. This suggests that 2B4/CD48 interactions can occur directly between T cells. Furthermore, when activated 2B4+CD8+ T cells were mixed with 2B4−CD8+ TCR-transgenic T cells and specific peptide-loaded APC, the proliferation of the latter T cells was inhibited by anti-2B4 Ab. Taken together, this suggests that 2B4 on activated/memory T cells serves as a ligand for CD48, and by its ability to interact with CD48 provides costimulatory-like function for neighboring T cells.
Cutting Edge: Secondary Lymphoid Organs Are Essential for Maintaining the CD4, But Not CD8, Naive T Cell Pool
Authors: Dai Zhenhua , Lakkis Fadi G.
Published: April 2014

Despite declining thymic output with age, the peripheral naive T cell pool of an adult animal remains remarkably stable. Therefore, a central question in immunology is how the naive T cell pool is maintained. Here we show that the maintenance of the naive CD4, but not CD8, T cell population in the thymectomized adult mouse is dependent on the presence of secondary lymphoid tissues. This finding is explained by the inability of naive CD4 T cells to sustain normal levels of the survival molecule Bcl-2 or to undergo homeostatic proliferation in the absence of secondary lymphoid organs. Thus, naive CD4 T cells must traffic through secondary lymphoid organs to maintain a stable CD4 pool while naive CD8 T cells encounter their survival and proliferation signals outside the organized structures of secondary lymphoid tissues.
Multiple Levels of Activation of Murine CD8+ Intraepithelial Lymphocytes Defined by OX40 (CD134) Expression: Effects on Cell-Mediated Cytotoxicity, IFN-γ, and IL-10 Regulation
Authors: Wang Heuy-Ching , Klein John R.
Published: April 2014

The involvement of OX40 (CD134) in the activation of CD8+ intestinal intraepithelial lymphocytes (IELs) has been studied using freshly isolated IELs and in vitro CD3-stimulated IELs. Although freshly isolated CD8+ IELs exhibited properties of activated T cells (CD69 expression and ex vivo cytotoxicity), virtually all CD8+ IELs from normal mice were devoid of other activation-associated properties, including a lack of expression of OX40 and the ligand for OX40 (OX40L) and an absence of intracellular IFN-γ staining. However, OX40 and OX40L expression were rapidly up-regulated on CD8 IELs following CD3 stimulation, indicating that both markers on IELs reflect activation-dependent events. Unlike IELs, activated lymph node T cells did not express OX40L, thus indicating that OX40-OX40L communication in the intestinal epithelium is part of a novel CD8 network. Functionally, OX40 expression was exclusively associated with IELs with active intracellular IFN-γ synthesis and markedly enhanced cell-mediated cytotoxicity. However, OX40 costimulation during CD3-mediated activation significantly suppressed IL-10 synthesis by IELs, whereas blockade of OX40-OX40L by anti-OX40L mAb markedly increased IL-10 production. These findings indicate that: 1) resident CD69+OX40− IELs constitute a population of partially activated T cells poised for rapid delivery of effector activity, 2) OX40 and OX40L expression defines IELs that have undergone recent immune activation, 3) OX40+ IELs are significantly more efficient CTL than are OX40− IELs, and 4) the local OX40/OX40L system plays a critical role in regulating the magnitude of cytokine responses in the gut epithelium.
Soluble CD26/Dipeptidyl Peptidase IV Induces T Cell Proliferation Through CD86 Up-Regulation on APCs
Authors: Ohnuma Kei , Munakata Yasuhiko , Ishii Tomonori , Iwata Satoshi , Kobayashi Seiji , Hosono Osamu , Kawasaki Hiroshi , Dang Nam H. , Morimoto Chikao
Published: April 2014

CD26 is a T cell costimulatory molecule with dipeptidyl peptidase IV enzyme activity in its extracellular region. We have previously reported that the addition of soluble CD26 (sCD26) resulted in enhanced proliferation of peripheral blood T lymphocytes induced by the recall Ag, tetanus toxoid (TT). However, the mechanism involved in this immune enhancement has not yet been elucidated. In this paper, we demonstrate that the enhancing effect of sCD26 on TT-induced T cell proliferation occurred in the early stages of immune response. The cells directly affected by exogenously added sCD26 are the CD14-positive monocytes in the peripheral blood. Mannose-6 phosphate interfered with the uptake of sCD26 into monocytes, suggesting that mannose-6 phosphate/insulin-like growth factor II receptor plays a role in the transportation of sCD26 into monocytes. When sCD26 was added after Ag presentation had taken place, enhancement in TT-induced T cell proliferation was not observed. In addition, enhancement of TT-mediated T cell proliferation by sCD26 does not result from trimming of the MHC-bound peptide on the surface of monocytes. Importantly, we also showed that exogenously added sCD26 up-regulated the expression of the costimulatory molecule CD86 on monocytes through its dipeptidyl peptidase IV activity, and that this increased expression of CD86 was observed at both protein and mRNA level. Therefore, our findings suggest that sCD26 enhances T cell immune response to recall Ag via its direct effect on APCs.