bluuebunny/crossref_metadata_2025 huggingface dataset:
Photoaffinity Antigens for Human γδ T Cells
Authors: Sarikonda Ghanashyam , Wang Hong , Puan Kia-Joo , Liu Xiao-hui , Lee Hoi K. , Song Yongcheng , Distefano Mark D. , Oldfield Eric , Prestwich Glenn D. , Morita Craig T.
Published: April 2014

Vγ2Vδ2 T cells comprise the major subset of peripheral blood γδ T cells in humans and expand during infections by recognizing small nonpeptide prenyl pyrophosphates. These molecules include (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMBPP), a microbial isoprenoid intermediate, and isopentenyl pyrophosphate, an endogenous isoprenoid intermediate. Recognition of these nonpeptide Ags is mediated by the Vγ2Vδ2 T cell Ag receptor. Several findings suggest that prenyl pyrophosphates are presented by an Ag-presenting molecule: contact between T cells and APC is required, the Ags do not bind the Vγ2Vδ2 TCR directly, and Ag recognition is abrogated by TCR mutations in CDRs distant from the putative Ag recognition site. Identification of the putative Ag-presenting molecule, however, has been hindered by the inability to achieve stable association of nonpeptide prenyl pyrophosphate Ags with the presenting molecule. In this study, we show that photoaffinity analogues of HMBPP, meta/para-benzophenone-(methylene)-prenyl pyrophosphates (m/p-BZ-(C)-C5-OPP), can crosslink to the surface of tumor cell lines and be presented as Ags to γδ T cells. Mutant tumor cell lines lacking MHC class I, MHC class II, β2-microglobulin, and CD1, as well as tumor cell lines from a variety of tissues and individuals, will all crosslink to and present m-BZ-C5-OPP. Finally, pulsing of BZ-(C)-C5-OPP is inhibited by isopentenyl pyrophosphate and an inactive analog, suggesting that they bind to the same molecule. Taken together, these results suggest that nonpeptide Ags are presented by a novel-Ag-presenting molecule that is widely distributed and nonpolymorphic, but not classical MHC class I, MHC class II, or CD1.
Coexpression of TGF-β1 and IL-10 Enables Regulatory T Cells to Completely Suppress Airway Hyperreactivity
Authors: Presser Katrin , Schwinge Dorothee , Wegmann Michael , Huber Samuel , Schmitt Steffen , Quaas Alexander , Maxeiner Joachim H. , Finotto Susetta , Lohse Ansgar W. , Blessing Manfred , Schramm Christoph
Published: April 2014

In allergic airway disease, Treg may play an important role in the modulation of airway hyperreactivity (AHR) and inflammation. We therefore investigated the therapeutic potential of Treg in an Ag-dependent murine asthma model. We here describe that AHR can be completely suppressed by adoptive transfer of Treg overexpressing active TGF-β1. Using mice with impaired TGF-β signaling in T cells, we could demonstrate that TGF-β signaling in recipient effector T cells or transferred Treg themselves is not required for the protective effects on AHR. However, the expression of IL-10 by Treg was found to be essential for the suppression of AHR, since Treg overexpressing active TGF-β1 but deficient in IL-10 lacked protective effects. Airway inflammation could not be significantly suppressed by wild-type or transgenic Treg. In conclusion, modulation of cytokine expression by Treg may have therapeutic potential for the treatment of AHR in asthma. The mechanisms of the effects of Treg on airway inflammation require further clarification.
T Cell-Independent Spontaneous Loss of Tolerance by Anti-Double-Stranded DNA B Cells in C57BL/6 Mice
Authors: Tsao Patricia Y. , Jiao Jing , Ji Mei Qing , Cohen Philip L. , Eisenberg Robert A.
Published: April 2014

Systemic lupus erythematosus is characterized by loss of tolerance to DNA and other nuclear Ags. To understand the role of T cells in the breaking of tolerance, an anti-DNA site-specific transgenic model of spontaneous lupus, B6.56R, was studied. T cells were eliminated by crossing B6.56R with CD4−/− or TCRβ−/−δ−/− mice, and the effects on anti-dsDNA serum levels, numbers of anti-dsDNA Ab-secreting cells, and isotypes of anti-dsDNA were analyzed. In addition, the development and activation of B cells in these mice were examined. Surprisingly, the presence of T cells made little difference in the development and character of the serum anti-dsDNA Ab in B6.56R mice. At 1 mo of age, anti-dsDNA Abs were somewhat lower in mice deficient in αβ and γδ T cells. Levels of Abs later were not affected by T cells, nor was autoantibody class switching. B cell activation was somewhat diminished in T cell-deficient mice. Thus, in the B6 background, the presence of an anti-dsDNA transgene led the production of autoantibodies with a specificity and isotype characteristic of murine systemic lupus erythematosus with little influence from T cells. TLR9 also did not appear to play a role. Although we do not yet understand the mechanism of this failure of immunoregulation, these results suggest that similar processes may influence autoimmunity associated with clinical disease.
Egr2 Is Required for Bcl-2 Induction during Positive Selection
Authors: Lauritsen Jens-Peter Holst , Kurella Sridevi , Lee Sang-Yun , Lefebvre Juliette M. , Rhodes Michele , Alberola-Ila José , Wiest David L.
Published: April 2014

The repertoire of TCR specificities is established by a selection process in the thymus, during which precursor survival and maturation is dictated by the nature of the TCR signals. The differences in signals that determine whether precursors will survive and mature or be induced to die remain poorly understood. Among the molecular effectors involved in executing the differentiation process initiated by TCR-ligand interactions is a family of Zn-finger transcription factors termed early growth response genes (Egr). Indeed, ablation of the Egr1 gene impairs ligand-induced maturation (positive selection) but not ligand-induced deletion (negative selection). The partial impairment of positive selection by Egr1 deficiency is not enhanced by simultaneous deletion of another Egr family member, Egr3. Accordingly, we asked whether this results from compensation by another family member, Egr2. In this manuscript, we demonstrate that deletion of Egr2 impairs positive selection of both CD4 and CD8 single-positive thymocytes. Interestingly, many of the genes involved in positive selection and T cell differentiation are up-regulated normally in the Egr2-deficient thymocytes. However, Bcl-2 up-regulation is not sustained during late stages of positive selection. This defect is at least partially responsible for the developmental blockade in Egr2-deficient thymocytes, as enforced expression of Bcl-2 rescues T cell development in Egr2−/− thymocytes. Taken together, these data suggest that Egr2 plays a central role in the up-regulation of the survival molecule Bcl-2 during positive selection.
TCR Down-Regulation Controls Virus-Specific CD8+ T Cell Responses
Authors: Bonefeld Charlotte Menné , Haks Mariëlle , Nielsen Bodil , von Essen Marina , Boding Lasse , Hansen Ann Kathrine , Larsen Jeppe Madura , Ødum Niels , Krimpenfort Paul , Kruisbeek Ada , Christensen Jan Pravsgaard , Thomsen Allan Randrup , Geisler Carsten
Published: April 2014

The CD3γ di-leucine-based motif plays a central role in TCR down-regulation. However, little is understood about the role of the CD3γ di-leucine-based motif in physiological T cell responses. In this study, we show that the expansion in numbers of virus-specific CD8+ T cells is impaired in mice with a mutated CD3γ di-leucine-based motif. The CD3γ mutation did not impair early TCR signaling, nor did it compromise recruitment or proliferation of virus-specific T cells, but it increased the apoptosis rate of the activated T cells by increasing down-regulation of the antiapoptotic molecule Bcl-2. This resulted in a 2-fold reduction in the clonal expansion of virus-specific CD8+ T cells during the acute phase of vesicular stomatitis virus and lymphocytic choriomeningitis virus infections. These results identify an important role of CD3γ-mediated TCR down-regulation in virus-specific CD8+ T cell responses.
TNF Receptor-Associated Factor 5 Is Required for Optimal T Cell Expansion and Survival in Response to Infection
Authors: Kraus Zachary J. , Haring Jodie S. , Bishop Gail A.
Published: April 2014

Receptors belonging to the TNF-receptor (TNF-R) superfamily include important costimulatory molecules, many of which specifically affect T cell activation. TNF receptor-associated factors (TRAFs) are recruited to many TNF-R superfamily members and are important modulators of the proximal signaling events that occur at the time of receptor engagement and activation. TRAF5 has been shown to be a positive regulator of a number of these receptors that are involved in T cell costimulation. However, the potential importance of TRAF5 in cellular immune responses to infection or in T cell expansion and memory have not been studied. We report in this study that TRAF5 was required for optimal CD8+ T cell responses following infection with Listeria monocytogenes expressing OVA (LM-OVA). TRAF5 was necessary for optimal T cell expansion following primary infection with LM-OVA, and its absence resulted in fewer memory CD8+ T cells following LM-OVA infection, together with higher bacterial loads in the liver. The effect of TRAF5 on CD8+ T cell expansion was T cell intrinsic and not due to effects of TRAF5 deficiency on APCs. Although their proliferative ability remained intact, CD8+ T cells from TRAF5−/− mice were more sensitive to apoptosis and were unresponsive to the prosurvival effects of the TNF-R superfamily costimulator CD27. Collectively, these studies identify TRAF5 as an important positive signaling element that enhances T cell expansion and pathogen containment by providing a survival advantage to responding Ag-specific CD8+ T cells during infection.
Granzyme C Supports Efficient CTL-Mediated Killing Late in Primary Alloimmune Responses
Authors: Getachew Yonas , Stout-Delgado Heather , Miller Bonnie C. , Thiele Dwain L.
Published: April 2014

It is well established that granzymes A and B play a role in CTL killing of target cells by the perforin-dependent granule exocytosis pathway. The functions of multiple additional granzymes expressed in CTL are less well defined. In the present studies, CTL generated from mice deficient in dipeptidyl peptidase 1 (DPP1) were used to investigate the contribution of granzyme C to CTL killing of allogeneic target cells. DPP1 is required for activation of granzymes A and B by proteolytic removal of their N-terminal dipeptide prodomains while a significant portion of granzyme C is processed normally in the absence of DPP1. Cytotoxicity of DPP1−/− CTL generated in early (5-day) MLC in vitro and in peritoneal exudate cells 5 days after initial allogeneic sensitization in vivo was significantly impaired compared with wild-type CTL. Following 3 days of restimulation with fresh allogeneic stimulators however, cytotoxicity of these DPP1−/− effector cells was comparable to that of wild-type CTL. Killing mediated by DPP1−/− CTL following restimulation was rapid, perforin dependent, Fas independent and associated with early mitochondrial injury, phosphatidyl serine externalization, and DNA degradation, implicating a granzyme-dependent apoptotic pathway. The increased cytotoxicity of DPP1−/− CTL following restimulation coincided with increased expression of granzyme C. Moreover, small interfering RNA inhibition of granzyme C expression during restimulation significantly decreased cytotoxicity of DPP1−/− but not wild-type CTL. These results indicate that during late primary alloimmune responses, granzyme C can support CTL-mediated killing by the granule exocytosis pathway in the absence of functional granzymes A or B.
An Activation-Induced Cytidine Deaminase-Independent Mechanism of Secondary VH Gene Rearrangement in Preimmune Human B Cells
Authors: Longo Nancy S. , Grundy Gabrielle J. , Lee Jisoo , Gellert Martin , Lipsky Peter E.
Published: April 2014

VH replacement is a form of IgH chain receptor editing that is believed to be mediated by recombinase cleavage at cryptic recombination signal sequences (cRSS) embedded in VH genes. Whereas there are several reports of VH replacement in primary and transformed human B cells and murine models, it remains unclear whether VH replacement contributes to the normal human B cell repertoire. We identified VH→VH(D)JH compound rearrangements from fetal liver, fetal bone marrow, and naive peripheral blood, all of which involved invading and recipient VH4 genes that contain a cryptic heptamer, a 13-bp spacer, and nonamer in the 5′ portion of framework region 3. Surprisingly, all pseudohybrid joins lacked the molecular processing associated with typical VH(D)JH recombination or nonhomologous end joining. Although inefficient compared with a canonical recombination signal sequences, the VH4 cRSS was a significantly better substrate for in vitro RAG-mediated cleavage than the VH3 cRSS. It has been suggested that activation-induced cytidine deamination (AICDA) may contribute to VH replacement. However, we found similar secondary rearrangements using VH4 genes in AICDA-deficient human B cells. The data suggest that VH4 replacement in preimmune human B cells is mediated by an AICDA-independent mechanism resulting from inefficient but selective RAG activity.
Induction of A:T Mutations Is Dependent on Cellular Environment but Independent of Mutation Frequency and Target Gene Location
Authors: Ukai Akiko , Ishimaru Konomi , Ouchida Rika , Mori Hiromi , Kano Chie , Moritan Toshiyuki , Wang Ji-Yang
Published: April 2014

Based on its substrate specificity, activation-induced cytidine deaminase can directly induce C:G mutations in Ig genes. However the origin of A:T mutations, which occur in a similar proportion in germinal center (GC) B cells, is unclear. Genetic evidence suggests that the induction of A:T mutations requires the components of the mismatch repair system and DNA polymerase η (POLH). We found that fibroblasts and GC B cells expressed similar levels of the mismatch repair components, but nonetheless the fibroblasts failed to generate a significant proportion of A:T mutations in a GFP reporter gene even after POLH overexpression. To investigate whether the ability to generate A:T mutations is dependent on the cellular environment (i.e., GC B cell or fibroblast) or the target gene (i.e., Ig or GFP), we developed a mutation detection system in a human GC-like cell line. We introduced a GFP gene with a premature stop codon into Ramos cells and compared the activation-induced cytidine deaminase-induced mutations in the endogenous VH and the transgenic GFP genes. Remarkably, a high proportion of A:T mutations was induced in both genes. Ectopic expression of POLH did not further increase the proportion of A:T mutations but diminished the strand bias of these mutations that is normally observed in VH genes. Intriguingly, the total mutation frequency in the GFP gene was consistently one-fifth of that in the VH gene. These results demonstrate that the ability to generate A:T mutations is dependent on the GC B cell environment but independent of the mutation frequency and target gene location.
RGS13 Controls G Protein-Coupled Receptor-Evoked Responses of Human Mast Cells
Authors: Bansal Geetanjali , DiVietro Jeffrey A. , Kuehn Hye Sun , Rao Sudhir , Nocka Karl H. , Gilfillan Alasdair M. , Druey Kirk M.
Published: April 2014

IgE-mediated mast cell degranulation and release of vasoactive mediators induced by allergens elicits allergic responses. Although G protein-coupled receptor (GPCR)-induced signals may amplify IgE-dependent degranulation, how GPCR signaling in mast cells is regulated remains incompletely defined. We investigated the role of regulator of G protein signaling (RGS) proteins in the modulation of these pathways in human mast cells. Several RGS proteins were expressed in mast cells including RGS13, which we previously showed inhibited IgE-mediated mast cell degranulation and anaphylaxis in mice. To characterize how RGS13 affects GPCR-mediated functions of human mast cells, we analyzed human mast cell lines (HMC-1 and LAD2) depleted of RGS13 by specific small interfering RNA or short hairpin RNA and HMC-1 cells overexpressing RGS13. Transient RGS13 knockdown in LAD2 cells lead to increased degranulation to sphingosine-1-phosphate but not to IgE-Ag or C3a. Relative to control cells, HMC-1 cells stably expressing RGS13-targeted short hairpin RNA had greater Ca2+ mobilization in response to several natural GPCR ligands such as adenosine, C5a, sphingosine-1-phosphate, and CXCL12 than wild-type cells. Akt phosphorylation, chemotaxis, and cytokine (IL-8) secretion induced by CXCL12 were also greater in short hairpin RGS13-HMC-1 cells compared with control. RGS13 overexpression inhibited CXCL12-evoked Ca2+ mobilization, Akt phosphorylation and chemotaxis. These results suggest that RGS13 restricts certain GPCR-mediated biological responses of human mast cells.
Complement Receptor of the Ig Superfamily Enhances Complement-Mediated Phagocytosis in a Subpopulation of Tissue Resident Macrophages
Authors: Gorgani Nick N. , He Jeannie Q. , Katschke Kenneth J. , Helmy Karim Y. , Xi Hongkang , Steffek Micah , Hass Philip E. , van Lookeren Campagne Menno
Published: April 2014

An important function of the complement cascade is to coat self and foreign particles with C3-proteins that serve as ligands for phagocytic receptors. Although tissue resident macrophages play an important role in complement-mediated clearance, the receptors coordinating this process have not been well characterized. In the present study, we identified a subpopulation of resident peritoneal macrophages characterized by high expression of complement receptor of the Ig superfamily (CRIg), a recently discovered complement C3 receptor. Macrophages expressing CRIg showed significantly increased binding and subsequent internalization of complement-opsonized particles compared with CRIg negative macrophages. CRIg internalized monovalent ligands and was able to bind complement-opsonized targets in the absence of Ca2+ and Mg2+, which differs from the β2-integrin CR3 that requires divalent cations and polyvalent ligands for activation of the receptor. Although CRIg dominated in immediate binding of complement-coated particles, CRIg and CR3 contributed independently to subsequent particle phagocytosis. CRIg thus identifies a subset of tissue resident macrophages capable of increased phagocytosis of complement C3-coated particles, a function critical for immune clearance.
A Novel IFN Regulatory Factor 3-Dependent Pathway Activated by Trypanosomes Triggers IFN-β in Macrophages and Fibroblasts
Authors: Chessler Anne-Danielle C. , Ferreira Ludmila R. P. , Chang Tun-Han , Fitzgerald Katherine A. , Burleigh Barbara A.
Published: April 2014

Innate immune recognition of intracellular pathogens involves both extracellular and cytosolic surveillance mechanisms. The intracellular protozoan parasite Trypanosoma cruzi triggers a robust type I IFN response in both immune and nonimmune cell types. In this study, we report that signaling through TBK1 and IFN regulatory factor 3 is required for T. cruzi-mediated expression of IFN-β. The TLR adaptors MyD88 and TRIF, as well as TLR4 and TLR3, were found to be dispensable, demonstrating that T. cruzi induces IFN-β expression in a TLR-independent manner. The potential role for cytosolic dsRNA sensing pathways acting through RIG-I and MDA5 was ruled out because T. cruzi was shown to trigger robust expression of IFN-β in macrophages lacking the MAVS/IPS1/VISA/CARDif adaptor protein. The failure of T. cruzi to activate HEK293-IFN-β-luciferase cells, which are highly sensitive to cytosolic triggers of IFN-β expression including Listeria, Sendai virus, and transfected dsRNA and dsDNA, further indicates that the parasite does not engage currently recognized cytosolic surveillance pathways. Together, these findings identify the existence of a novel TLR-independent pathogen-sensing mechanism in immune and nonimmune cells that converges on TBK1 and IFN regulatory factor 3 for activation of IFN-β gene expression.
Multimerization of Surfactant Protein D, but Not Its Collagen Domain, Is Required for Antiviral and Opsonic Activities Related to Influenza Virus
Authors: White Mitchell , Kingma Paul , Tecle Tesfaldet , Kacak Nilgun , Linders Bruce , Heuser John , Crouch Erika , Hartshorn Kevan
Published: April 2014

Surfactant protein D (SP-D) plays important roles in the initial innate defense against influenza A virus (IAV). The collagen domain of SP-D is probably critical for its homeostatic functions in vivo and has been implicated in the modulation of macrophage responses to SP-D-ligand complexes. For the current studies, we used a panel of rat SP-D mutants lacking all or part of the collagen domain to more specifically evaluate the contributions of this domain to viral interactions. SP-D multimers lacking the collagenous sequence efficiently neutralized Phil82 IAV, promoted neutrophil uptake of IAV, and also potentiated the IAV-induced neutrophil respiratory burst response. A dodecameric mutant with shortened collagenous arms showed enhanced viral aggregation and neuraminidase inhibition, and an increased capacity to inhibit a partially collectin-resistant strain of IAV. By contrast, truncated molecules lacking an N-terminal and collagen domain showed no detectable antiviral and opsonizing activity, despite preservation of lectin activity and detectable viral binding. Thus, multimerization, which is mediated by the N-peptide, is more important than the collagen domain for efficient viral neutralization and opsonization. However, the structure of the collagen domain significantly influences the anti-viral activity of multimerized forms of SP-D.
Identification of Human T Cell Antigens for the Development of Vaccines against Mycobacterium tuberculosis
Authors: Bertholet Sylvie , Ireton Gregory C. , Kahn Maria , Guderian Jeffrey , Mohamath Raodoh , Stride Nicole , Laughlin Elsa M. , Baldwin Susan L. , Vedvick Thomas S. , Coler Rhea N. , Reed Steven G.
Published: April 2014

Development of a subunit vaccine for Mycobacterium tuberculosis (Mtb) depends on the identification of Ags that induce appropriate T cell responses. Using bioinformatics, we selected a panel of 94 Mtb genes based on criteria that included growth in macrophages, up- or down-regulation under hypoxic conditions, secretion, membrane association, or because they were members of the PE/PPE or EsX families. Recombinant proteins encoded by these genes were evaluated for IFN-γ recall responses using PBMCs from healthy subjects previously exposed to Mtb. From this screen, dominant human T cell Ags were identified and 49 of these proteins, formulated in CpG, were evaluated as vaccine candidates in a mouse model of tuberculosis. Eighteen of the individual Ags conferred partial protection against challenge with virulent Mtb. A combination of three of these Ags further increased protection against Mtb to levels comparable to those achieved with bacillus Calmette-Guérin vaccination. Vaccine candidates that led to reduction in lung bacterial burden following challenge-induced pluripotent CD4 and CD8 T cells, including Th1 cell responses characterized by elevated levels of Ag-specific IgG2c, IFN-γ, and TNF. Priority vaccine Ags elicited pluripotent CD4 and CD8 T responses in purified protein derivative-positive donor PBMCs. This study identified numerous novel human T cell Ags suitable to be included in subunit vaccines against tuberculosis.
The Number of Respiratory Syncytial Virus (RSV)-Specific Memory CD8 T Cells in the Lung Is Critical for Their Ability to Inhibit RSV Vaccine-Enhanced Pulmonary Eosinophilia
Authors: Olson Matthew R. , Hartwig Stacey M. , Varga Steven M.
Published: April 2014

Children that were administered a formalin-inactivated respiratory syncytial virus (FI-RSV) vaccine experienced enhanced respiratory disease, including pulmonary eosinophilia, after contracting a natural RSV infection. RSV vaccine-enhanced disease can be mimicked in BALB/c mice immunized with either FI-RSV or with a recombinant vaccinia virus (vacv) expressing the RSV attachment (G) protein. We have recently demonstrated that memory CD8 T cells directed against the RSV immunodominant M282–90 epitope inhibit the development of pulmonary eosinophilia in either vacvG- or FI-RSV-immunized mice by reducing the total number of Th2 cells in the lung after RSV challenge. In this study, we show that memory CD8 T cells specific to a subdominant epitope within the RSV fusion (F) protein fail to inhibit the development of pulmonary eosinophilia after RSV challenge of mice previously co-immunized with vacvF and with either vacvG or FI-RSV. We observed that the inability of RSV F85-specific memory CD8 T cells to inhibit the development of pulmonary eosinophilia was largely due to an inadequate total number of F85-specific memory CD8 T cells in the lung at early times after RSV challenge. Increasing the number of F85-specific memory CD8 T cells after immunization grants these cells the ability to inhibit RSV vaccine-enhanced pulmonary eosinophilia. Moreover, we demonstrate that RSV-specific memory CD8 T cells, when present in sufficient numbers, inhibit the production of the Th2-associated chemokines CCL17 and CCL22. Taken together, these results indicate that RSV-specific memory CD8 T cells may alter the trafficking of Th2 cells and eosinophils into the lung.
Factor C Acts as a Lipopolysaccharide-Responsive C3 Convertase in Horseshoe Crab Complement Activation
Authors: Ariki Shigeru , Takahara Shusaku , Shibata Toshio , Fukuoka Takaaki , Ozaki Aya , Endo Yuichi , Fujita Teizo , Koshiba Takumi , Kawabata Shun-ichiro
Published: April 2014

The complement system in vertebrates plays an important role in host defense against and clearance of invading microbes, in which complement component C3 plays an essential role in the opsonization of pathogens, whereas the molecular mechanism underlying C3 activation in invertebrates remains unknown. In an effort to understand the molecular activation mechanism of invertebrate C3, we isolated and characterized an ortholog of C3 (designated TtC3) from the horseshoe crab Tachypleus tridentatus. Flow cytometric analysis using an Ab against TtC3 revealed that the horseshoe crab complement system opsonizes both Gram-negative and Gram-positive bacteria. Evaluation of the ability of various pathogen-associated molecular patterns to promote the proteolytic conversion of TtC3 to TtC3b in hemocyanin-depleted plasma indicated that LPS, but not zymosan, peptidoglycan, or laminarin, strongly induces this conversion, highlighting the selective response of the complement system to LPS stimulation. Although originally characterized as an LPS-sensitive initiator of hemolymph coagulation stored within hemocytes, we identified factor C in hemolymph plasma. An anti-factor C Ab inhibited various LPS-induced phenomena, including plasma amidase activity, the proteolytic activation of TtC3, and the deposition of TtC3b on the surface of Gram-negative bacteria. Moreover, activated factor C present on the surface of Gram-negative bacteria directly catalyzed the proteolytic conversion of the purified TtC3, thereby promoting TtC3b deposition. We conclude that factor C acts as an LPS-responsive C3 convertase on the surface of invading Gram-negative bacteria in the initial phase of horseshoe crab complement activation.
Inhibitors of TLR8 Reduce TNF Production from Human Rheumatoid Synovial Membrane Cultures
Authors: Sacre Sandra M. , Lo Alexandra , Gregory Bernard , Simmonds Rachel E. , Williams Lynn , Feldmann Marc , Brennan Fionula M. , Foxwell Brian M.
Published: April 2014

The advent of anti-TNF biologicals has been a seminal advance in the treatment of rheumatoid arthritis (RA) and has confirmed the important role of TNF in disease pathogenesis. However, it is unknown what sustains the chronic production of TNF. In this study, we have investigated the anti-inflammatory properties of mianserin, a serotonin receptor antagonist. We discovered mianserin was able to inhibit the endosomal TLRs 3, 7, 8, and 9 in primary human cells and inhibited the spontaneous release of TNF and IL-6 from RA synovial membrane cultures. This suggested a role for these TLRs in production of TNF and IL-6 from RA which was supported by data from chloroquine, an inhibitor of endosomal acidification (a prerequisite for TLRs 3, 7, 8, and 9 activation) which also inhibited production of these cytokines from RA synovial cultures. Only stimulation of TLR 3 or 8 induced TNF from these cultures, indicating that TLR7 and TLR9 were of less consequence in this model. The key observation that indicated the importance of TLR8 was the inhibition of spontaneous TNF production by imiquimod, which we discovered to be an inhibitor of TLR8. Together, these data suggest that TLR8 may play a role in driving TNF production in RA. Because this receptor can be inhibited by small m.w. molecules, it may prove to be an important therapeutic target.
Transgenic Expression of Cyclooxygenase-2 in Hepatocytes Accelerates Endotoxin-Induced Acute Liver Failure
Authors: Han Chang , Li Guiying , Lim Kyu , DeFrances Marie C. , Gandhi Chandrashekhar R. , Wu Tong
Published: April 2014

Bacterial LPS (endotoxin) is implicated in the pathogenesis of acute liver failure and several chronic inflammatory liver diseases. To evaluate the effect of hepatocyte cyclooxygenase (COX)-2 in LPS-induced liver injury, we generated transgenic mice with targeted expression of COX-2 in the liver by using the albumin promoter-enhancer driven vector and the animals produced were subjected to a standard experimental protocol of LPS-induced acute fulminant hepatic failure (i.p. injection of low dose of LPS in combination with d-galactosamine (d-GalN)). The COX-2 transgenic mice exhibited earlier mortality, higher serum aspartate aminotransferase and alanine aminotransferase levels and more prominent liver tissue damage (parenchymal hemorrhage, neutrophilic inflammation, hepatocyte apoptosis, and necrosis) than wild-type mice. Western blot analysis of the liver tissues showed that LPS/d-GalN treatment for 4 h induced much higher cleavage of poly(ADP-ribose) polymerase, caspase-3, and caspase-9 in COX-2 transgenic mice than in wild-type mice. Increased hepatic expression of JNK-2 in COX-2 transgenic mice suggest that up-regulation of JNK-2 may represent a potential mechanism for COX-2-mediated exacerbation of liver injury. Blocking the prostaglandin receptor, EP1, prevented LPS/d-GalN-induced liver injury and hepatocyte apoptosis in COX-2 transgenic mice. Accordingly, the mice with genetic ablation of EP1 showed less LPS/d-GalN-induced liver damage and less hepatocyte apoptosis with prolonged survival when compared with the wild-type mice. These findings demonstrate that COX-2 and its downstream prostaglandin receptor EP1 signaling pathway accelerates LPS-induced liver injury. Therefore, blocking COX-2-EP1 pathway may represent a potential approach for amelioration of LPS-induced liver injury.
CD40-Induced Signaling in Human Endothelial Cells Results in mTORC2- and Akt-Dependent Expression of Vascular Endothelial Growth Factor In Vitro and In Vivo
Authors: Dormond Olivier , Contreras Alan G. , Meijer Esther , Datta Dipak , Flynn Evelyn , Pal Soumitro , Briscoe David M.
Published: April 2014

We have examined CD40-dependent signals in endothelial cells (EC) mediating the expression of vascular endothelial growth factor (VEGF) and VEGF-induced angiogenesis. We treated confluent cultures of EC with soluble CD40L (sCD40L), and by Western blot found a marked increase in the phosphorylation of Akt, 4EBP-1, and S6K1, compared with untreated cells. EC were transfected with a full-length VEGF promoter-luciferase construct and cultured in the absence or presence of rapamycin and sCD40L. We found that rapamycin, which blocks mTORC1 and mTORC2 signaling, inhibited sCD40L-mediated transactivation of VEGF. In addition, by Western blot, we found that the transfection of EC with small interfering RNA (siRNA) to rictor (to inhibit mTORC2), and not raptor (to inhibit mTORC1), inhibited sCD40L-dependent protein expression of VEGF. In additions, we found that basal levels of phosphorylated Akt as well as VEGF were increased in EC transfected with the raptor siRNA. Also, rapamycin failed to inhibit VEGF promoter activation, as well as VEGF protein expression in EC transfected with a constitutively active construct of Akt, further demonstrating that mTORC1 is not necessary for CD40- and Akt-induced expression of VEGF. Finally, we injected human CD40L-transfected fibroblasts or mock transfectants into human skin on SCID mice. We found that the injection of CD40L transfectants, but not mock cells, resulted in VEGF expression and mediated a marked angiogenesis reaction, and this response was reduced in mice treated with rapamycin. Together, these observations indicate that mTORC2 and Akt facilitate CD40-inducible expression of VEGF in EC, which is of clinical importance in tumor growth and the progression of chronic inflammatory diseases.
Promiscuous Interaction between Gold-Specific T Cells and APCs in Gold Allergy
Authors: Hashizume Hideo , Seo Naohiro , Ito Taisuke , Takigawa Masahiro , Yagi Hiroaki
Published: April 2014

Gold compounds clinically used as immunomodulators have high potential to evoke hypersensitivity reactions as an adverse effect. To explore the mechanism of gold allergy, we immunologically characterized T cells infiltrating skin rashes and generated 44 gold-specific T cell clones and lines from a rheumatoid arthritis patient who developed skin rashes and systemic symptoms after gold treatment. CD4+ and CD8+ cells predominantly infiltrating the skin rashes and some of the T cell clones and lines shared common Vβs. These cells exhibited Th0-like, Th2-like, and Tc1-like cytokine profiles, and showed chemotactic activities for thymus and activation-regulated chemokine and IFN-γ-inducible protein-10 corresponding to the cytokine profiles. T cell recognition of gold consisted of MHC-restricted and MHC-independent pathways. Blocking studies with anti-MHC Abs indicated that the groove of MHC in APCs, where Ags should ordinarily be settled, did not serve as a conjugating site of gold for these T cells in certain cases. These observations raise the possibility that gold-specific CD4+ and CD8+ T cells and APCs promiscuously interact under stimulation with gold, resulting in various clinical manifestations in gold allergy.
Deficiency of HIV-Gag-Specific T Cells in Early Childhood Correlates with Poor Viral Containment
Authors: Huang SiHong , Dunkley-Thompson Jacqueline , Tang YanHua , Macklin Eric A. , Steel-Duncan Julianne , Singh-Minott Indira , Ryland Elizabeth G. , Smikle Monica , Walker Bruce D. , Christie Celia D. C. , Feeney Margaret E.
Published: April 2014

Perinatal HIV infection is characterized by a sustained high-level viremia and a high risk of rapid progression to AIDS, indicating a failure of immunologic containment of the virus. We hypothesized that age-related differences in the specificity or function of HIV-specific T cells may influence HIV RNA levels and clinical outcome following perinatal infection. In this study, we defined the HIV epitopes targeted by 76 pediatric subjects (47 HIV infected and 29 HIV exposed, but uninfected), and assessed the ability of HIV-specific CD8 and CD4 T cells to degranulate and produce IFN-γ, TNF-α, and IL-2. No responses were detected among HIV-uninfected infants, whereas responses among infected subjects increased in magnitude and breadth with age. Gag-specific responses were uncommon during early infancy, and their frequency was significantly lower among children younger than 24 mo old (p = 0.014). Importantly, Gag responders exhibited significantly lower HIV RNA levels than nonresponders (log viral load 5.8 vs 5.0; p = 0.005). Both the total and Gag-specific T cell frequency correlated inversely with viral load after correction for age, whereas no relationship with targeting of other viral proteins was observed. Functional assessment of HIV-specific T cells by multiparameter flow cytometry revealed that polyfunctional CD8 cells were less prevalent in children before 24 mo of age, and that HIV-specific CD4 cell responses were of universally low frequency among antiretroviral-naive children and absent in young infants. These cross-sectional data suggest that qualitative differences in the CD8 response, combined with a deficiency of HIV-specific CD4 cells, may contribute to the inability of young infants to limit replication of HIV.
Binding of Rituximab, Trastuzumab, Cetuximab, or mAb T101 to Cancer Cells Promotes Trogocytosis Mediated by THP-1 Cells and Monocytes
Authors: Beum Paul V. , Mack David A. , Pawluczkowycz Andrew W. , Lindorfer Margaret A. , Taylor Ronald P.
Published: April 2014

More than 20 years ago clinical investigations in the immunotherapy of cancer revealed that infusion of certain immunotherapeutic mAbs directed to tumor cells induced loss of targeted epitopes. This phenomenon, called antigenic modulation, can compromise mAb-based therapies. Recently we reported that rituximab (RTX) treatment of chronic lymphocytic leukemia patients induced substantial loss of targeted CD20 on B cells found in the circulation after RTX infusion; this “shaving” of RTX-CD20 complexes from B cells is also promoted in vitro by THP-1 monocytes and by PBMC in a reaction mediated by Fcγ receptors. The mechanism responsible for shaving appears to be trogocytosis, a process in which receptors on effector cells remove and internalize cognate ligands and cell membrane fragments from target cells. We now report that three therapeutic mAbs approved by the U.S. Food and Drug Administration for the treatment of cancer, RTX, cetuximab, and trastuzumab, as well as mAb T101, which has been shown to induce antigenic modulation in the clinic, promote trogocytosis in vitro upon binding to their respective target cells. Trogocytosis of the mAb-opsonized cells is mediated by THP-1 monocytes and by primary monocytes isolated from PBMC. In view of these results, it is likely that these mAbs and possibly other anticancer mAbs now used in the clinic may promote trogocytic removal of the therapeutic mAbs and their cognate Ags from tumor cells in vivo. Our findings may have important implications with respect to the use of mAbs in cancer immunotherapy.
Restoration of Adenosine Deaminase-Deficient Human Thymocyte Development In Vitro by Inhibition of Deoxynucleoside Kinases
Authors: Joachims Michelle L. , Marble Patrick A. , Laurent Aletha B. , Pastuszko Peter , Paliotta Marco , Blackburn Michael R. , Thompson Linda F.
Published: April 2014

Mutations in the gene encoding adenosine deaminase (ADA), a purine salvage enzyme, lead to immunodeficiency in humans. Although ADA deficiency has been analyzed in cell culture and murine models, information is lacking concerning its impact on the development of human thymocytes. We have used chimeric human/mouse fetal thymic organ culture to study ADA-deficient human thymocyte development in an “in vivo-like” environment where toxic metabolites accumulate in situ. Inhibition of ADA during human thymocyte development resulted in a severe reduction in cellular expansion as well as impaired differentiation, largely affecting mature thymocyte populations. Thymocyte differentiation was not blocked at a discrete stage; rather, the paucity of mature thymocytes was due to the induction of apoptosis as evidenced by activation of caspases and was accompanied by the accumulation of intracellular dATP. Inhibition of adenosine kinase and deoxycytidine kinase prevented the accumulation of dATP and restored thymocyte differentiation and proliferation. Our work reveals that multiple deoxynucleoside kinases are involved in the phosphorylation of deoxyadenosine when ADA is absent, and suggests an alternate therapeutic strategy for treatment of ADA-deficient patients.
Cutting Edge: Notch Signaling Induces a Distinct Cytokine Profile in Dendritic Cells That Supports T Cell-Mediated Regulation and IL-2-Dependent IL-17 Production
Authors: Bugeon Laurence , Gardner Leanne M. , Rose Anna , Gentle Madeleine , Dallman Margaret J.
Published: April 2014

Recently it has been shown that dendritic cells (DC) express both Notch and Notch ligands, allowing for the possibility that Notch signaling may influence their maturation. We show that although both Jagged (Jgd) and Delta-like (DlL) ligands were able to activate the canonical Notch pathway in mouse DC, only Jgd1 could induce the production of certain cytokines. Maturation of DC via Jgd1 resulted in an entirely different maturation program from that induced through TLR (via LPS) signaling, promoting the production of high levels of IL-2 and IL-10. DC matured by Jgd1 (Jgd1-conditioned DC) promoted the survival and proliferation of CD4+CD25+ regulatory T cells that were able to suppress efficiently the proliferation of CD25− cells. Further, CD25+ cells cultured with Jgd1-conditioned DC produced very high levels of IL-17 in an IL-2-dependent fashion. Our data suggest a new and important role for the Notch pathway in the regulation of the DC phenotype.
Cutting Edge: Thymic Crosstalk Regulates Delta-Like 4 Expression on Cortical Epithelial Cells
Authors: Fiorini Emma , Ferrero Isabel , Merck Estelle , Favre Stéphanie , Pierres Michel , Luther Sanjiv A. , MacDonald H. Robson
Published: April 2014

Interactions between Notch1 receptors on lymphoid progenitors and Delta-like 4 (DL4) ligands on cortical thymic epithelial cells (cTEC) are essential for T cell lineage commitment, expansion, and maturation in the thymus. Using a novel mAb against DL4, we show that DL4 levels on cTEC are very high in the fetal and neonatal thymus when thymocyte expansion is maximal but decrease dramatically in the adult when steady-state homeostasis is attained. Analysis of mutant mouse strains where thymocyte development is blocked at different stages indicates that lymphostromal interactions (“thymus crosstalk”) are required for DL4 down-regulation on cTEC. Reconstitution of thymocyte development in these mutant mice further suggests that maturation of thymocytes to the CD4+CD8+ stage and concomitant expansion are needed to promote DL4 down-regulation on cTEC. Collectively, our data support a model where thymic crosstalk quantitatively regulates the rate of Notch1-dependent thymopoiesis by controlling DL4 expression levels on cTEC.
Cutting Edge: A T-bet-Independent Role for IFN-α/β in Regulating IL-2 Secretion in Human CD4+ Central Memory T Cells
Authors: Davis Ann M. , Ramos Hilario J. , Davis Laurie S. , Farrar J. David
Published: April 2014

IL-2 is a hallmark cytokine secreted by central memory CD4+ T cells (TCM). Although naive cells rapidly secrete IL-2 in response to Ag stimulation, IL-12 inhibits IL-2 secretion in daughter cells as they differentiate into Th1 cells. In this study, we uncover a unique role for IFN-α in regulating IL-2 secretion by human TCM cells. IFN-α synergized with IL-12 to enhance a subset of cells that secreted high and sustained levels of IL-2. These IL-2-secreting cells displayed phenotypic and functional characteristics of TCM and were capable of generating IFN-γ-secreting effectors upon secondary activation. T-bet has been implicated in negatively regulating IL-2 secretion in murine T cells; however, T-bet expression did not inhibit IFN-α-dependent IL-2 secretion in human TCM cells. Thus, our results highlight a unique role for IFN-α in regulating the development of IL-2-secreting human TCM cells.
Cutting Edge: Antigen-Specific TGFβ-Induced Regulatory T Cells Suppress Th17-Mediated Autoimmune Disease
Authors: Huter Eva N. , Stummvoll Georg H. , DiPaolo Richard J. , Glass Deborah D. , Shevach Ethan M.
Published: April 2014

CD4+ T cells from the TCR transgenic TxA23 mouse recognize a peptide from the H/K-ATPase α-chain. When TxA23 CD4+ thymocytes are differentiated into Th1, Th2, and Th17 lines, all three subpopulations induced autoimmune gastritis (AIG) upon transfer into nu/nu recipients. The induction of AIG by naive T cells or by Th1 or Th2 cell lines could be prevented by the cotransfer of polyclonal Foxp3+ T regulatory cells (nTreg), whereas Th17-induced AIG was resistant to suppression. We compared the capacity of different types of Treg to suppress Th17-mediated AIG. Cotransfer of either nTreg or polyclonal TGFβ-induced Treg (iTreg) did not prevent AIG, while cotransfer of TGFβ-induced Ag-specific TxA23 iTreg completely prevented the development of disease. Ag-specific iTreg were able to suppress Th17-mediated disease when injected 6 days after the Th17 effectors. The implications of these results for the use of Treg for the cellular biotherapy of autoimmune disease are discussed.
High-Programmed Death-1 Levels on Hepatitis C Virus-Specific T Cells during Acute Infection Are Associated with Viral Persistence and Require Preservation of Cognate Antigen during Chronic Infection
Authors: Rutebemberwa Alleluiah , Ray Stuart C. , Astemborski Jacquie , Levine Jordana , Liu Lin , Dowd Kimberly A. , Clute Shalyn , Wang Changyu , Korman Alan , Sette Alessandro , Sidney John , Pardoll Drew M. , Cox Andrea L.
Published: April 2014

Hepatitis C virus (HCV) is an important human pathogen that represents a model for chronic infection given that the majority of infected individuals fail to clear the infection despite generation of virus-specific T cell responses during the period of acute infection. Although viral sequence evolution at targeted MHC class I-restricted epitopes represents one mechanism for immune escape in HCV, many targeted epitopes remain intact under circumstances of viral persistence. To explore alternative mechanisms of HCV immune evasion, we analyzed patterns of expression of a major inhibitory receptor on T cells, programmed death-1 (PD-1), from the time of initial infection and correlated these with HCV RNA levels, outcome of infection, and sequence escape within the targeted epitope. We show that the level of PD-1 expression in early HCV infection is significantly higher on HCV-specific T cells from subjects who progress to chronic HCV infection than from those who clear infection. This correlation is independent of HCV RNA levels, compatible with the notion that high PD-1 expression on HCV-specific CD8 T cells during acute infection inhibits viral clearance. Viral escape during persistent infection is associated with reduction in PD-1 levels on the surface of HCV-specific T cells, supporting the necessity of ongoing antigenic stimulation of T cells for maintenance of PD-1 expression. These results support the idea that PD-1 expression on T cells specific for nonescaped epitopes contributes to viral persistence and suggest that PD-1 blockade may alter the outcome of HCV infection.
Dendritic Cell Differentiation Signals Induce Anti-Inflammatory Properties in Human Adult Microglia
Authors: Lambert Caroline , Desbarats Julie , Arbour Nathalie , Hall Jeffery A. , Olivier Andre , Bar-Or Amit , Antel Jack P.
Published: April 2014

Microglia are resident cells of the CNS that belong to the myeloid cell lineage. In experimental models of neuroinflammation, they have limited capacity to function as APCs when compared with dendritic cells (DCs). Human peripheral blood monocytes have the plasticity to differentiate into mature DCs when exposed to GM-CSF and IL-4 followed by LPS. In this study we addressed the potential of human microglia to acquire phenotypic and functional properties of mature DCs under similar inducing conditions. Treated adult and fetal microglia became CD14low and acquired limited expression of CD209 (DC-SIGN); they remained CD1a− and CD83−, and decreased MHCII expression, suggesting that they had not achieved a complete DC phenotype. The monocyte-derived DCs efficiently promoted CD4 T cell proliferation in an allogeneic MLR, whereas differentiated adult microglia had a decreased ability to stimulate CD4 T cell proliferation compared with their untreated counterparts. Differentiated fetal microglia did support CD4 T cell proliferation, whereas untreated cells could not. Fetal and adult microglia produced significant amounts of IL-10 following differentiation but no detectable IL-12 p70, in contrast to differentiated monocytes that produced IL-12 p70. Our data indicate that neither adult nor fetal microglia acquired the full characteristic phenotype of mature stimulatory DCs when treated with DC-inducing cytokines in vitro. Moreover, such treatment, especially of adult microglia, induces functional responses that could promote an antiinflammatory environment in the CNS.