bluuebunny/crossref_metadata_2025 huggingface dataset:
BLyS and APRIL Form Biologically Active Heterotrimers That Are Expressed in Patients with Systemic Immune-Based Rheumatic Diseases
Authors: Roschke Viktor , Sosnovtseva Svetlana , Ward Christopher D. , Hong June S. , Smith Rodger , Albert Vivian , Stohl William , Baker Kevin P. , Ullrich Stephen , Nardelli Bernardetta , Hilbert David M. , Migone Thi-Sau
Published: April 2014

BLyS and APRIL are two members of the TNF superfamily that are secreted by activated myeloid cells and have costimulatory activity on B cells. BLyS and APRIL share two receptors, TACI and BCMA, whereas a third receptor, BAFF-R, specifically binds BLyS. Both BLyS and APRIL have been described as homotrimeric molecules, a feature common to members of the TNF superfamily. In this study, we show that APRIL and BLyS can form active heterotrimeric molecules when coexpressed and that circulating heterotrimers are present in serum samples from patients with systemic immune-based rheumatic diseases. These findings raise the possibility that active BLyS/APRIL heterotrimers may play a role in rheumatic and other autoimmune diseases and that other members of the TNF ligand superfamily may also form active soluble heterotrimers.
Amelioration of Collagen-Induced Arthritis by Blockade of Inducible Costimulator-B7 Homologous Protein Costimulation
Authors: Iwai Hideyuki , Kozono Yuko , Hirose Sachiko , Akiba Hisaya , Yagita Hideo , Okumura Ko , Kohsaka Hitoshi , Miyasaka Nobuyuki , Azuma Miyuki
Published: April 2014

B7 homologous protein (B7h)/B7-related protein 1 (B7RP-1) is a new member of the B7 family of costimulatory molecules that specifically interacts with inducible costimulator (ICOS) expressed on activated T cells. Collagen type II (CII)-induced arthritis (CIA) is an experimental model of arthritis that has been used to dissect the pathogenesis of human rheumatoid arthritis. In this study, we have investigated the effect of neutralizing anti-B7h mAb on the development and disease progression of CIA. Administration of anti-B7h mAb significantly ameliorated the disease as assessed by clinical arthritis score and histology in the joints, and a beneficial effect was also obtained by a delayed treatment after the onset of disease. Expression of ICOS and B7h was observed in the inflamed synovial tissue as well as in the draining lymph nodes (LNs) and expansion of ICOS+ T cells in the LN was reduced by the anti-B7h mAb treatment. Expression of mRNA for proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 in the joints was inhibited by the treatment. Proliferative responses and production of IFN-γ and IL-10 upon restimulation with CII in vitro were significantly inhibited in LN cells from the anti-B7h mAb-treated mice. Serum anti-CII IgG1, IgG2a, and IgG2b levels were also reduced. Our present results showed a beneficial effect of the B7h blockade on CIA through anti-inflammatory actions and inhibition of both Th1- and Th2-mediated immune responses, suggesting that the ICOS-B7h interaction plays an important role in the pathogenesis of CIA and thus the blockade of this pathway may be beneficial for the treatment of human rheumatoid arthritis.
Src Homology 2 Domain-Containing Inositol Polyphosphate Phosphatase Regulates NF-κB-Mediated Gene Transcription by Phagocytic FcγRs in Human Myeloid Cells
Authors: Tridandapani Susheela , Wang Yijie , Marsh Clay B. , Anderson Clark L.
Published: April 2014

FcγR-mediated phagocytosis is accompanied by the generation of tissue-damaging products such as inflammatory cytokines and reactive oxygen species. Hence, the phagocytic response must be a tightly regulated process. Recent studies have established that clustering FcγR on human myeloid cells causes tyrosine phosphorylation of Src homology 2 domain-containing inositol polyphosphate phosphatase (SHIP). However, it is not known how these immunoreceptor tyrosine-based activation motif (ITAM)-bearing phagocytic FcγR activate SHIP, or whether the activation of SHIP by ITAMs has any functional relevance. Experiments addressing the mechanism of SHIP association with ITAMs have been done in in vitro systems using phosphopeptides. In this study we undertook to dissect the molecular mechanism by which SHIP associates with the native ITAM-FcγR and becomes phosphorylated. In this report we provide evidence that first, SHIP is indeed phosphorylated by ITAM-FcγR, using cell systems that lack FcγRIIb expression; second, coimmunoprecipitation experiments demonstrate that SHIP associates with native ITAM-bearing FcγRIIa in vivo; and third, phosphorylation of SHIP by FcγRIIa is inhibited by overexpressing either the SHIP Src homology 2 domain or a dominant negative mutant of Shc. In contrast, SHIP phosphorylation was not inhibited by a dominant negative mutant of Grb2. We extend these observations to show that SHIP activation by ITAM-FcγR down-regulates NF-κB-induced gene transcription. These findings both provide a molecular mechanism for SHIP association with native ITAM-bearing receptors and demonstrate that SHIP association with ITAM-FcγR serves to regulate gene expression during the phagocytic process.
Enhancement of Complement Activation and Opsonophagocytosis by Complexes of Mannose-Binding Lectin with Mannose-Binding Lectin-Associated Serine Protease After Binding toStaphylococcus aureus
Authors: Neth Olaf , Jack Dominic L. , Johnson Marina , Klein Nigel J. , Turner Malcolm W.
Published: April 2014

Human mannose-binding lectin (MBL) is a serum protein of the innate immune system that circulates as a complex with a group of so-called MBL-associated serine proteases (MASP-1, MASP-2, and MASP-3). Complexes of MBL-MASP2 are able to activate the complement system in an Ab and C1-independent fashion after binding of the lectin to appropriate microbial sugar arrays. We have evaluated the additive effect of the lectin pathway relative to other complement activation pathways and the subsequent effect on neutrophil phagocytosis. Complement activation in the sera of MBL-deficient individuals was studied with and without the addition of exogenous MBL-MASP. Flow cytometry was used to measure the deposition of C4, factor B, C3b, and iC3b on Staphylococcus aureus. Deposition of the first cleavage product of the lectin pathway, C4b, was increased using the sera of three different MBL-deficient individuals when exogenous MBL-MASP was added. Factor B was deposited in association with C4, but there was no evidence of independent alternative pathway activation. Similar enhancement of C3b deposition was also observed, with evidence of elevated amounts of C3b processed to iC3b. The increase in opsonic C3 fragments mediated by MBL was associated with a significant increase in the uptake of organisms by neutrophils. We also observed significant increases in phagocytosis with MBL-MASPs that were independent of complement activation. We conclude that MBL-MASP makes a major contribution to complement-mediated host defense mechanisms.
Neutrophil Influx in Response to a Peritoneal Infection with Salmonella Is Delayed in Lipopolysaccharide-Binding Protein or CD14-Deficient Mice
Authors: Yang Kang K. , Dorner Brigitte G. , Merkel Ulrike , Ryffel Bernard , Schütt Christine , Golenbock Douglas , Freeman Mason W. , Jack Robert S.
Published: April 2014

The induction of an adaptive immune response to a previously unencountered pathogen is a time-consuming process and initially the infection must be held in check by the innate immune system. In the case of an i.p. infection with Salmonella typhimurium, survival requires both CD14 and LPS-binding protein (LBP) which, together with Toll-like receptor 4 and myeloid differentiation protein 2, provide a sensitive means to detect bacterial LPS. In this study, we show that in the first hours after i.p. infection with Salmonella a local inflammatory response is evident and that concomitantly neutrophils flood into the peritoneum. This rapid neutrophil influx is dependent on TNF since it is 1) abolished in TNF KO mice and 2) can be induced by i.p. injection of TNF in uninfected animals. Neutrophil influx is not strictly dependent on the presence of either LBP or CD14. However, in their absence, no local inflammatory response is evident, neutrophil migration is delayed, and the mice succumb to the infection. Using confocal microscopy, we show that the neutrophils which accumulate in CD14 and LBP null mice, albeit with delayed kinetics, are nevertheless fully capable of ingesting the bacteria. We suggest that the short delay in neutrophil influx gives the pathogen a decisive advantage in this infection model.
Comparison of the Pro-Oxidative and Proinflammatory Effects of Organic Diesel Exhaust Particle Chemicals in Bronchial Epithelial Cells and Macrophages
Authors: Li Ning , Wang Meiying , Oberley Terry D. , Sempf Joan M. , Nel Andre E.
Published: April 2014

Inhaled diesel exhaust particles (DEP) exert proinflammatory effects in the respiratory tract. This effect is related to the particle content of redox cycling chemicals and is involved in the adjuvant effects of DEP in atopic sensitization. We demonstrate that organic chemicals extracted from DEP induce oxidative stress in normal and transformed bronchial epithelial cells, leading to the expression of heme oxygenase 1, activation of the c-Jun N-terminal kinase cascade, IL-8 production, as well as induction of cytotoxicity. Among these effects, heme oxygenase 1 expression is the most sensitive marker for oxidative stress, while c-Jun N-terminal kinase activation and induction of apoptosis-necrosis require incremental amounts of the organic chemicals and increased levels of oxidative stress. While a macrophage cell line (THP-1) responded in similar fashion, epithelial cells produced more superoxide radicals and were more susceptible to cytotoxic effects than macrophages. Cytotoxicity is the result of mitochondrial damage, which manifests as ultramicroscopic changes in organelle morphology, a decrease in the mitochondrial membrane potential, superoxide production, and ATP depletion. Epithelial cells also differ from macrophages in not being protected by a thiol antioxidant, N-acetylcysteine, which effectively protects macrophages against cytotoxic DEP chemicals. These findings show that epithelial cells exhibit a hierarchical oxidative stress response that differs from that of macrophages by more rapid transition from cytoprotective to cytotoxic responses. Moreover, epithelial cells are not able to convert N-acetylcysteine to cytoprotective glutathione.
Enhanced Activity of Human IL-10 After Nitration in Reducing Human IL-1 Production by Stimulated Peripheral Blood Mononuclear Cells
Authors: Freels Jon L. , Nelson Dan K. , Hoyt Jeffrey C. , Habib Michael , Numanami Hiroki , Lantz R. Clark , Robbins Richard A.
Published: April 2014

Nitric oxide and superoxide form the unstable compound, peroxynitrite, which can nitrate proteins and compromise function of proinflammatory cytokines at sites of inflammation. Reduced function of proinflammatory proteins such as IL-8, macrophage inflammatory protein-1α, and eotaxin suggest an anti-inflammatory effect of nitration. The effects of nitration on anti-inflammatory cytokines such as IL-10 are unknown. We hypothesized that peroxynitrite would modify the function of anti-inflammatory cytokines like IL-10. To test this hypothesis, the capacity of recombinant human IL-10 to inhibit production of human IL-1β (IL-1) from LPS-stimulated human PBMC was evaluated. Human IL-10 was nitrated by incubation with peroxynitrite or by incubation with 3-morpholinosydnonimine, a peroxynitrite generator, for 2 h and then incubated with LPS-stimulated PBMC for 6 h, and IL-1 was measured in the culture supernatant fluids. Human IL-1 production was significantly lower in the peroxynitrite- or 3-morpholinosydnonimine-nitrated IL-10 group than in the IL-10 controls (p < 0.05, all comparisons). This finding demonstrates that although peroxynitrite inhibits proinflammatory cytokines, it may augment anti-inflammatory cytokines and further point to an important role for peroxynitrite in the regulation of inflammation.
House Dust Mite Allergens Induce Proinflammatory Cytokines from Respiratory Epithelial Cells: The Cysteine Protease Allergen, Der p 1, Activates Protease-Activated Receptor (PAR)-2 and Inactivates PAR-1
Authors: Asokananthan Nithiananthan , Graham Peter T. , Stewart David J. , Bakker Anthony J. , Eidne Karin A. , Thompson Philip J. , Stewart Geoffrey A.
Published: April 2014

In previous studies, we demonstrated that allergenic house dust mite proteases are potent inducers of proinflammatory cytokines from the respiratory epithelium, although the precise mechanisms involved were unclear. In this study, we investigated whether this was achieved through activation of protease-activated receptor (PAR)-1 or -2. Pretreatment of A549 respiratory epithelial cells with the clinically important cysteine protease allergen, Der p 1, ablated subsequent PAR-1, but not PAR-2 agonist peptide-induced IL-6 and IL-8 release. HeLa cells transfected with the plasmid coding for PAR-2, in contrast to PAR-1, released significant concentration of IL-6 after exposure to Der p 1. Exposure of HeLa cells transfected with either PAR-1/enhanced yellow fusion protein or PAR-2/enhanced yellow fusion protein to Der p 1 caused receptor internalization in the latter cells only, as judged by confocal microscopy with re-expression of the receptor within 120-min postenzyme exposure. Der p 1-induced cytokine release from both A549 and transfected HeLa cells was accompanied by changes in intracellular Ca2+ concentrations. Desensitization studies showed that Der p 1 pretreatment of the A549 cells resulted in the abolition of both trypsin- and PAR-2 agonist peptide-induced Ca2+ release, but not that induced by subsequent exposure to either thrombin or PAR-1 agonist peptide. These data indicate for the first time that the house dust mite allergen Der p 1-induced cytokine release from respiratory epithelial cells is, in part, mediated by activation of PAR-2, but not PAR-1.
TGF-β and IL-13 Synergistically Increase Eotaxin-1 Production in Human Airway Fibroblasts
Authors: Wenzel Sally E. , Trudeau John B. , Barnes Steve , Zhou XiuXia , Cundall Meghan , Westcott Jay Y. , McCord Kelly , Chu Hong Wei
Published: April 2014

Chronic diseases may involve an “innate” response followed by an adaptive immune response, of a Th1 or Th2 variety. Little is known regarding the interactions of these responses. We hypothesized that TGF-β1 (innate response factor associated with wound repair) in combination with IL-13 (Th2 factor) might augment inflammatory processes associated with asthma. Airway fibroblasts were cultured from asthmatic subjects and normal controls. These fibroblasts were exposed to TGF-β1 and IL-13 alone or in combination, and eotaxin-1 expression and production were evaluated. At 48 h, eotaxin-1 production was markedly increased with the combination of TGF-β1 and IL-13 (p < 0.0001) compared with either stimulus alone. mRNA increased slightly at 1 h with IL-13 or TGF-β1 plus IL13, peaked, and became significantly increased over IL-13 alone at 24 h. Protein was measurable from 6 h with IL-13 and TGF-β1 plus IL-13, but greater levels were measured over time with the combination. Actinomycin ablated the increase in mRNA and protein seen with IL-13 alone and with TGF-β1 plus IL-13. Cycloheximide blocked the increase in mRNA at 6 h in both conditions, but also blocked the increase at 24 h with TGF-β1 plus IL-13. STAT-6 was rapidly activated with both IL-13 and the combination, without difference. Finally, eotaxin-1-positive fibroblasts were identified in severe asthma biopsies in greater numbers than in normals. These results support the concept that interactions of innate and adaptive immune systems may be important in promoting the tissue eosinophilia of asthma, particularly in those with more severe disease.
A Transgenic Mouse Model of Autoimmune Glomerulonephritis and Necrotizing Arteritis Associated with Cryoglobulinemia
Authors: Kikuchi Shuichi , Pastore Yves , Fossati-Jimack Liliane , Kuroki Aki , Yoshida Haruyoshi , Fulpius Thierry , Araki Kimi , Takahashi Satoru , Lemoine Robert , Reininger Luc , Izui Shozo
Published: April 2014

Mice implanted with hybridoma secreting 6-19 IgG3 anti-IgG2a rheumatoid factor (RF) with cryoglobulin activity develop acute glomerulonephritis and cutaneous leukocytoclastic vasculitis. As the RF activity is implicated in the skin, but not glomerular lesions, it is still unclear whether the renal pathogenicity is determined by 6-19 H chains alone or their combination with L chains. To address this question, we have generated transgenic mice expressing only the H chain gene or both H and L chain genes of the 6-19 IgG3 anti-IgG2a RF and determined the development of glomerular and vascular lesions. H-single and H/L-double transgenic mice displayed comparable high amounts of IgG3 cryoglobulins, but only H/L-double transgenic mice having 10-fold higher levels of IgG3 anti-IgG2a RF progressively developed chronic, lethal glomerulonephritis. The severe glomerular lesions observed at 8–10 mo of age were very heterogeneous (membranoproliferative changes, crescents, and sclerosis); in addition, one-third of them had necrotizing arteritis in the kidneys and skeletal muscles. These renal and vascular changes were very different from those observed in the acute cryoglobulinemia, characterized by mainly “wire-loop” glomerular lesions and a cutaneous leukocytoclastic form of vasculitis. Thus, our data demonstrate the importance of a unique combination of the H and L chains for the expression of the pathogenic activity of IgG3 cryoglobulins and that a single autoantibody is able to induce different types of glomerular and vascular complications, depending on its production levels and kinetics.
Cutting Edge: Tyk2 Is Required for the Induction and Nuclear Translocation of Daxx Which Regulates IFN-α-Induced Suppression of B Lymphocyte Formation
Authors: Shimoda Kazuya , Kamesaki Kenjirou , Numata Akihiko , Aoki Kenichi , Matsuda Tadashi , Oritani Kenji , Tamiya Sadafumi , Kato Kouji , Takase Ken , Imamura Rie , Yamamoto Tetsuya , Miyamoto Toshihiro , Nagafuji Koji , Gondo Hisashi , Nagafuchi Seiho , Nakayama Kei-Ichi , Harada Mine
Published: April 2014

IFN-α inhibits B lymphocyte development, and the nuclear protein Daxx has been reported to be essential for this biological activity. We show in this study that IFN-α inhibits the clonal proliferation of B lymphocyte progenitors in response to IL-7 in wild-type, but not in tyk2-deficient, mice. In addition, the IFN-α-induced up-regulation and nuclear translocation of Daxx are completely abrogated in the absence of tyk2. Therefore, tyk2 is directly involved in IFN-α signaling for the induction and translocation of Daxx, which may result in B lymphocyte growth arrest and/or apoptosis.
TRAIL: A Mechanism of Tumor Surveillance in an Immune Privileged Site
Authors: Lee Hae-ock , Herndon John M. , Barreiro Ramon , Griffith Thomas S. , Ferguson Thomas A.
Published: April 2014

TRAIL is a recently described member of the TNF superfamily. The ability of TRAIL to induce apoptosis in a large number of tumors has stimulated interest in TRAIL as a tumor therapeutic agent. Although TRAIL mRNA is expressed in a number of tissues, its functional significance to various organs is unknown. Because tumors rarely develop in the eye, we have examined this organ for functional TRAIL expression. Our analysis revealed that TRAIL mRNA and protein are constitutively expressed on numerous ocular structures, including the cornea and retina. More importantly, ocular tissue displays functional TRAIL as determined by in vitro killing of TRAIL-sensitive tumor cell lines. Previous studies have shown that ocular tissue also expresses functional Fas ligand (FasL). To assess the contribution of TRAIL and FasL for tumor cell killing in the eye, cell lines susceptible to both TRAIL and FasL were examined. The results show that ocular tissue kills via either ligand, suggesting a compensatory mechanism between TRAIL and FasL. Collectively, these results provide physiological evidence for ocular TRAIL expression, and suggest a role for this molecule in tumor surveillance in an immune privileged site.
Histidine-Rich Glycoprotein Binds to DNA and FcγRI and Potentiates the Ingestion of Apoptotic Cells by Macrophages
Authors: Gorgani Nick N. , Smith Brian A. , Kono Dwight H. , Theofilopoulos Argyrios N.
Published: April 2014

Histidine-rich glycoprotein (HRG) is an abundant serum protein that exhibits many functions in diverse biological systems. In this study, we show that HRG potentiates the ingestion of apoptotic cells by mature human monocyte-derived macrophages (HMDM). HRG bound specifically to apoptotic Jurkat cells and mature HMDM in a saturable and concentration-dependent manner. Purified HRG or HRG in sera increased the number of HMDM-containing apoptotic cells and accelerated the ingestion, while neutralization or depletion of HRG from sera reduced this effect. Anti-FcγRI mAb inhibited HRG binding to HMDM, while DNA, but not chromatin, inhibited HRG binding to apoptotic cells, and either anti-FcγRI or DNA abrogated the HRG-dependent ingestion. The findings indicate that HRG, by acting as a bridge between DNA on apoptotic cells and FcγRI on HMDM, is a key physiological mediator of apoptotic cell clearance by macrophages.
High-Affinity HLA-A(*)02.01 Peptides from Parathyroid Hormone-Related Protein Generate In Vitro and In Vivo Antitumor CTL Response Without Autoimmune Side Effects
Authors: Francini Guido , Scardino Antonio , Kosmatopoulos Kostas , Lemonnier François A. , Campoccia Giuseppe , Sabatino Marianna , Pozzessere Daniele , Petrioli Roberto , Lozzi Luisa , Neri Paolo , Fanetti Giuseppe , Cusi Maria Grazia , Correale Pierpaolo
Published: April 2014

Parathyroid hormone-related protein (PTH-rP), a protein produced by prostate carcinoma and other epithelial cancers, is a key agent in the development of bone metastases. We investigated whether the protein follows the self-tolerance paradigm or can be used as a target Ag for anticancer immunotherapy by investigating the immunogenicity of two HLA-A(*)02.01-binding PTH-rP-derived peptides (PTR-2 and -4) with different affinity qualities. PTH-rP peptide-specific CTL lines were generated from the PBMC of two HLA-A(*)02.01+ healthy individuals, stimulated in vitro with PTH-rP peptide-loaded autologous dendritic cells and IL-2. The peptide-specific CTLs were able to kill PTH-rP+HLA-A(*)02.01+ breast and prostate carcinoma cell lines. The two peptides were also able to elicit a strong antitumor PTH-rP-specific CTL response in HLA-A(*)02.01 (HHD) transgenic mice. The vaccinated mice did not show any sign of side effects due to cell-mediated autoimmunity or toxicity. In this study we describe two immunogenic and toxic-free PTH-rP peptides as valid candidates for the design of peptide-based vaccination strategies against prostate cancer and bone metastases from the most common epithelial malignancies.
Reduction of Antiviral CD8 Lymphocytes In Vivo with Dendritic Cells Expressing Fas Ligand—Increased Survival of Viral (Lymphocytic Choriomeningitis Virus) Central Nervous System Infection
Authors: Wolfe Tom , Asseman Chrystelle , Hughes Anna , Matsue Hiroyuki , Takashima Akira , von Herrath Matthias G.
Published: April 2014

In vivo administration of APC expressing Fas ligand (Fas-L+ dendritic cells (DCs)) has shown promise in dampening allergic reactions and transplant rejection. Since the effect in these studies was mainly on CD4 lymphocytes, our goal was to evaluate the ability of such killer DCs to eliminate antiviral CD8 lymphocytes and in this way ameliorate viral immunopathology or, conversely, impede viral clearance. Intravenous administration of Fas-L+ DCs resulted in a 50% reduction of lytic CD8 precursors following intracerebral infection with lymphocytic choriomeningitis virus (LCMV), and accordingly, immunopathology and survival of LCMV meningitis were improved, whereas viral clearance remained unaffected. In transfer studies the effect of the Fas-L+ DCs was only quantifiable on experienced, not naive, CD8 lymphocytes. Importantly, loading of Fas-L+ DCs with viral Ag before therapy was not necessary to achieve this effect, indicating that non-LCMV-infected Fas-L+ DCs acquired viral Ag during acute LCMV infection in vivo. Our studies delineate important aspects for the clinical use of Fas-L+ DCs in vivo. One should expect that they acquire viral Ags and suppress antiviral CD8 responses to some degree when given while an acute infection is ongoing. In terms of safety it is encouraging that resolution of the infection, at least in the case of LCMV, is not inhibited.
Systemic Administration of IL-15 Augments the Antigen-Specific Primary CD8+ T Cell Response Following Vaccination with Peptide-Pulsed Dendritic Cells
Authors: Rubinstein Mark P. , Kadima Andre N. , Salem Mohamed L. , Nguyen Christophe L. , Gillanders William E. , Cole David J.
Published: April 2014

The systemic administration of IL-2 can act as a potent adjuvant for T cell-directed vaccine strategies. However, not only is the administration of IL-2 potentially toxic, but recent evidence suggests that it may also paradoxically limit the duration and magnitude of the cytotoxic T cell response. A recently identified cytokine, IL-15, shares many properties with IL-2 and may provide a preferential means of augmenting T cell-directed vaccine responses. Although well characterized in vitro, there are few data on the ability of IL-15 to augment T cell-mediated responses in vivo. We therefore evaluated the ability of systemic IL-15 to function as a T cell adjuvant in a murine vaccine model. To establish a population of easily identifiable Ag-responsive T cells, naive CD8+ (OT-1) T cells were first adoptively transferred into mice. Vaccination with peptide-pulsed dendritic cells induced a modest expansion of OT-1 T cells. The addition of systemic IL-15 for 7 days following vaccination resulted in a significant increase in the expansion of responding T cells in the PBL, spleen, and lymph nodes. Importantly, the responding T cells were cytotoxic and maintained a Tc1-biased phenotype. We did not observe either enhanced resistance to activation-induced cell death or preferential generation of memory T cells as a result of treatment with IL-15 compared with IL-2. These studies show for the first time that IL-15 is capable of augmenting the primary CD8+ T cell response to vaccination and contribute to the basis for future experiments exploring the clinical role of IL-15.
Quantitation of CD8+ T Cell Expansion, Memory, and Protective Immunity After Immunization with Peptide-Coated Dendritic Cells
Authors: Hamilton Sara E. , Harty John T.
Published: April 2014

Dendritic cells (DCs) are potent APCs for naive CD8+ T cells and are being investigated as vaccine delivery vehicles. In this study, we examine the CD8+ T cell response to defined peptides from Listeria monocytogenes (LM), lymphocytic choriomeningitis virus, and murine CMV coated singly and in combination onto mature bone marrow-derived DCs (BMDCs). We show that immunization of mice with 2 × 105 mature BMDCs coated with multiple MHC class I peptides generates a significant Ag-specific CD8+ T cell response in both the spleen and nonlymphoid organs. This immunization resulted in a peptide-specific hierarchy in the magnitude of CD8+ T cell priming and noncoordinate kinetics in response to different peptide epitopes. Kinetics were not exclusively due to specific characteristics of the MHC class I molecule, and were not altered in an Ag-independent manner by concurrent LM infection. Mice immunized with listeriolysin O 91–99-coated BMDCs are protected against high dose challenge with virulent LM. This protection was enhanced by diversifying the memory CD8+ T cell compartment, even in the absence of a large increase in Ag-specific CD8+ memory T cells.
Long-Term Maintenance of Virus-Specific Effector Memory CD8+ T Cells in the Lung Airways Depends on Proliferation
Authors: Hogan Robert J. , Cauley Linda S. , Ely Kenneth H. , Cookenham Tres , Roberts Alan D. , Brennan Jean W. , Monard Simon , Woodland David L.
Published: April 2014

Recent studies have shown that virus-specific effector memory T cells can be recovered from the lung airways long after clearance of a respiratory virus infection. These cells are thought to play an important role in the recall response to secondary viral infection. It is currently unclear whether these cells actually persist at this site or are maintained by continual proliferation and recruitment. In this study, we have analyzed the mechanisms underlying the persistence of memory CD8+ T cells in the lung airway lumina following recovery from a respiratory virus infection. The data identify two distinct populations of memory cells. First, a large population Ag-specific CD8+ T cells is deposited in the airways during the acute response to the virus. These cells persist in a functional state for several weeks with minimal further division. Second, a smaller population of Ag-specific CD8+ T cells is maintained in the lung airways by homeostatic proliferation and migration to lung airways after viral clearance. This rate of proliferation is identical to that observed in the spleen, suggesting that these cells may be recent immigrants from the lymphoid organs. These data have significant implications for vaccines designed to promote cellular immunity at mucosal sites such as the lung.
The Immunosuppressive Agent 15-Deoxyspergualin Functions by Inhibiting Cell Cycle Progression and Cytokine Production Following Naive T Cell Activation
Authors: Holcombe Hilda , Mellman Ira , Janeway Charles A. , Bottomly Kim , Dittel Bonnie N.
Published: April 2014

Immunosuppressive agents are commonly used in the prevention of graft rejection following transplantation and in the treatment of autoimmunity. In this study, we examined the immunosuppressive mechanism of the drug 15-deoxyspergualin (DSG), which has shown efficacy in the enhancement of graft survival and in the treatment of autoimmunity. Using a murine model of chronic relapsing and remitting experimental autoimmune encephalomyelitis, we were able to demonstrate that DSG both delayed and reduced the severity of experimental autoimmune encephalomyelitis. Subsequent in vitro studies to examine the mechanism of immune suppression showed that DSG was not able to inhibit early activation of naive CD4 T cells, but DSG did effectively inhibit the growth of naive CD4 T cells after activation. An analysis of cell proliferation and cell cycle showed that DSG treatment led to a block in cell cycle progression 2–3 days following Ag stimulation. In addition, DSG treatment inhibited the production of IFN-γ by Th1 effector T cells. These studies suggest that CD4 T cells are a predominant target for DSG and the immunosuppressive effects of the drug may result from reduced CD4 T cell expansion and decreased polarization into IFN-γ-secreting Th1 effector T cells in the induction of certain autoimmune disorders.
Murine Neonatal CD4+ Lymph Node Cells Are Highly Deficient in the Development of Antigen-Specific Th1 Function in Adoptive Adult Hosts
Authors: Adkins Becky , Bu Yurong , Guevara Patricia
Published: April 2014

It is well established that murine neonates are biased toward Th2 responses. Th2-dominant responses are observed following immunization with a variety of Ags, using different carrier/adjuvant systems, and are seen in both BALB/c and C57BL/6 mice. Therefore, Th2 skewing appears to be a universal phenomenon unique to the neonatal period. One important question about this phenomenon is whether these responses are due to T cell intrinsic properties or are regulated by the neonatal environment. Here we have addressed this issue by transferring neonatal or adult CD4+ lymph node cells to adoptive adult recombinase-activating gene 2−/− hosts and studied the development of Th responses. Neonatal CD4+ cells were highly deficient in the development of both primary and secondary Ag-specific Th1 responses. This did not appear to be due to anergy of a developed population, since exogenous IL-2 only marginally increased production of the Th1 cytokine IFN-γ. This profound Th1 deficiency was observed despite similar proliferation by neonatal and adult cells within the recombinase-activating gene 2−/− hosts. Moreover, neonatal CD4+ cells up-regulated activation markers in a manner similar to adult CD4+ cells. Therefore, although their proliferation and phenotypic maturation proceeded normally, neonatal CD4+ cells appeared to be intrinsically deficient in the functional maturation of Th1 lineage cells. These results offer a candidate explanation for the reduced graft-vs-host responses observed following transplantation of cord blood cells or murine neonatal lymphoid cells to allogeneic adult hosts.
Age-Dependent Defects in TCR-Triggered Cytoskeletal Rearrangement in CD4+ T Cells
Authors: Garcia Gonzalo G. , Miller Richard A.
Published: April 2014

Previous research has shown that many of the CD4 T cells from older mice do not form functional immune synapses after conjugation with peptide-pulsed APC. We now show that the defect lies at a very early stage in the cytoskeletal reorganization that precedes movement of protein kinases and their substrates to the TCR/APC interface. Antagonist peptides presented to T cells from young mice induce migration of talin (but not paxillin, vinculin, or F-actin) to the APC contact zone, but CD4 T cells from older donors typically fail to show the talin polarization response. A spreading assay in which contact with anti-CD3-coated slides induces CD4 T cells to assume a conical shape and develop lammelopodia also shows a decline with age in the proportion of T cells that can initiate cytoskeletal changes in response to this simplified stimulus. Finally, the transition from detergent-soluble to cytoskeletal forms of the p16, p21, and p23 isoforms of CD3ζ in response to CD3/CD4/CD28 cross-linking is much stronger in young than in old T cells. Thus, defects in cytoskeletal reorganization triggered by initial contact between TCR and peptide-bearing APC precede, and presumably contribute to, defective activation of protein kinase-mediated signals in the first few minutes of the activation cascade in T cells from aged mice.
Transcriptional Regulation of the MHC Class II Trans-Activator (CIITA) Promoter III: Identification of a Novel Regulatory Region in the 5′-Untranslated Region and an Important Role for cAMP-Responsive Element Binding Protein 1 and Activating Transcription Factor-1 in CIITA-Promoter III Transcriptional Activation in B Lymphocytes
Authors: van der Stoep Nienke , Quinten Edwin , van den Elsen Peter J.
Published: April 2014

The class II trans-activator (CIITA), which acts as a master regulator for expression of MHC class II genes, is expressed constitutively in mature B cells. This constitutive expression of CIITA is driven by CIITA promoter III (CIITA-PIII). However, little is known about the factors that control the B cell-mediated trans-activation of CIITA-PIII. In this study using B cells we have identified several cAMP-responsive elements (CREs) in the proximal promoter and in the 5′-untranslated region (5′-UTR) that are involved in the activation of CIITA-PIII. We show that activating transcription factor (ATF)/CRE binding protein (CREB) factors bind to the CREs in vitro and in vivo. Notably, our results also reveal that the 5′-UTR of CIITA-PIII functions as an important regulatory region in B lymphocytes. Furthermore, transient cotransfections of a CIITA-PIII luciferase reporter construct with either CREB-1 or ATF-1 boost CIITA-PIII trans-activation in a dose-dependent manner, which was further enhanced by addition of general coactivator CREB-binding protein. Transient transfections using mutant CIITA-PIII luciferase reporter constructs that either lack the (5′-UTR) or abolish binding of CREB-1 and ATF-1 to the CRE located in activation response element-2, displayed severely reduced promoter activity in B cells. A similar successive deletion of the CREs resulted in a subsequent reduction of CREB-1-induced activity of CIITA-PIII in B cells. Together our results argue for an important role of ATF/CREB factors and the 5′-UTR of CIITA-PIII in the trans-activation of CIITA-PIII in B cells.
Structural Factors Contributing to DM Susceptibility of MHC Class II/Peptide Complexes
Authors: Belmares Michael P. , Busch Robert , Wucherpfennig Kai W. , McConnell Harden M. , Mellins Elizabeth D.
Published: April 2014

Peptide loading of MHC class II (MHCII) molecules is assisted by HLA-DM, which releases invariant chain peptides from newly synthesized MHCII and edits the peptide repertoire. Determinants of susceptibility of peptide/MHCII complexes to DM remain controversial, however. Here we have measured peptide dissociation in the presence and the absence of DM for 36 different complexes of varying intrinsic stability. We found large variations in DM susceptibility for different complexes using either soluble or full-length HLA-DM. The DM effect was significantly less for unstable complexes than for stable ones, although this correlation was modest. Peptide sequence- and allele-dependent interactions along the entire length of the Ag binding groove influenced DM susceptibility. We also observed differences in DM susceptibility during peptide association. Thus, the peptide repertoire displayed to CD4+ T cells is the result of a mechanistically complicated editing process and cannot be simply predicted from the intrinsic stability of the complexes in the absence of DM.
Killer Ig-Like Receptor Haplotype Analysis by Gene Content: Evidence for Genomic Diversity with a Minimum of Six Basic Framework Haplotypes, Each with Multiple Subsets
Authors: Hsu Katharine C. , Liu Xiao-Rong , Selvakumar Annamalai , Mickelson Eric , O’Reilly Richard J. , Dupont Bo
Published: April 2014

Killer Ig-like receptor (KIR) genes constitute a multigene family whose genomic diversity is achieved through differences in gene content and allelic polymorphism. KIR haplotypes containing a single activating KIR gene (A-haplotypes), and KIR haplotypes with multiple activating receptor genes (B-haplotypes) have been described. We report the evaluation of KIR gene content in extended families, sibling pairs, and an unrelated Caucasian panel through identification of the presence or absence of 14 KIR genes and 2 pseudogenes. Haplotype definition included subtyping for the expressed and nonexpressed KIR2DL5 variants, for two alleles of pseudogene 3DP1, and for two alleles of 2DS4, including a novel 2DS4 allele, KIR1D. KIR1D appears functionally homologous to the rhesus monkey KIR1D and likely arose as a consequence of a 22 nucleotide deletion in the coding sequence of 2DS4, leading to disruption of Ig-domain 2D and a premature termination codon following the first amino acid in the putative transmembrane domain. Our investigations identified 11 haplotypes within 12 families. From 49 sibling pairs and 17 consanguineous DNA samples, an additional 12 haplotypes were predicted. Our studies support a model for KIR haplotype diversity based on six basic gene compositions. We suggest that the centromeric half of the KIR genomic region is comprised of three major combinations, while the telomeric half can assume a short form with either 2DS4 or KIR1D or a long form with multiple combinations of several stimulatory KIR genes. Additional rare haplotypes can be identified, and may have arisen by gene duplication, intergenic recombination, or deletions.
Ly-6 Superfamily Members Ly-6A/E, Ly-6C, and Ly-6I Recognize Two Potential Ligands Expressed by B Lymphocytes
Authors: Pflugh David L. , Maher Stephen E. , Bothwell Alfred L. M.
Published: April 2014

Most hemopoietic cells express one or more members of the Ly-6 supergene family of small glycosylphosphatidylinositol-linked proteins. Although levels of Ly-6 proteins vary with stages of differentiation and activation, their function largely remains unknown. To ascertain whether ligands for Ly-6 proteins exist, chimeric proteins were constructed in which Ly-6E, Ly-6C, and Ly-6I were fused to the murine IgM heavy chain. These chimeras specifically stained both developing and mature B lymphocytes, as assessed by flow cytometry. Analysis of variants of the CH27 B cell lymphoma revealed that Ly-6A/E and Ly-6I recognized different molecules. CH27 cells with low levels of Ly-6A/E ligand activity also lost expression of CD22, and cells transfected with CD22 gained the ability to bind the Ly-6A/E chimera and, to a lesser extent, the Ly-6C and Ly-6I chimeric proteins. As many mature B cells coexpress Ly-6A/E and CD22, the function of Ly-6 molecules may be to associate with other membrane proteins, possibly concentrating these ligands in lipid rafts, rather than acting directly as cell:cell adhesion molecules.
C-Terminal Cysteine Residues Determine the IgE Binding ofAspergillus fumigatusAllergen Asp f 2
Authors: Banerjee Banani , Kurup Viswanath P. , Greenberger Paul A. , Kelly Kevin J. , Fink Jordan N.
Published: April 2014

The knowledge of the structure function relationship of the allergen is essential to design allergenic variants with reduced IgE binding capacity but intact T cell reactivity. Asp f 2 is a major allergen from the fungus Aspergillus fumigatus and >90% of A. fumigatus-sensitized individuals displayed IgE binding to Asp f 2. In the present study, we evaluated the involvement of C-terminal cysteine residues in IgE binding conformation of Asp f 2. The deletion mutants were constructed by adding three C-terminal cysteines of the native Asp f 2 one at a time to the non-IgE binding Asp f 2 (68–203). The point mutants of Asp f 2 (68–268) with C204A and C257A substitutions were constructed to study the role of C-terminal cysteines in IgE binding. Immunological evaluation of reduced and alkylated Asp f 2 and its mutants were conducted to determine the contribution of free sulfhydryl groups as well as the disulfide bonds in allergen Ab interaction. Four-fold increase in IgE Ab binding of Asp f 2 (68–267) compared with Asp f 2 (68–266) and complete loss in IgE binding of C204A mutant of Asp f 2 (68–268) indicate the involvement of C204 and C267 in IgE binding conformation of Asp f 2. A significant reduction in IgE binding of wild and mutated Asp f 2 after reduction and alkylation emphasizes the importance of cysteine disulfide bonds in epitope Ab interaction. The hypoallergenic variants may be explored further to develop safe immunotherapeutic strategy for allergic disorders.
The Structure of HLA-B8 Complexed to an Immunodominant Viral Determinant: Peptide-Induced Conformational Changes and a Mode of MHC Class I Dimerization
Authors: Kjer-Nielsen Lars , Clements Craig S. , Brooks Andrew G. , Purcell Anthony W. , Fontes Marcos R. , McCluskey James , Rossjohn Jamie
Published: April 2014

EBV is a ubiquitous human pathogen that chronically infects up to 90% of the population. Persistent viral infection is characterized by latency and periods of viral replication that are kept in check by a strong antiviral CTL response. Despite the size of the EBV genome, CTL immunity focuses on only a few viral determinants but expands a large primary and memory response toward these epitopes. In unrelated HLA-B8+ individuals, the response to the immunodominant latent Ag FLRGRAYGL from Epstein Barr nuclear Ag 3A is largely comprised of CTL clones with identical conserved αβ TCR structures. To better understand the structural correlates of Ag immunodominance and TCR selection bias, we have solved the crystal structure of the HLA-B8-FLRGRAYGL peptide complex to a resolution of 1.9 Å. The structure confirms the importance of P3-Arg, P5-Arg, and P9-Leu as dominant anchor residues involved in peptide binding to HLA-B8. A bulged conformation of the bound peptide provides a structural basis for the critical role of the P7-Tyr residue in T cell recognition. The peptide also induces backbone and side-chain conformational changes in HLA-B8 that are transmitted along the peptide-binding groove in a domino effect. The HLA-B8-FLRGRAYGL complex crystallizes as a dimer in the asymmetric unit and is oriented such that both peptide ligands are projected in the same plane suggesting a higher order arrangement of MHC-peptide complexes that could be involved in formation of the class I Ag-loading complex or in T cell activation.
Increasing the Affinity of a Human IgG1 for the Neonatal Fc Receptor: Biological Consequences
Authors: Acqua William F. Dall’ , Woods Robert M. , Ward E. Sally , Palaszynski Susan R. , Patel Nita K. , Brewah Yambasu A. , Wu Herren , Kiener Peter A. , Langermann Solomon
Published: April 2014

Many biological functions, including control of the homeostasis and maternofetal transfer of serum γ-globulins, are mediated by the MHC class I-related neonatal FcR (FcRn). A correlation exists in mice between the binding affinity of IgG1/Fc fragments to FcRn at pH 6.0 and their serum t1/2. To expand this observation, phage display of mutagenized Fc fragments derived from a human IgG1 was used to increase their affinity to both murine and human FcRn. Ten variants were identified that have a higher affinity toward murine and human FcRn at pH 6.0, with ΔΔG (ΔGwild type − ΔGmutant) from 1.0 to 2.0 kcal/mol and from 0.6 to 2.4 kcal/mol, respectively. Those variants exhibit a parallel increase in binding at pH 7.4 to murine, but not human, FcRn. Although not degraded in blood in vitro, accumulated in tissues, nor excreted in urine, their serum concentration in mice is decreased. We propose that higher affinity to FcRn at pH 7.4 adversely affects release into the serum and offsets the benefit of the enhanced binding at pH 6.0.
Critical Role of Mitochondrial Damage in Determining Outcome of Macrophage Infection with Mycobacterium tuberculosis
Authors: Duan Lei , Gan Huixian , Golan David E. , Remold Heinz G.
Published: April 2014

Human macrophages (Mφ) respond to Mycobacterium tuberculosis (Mtb) infection by undergoing apoptosis, a cornerstone of effective antimycobacterial host defense. Virulent mycobacteria override this reaction by inducing necrosis leading to uncontrolled Mtb replication. Accordingly, Mφ death induced by inoculation with Mtb had the characteristics of apoptosis and necrosis and correlated with moderate increase of mitochondrial permeability transition (MPT), mitochondrial cytochrome c release, and caspase-9 and -3 activation. We hypothesized that changes in intramitochondrial Ca2+ concentration ([Ca2+]m) determine whether Mφ undergo either apoptosis or necrosis. Therefore, we induced mechanism(s) leading to predominant apoptosis or necrosis by modulating [Ca2+]m and examined their physiological consequences. Adding calcium ionophore A23187 to Mφ inoculated with Mtb further increased calcium flux into the cells which is thought to lead to increased [Ca2+]m, blocked necrosis, stabilized MPT, decreased mitochondrial cytochrome c release, lowered caspase activation, and accompanied effective antimycobacterial activity. In contrast, Mφ infected with Mtb in presence of the mitochondrial calcium uniporter inhibitor ruthenium red showed increased mitochondrial swelling and cytochrome c release and decreased MPT and antimycobacterial activity. Thus, in Mtb-infected Mφ, high levels of mitochondrial membrane integrity, low levels of caspase activation, and diminished mitochondrial cytochrome c release are hallmarks of apoptosis and effective antimycobacterial activity. In contrast, breakdown of mitochondrial membrane integrity and increased caspase activation are characteristic of necrosis and uncontrolled Mtb replication.