bluuebunny/crossref_metadata_2025 huggingface dataset:
Bacterial Superantigens Induce Down-Modulation of CC Chemokine Responsiveness in Human Monocytes Via an Alternative Chemokine Ligand-Independent Mechanism
Authors: Rahimpour Rahbar , Mitchell Gordon , Khandaker Masud H. , Kong Chen , Singh Bhagirath , Xu Luoling , Ochi Atsuo , Feldman Ross D. , Pickering J. Geoffrey , Gill Bruce M. , Kelvin David J.
Published: January 2023

Staphylococcal superantigens (SAgs) are very potent T cell mitogens, but they can also activate monocytes by binding directly to MHC class II molecules in a manner independent of TCR coengagement. Induction of proinflammatory cytokines and chemokine expression in monocytes by superantigens has recently been reported. Here we report that superantigen stimulation of human peripheral blood monocytes results in a rapid, dose-dependent, and specific down-regulation of chemokine (macrophage inflammatory protein-1α (MIP-1α), monocyte chemotactic protein-1 and MIP-1β) binding sites (e.g., CCR1, CCR2, and CCR5), which correlates with a concomitant hyporesponsiveness of human monocytes to these CC chemokine ligands. This down-regulation occurs 15–30 min following superantigen stimulation and is specific to chemokine receptors, in that binding and responsiveness of monocytes to the chemoattractant formyl-tripeptide FMLP are not affected. We further demonstrate that SAg-induced down-modulation of chemokine binding and monocyte hyporesponsiveness to the chemokines MIP-1α, monocyte chemotactic protein-1, and MIP-1β is mediated through cellular protein tyrosine kinases, and the down-modulation can be mimicked by an MHC class II-specific mAb. Additionally, our observations indicate that SAg-induced loss of chemokine binding and monocyte responsiveness is probably mediated by secreted serine proteinases. Bacterial SAg-induced down-modulation of chemokine responsiveness represents a previously unrecognized strategy by some bacteria to subvert immune responses by affecting the intricate balance between chemokine and chemokine receptor expression and function.
Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide Inhibit Endotoxin-Induced TNF-α Production by Macrophages: In Vitro and In Vivo Studies
Authors: Delgado Mario , Pozo David , Martinez Carmen , Leceta Javier , Calvo Juan Ramon , Ganea Doina , Gomariz Rosa P.
Published: January 2023

Vasoactive intestinal peptide (VIP) is a neuropeptide synthesized by immune cells that can modulate several immune aspects, including the function of cells involved in the inflammatory response, such as macrophages and monocytes. The production and release of cytokines by activated phagocytes are important events in the pathogenesis of ischemia-reperfusion injury. There is abundant evidence that the proinflammatory cytokine TNF-α is an important mediator of shock and organ failure complicating Gram-negative sepsis. VIP has been shown to attenuate the deleterious consequences of this pathologic phenomenon. In this study we have investigated the effects of VIP and the structurally related neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP38) on the production of TNF-α by endotoxin-activated murine peritoneal macrophages. Both neuropeptides rapidly and specifically inhibit the LPS-stimulated production of TNF-α, exerting their action through the binding to VPAC1 receptor and the subsequent activation of the adenylate cyclase system. VIP and PACAP regulate the production of TNF-α at a transcriptional level. In vitro results were correlated with an inhibition of both TNF-α expression and release in endotoxemic mice in vivo. The immunomodulatory role of VIP in vivo is supported by the up-regulation of VIP release in serum and peritoneal fluid by LPS and proinflammatory cytokines such as TNF-α, IL-1β, and IL-6. These findings support the idea that under toxicity conditions associated with high LPS doses, VIP and PACAP could act as protective mediators that regulate the excessive release of TNF-α to reduce inflammation or shock.
Immunostimulatory CpG-Oligodeoxynucleotides Cause Extramedullary Murine Hemopoiesis
Authors: Sparwasser Tim , Hültner Lothar , Koch Eva Sophie , Luz Arne , Lipford Grayson B. , Wagner Hermann
Published: January 2023

Bacterial DNA and the synthetic CpG-oligodeoxynucleotides (ODNs) derived thereof have attracted attention because they activate cells of the adaptive immune system (lymphocytes) and the innate immune system (APCs) in a sequence-dependent manner. Here, we addressed whether CpG-ODNs affect hemopoiesis. Challenging mice with immunostimulatory CpG-ODN sequences led to transient splenomegaly, with a maximum increase of spleen weight at day 6. The induction of splenomegaly by CpG-ODNs was sequence-specific, dose-dependent, and associated with an increase in splenic cell count, in numbers of granulocyte-macrophage CFUs (GM-CFUs), and early erythroid progenitors (burst-forming units-erythroid). The transfer of spleen cells from CpG-ODN-pretreated animals into lethally irradiated syngeneic mice yielded an increase of spleen CFUs. Furthermore, the challenge of sublethally irradiated mice with CpG-ODNs caused radioprotective effects, in that recovery of GM-CFUs and cytotoxic T cell function was enhanced. The increase in GM-CFU and CTL function correlated with an enhanced resistance to Listeria infection in irradiated mice. We conclude from these data that CpG-ODNs trigger extramedullary hemopoiesis, and that this finding could be of therapeutic relevance in myelosuppression.
A Conformation-Dependent Epitope in Addison’s Disease and Other Endocrinological Autoimmune Diseases Maps to a Carboxyl-Terminal Functional Domain of Human Steroid 21-Hydroxylase
Authors: Nikoshkov Andrej , Falorni Alberto , Lajic Svetlana , Laureti Stefano , Wedell Anna , Lernmark Åke , Luthman Holger
Published: January 2023

Idiopathic Addison’s disease develops as a consequence of autoimmune destruction of steroid-producing cells in the adrenal gland. A major autoantigen is 21-hydroxylase (21OH; P450c21), which is involved in the biosynthesis of cortisol and aldosterone in the adrenal cortex. We selected a number of functionally important 21OH amino acid substitutions, found in patients with congenital adrenal hyperplasia, to study their effects on the binding of 21OH autoantibodies (21OHAb) to 21OH. The ability of 21OHAb to bind in vitro transcribed and translated wild-type 21OH and five different 21OH mutant proteins was quantified by liquid-phase assays. Sera from 21OHAb-positive patients with idiopathic Addison’s disease (n = 24), Graves’ disease (n = 3), and insulin-dependent diabetes mellitus (n = 1) were used. While the P105L, delE196, and G291S mutations had no effect on autoantibody binding, the P453S mutation had a considerable effect, and the R483P mutation almost completely abolished binding. Synthetic peptides corresponding to linear epitopes defined by amino acids 447–461 and 477–491 were unable to compete with wild-type 21OH for binding to autoantibodies. Direct 21OH DNA sequencing could not reveal any specific genetic variation in alleles found in 21OHAb-positive patients. We conclude that the region involving R483 plays a key role in the formation of a three-dimensional epitope in a functionally important C-terminal domain of the enzyme.
Cutting Edge: Negative Selection of Immature Thymocytes by a Few Peptide-MHC Complexes: Differential Sensitivity of Immature and Mature T Cells
Authors: Peterson Daniel A. , DiPaolo Richard J. , Kanagawa Osami , Unanue Emil R.
Published: January 2023

We quantitated the number of peptide-class II MHC complexes required to affect the deletion or activation of 3A9 TCR transgenic thymocytes. Deletion of immature double positive thymocytes was very sensitive, taking place with approximately three peptide-MHC complexes per APC. However, the activation of mature CD4+ thymocytes required 100-fold more complexes per APC. Therefore, a “biochemical margin of safety” exists at the level of the APC. To be activated, autoreactive T cells in peripheral lymphoid tissues require a relatively high level of peptide-MHC complexes.
Tetracycline Up-Regulates COX-2 Expression and Prostaglandin E2 Production Independent of Its Effect on Nitric Oxide
Authors: Attur Mukundan G. , Patel Rajesh N. , Patel Prakash D. , Abramson Steven B. , Amin Ashok R.
Published: January 2023

Tetracyclines (doxycycline and minocycline) augmented (one- to twofold) the PGE2 production in human osteoarthritis-affected cartilage (in the presence or absence of cytokines and endotoxin) in ex vivo conditions. Similarly, bovine chondrocytes stimulated with LPS showed (one- to fivefold) an increase in PGE2 accumulation in the presence of doxycycline. This effect was observed at drug concentrations that did not affect nitric oxide (NO) production. In murine macrophages (RAW 264.7) stimulated with LPS, tetracyclines inhibited NO release and increased PGE2 production. Tetracycline(s) and l-N-monomethylarginine (l-NMMA) (NO synthase inhibitor) showed an additive effect on inhibition of NO and PGE2 accumulation, thereby uncoupling the effects of tetracyclines on NO and PGE2 production. The enhancement of PGE2 production in RAW 264.7 cells by tetracyclines was accompanied by the accumulation of both cyclooxygenase (COX)-2 mRNA and cytosolic COX-2 protein. In contrast to tetracyclines, l-NMMA at low concentrations (≤100 μM) inhibited the spontaneous release of NO in osteoarthritis-affected explants and LPS-stimulated macrophages but had no significant effect on the PGE2 production. At higher concentrations, l-NMMA (500 μM) inhibited NO release but augmented PGE2 production. This study indicates a novel mechanism of action of tetracyclines to augment the expression of COX-2 and PGE2 production, an effect that is independent of endogenous concentration of NO.
Early Membrane Rupture Events During Neutrophil-Mediated Antibody-Dependent Tumor Cell Cytolysis
Authors: Kindzelskii Andrei L. , Petty Howard R.
Published: January 2023

Although cell-mediated cytolysis is a fundamental immune effector response, its mechanism remains poorly understood at the cellular level. In this report, we image for the first time transient ruptures, as inferred by cytoplasmic marker release, in tumor cell membranes during Ab-dependent cellular cytolysis. The cytosol of IgG-opsonized YAC tumor cells was labeled with tetramethylrhodamine diacetate followed by the formation of tumor cell-neutrophil conjugates. We hypothesized that tumor cell cytolysis proceeds via a series of discrete membrane rupture/resealing events that contribute to marker release. To test this hypothesis, we occluded the fluorescence image of the labeled tumor cells by passing an opaque disk into a field-conjugated plane between the light source and the sample. Multiple small bursts of fluorescent label release from tumor cells could be detected using a photomultiplier tube. Similarly, multiple fluorescent plumes were observed at various sites around the perimeter of a target. These findings support a multihit model of target cytolysis and suggest that cytolytic release is not focused at specific sites. Cytolytic bursts were generally observed at 20-s intervals, which match the previously described reduced nicotinamide-adenine dinucleotide phosphate and superoxide release oscillation periods for neutrophils; we speculate that metabolic oscillations of the effector cell drive the membrane damage of the target.
IL-2-Dependent Expression of Genes Involved in Cytoskeleton Organization, Oncogene Regulation, and Transcriptional Control
Authors: Herblot Sabine , Chastagner Patricia , Samady Laila , Moreau Jean-Louis , Demaison Christophe , Froussard Patrick , Liu Xinyuan , Bonnet Jacques , Thèze Jacques
Published: January 2023

IL-2 induces growth, differentiation, and/or apoptosis of lymphoid cells. To study further the molecular basis of IL-2 function, we used a cDNA subtraction approach involving a cell line grown in IL-2 or IL-4. From the corresponding library, 66 nonredundant sequences were characterized; 16 of them encode identified proteins. The kinetics of in vitro expression of 8 selected sequences, the functions of which could be associated with IL-2-induced T cell activation/differentiation, was investigated using an IL-2-dependent T cell line. IL-2 increased the expression of cytoskeleton proteins (α-tubulin), oncogene-regulating proteins (CCCTC-binding factor, Jun inhibitor factor-1), and transcription factors (E2F-4, cyclic AMP-responsive element-binding protein, zhx-1). IL-2 also regulated the expression of genes coding for multifunctional proteins, e.g., β-catenin and nucleolin. These results were verified using Con A-induced T cell blasts stimulated or not by IL-2. The in vivo expression of four of these genes was also analyzed in spleen and lymph node cells of IL-2-deficient and MRL/lpr mice, which both have high numbers of activated cells, but the latter have intact IL-2 expression. The expression of β-catenin, CCCTC-binding factor, Jun inhibitor factor-1, and nucleolin was significantly higher in MRL/lpr animals. A similar analysis of thymocytes from IL-2−/− and IL-2+/− mice demonstrated the same expression patterns of the 4 sequences in these strains. The expression of the IL-2-induced genes described herein is similar to the regulatory pattern of IL-2Rα. Taken together, our data provide additional evidence for the pleiotropic action of IL-2 in the periphery and IL-2 independence of molecular processes involved in thymocyte differentiation.
Enumeration of Antigen-Presenting Cells in Mice Infected with Sendai Virus
Authors: Usherwood Edward J. , Hogg Twala L. , Woodland David L.
Published: January 2023

Substantial progress has been made in understanding Ag presentation to T cells; however, relatively little is known about the location and frequency of cells presenting viral Ags during a viral infection. Here, we took advantage of a highly sensitive system using lacZ-inducible T cell hybridomas to enumerate APCs during the course of respiratory Sendai virus infection in mice. Using lacZ-inducible T cell hybridomas specific for the immunodominant hemagglutinin-neuraminidase HN421–436/I-Ab and nucleoprotein NP324–332/Kb epitopes, we detected APCs in draining mediastinal lymph nodes (MLNs), in cervical lymph nodes, and also in the spleen. HN421–436/I-Ab- and NP324–332/Kb-presenting cells were readily detectable between days 3 and 9 postinfection, with more APCs present in the MLN than in the cervical lymph nodes. Interestingly, no infectious virus was detected in lymphoid tissue beyond day 6, suggesting that a depot of noninfectious viral Ag survives, in some form, for 2–3 days after viral clearance. Fractionation of the MLN demonstrated that APC frequency was enriched in dendritic cells and macrophages but depleted in the B cell population, suggesting that B cells do not form a large population of APCs during the primary response to this virus.
CD86 (B7-2) Can Function to Drive MHC-Restricted Antigen-Specific CTL Responses In Vivo
Authors: Agadjanyan Michael G. , Kim Jong J. , Trivedi Neil , Wilson Darren M. , Monzavi-Karbassi Behjatolah , Morrison Lake D. , Nottingham Liesl K. , Dentchev Tzvete , Tsai Anthony , Dang Kesen , Chalian Ara A. , Maldonado Michael A. , Williams William V. , Weiner David B.
Published: January 2023

Activation of T cells requires both TCR-specific ligation by direct contact with peptide Ag-MHC complexes and coligation of the B7 family of ligands through CD28/CTLA-4 on the T cell surface. We recently reported that coadministration of CD86 cDNA along with DNA encoding HIV-1 Ags i.m. dramatically increased Ag-specific CTL responses. We investigated whether the bone marrow-derived professional APCs or muscle cells were responsible for the enhancement of CTL responses following CD86 coadministration. Accordingly, we analyzed CTL induction in bone marrow chimeras. These chimeras are capable of generating functional viral-specific CTLs against vaccinia virus and therefore represent a useful model system to study APC/T cell function in vivo. In vaccinated chimeras, we observed that only CD86 + Ag + MHC class I results in 1) detectable CTLs following in vitro restimulation, 2) detectable direct CTLs, 3) enhanced IFN-γ production in an Ag-specific manner, and 4) dramatic tissue invasion of T cells. These results support that CD86 plays a central role in CTL induction in vivo, enabling non-bone marrow-derived cells to prime CTLs, a property previously associated solely with bone marrow-derived APCs.
Structural Deficiencies in Granuloma Formation in TNF Gene-Targeted Mice Underlie the Heightened Susceptibility to Aerosol Mycobacterium tuberculosis Infection, Which Is Not Compensated for by Lymphotoxin
Authors: Bean Andrew G. D. , Roach Daniel R. , Briscoe Helen , France Malcolm P. , Korner Heinrich , Sedgwick Jonathon D. , Britton Warwick J.
Published: January 2023

TNF and lymphotoxin-α (LTα) may act at various stages of the host response to Mycobacterium tuberculosis. To dissect the effects of TNF independent of LTα, we have used C57BL/6 mice with a disruption of the TNF gene alone (TNF−/−). Twenty-one days following aerosol M. tuberculosis infection there was a marked increase in the number of organisms in the lungs of TNF−/− mice, and by 28–35 days all animals had succumbed, with widespread dissemination of M. tuberculosis. In comparison with the localized granulomas containing activated macrophages and T cells in lungs and livers of C57BL/6 wild-type (wt) mice, cellular infiltrates in TNF−/− mice were poorly formed, with extensive regions of necrosis and neutrophilic infiltration of the alveoli. Phenotypic analysis of lung homogenates demonstrated similar numbers of CD4+ and CD8+ T cells in TNF−/− and wt mice, but in TNF-deficient mice the lymphocytes were restricted to perivascular and peribronchial areas rather than colocated with macrophages in granulomas. T cells from TNF−/− mice retained proliferative and cytokine responses to purified protein derivative, and delayed-type hypersensitivity to purified protein derivative was demonstrable. Macrophages within the lungs of TNF−/− and wt mice showed similar levels of MHC class II and inducible nitric oxide synthase expression, and levels of serum nitrite were comparable. Thus, the enhanced susceptibility of TNF−/− is not compensated for by the presence of LTα, and the critical role of TNF is not in the activation of T cells and macrophages but in the local organization of granulomas.
Successful Adoptive Immunotherapy of MurinePoorly ImmunogenicTumor with Specific Effector Cells Generated from Gene-Modified Tumor-Primed Lymph Node Cells
Authors: Tanaka Hiroshi , Yoshizawa Hirohisa , Yamaguchi Yoshifumi , Ito Kazuhisa , Kagamu Hiroshi , Suzuki Eiichi , Gejyo Fumitake , Hamada Hirofumi , Arakawa Masaaki
Published: January 2023

We previously reported that cytokine gene transfer into weakly immunogenic tumor cells could enhance the generation of precursor cells of tumor-reactive T cells and subsequently augment antitumor efficacy of adoptive immunotherapy. We investigated whether such potent antitumor effector T cells could be generated from mice bearing poorly immunogenic tumors. In contrast to similarly modified weakly immunogenic tumors, MCA102 cells, which are chemically induced poorly immunogenic fibrosarcoma cells transfected with cDNA for IL-2, IL-4, IL-6, IFN-γ, failed to augment the host immune reaction. Because priming of antitumor effector T cells in vivo requires two important signals provided by tumor-associated Ags and costimulatory molecules, these tumor cells were cotransfected with a B7-1 cDNA. Transfection of both IFN-γ and B7-1 (MCA102/B7-1/IFN-γ) resulted in regression of s.c. tumors, while tumor transfected with other combinations of cytokine and B7-1 showed progressive growth. Cotransfection of IFN-γ and B7-1 into other poorly immunogenic tumor B16 and LLC cells also resulted in the regression of s.c. tumors. Cells derived from lymph nodes draining MCA102/B7-1/IFN-γ tumors showed potent antitumor efficacy, eradicating established pulmonary metastases, but this effect was not seen with parental tumors. This mechanism of enhanced antitumor efficacy was further investigated, and T cells with down-regulated L-selectin expression, which constituted all the in vivo antitumor reactivity, were significantly increased in lymph nodes draining MCA102/B7-1/IFN-γ tumors. These T cells developed into potent antitumor effector cells after in vitro activation with anti-CD3/IL-2. The strategy presented here may provide a basis for developing potent immunotherapy for human cancers.
Soluble Fas Ligand Is Chemotactic for Human Neutrophilic Polymorphonuclear Leukocytes
Authors: Ottonello Luciano , Tortolina Giuseppe , Amelotti Massimo , Dallegri Franco
Published: January 2023

It has been recently shown that Fas ligand (FasL) expression on islet β grafts results in neutrophilic infiltration and graft rejection. In this study, we show that human recombinant soluble FasL is endowed with potent chemotactic properties toward human neutrophilic polymorphonuclear leukocytes (neutrophils) at concentrations incapable of inducing cell apoptosis. Furthermore, neutrophils exposed to soluble FasL did not display detectable change of intracellular Ca2+ and did not undergo superoxide production or exocytosis of primary and secondary granules. Our results show that FasL is a potent chemoattractant for human neutrophils without evoking their secretory responses. This finding suggests a novel proinflammatory function for this ligand and may help to clarify the mechanism governing FasL-mediated graft rejection, thereby offering rational bases for controlling and modulating FasL-based immunotherapies.
Adenoviral Transfer of the Viral IL-10 Gene Periarticularly to Mouse Paws Suppresses Development of Collagen-Induced Arthritis in Both Injected and Uninjected Paws
Authors: Whalen Janey D. , Lechman Eric L. , Carlos Casey A. , Weiss Kurt , Kovesdi Imre′ , Glorioso Joseph C. , Robbins Paul D. , Evans Christopher H.
Published: January 2023

Gene therapy is a promising new approach in the treatment of rheumatoid arthritis. Gene delivery to diseased joints offers the prospect of achieving high, local concentrations of a therapeutic gene product in a sustained manner, while minimizing exposure of nontarget organs. We report that a single administration of a modified adenovirus encoding the Epstein-Barr-derived homologue of IL-10 can suppress the development of disease for extended periods of time when injected locally within the periarticular tissue surrounding the ankle joints of mice with collagen type II-induced arthritis. Furthermore, we show that injection of an adenoviral vector carrying the IL-10 gene into a single paw can suppress development of arthritis in other, noninjected paws of the same individual. The systemic protection resulting from local gene therapy occurred in the absence of detectable levels of viral IL-10 in the serum. Circulating Ab levels to heterologous collagen were unaffected; however, treatment with viral IL-10 significantly suppressed the development of Abs to autologous mouse type II collagen. Thus, the treatment of a single joint by local delivery of the vIL-10 gene may protect multiple joints of the same individual while avoiding deleterious side effects often associated with systemic therapy.
Endotoxin Fails to Induce IFN-γ in Endotoxin-Tolerant Mice: Deficiencies in Both IL-12 Heterodimer Production and IL-12 Responsiveness
Authors: Balkhy Hanan H. , Heinzel Frederick P.
Published: January 2023

Mice exposed to sublethal endotoxemia develop short-term endotoxin tolerance, a state characterized by decreased monokine production and enhanced protection against endotoxic lethality. We confirmed that TNF-α production is markedly impaired in endotoxin-tolerant mice and additionally found 2- to 6-fold decreases in serum IFN-γ in these animals following endotoxin challenge. The IFN-γ deficiency of endotoxin tolerance correlated with 8-fold decreases in the bioactive p40/p35 heterodimeric form of IL-12. In contrast, total circulating IL-12 p40 was reduced by only 30–50%. Endotoxin-tolerant mice were less responsive to IL-12 than control mice, as evidenced by 3-fold lower levels of IFN-γ inducible in vivo when rIL-12 was administered at the time of endotoxin challenge. Similarly, spleen cell cultures of endotoxin-tolerant mice produced 3-fold less IFN-γ in the presence of optimal concentrations of both IL-12 and IL-18. Finally, levels of IL-12R β2 subunit mRNA and the percent composition of NK lymphocytes in the spleen were both decreased in endotoxin-tolerant mice relative to controls. We conclude that endotoxin-tolerant mice are profoundly impaired in their ability to produce IFN-γ in response to endotoxin and that this is associated with acquired defects in both the production of circulating IL-12 heterodimer response and the response to IL-12 by NK cells.
Kinetics of the Changes of Lymphocyte Subsets Defined by Cytokine Production at Single Cell Level During Highly Active Antiretroviral Therapy for HIV-1 Infection
Authors: Sousa Ana E. , Chaves Ana F. , Doroana Manuela , Antunes Francisco , Victorino Rui M. M.
Published: January 2023

The effects of highly active antiretroviral therapy on cytokine imbalances associated with HIV-1 infection have not been characterized. Using single cell analysis by flow cytometry, we show that a significant recovery in the frequency of IL-2-producing cells was only observed in patients with a sustained control of viral replication and that the overexpanded CD8 T cell population of CD28− IFN-γ+ cells was not significantly reduced after 1 yr of effective therapy. Moreover, a detrimental role of IL-4 is suggested by the association between an enhanced proportion of IL-4-producing cells within the CD4 and particularly the CD8 subset and viral load rebound. Finally, the kinetics of changes of cell subsets assessed for simultaneous production of different cytokines supports the view that cell reconstitution during highly active antiretroviral therapy is initially due to redistribution of terminally differentiated cells, followed by peripheral expansion of less differentiated ones and a late progressive increase of the proportion of functionally defined naive/memory precursor lymphocytes. These data bring new support for the role of cytokine imbalances in AIDS pathogenesis and may be relevant for the definition of immunointervention targets.
Factors Associated with the Development of Neonatal Tolerance After the Administration of a Plasmid DNA Vaccine
Authors: Ichino Motohide , Mor Gil , Conover Jackie , Weiss Walter R. , Takeno Mitsuhiro , Ishii Ken J. , Klinman Dennis M.
Published: January 2023

A plasmid DNA vaccine encoding the circumsporozoite protein of malaria (pCSP) induces tolerance rather than immunity when administered to newborn mice. We find that this tolerance persists for >1 yr after neonatal pCSP administration and interferes with the induction of protective immunity in animals challenged with live sporozoites. Susceptibility to tolerance induction wanes rapidly with age, disappearing within 1 wk of birth. Higher doses of plasmid are more tolerogenic, and susceptibility to tolerance is not MHC-restricted. CD8+ T cells from tolerant mice suppress the in vitro Ag-specific immune response of cells from adult mice immunized with pCSP. Similarly, CD8+ T cells from tolerant mice transfer nonresponsiveness to naive syngeneic recipients. These findings clarify the cellular basis and factors contributing to the development of DNA vaccine-induced neonatal tolerance.
The CC Chemokine Receptor-7 Ligands 6Ckine and Macrophage Inflammatory Protein-3β Are Potent Chemoattractants for In Vitro- and In Vivo-Derived Dendritic Cells
Authors: Kellermann Sirid-Aimée , Hudak Susan , Oldham Elizabeth R. , Liu Yong-Jun , McEvoy Leslie M.
Published: January 2023

Dendritic cell migration to secondary lymphoid tissues is critical for Ag presentation to T cells necessary to elicit an immune response. Despite the importance of dendritic cell trafficking in immunity, at present little is understood about the mechanisms that underlie this phenomenon. Using a novel transwell chemotaxis assay system, we demonstrate that the CC chemokine receptor-7 (CCR7) ligands 6Ckine and macrophage inflammatory protein (MIP)-3β are selective chemoattractants for MHC class IIhigh B7-2high bone marrow-derived dendritic cells at a potency 1000-fold higher than their known activity on naive T cells. Furthermore, these chemokines stimulate the chemotaxis of freshly isolated lymph node dendritic cells, as well as the egress of skin dendritic cells ex vivo. Because these chemokines are expressed in lymphoid organs and 6Ckine has been localized to high endothelial venules and lymphatic endothelium, we propose that they may play an important role in the homing of dendritic cells to lymphoid tissues.
Absence of Peyer’s Patches and Abnormal Lymphoid Architecture in Chronic Proliferative Dermatitis (cpdm/cpdm) Mice
Authors: HogenEsch Harm , Janke Sarah , Boggess Dawnalyn , Sundberg John P.
Published: January 2023

The chronic proliferative dermatitis (cpdm) mutation causes inflammation in multiple organs, most prominently in the skin. Examination of the immune system revealed severe abnormalities in the architecture of lymphoid tissues. Peyer’s patches were absent. In contrast, the spleen, lymph nodes, and nasal-associated lymphoid tissues were present. The spleen had normal numbers of T and B cells, but the spleen, lymph nodes, and nasal-associated lymphoid tissues had poorly defined follicles and lacked germinal centers and follicular dendritic cells. The marginal zone in the spleen was absent. The total concentration of serum IgG, IgA, and IgE in cpdm/cpdm mice was significantly decreased, whereas serum IgM was normal. Fecal IgA was low to undetectable in mutant mice, and the concentration of fecal IgM was increased. The titer of DNP-specific Abs following immunization with DNP-keyhole limpet hemocyanin was significantly decreased for all IgG subclasses. In contrast, T cell function appeared normal as assessed by evaluation of the contact hypersensitivity response in cpdm/cpdm mice. The cpdm mutation causes a complex phenotype that is characterized by multiorgan inflammation and the defective development of lymphoid tissues. The cpdm/cpdm mouse may be a useful model to study the factors that control the development of lymphoid tissues, in particular the Peyer’s patches, and the mechanisms that control the humoral immune response.
Utilization of MHC Class I Transgenic Mice for Development of Minigene DNA Vaccines Encoding Multiple HLA-Restricted CTL Epitopes
Authors: Ishioka Glenn Y. , Fikes John , Hermanson Gary , Livingston Brian , Crimi Claire , Qin Mingsheng , France del Guercio Marie- , Oseroff Carla , Dahlberg Carol , Alexander Jeff , Chesnut Robert W. , Sette Alessandro
Published: January 2023

We engineered a multiepitope DNA minigene encoding nine dominant HLA-A2.1- and A11-restricted epitopes from the polymerase, envelope, and core proteins of hepatitis B virus and HIV, together with the PADRE (pan-DR epitope) universal Th cell epitope and an endoplasmic reticulum-translocating signal sequence. Immunization of HLA transgenic mice with this construct resulted in: 1) simultaneous CTL induction against all nine CTL epitopes despite their varying MHC binding affinities; 2) CTL responses that were equivalent in magnitude to those induced against a lipopeptide known be immunogenic in humans; 3) induction of memory CTLs up to 4 mo after a single DNA injection; 4) higher epitope-specific CTL responses than immunization with DNA encoding whole protein; and 5) a correlation between the immunogenicity of DNA-encoded epitopes in vivo and the in vitro responses of specific CTL lines against minigene DNA-transfected target cells. Examination of potential variables in minigene construct design revealed that removal of the PADRE Th cell epitope or the signal sequence, and changing the position of selected epitopes, affected the magnitude and frequency of CTL responses. Our results demonstrate the simultaneous induction of broad CTL responses in vivo against multiple dominant HLA-restricted epitopes using a minigene DNA vaccine and underline the utility of HLA transgenic mice in development and optimization of vaccine constructs for human use.
Antibodies to the IL-12 Receptor β2 Chain Mark Human Th1 But Not Th2 Cells In Vitro and In Vivo
Authors: Rogge Lars , Papi Alberto , Presky David H. , Biffi Mauro , Minetti Lisa J. , Miotto Deborah , Agostini Carlo , Semenzato Gianpietro , Fabbri Leonardo M. , Sinigaglia Francesco
Published: January 2023

Great attention has been placed on the possibility of distinguishing Th1 from Th2 cells on the basis of differential expression of surface receptors. We have recently shown that the differential expression of the IL-12Rβ2 chain in Th1 and Th2 cells, as measured at the mRNA level, accounts for an important regulatory mechanism in the differentiation of the two cell subsets. In this study, we identify IL-12R expression at the protein level. We have generated an anti-IL-12Rβ2-specific mAb and analyzed IL-12Rβ2 expression on polarized Th cell populations generated in vitro and on T cells derived from patients with Th1- or Th2-mediated inflammatory conditions. Although IL-12Rβ2 was absent in freshly isolated PBMC and in cord blood cells, we were able to detect IL-12Rβ2 expression selectively in differentiated Th1 and T cytotoxic 1, but not Th2 or T cytotoxic 2 cells. In the presence of IL-12, cell surface expression of the IL-12Rβ2 subunit was readily detected on T cells after 24 h, reached the maximum at day 5, and declined thereafter. Most importantly, the anti-IL-12Rβ2 mAb recognizes lung T cells from patients with sarcoidosis, a disease characterized by a typical cell-mediated, Th1-type inflammatory response. In contrast, IL-12Rβ2 was absent in lung T cells from patients with allergic asthma, a disease characterized by a Th2-type inflammatory response. The mAb reported in this study should represent a powerful tool to investigate the role of Th1 and Th2 cells in inflammatory conditions and to monitor therapies aimed at altering the balance of Th cell subsets.
Human T Cells Express the C5a Receptor and Are Chemoattracted to C5a
Authors: Nataf Serge , Davoust Nathalie , Ames Robert S. , Barnum Scott R.
Published: January 2023

The anaphylatoxin C5a is a potent mediator of inflammation that exerts a broad range of activity on cells of the myeloid lineage. In this study, we present the first evidence that human T cells express the C5a receptor (C5aR) and are chemotactic to C5a. Using FACS analysis, we found that the C5aR was expressed at a low basal level on unstimulated T cells and was strikingly up-regulated upon PHA stimulation in a time- and dose-dependent manner. CD3+ sorted T cells as well as Jurkat T cells were shown to express C5aR mRNA as assessed by RT-PCR. Moreover, semiquantitative RT-PCR analysis demonstrated that C5aR mRNA was down-regulated in purified T cells upon long-term PHA stimulation. To demonstrate that C5a was biologically active on T cells, we investigated the chemotactic activity of C5a and observed that purified CD3+ T cells are chemotactic to C5a at nanomolar concentrations. Finally, using a combination of in situ hybridization and immunohistochemistry, we showed that the T cells infiltrating the central nervous system during experimental allergic encephalomyelitis express the C5aR mRNA. In summary, these results suggest that C5a exerts direct effects on T cells and could be involved in the trafficking of T cells under physiological and pathological conditions, including inflammatory diseases of the central nervous system.
Regulation of CD154 (CD40 Ligand) mRNA Stability During T Cell Activation
Authors: Ford Gregory S. , Barnhart Bryan , Shone Scott , Covey Lori R.
Published: January 2023

The CD154 protein (CD40 ligand), which is critical to the regulation of both humoral and cellular immune responses, is expressed transiently on the surface of activated CD4+ T cells. To determine whether control of mRNA stability contributes to the highly regulated expression of CD154 during T cell activation, CD4+ T cells were isolated from human peripheral blood and stimulated for various lengths of time with plate-bound anti-CD3 mAb. At early times after anti-CD3 activation, the CD154 message was found to be very unstable, however, the stability measurably increased after 24–48 h of activation. Similar analyses of TNF-α and c-myc mRNA decay throughout a time course of T cell activation revealed patterns of regulation that were distinct from CD154. Similar to the effect on TNF-α mRNA, stimulation of T cells with PMA + ionomycin greatly increased the stability of CD154 message. However, CD154 message stability was only modestly increased in T cells coactivated with anti-CD3 and anti-CD28 at 5 h and not increased by costimulation at 24 h. Finally, an analysis of both mRNA and surface protein expression over a time course of T cell activation with anti-CD3 revealed a rapid induction of expression early after activation. This induction was followed by a more gradual decrease in expression over the next 48 h. Together, these data support a role for posttranscriptional regulation in the control and overall expression of CD154 in activated T cells.
Molecular Cloning of a Glycosylphosphatidylinositol-Anchored Molecule CDw108
Authors: Yamada Akira , Kubo Keisuke , Takeshita Toshikazu , Harashima Nanae , Kawano Koichiro , Mine Takashi , Sagawa Kimitaka , Sugamura Kazuo , Itoh Kyogo
Published: January 2023

CDw108, also known as the John-Milton-Hagen human blood group Ag, is an 80-kDa glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein that is preferentially expressed on activated lymphocytes and E. The molecular characteristics and biological function of the CDw108 were not clarified previously. In this manuscript, we identify the cDNA clone containing the entire coding sequence of the CDw108 gene and report its molecular characteristics. The 1998-base pairs of the open reading frame of the cloned cDNA encoded a protein of 666 amino acids (aa), including the 46 aa of the signal peptide and the 19 aa of the GPI-anchor motif. Thus, the membrane-anchoring form of CDw108 was the 602 aa, and the estimated molecular mass of the unglycosylated form was 68 kDa. The RGD (Arg-Gly-Asp) cell attachment sequence and the five potential N-linked glycosylation sites were located on the membrane-anchoring form. Flow cytometric and immunoprecipitation analyses of the CDw108 cDNA transfectants confirmed that the cloned cDNA encoded the native form of CDw108. The CDw108 mRNA was expressed in activated PBMCs as well as in the spleen, thymus, testis, placenta, and brain, but was not expressed in any other tissues tested. Radiation hybrid mapping indicated that the CDw108 gene was located in the middle of the long arm of chromosome 15 (15q23–24). This molecular information will be critical for understanding the biological function of the CDw108 Ag.
B7-2 Is Required for the Progression But Not the Initiation of the Type 2 Immune Response to a Gastrointestinal Nematode Parasite
Authors: Greenwald R. J. , Urban J. F. , Ekkens M. J. , Chen S.-J. , Nguyen D. , Fang H. , Finkelman F. D. , Sharpe A. H. , Gause W. C.
Published: January 2023

T cells require CD28/CTLA-4 costimulatory molecule interactions in addition to Ag-specific signals through the TCR for in vivo effector Th cell function. Some studies have suggested that the ligands for these costimulatory molecules may differentially influence effector T cell function with B7-2 favoring a type 2 response and B7-1 favoring a type 1 response, while other studies have suggested that these molecules may be redundant. The recent development of B7-2-deficient mice permits the direct analysis of the requirement of B7-2 during a type 2 immune response to an infectious pathogen. We have examined, in B7-2-deficient mice, effector Th cell function and the associated type 2 immune response following infection with Heligmosomoides polygyrus, a natural murine parasitic nematode. Elevations in cytokine gene expression and protein secretion were pronounced and comparable in inoculated B7-2−/− and B7-2+/+ mice at day 8 after H. polygyrus inoculation. However, by day 14 after infection, increases in T cell cytokine expression were markedly inhibited in H. polygyrus-inoculated B7-2−/− mice. Furthermore, elevations in serum IgE and germinal center formation were inhibited at later stages of the immune response, while elevations in serum IgG1 persisted. These findings suggest that certain T-dependent components vary in their B7-2-dependency during the type 2 immune response. They further demonstrate that B7-2 interactions are not necessary for the initiation of the type 2 immune response, but are instead required for its progression after the development of effector T cells.
Therapy of Murine Tumors with Recombinant Bordetella pertussis Adenylate Cyclase Carrying a Cytotoxic T Cell Epitope
Authors: Fayolle Catherine , Ladant Daniel , Karimova Gouzel , Ullmann Agnès , Leclerc Claude
Published: January 2023

Bordetella pertussis secretes an invasive adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain into the cytosol of eukaryotic target cells directly through the cytoplasmic membrane. We have shown previously that recombinant CyaA can be used to deliver viral CD8+ T cell epitopes to the MHC-class I presentation pathway to trigger specific CTL responses in vivo. In the present study, we show that mice immunized with a detoxified but still invasive CyaA carrying a CD8+ T cell epitope of OVA developed strong epitope-specific CTL responses, which kill tumor cells expressing this Ag. Treating mice with this recombinant molecule after the graft of melanoma cells expressing OVA induced a strong survival advantage compared with control animals. To our knowledge, this study represents the first demonstration that a nonreplicative and nontoxic vector carrying a single CTL epitope can stimulate efficient protective and therapeutic antitumor immunity.
Induction and Regulation of Macrophage Metalloelastase by Hyaluronan Fragments in Mouse Macrophages
Authors: Horton Maureen R. , Shapiro Steven , Bao Clare , Lowenstein Charles J. , Noble Paul W.
Published: January 2023

Although the metalloproteinase murine metalloelastase (MME) has been implicated in lung disorders such as emphysema and pulmonary fibrosis, the mechanisms regulating MME expression are unclear. Low m.w. fragments of the extracellular matrix component hyaluronan (HA) that accumulate at sites of lung inflammation are capable of inducing inflammatory gene expression in macrophages (Mφ). The purpose of this study was to examine the effect of HA fragments on the expression of MME in alveolar Mφ. The mouse alveolar Mφ cell line MH-S was stimulated with HA fragments over time, total RNA was isolated, and Northern blot analysis was performed. HA fragments induced MME mRNA in a time-dependent fashion, with maximal levels at 6 h. HA fragments also induced MME protein expression as well as enzyme activity. The induction of MME gene expression was specific for low m.w. HA fragments and dependent upon new protein synthesis; it occurred at the level of gene transcription. We also examined the effect of HA fragments on MME expression in inflammatory alveolar Mφ from bleomycin-injured rat lungs. Although normal rat alveolar Mφ did not express MME mRNA in response to HA fragments, alveolar Mφ from the bleomycin-treated rats responded to HA fragment stimulation by increasing MME mRNA levels. Furthermore, baseline and HA fragment-induced MME gene expression in alveolar Mφ from bleomycin-treated rats was inhibited by IFN-γ. These data suggest that HA fragments may be an important mechanism for the expression of MME by Mφ in inflammatory lung disorders.
Mutations in Immunodominant T Cell Epitopes Derived from the Nonstructural 3 Protein of Hepatitis C Virus Have the Potential for Generating Escape Variants That May Have Important Consequences for T Cell Recognition
Authors: Wang Huiru , Eckels David D.
Published: January 2023

One of the most disturbing features of hepatitis C virus (HCV) is its long-term persistence in the host. One hypothesis to explain this phenomenon is that HCV escapes immune recognition through its intrinsic hypermutability. To determine whether immunodominant T cell epitopes derived from HCV nonstructural 3 (NS3) protein might be subject to sequence variations leading to escape mutants, we examined sequence variations of one IL-2-producing epitope, NS3358–375, and one IL-10-producing epitope, NS3505–521. By PCR amplification, cloning, and sequencing, we observed significant sequence variations in the two epitopes, although the selection intensity for each epitope was different. For NS3358–375, more variants were observed, and for NS3505–521, fewer mutations were observed. Moreover, functional studies revealed that three NS3358–375 and one NS3505–521 variants failed to stimulate T cell proliferation, and two other NS3358–375 and NS3505–521 variants weakly stimulated T cell responses. Our results are consistent with immune selection of viral variants at the epitope level, which may enable HCV to evade host defenses over time.
TNF-α Regulates Corneal Langerhans Cell Migration
Authors: Dekaris Iva , Zhu Su-Ning , Dana M. Reza
Published: January 2023

Langerhans cells (LC) belong to the dendritic cell family and mediate Ag presentation in the cornea and ocular surface. Under normal physiological conditions, the central cornea is devoid of LC. Centripetal migration of LC plays a critical role in promoting immunoinflammatory responses in the eye including allograft rejection and herpetic keratitis. The molecular mechanisms responsible for ocular LC migration are poorly understood. To examine whether TNF-α mediates corneal LC migration and to establish the interaction of IL-1 and TNF-α in regulating LC migratory capacity, we utilized gene-targeted knockout mice lacking IL-1 receptor I (IL-1RI−/−), TNF receptor I (p55−/−), TNF receptor II (p75−/−), or both (p55−/−p75−/−). LC migration was induced by thermal cautery or cytokine injection and enumerated by an immunofluorescence assay. Migration of LC after cauterization and TNF-α injection was significantly depressed in both p55−/− and p75−/− mice. Similarly, in the first 72 h after intracorneal injection of IL-1α, LC migration was reduced in p55−/−, p75−/−, and p55−/−p75−/− mice. In contrast, injection of TNF-α in IL-1RI−/− mice led to normal migration of corneal LC indistinguishable from wild-type controls. These results suggest that the IL-1 induction of corneal LC migration is largely mediated by TNFR function, whereas TNF-α induction of LC migration is independent of IL-1RI activity. Moreover, the data suggest that both p55 and p75 signaling pathways are important in mediating LC migration in the cornea.
Endocytosis of Heparin-Binding Protein (CAP37) Is Essential for the Enhancement of Lipopolysaccharide-Induced TNF-α Production in Human Monocytes
Authors: Heinzelmann Michael , Platz Andreas , Flodgaard Hans , Polk Hiram C. , Miller Frederick N.
Published: January 2023

Heparin-binding protein (HBP), also known as CAP37, is a proteolytically inactive serine protease homologue that is released from activated granulocytes. However, HBP is not a biologically inactive molecule but rather a multifunctional protein with properties that include the enhancement of LPS-induced TNF-α production from monocytes. We have previously demonstrated that HBP is internalized in monocytes. In the current study, we hypothesize that HBP is internalized in monocytes via endocytosis, and this internalization is an important mechanism by which HBP enhances LPS-induced TNF-α release. Using whole blood from healthy donors and flow cytometry, we found that colchicine (0.1–10 mM), cytochalasin D (1000 μM), NH4Cl (10–50 mM), and bafilomycin A1 (0.1–3 μM) significantly reduced the affinity of FITC-HBP for CD14-positive monocytes. Using isolated human monocytes and ELISA, we found that colchicine (0.1 mM), cytochalasin D (30 and 300 μM), NH4Cl (30 mM), and bafilomycin A1 (1 μM) significantly reduced the effect of HBP (10 μg/ml) to enhance LPS (10 ng/ml)-induced TNF-α release after 24 h. These findings demonstrate that internalization of HBP in monocytes is essential for the enhancement of LPS-induced TNF-α release. Transport of HBP to an activating compartment depends on intact F-actin polymerization and endosomal acidification, an important mechanism for endosomal protein sorting and trafficking.