bluuebunny/crossref_metadata_2025 huggingface dataset:
Active Sites in Complement Component C3 Mapped by Mutations at Indels
Authors: Ogata Ronald T. , Ai Rong , Low Pamela J.
Published: December 2022

Engineered mutants of human complement component C3 were used to test the idea that sites of length polymorphisms in protein families (indels) can guide a search for protein:protein interaction sites. Sequence changes were introduced at each of the 27 indels in the C3/4/5 protein family, and mutants at 26 indels were expressed by transiently transfected COS cells. Expressed proteins were assayed 1) for concentration, by ELISA and by autoradiography of radiolabeled protein; 2) for classical pathway hemolytic activity; 3) for susceptibility to proteolytic activation by the alternative pathway and cobra venom factor C3 convertases; and 4) for susceptibility to complement factor I in the presence of factor H. Most of the mutations did not appreciably alter expression or activity relative to wild-type C3, consistent with the idea that most indels occur at the protein surface. Mutations at four indels severely damaged C3 functional activity, but did not affect the stability or structure of the protein, as assessed by their effects on expression by COS cells and on susceptibility to cleavage by C3 convertases and factor I. These indels are therefore near functionally important amino acid residues; they represent good candidates for sites of protein:protein interactions. Mutation of the sequence at a fifth indel altered the equilibrium between the latent and reacted C3 conformations, and mutations at 4 other indels substantially decreased both protein activity and expression. The mutants provided an overview of the structural and functional roles played by different parts of C3.
Mef2 Proteins, Required for Muscle Differentiation, Bind an Essential Site in the Ig λ Enhancer
Authors: Satyaraj Ebenezer , Storb Ursula
Published: December 2022

The Ig λ light chain gene enhancer has two unique essential motifs, λA and λB. The transcription factors that bind the λB motif have been identified as Pu.1 and Pu.1-interacting partner (Pip). We report here that the λA site includes a binding site for the myocyte-specific enhancer factor 2 (Mef2) family of transcription factors. Mef2 proteins were first described in muscle cells and, in vertebrates, include four known members designated A to D. Using a λA electrophoretic-mobility shift assay (EMSA), in conjunction with a high affinity Mef2 binding site and anti-Mef2 Abs, we show that members of the Mef2 family are present in nuclear extracts of λ-producing B cells and bind the λA site. Functional assays using the chloramphenicol acetyltransferase (CAT) reporter construct containing three copies of the λA motif demonstrate that the λA sequence can function as an enhancer in conjunction with the thymidine kinase (TK) promoter and is regulated by Mef2 proteins. Extrapolating from other systems where transcriptional regulation by Mef2 has been studied, other transcription factors may be involved along with Mef2 in transcriptional regulation at the λA site.
Proapoptotic Activity of aTrypanosoma cruziCeramide-Containing Glycolipid Turned on in Host Macrophages by IFN-γ
Authors: Freire-de-Lima Célio G. , Nunes Marise P. , Corte-Real Suzana , Soares Milena P. , Previato José O. , Mendonça-Previato Lúcia , DosReis George A.
Published: December 2022

The effects of glycoinositolphospholipid (GIPL), from the pathogenic protozoan Trypanosoma cruzi, and its isolated glycan and lipid (dihydroceramide) components, were investigated in J774 cells and primary macrophages. Isolated GIPL ceramide, but not intact GIPL or its glycan, induced intense fluid phase endocytosis when added exogenously. In the presence of the cytokine IFN-γ, GIPL ceramide induced marked apoptosis in J774 cells and macrophages, independent of nitric oxide secretion. When cells were preincubated with the GIPL-derived glycan chain, addition of intact GIPL induced macrophage apoptosis in the presence of IFN-γ. Synthetic C2-dihydroceramide also induced apoptosis in the presence of IFN-γ. Induction of apoptosis in T. cruzi-infected macrophages by GIPL ceramide plus IFN-γ led to increased parasite release compared with IFN-γ treatment alone. Viable parasites released comprised both infective trypomastigote and spheromastigote forms. These results identify a novel pathway by which T. cruzi glycosylphosphatidylinositol family molecules affect host macrophages, with implications for the infectious process.
Novel C-Terminally Truncated Isoforms of the CXC Chemokine β-Thromboglobulin and Their Impact on Neutrophil Functions
Authors: Ehlert Jan Erik , Gerdes Johannes , Flad Hans-Dieter , Brandt Ernst
Published: December 2022

The neutrophil agonist neutrophil-activating peptide-2 (NAP-2) arises through proteolytic processing of platelet-derived N-terminally extended inactive precursors, the most abundant one being connective tissue-activating peptide-III (CTAP-III). Apart from N-terminal processing, C-terminal processing also appears to participate in the functional regulation of NAP-2, as indicated by our recent identification of an isoform missing four C-terminal amino acids, NAP-2 (1–66), which was about threefold more potent than full-size NAP-2. In the present study, we report on the discovery and identification of natural NAP-2 (1–63), an isoform truncated by seven C-terminal residues. Functional and receptor-binding analyses demonstrated that NAP-2 (1–63) represents the most active isoform, being about fivefold more potent than full-size NAP-2. Analyses of rNAP-2 isoforms successively truncated at the C terminus by up to eight residues suggest functionally important roles for acidic residues and for the leucine at position 63, a residue highly conserved within CXC chemokines. Finally, we report on a novel C-terminally truncated isoform of CTAP-III (CTAP-III (1–81)) representing the potential precursor of NAP-2 (1–66). We show that C-terminal truncation in CTAP-III enhances its potency to desensitize chemokine-induced neutrophil activation, indicating that C-terminal processing might not only serve to enhance neutrophil activation, but might as well participate in the down-regulation of an inflammatory response.
Fusion of Azurophil Granules with Phagosomes and Activation of the Tyrosine Kinase Hck Are Specifically Inhibited During Phagocytosis of Mycobacteria by Human Neutrophils
Authors: N’Diaye Elsa-Noah , Darzacq Xavier , Astarie-Dequeker Catherine , Daffé Mamadou , Calafat Jero , Maridonneau-Parini Isabelle
Published: December 2022

Pathogenic mycobacteria parasitize macrophages and reside within phagosomes, which do not fuse with lysosomal granules. Mycobacteria are also internalized by neutrophils, which possess at least two types of granules, specific and azurophil granules, the latter being specialized lysosomes. Here, we investigated the ability of mycobacteria to inhibit the fusion of these granules with their phagosomes in human neutrophils. It was found that when pathogenic (Mycobacterium kansasii and Mycobacterium avium) or nonpathogenic (Mycobacterium smegmatis and Mycobacterium phlei) mycobacteria were internalized by neutrophils, they induced the inhibition of azurophil granule fusion with phagosomes even when they were serum opsonized. In contrast, secretion of specific granule content and production of O2−, both of which contribute to the neutrophil bactericidal response, were triggered. Hck is a Src family tyrosine kinase associated with azurophil granules. During internalization of zymosan, azurophil granules fused with phagosomes and Hck was activated and translocated to the phagosomal membrane, whereas in neutrophils engulfing mycobacteria, Hck did not translocate and remained unactivated. The activation of the tyrosine kinase Fgr was not affected. These results indicate that 1) pathogenic and nonpathogenic mycobacteria trigger similar bactericidal responses in neutrophils, 2) phagocytosis and fusion of azurophil granules can be uncoupled by mycobacteria, and 3) Hck could be one of the key elements of the azurophil secretory pathway that are altered during phagocytosis of mycobacteria.
Role of IL-6 and the Soluble IL-6 Receptor in Inhibition of VCAM-1 Gene Expression
Authors: Oh Jae-Wook , Van Wagoner Nicholas J. , Rose-John Stefan , Benveniste Etty N.
Published: December 2022

Adhesion molecules such as VCAM-1 and ICAM-1 are increased in the central nervous system (CNS) during inflammatory responses and contribute to extravasation of leukocytes across the blood-brain barrier (BBB) and into CNS parenchyma. Astrocytes contribute to the structural integrity of the BBB and can be induced to express VCAM-1 and ICAM-1 in response to cytokines such as TNF-α, IL-1β, and IFN-γ. In this study, we investigated the influence of IL-6 on astroglial adhesion molecule expression. IL-6, the soluble form of the IL-6R (sIL-6R), or both IL-6 plus sIL-6R, had no effect on VCAM-1 or ICAM-1 gene expression. Interestingly, the IL-6/sIL-6R complex inhibited TNF-α-induced VCAM-1 gene expression but did not affect TNF-α-induced ICAM-1 expression. The inhibitory effect of IL-6/sIL-6R complex was reversed by the inclusion of anti-IL-6R and gp130 Abs, demonstrating the specificity of the response. A highly active fusion protein of sIL-6R and IL-6, covalently linked by a flexible peptide, which is designated H-IL-6, also inhibited TNF-α-induced VCAM-1 expression. sIL-6R alone was an effective inhibitor of TNF-α-induced VCAM-1 due to endogenous IL-6 production. These results indicate that the IL-6 system has an unexpected negative effect on adhesion molecule expression in glial cells and may function as an immunosuppressive cytokine within the CNS.
Oncostatin M Up-Regulates Tissue Inhibitor of Metalloproteinases-3 Gene Expression in Articular Chondrocytes via De Novo Transcription, Protein Synthesis, and Tyrosine Kinase- and Mitogen-Activated Protein Kinase-Dependent Mechanisms
Authors: Liand Wen Qing , Zafarullah Muhammad
Published: December 2022

Cytokines and growth factors regulate physiologic and pathologic turn-over of cartilage extracellular matrix (ECM) by altering the balance between tissue inhibitors of metalloproteinases (TIMPs) and matrix metalloproteinases (MMPs). Oncostatin M (OSM) is a cytokine of the IL-6 family whose levels are increased in the serum and synovial fluids of patients with rheumatoid arthritis. We examined responsiveness of the TIMP-3 gene to OSM in articular chondrocytes and studied the regulatory and signaling mechanisms of this response. OSM induced TIMP-3 mRNA and protein expression in a dose- and time-dependent fashion. Concomitantly, stromelysin-1 and collagenase-1 RNA and activities were also induced. A cartilage matrix growth factor, TGF-β, induced TIMP-3, but combined OSM and TGF-β did not further increase the extent of induction, suggesting a lack of synergy between the two. OSM induction of TIMP-3 gene expression was dependent upon de novo protein synthesis and transcription. RNA decay time-courses suggested that the OSM-mediated increase of TIMP-3 RNA was not due to enhanced message stability and, along with inhibition by actinomycin-D, suggested a transcriptional control. The antiinflammatory glucocorticoid, dexamethasone, down-regulated this augmentation. Investigation of the signaling mechanisms revealed that protein tyrosine kinase inhibitors genistein and herbimycin A, as well as the specific mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059, suppressed OSM-induced TIMP-3 message expression, suggesting the involvement of tyrosine kinases and mitogen-activated protein kinase cascades in the signaling of OSM leading to TIMP-3 RNA enhancement. Thus OSM can potentially alter the cartilage matrix metabolism by regulating genes like TIMP-3 and matrix metalloproteinases.
Transgenic Expression of IFN-α in the Central Nervous System of Mice Protects Against Lethal Neurotropic Viral Infection but Induces Inflammation and Neurodegeneration
Authors: Akwa Yvette , Hassett Daniel E. , Eloranta Maija-Leena , Sandberg Kristian , Masliah Eliezer , Powell Henry , Whitton J. Lindsay , Bloom Floyd E. , Campbell Iain L.
Published: December 2022

Type I IFNs, which include IFN-α, appear to have complex and broad-ranging actions in the central nervous system (CNS) that may result in protection or injury. To better understand these issues, we generated transgenic mice that produce IFN-α1 chronically from astrocytes. These glial fibrillary acidic protein-IFN-α transgenic mice developed a progressive inflammatory encephalopathy, with marked calcium mineralization, meninoencephalitis, gliosis, and neurodegeneration. Many features of this murine encephalopathy resembled those found in certain human encephalopathies of unknown etiology; these diseases, exemplified by Aicardi-Goutières syndrome and some viral encephalopathies, show increased intrathecal production of IFN-α. Our data suggest that IFN-α overproduction may be the primary factor initiating these human diseases. Following intracerebral infection with lymphocytic choriomeningitis virus, glial fibrillary acidic protein-IFN-α mice had significantly increased survival rates associated with markedly reduced virus titers and immune pathology in the brain but normal peripheral CTL responses. Therefore, the production of IFN-α in the CNS can be a two-edged sword that on the one hand confers protection against a lethal viral infection but on the other causes significant injury to the brain. These transgenic mice provide a novel animal model in which to further evaluate the mechanisms that underlie the diverse actions of type I IFNs in the intact CNS.
Antibodies Against IL-12 Prevent Superantigen-Induced and Spontaneous Relapses of Experimental Autoimmune Encephalomyelitis
Authors: Constantinescu Cris S. , Wysocka Maria , Hilliard Brendan , Ventura Elvira S. , Lavi Ehud , Trinchieri Giorgio , Rostami Abdolmohamad
Published: December 2022

Immunization of (PL/J × SJL/J)F1 mice with myelin basic protein (MBP) induces relapsing experimental autoimmune encephalomyelitis (EAE). Relapses occur 7 to 10 days after recovery from the initial paralysis. Staphylococcal enterotoxins (SE) A or B, administered after recovery from the initial paralysis, induce immediate relapses. IL-12 is involved in the induction of EAE. Here, we show that SEA and SEB induce IL-12 in splenocytes from (PL/J × SJL/J)F1 mice in vitro and increase the level of IL-12 in the sera of mice treated with these superantigens. IL-12 administration mimics SE in inducing spontaneous relapses and in enhancing the severity and frequency of spontaneous relapses. IL-12 neutralization blocks SE-induced and subsequent relapses of EAE, and, when instituted after recovery from the initial attack, prevents spontaneous relapse. This is the first report of prevention of relapses of EAE with anti-IL-12 Ab, an approach which may prove useful in the prevention of exacerbations in multiple sclerosis.
A Single Specific Amino Acid Residue in Peptide Antigens Is Sufficient to Activate Memory CTL: Potential Role of Cross-Reactive Peptides in Memory T Cell Maintenance
Authors: Reali Eva , Guerrini Remo , Marastoni Mauro , Tomatis Roberto , Grazia Masucci Maria , Traniello Serena , Gavioli Riccardo
Published: December 2022

In the present study, we examined the structural requirements of peptide Ags for productive interactions with the TCR of CTL. For this purpose, we used as a model a previously identified immunodominant epitope that represents the target of EBV-specific HLA-A11-restricted CTL responses. By the use of peptides having minimal sequence homology with the wild-type epitope, we demonstrated that it is possible to selectively expand and reactivate memory CTL precursors without triggering the lytic mechanisms of wild-type specific effectors. In fact, stimulation of PBL from EBV-seropositive donors by polyalanine analogues, sharing only the putative TCR contact residue with the natural epitope, exclusively induced clonal expansion and reactivation of EBV-specific memory CTL precursors. Interestingly, these polyalanine peptides failed to trigger the cytotoxic function of CTLs specific for the wild-type viral epitope. This clearly indicates that reactivation of memory CTL precursors and triggering of the cytotoxic function have different requirements. The same phenomenon was observed using as stimulators naturally occurring peptides carrying the appropriate TCR contact residue. These data strongly suggest that cross-reactive peptides may play an important role in the expansion and reactivation of CTL clones from the memory T cell pool, and may be involved in long-term maintenance of T cell memory.
EBV-Induced Expression and HLA-DR-Restricted Presentation by Human B Cells of αB-Crystallin, a Candidate Autoantigen in Multiple Sclerosis
Authors: van Sechel Arianne C. , Bajramovic̀ Jeffrey J. , van Stipdonk Marianne J. B. , Persoon-Deen Carla , Geutskens Sacha B. , van Noort Johannes M.
Published: December 2022

The development of multiple sclerosis is most likely influenced by autoimmune responses to central nervous system myelin proteins as well as by infections with common viruses such as EBV and human herpesvirus-6. However, much remains to be established on how these factors interact. In this study, we show that upon EBV infection, human B cells start to express αB-crystallin, a small stress protein that was identified previously as an immunodominant Ag of CNS myelin in multiple sclerosis patients. EBV-induced expression of αB-crystallin in B cells leads to HLA-DR-restricted presentation of the protein and to activation of proinflammatory αB-crystallin-specific Th cells. While αB-crystallin is present in EBV-infected human B cells, the protein is absent from human lymphoid tissues under normal conditions. This is in sharp contrast to other stress proteins such as heat-shock protein (hsp)27 and hsp60 that are ubiquitously expressed in these tissues. In addition, the absence of αB-crystallin from lymphoid tissues in humans is unique as compared with other mammals. All other species examined, including rodents, sheep, and primates, showed constitutive expression of αB-crystallin in secondary lymphoid tissues and sometimes even in the thymus. Since constitutive lymphoid expression most likely results in immunologic tolerance, such a state of tolerance to αB-crystallin can be expected for all of these species, but not for humans. When taken together, our data provide evidence for a novel mechanism by which common viral infections can trigger myelin-directed autoimmunity in a way that is unique for humans.
Generation of Tumor Immunity by Bone Marrow-Derived Dendritic Cells Correlates with Dendritic Cell Maturation Stage
Authors: Labeur Marta S. , Roters Berthold , Pers Birgit , Mehling Annette , Luger Thomas A. , Schwarz Thomas , Grabbe Stephan
Published: December 2022

Bone marrow-derived dendritic cells (BmDC) are potent APC and can promote antitumor immunity in mice when pulsed with tumor Ag. This study aimed to define the culture conditions and maturation stages of BmDC that enable them to optimally function as APC in vivo. BmDC cultured under various conditions (granulocyte-macrophage CSF (GM-CSF) or GM-CSF plus IL-4 alone or in combination with Flt3 ligand, TNF-α, LPS, or CD40 ligand (CD40L)) were analyzed morphologically, phenotypically, and functionally and were tested for their ability to promote prophylactic and/or therapeutic antitumor immunity. Each of the culture conditions generated typical BmDC. Whereas cells cultured in GM-CSF alone were functionally immature, cells incubated with CD40L or LPS were mature BmDC, as evident by morphology, capacity to internalize Ag, migration into regional lymph nodes, IL-12 secretion, and alloantigen or peptide Ag presentation in vitro. The remaining cultures exhibited intermediate dendritic cell maturation. The in vivo Ag-presenting capacity of BmDC was compared with respect to induction of both protective tumor immunity and immunotherapy of established tumors, using the poorly immunogenic squamous cell carcinoma, KLN205. In correspondence to their maturation stage, BmDC cultured in the presence of CD40L exhibited the most potent immunostimulatory effects. In general, although not entirely, the capacity of BmDC to induce an antitumor immune response in vivo correlated to their degree of maturation. The present data support the clinical use of mature, rather than immature, tumor Ag-pulsed dendritic cells as cancer vaccines and identifies CD40L as a potent stimulus to enhance their in vivo Ag-presenting capacity.
A Theiler’s Virus Alternatively Initiated Protein Inhibits the Generation of H-2K-Restricted Virus-Specific Cytotoxicity
Authors: Lin Xiaoqi , Roos Raymond P. , Pease Larry R. , Wettstein Peter , Rodriguez Moses
Published: December 2022

In susceptible mouse strains, the wild-type Daniel’s (wt-DA) strain of Theiler’s murine encephalomyelitis virus induces a persistent central nervous system (CNS) infection with chronic demyelination. The virus is cleared from resistant mice with no resulting demyelination. We characterized the role of the DA L* protein in late demyelination and persistent infection. The DA genome has two alternative reading frames, encoding the virus polyprotein and L*, respectively. The mutant virus DAL*-1 fails to synthesize L* and does not persist in the CNS of wt-DA-susceptible SJL/J or B10.S mice. Since class I-restricted cytotoxicity has been shown to determine resistance to virus persistence and demyelination in this model, virus-specific cytotoxicity in the CNS of DA-resistant (B6 or B10) and -susceptible (SJL/J and B10.S) mice during the acute stage of DA and DAL*-1 infection was characterized. Following intracerebral inoculation with DAL*-1, virus-specific Db- and Kb-restricted CTLs were demonstrated in the CNS of resistant B10 mice, whereas only Db-restricted CTL were found in wt-DA-inoculated mice. CTLs specific to wt-DA or DAL*-1 recognized class I-presented peptides from either of the viruses. Of particular interest, Ks-restricted virus-specific cytotoxicity-restricted CTLs were identified in the CNS of susceptible SJL/J (H-2s) and B10.S (H-2s) mice inoculated with DAL*-1. In contrast, no virus-specific CTLs were identified in the CNS of SJL/J and B10.S mice inoculated with wt-DA. We propose that L* inhibits the generation of H-2K-restricted virus-specific cytotoxicity in the CNS, permitting a persistent infection in susceptible strains, with subsequent inflammatory demyelination in the CNS similar to that in human multiple sclerosis.
CC-Chemokine Receptor 6 Is Expressed on Diverse Memory Subsets of T Cells and Determines Responsiveness to Macrophage Inflammatory Protein 3α
Authors: Liao Fang , Rabin Ronald L. , Smith Craig S. , Sharma Geetika , Nutman Thomas B. , Farber Joshua M.
Published: December 2022

CC-chemokine receptor (CCR) 6 is the only known receptor for macrophage inflammatory protein (MIP)-3α, a CC chemokine chemotactic for lymphocytes and dendritic cells. Using anti-serum that we raised against the N-terminal residues of CCR6, we have characterized the surface expression of CCR6 on peripheral blood leukocytes and we have correlated CCR6 expression with responses to MIP-3α. We found that CCR6 was expressed only on memory T cells, including most α4β7 memory cells and cutaneous lymphocyte-associated Ag-expressing cells, and on B cells. Accordingly, chemotaxis of T cells to MIP-3α was limited to memory cells. Moreover, calcium signals on T cells in response to MIP-3α were confined to CCR6-expressing cells, consistent with CCR6 being the only MIP-3α receptor on peripheral blood T cells. Unlike many CC chemokines, MIP-3α produced a calcium signal on freshly isolated T cells, and CCR6 expression was not increased by up to 5 days of treatment with IL-2 or by cross-linking CD3. Despite their surface expression of CCR6, freshly isolated B cells did not respond to MIP-3α. In addition to staining peripheral blood leukocytes, our anti-serum detected CCR6 on CD34+ bone marrow cell-derived dendritic cells. Our data are the first to analyze surface expression of CCR6, demonstrating receptor expression on differentiated, resting memory T cells, indicating differences in receptor signaling on T cells and B cells and suggesting that CCR6 and MIP-3α may play a role in the physiology of resting memory T cells and in the interactions of memory T cells, B cells, and dendritic cells.
Presence or Absence of TGF-β Determines IL-4-Induced Generation of Type 1 or Type 2 CD8 T Cell Subsets
Authors: Erard Francois , Garcia-Sanz Jose A. , Moriggl Richard , Wild Marie-Therese
Published: December 2022

CD8+ T cells often differentiate into highly cytotoxic cells, secreting a Th1-like or type 1 cytokine pattern characterized by the production of IFN-γ. However, cytotoxic, and in some reports, noncytotoxic, type 2 cells that secrete IL-4, IL-5, or IL-10 instead of IFN-γ, can be generated when CD8+ T cells are primed in the presence of IL-4. Here, we show that IL-4 can also generate typical CD8 type 1 responses. Indeed, while presence of TGF-β biases the development of CD8 T cells that, then, produce little cytolytic activity and IFN-γ, addition of IL-4 results in the recovery of cytotoxicity and IFN-γ production. The cooperative effects of TGF-β and IL-4 imply dual functions, not only for IL-4, but also for TGF-β. Indeed, depending on the presence or absence of IL-4, TGF-β either inhibits or induces the generation of type 1 CD8+ T cells. Physiologically, the ratio of local IL-4/TGF-β concentration may therefore be a critical element in determining the outcome of T cell responses to pathogen and autoantigens. It allows CD8 T cells to switch from an immunotolerant state in the presence of only TGF-β or IL-4, to an immunocompetent proinflammatory type 1 state in the absence or presence of both cytokines.
TCR Activation Inhibits Chemotaxis Toward Stromal Cell-Derived Factor-1: Evidence for Reciprocal Regulation Between CXCR4 and the TCR
Authors: Peacock James W. , Jirik Frank R.
Published: December 2022

Stromal cell-derived factor-1 (SDF-1), a C-X-C family chemokine, is a potent T lymphocyte chemoattractant. We investigated the effects of T cell activation on the chemotactic response to SDF-1. Anti-CD3 Ab stimulation of either Jurkat T cells or murine peripheral CD4+ T lymphocytes produced a dramatic inhibition of SDF-1-induced chemotaxis. In contrast, the SDF-1 responses of Jurkat clones with deficiencies in key TCR signaling components (Lck, CD45, and TCR-β), were only marginally reduced by anti-CD3 stimulation. Similar to PMA treatment, which abolished both CXCR4 receptor expression and the chemotactic response of Jurkat cells to SDF-1, anti-CD3 Ab treatment reduced cell surface expression of CXCR4 to 65% of the control value, an effect that was blocked by protein kinase C inhibitors. Our data suggest that initial T cell activation events inhibit the response of Jurkat T cells to CXCR4 stimulation. In contrast, SDF-1 treatment resulted in a reduction of tyrosine phosphorylation of the TCR downstream effectors, ZAP-70, SLP-76, and LAT (linker for activation of T cells), suggesting that this chemokine potentially regulates the threshold for T cell activation.
The First Dose of aHaemophilus influenzaeType b Conjugate Vaccine Reactivates Memory B Cells: Evidence for Extensive Clonal Selection, Intraclonal Affinity Maturation, and Multiple Isotype Switches to IgA2
Authors: Hougs Lotte , Juul Lars , Ditzel Henrik J. , Heilmann Carsten , Svejgaard Arne , Barington Torben
Published: December 2022

The Ab response of a healthy adult to the first dose of a Haemophilus influenzae type b capsular polysaccharide (HibCP) conjugate vaccine was studied at the level of Ig gene usage by circulating Ab-secreting cells. Forty-one IgA and 17 IgG mRNA sequences were obtained. The major part of the response was confined to IgA Ab-secreting cells, and 72% of the IgA sequences were derived from the progeny of a single rearranged B cell. These sequences could be arranged in a genealogical tree showing multiple somatic mutations and at least two intraclonal isotype switches to IgA2. Fourteen somatic mutations were shared by this clonal progeny, indicating that extreme clonal selection had occurred early in the clonal development. Taking into account the frequency of somatic mutations and the clone size, it was evident that the responding cell population must have originated from a mutated, highly selected, and expanded population of cells existing before vaccination, i.e., memory B cells. The dominating heavy and light chains of the response were combined in a Fab that bound HibCP. It was shown that the shared heavy and light chain mutations increased the affinity for HibCP considerably, indicating that the clonal selection had been driven by affinity. Pre-existing memory cells in unvaccinated adults may explain several features of Ab responses to polysaccharide vaccines and may play a role in acquiring the ability to respond to pure polysaccharides during infancy.
Differential Effects of Type I IFNs on the Growth of WC1− CD8+ γδ T Cells and WC1+ CD8− γδ T Cells In Vitro
Authors: Tuo Wenbin , Bazer Fuller W. , Davis William C. , Zhu Daming , Brown Wendy C.
Published: December 2022

Type I IFNs have a broad array of immunoregulatory functions that include up-regulation of type 1 immune responses through enhancing differentiation and activation of CD8+ T cells and CD4+ Th1 cells. Ovine trophoblast IFN-τ is a recently described type I IFN with the potential for therapeutic use, based on its potent antiviral activity yet low toxicity. Studies were designed to determine the immunoregulatory effects of IFN-τ on Ag-stimulated T cells, and a novel effect of type I IFNs on γδ T cells was observed. In cultures of parasite Ag-stimulated bovine T cells that contained a mixture of αβ and γδ T cells, both IFN-τ and IFN-α suppressed the expansion of WC1+ CD2− CD6− CD8− γδ T cells, yet stimulated the growth of WC1− CD2+ CD6+ CD8+ γδ T cells and CD8+ αβ T cells. The CD8+ γδ T cell subset expressed high levels of the IL-2R α-chain. Furthermore, we showed that type I IFN enhanced IL-2 production by these Ag-stimulated T cell lines. In short term cultures of PBMC, IL-2 stimulated an expansion of WC1− CD6+ CD8+ γδ T cells, which was significantly increased by IFN-τ, even though IFN-τ alone did not support cell survival. These studies demonstrate for the first time that type I IFNs differentially modulate the proliferation of different subsets of γδ T cells, which appears to act in part via IL-2.
Intranasal Immunization with Plasmid DNA-Lipid Complexes Elicits Mucosal Immunity in the Female Genital and Rectal Tracts
Authors: Klavinskis Linda S. , Barnfield Christina , Gao Liquan , Parker Suezanne
Published: December 2022

The development of vaccines against pathogens transmitted across the genito-rectal mucosa that effectively stimulate both secretory IgA Abs and cytotoxic T lymphocytes in the genital tract and CTL in the draining lymph nodes (LN) has proven a major challenge. Here we report a novel, noninvasive approach of genetic vaccination via the intranasal route. Such vaccination elicits immune responses in the genital and rectal mucosa, draining LNs, and central lymphoid system. Intranasal immunization with plasmid DNA-lipid complexes encoding the model Ag firefly luciferase resulted in dissemination of the DNA and the encoded transcript throughout the respiratory and gastrointestinal tracts, draining LNs, and spleen. Complexing the plasmid DNA with the lipid DMRIE/DOPE enhanced expression of the encoded protein in the respiratory tract, increased specific secretory IgA Ab in the vaginal and rectal tracts, and increased the circulating levels of specific IgA and IgG. In addition, intranasal DNA immunization resulted in generation of Ag-specific CTL that were localized in the genital and cervical LNs and spleen. These results suggest that intranasal immunization with plasmid DNA-lipid complexes may represent a generic immunization strategy against pathogens transmitted across the genito-rectal and other mucosal surfaces.
High Level Expression of the Xlr Nuclear Protein in Immature Thymocytes and Colocalization with the Matrix-Associated Region-Binding SATB1 Protein
Authors: Escalier Denise , Allenet Bénédicte , Badrichani Anne , Garchon Henri-Jean
Published: December 2022

The X-linked lymphocyte-regulated (Xlr) protein is a 30,000 Mr nuclear protein bearing homology with meiosis-specific proteins and expressed in late stage B lymphoid cell lines. In the present study we investigated its expression in the T lymphoid lineage. In adults, a high level of expression was detected in CD4−CD8− thymocytes. Most remarkably, the peak of Xlr expression occurred early during thymus cell ontogeny, precisely on days 14–15 of gestation, and was associated with the first wave of pre-T cell differentiation. Its onset preceded the rearrangement of TCR genes, as Xlr expression was conserved in thymus cells from RAG10/0 mice. The lower expression of Xlr on day 13 of fetal development, the bright Thy1+ phenotype of Xlr-positive cells, their large size, and their absence from subcapsular areas suggest that Xlr expression must be turned on within the thymus and not in prethymic precursors. From day 16 of gestation, Xlr expression decreased markedly. At birth and later, Xlrhigh cells were mostly large cells scattered throughout the cortical area. As shown by confocal microscopy, expression of Xlr closely overlapped that of SATB1, which binds special AT-rich DNA sequences associated with the nuclear matrix and plays an important regulatory role for many genes. The remarkably regulated expression of Xlr in the lymphoid cell lineage and of its homologue Xmr in the germ cell lineage suggests that they might play an important role in chromatin metabolism at critical stages of differentiation during which the genome undergoes irreversible rearrangements.
Specific Recognition of HLA-E, But Not Classical, HLA Class I Molecules by Soluble CD94/NKG2A and NK Cells
Authors: Brooks Andrew G. , Borrego Francisco , Posch Phillip E. , Patamawenu Apisit , Scorzelli Christopher J. , Ulbrecht Matthias , Weiss Elizabeth H. , Coligan John E.
Published: December 2022

The CD94/NKG2 receptors expressed by subpopulations of NK cells and T cells have been implicated as receptors for a broad range of both classical and nonclassical HLA class I molecules. To examine the ligand specificity of CD94/NKG2 proteins, a soluble heterodimeric form of the receptor was produced and used in direct binding studies with cells expressing defined HLA class I/peptide complexes. We confirm that CD94/NKG2A specifically interacts with HLA-E and demonstrate that this interaction is dependent on the association of HLA-E with peptide. Moreover, no interaction between CD94/NKG2A and classical HLA class I molecules was observed, as assayed by direct binding of the soluble receptor or by functional assays using CD94/NKG2A+ NK cells. The role of the peptide associated with HLA-E in the interaction between HLA-E and CD94/NKG2A was also assessed. All class I leader sequence peptides tested bound to HLA-E and were recognized by CD94/NKG2A. However, amino acid variations in class I leader sequences affected the stability of HLA-E. Additionally, not all HLA-E/peptide complexes examined were recognized by CD94/NKG2A. Thus CD94/NKG2A recognition of HLA-E is controlled by peptide at two levels; first, peptide must stabilize HLA-E and promote cell surface expression, and second, the HLA-E/peptide complex must form the ligand for CD94/NKG2A.
A NF-κB/c-myc-Dependent Survival Pathway Is Targeted by Corticosteroids in Immature Thymocytes
Authors: Wang Weihong , Wykrzykowska Joanna , Johnson Todd , Sen Ranjan , Sen Jyoti
Published: December 2022

Glucocorticoid hormones modulate T cell maturation in vivo. While low levels of hormones are required for appropriate T cell development, high levels of glucocorticoid hormones target immature developing thymocytes for cell death during systemic stress. In this report, we propose a molecular mechanism for the induction of apoptosis in CD4+CD8+ double-positive thymocytes by dexamethasone in vivo. Dexamethasone injection induced the expression of IκBα and IκBβ in thymocytes and down-regulated NF-κB DNA binding activated by intrathymic signals. Down-regulation of NF-κB DNA binding preceded cell death, suggesting that NF-κB may be important for the survival of immature thymocytes. In addition, ex vivo treatment of thymocyte single-cell suspension with dexamethasone accelerated p65/RelA down-regulation and cell death. Conversely, NF-κB induction diminished dexamethasone-induced death. Expression of the c-myc proto-oncogene, a NF-κB target, was also reduced in thymocytes of dexamethasone-treated animals, and ectopic transgenic expression of c-myc in mice provided partial rescue of double-positive thymocytes from dexamethasone mediated cell death. These observations suggest that viability of CD4+CD8+ thymocytes may be maintained by an NF-κB/c-myc-dependent pathway in vivo.
Optimal Vaccination AgainstSchistosoma mansoniRequires the Induction of Both B Cell- and IFN-γ-Dependent Effector Mechanisms
Authors: Jankovic Dragana , Wynn Thomas A. , Kullberg Marika C. , Hieny Sara , Caspar Patricia , James Stephanie , Cheever Allen W. , Sher Alan
Published: December 2022

Mice immunized with radiation-attenuated cercariae of Schistosoma mansoni display resistance to challenge infection, which increases with multiple boosting. Protection in animals receiving a single vaccination is thought to involve a primarily cell-mediated, IFN-γ-dependent mechanism, while humoral immunity has been shown to contribute to challenge rejection in multiply (three times) immunized mice. To better understand the respective contribution of the B lymphocyte- and IFN-γ-dependent effector arms in host resistance, we compared vaccine-induced immunity in B cell-deficient (μMT) and IFN-γ knockout (GKO) animals. Unexpectedly, after a single vaccination, B cell knockout (KO) mice displayed reduced protection against challenge infection, although they developed a normal IFN-γ-dominated cytokine response. This defect in resistance was equivalent to that displayed by GKO animals. Moreover, whereas two additional vaccinations significantly increased the level of immunity in wild-type mice, the protection in B cell KO animals remained unchanged. In contrast, multiple vaccination resulted in increased but, nevertheless, defective resistance in GKO mice. Since FcR γ KO mice, which lack functional FcγRI, FcγRIII, and FcεRI, show no defects in vaccine-induced resistance after immunization either one or three times, the B cell-dependent mechanism of protection involved does not appear to require FcR signaling. Together, these findings indicate that effective vaccination against schistosomes depends on the simultaneous induction of both humoral and cell-mediated immunity, a conclusion that may explain the limited success of most subunit vaccine protocols designed to preferentially induce either B cell- or IFN-γ-dependent protective mechanisms.
Androgens Alter the Cytokine Profile and Reduce Encephalitogenicity of Myelin-Reactive T Cells
Authors: Bebo Bruce F. , Schuster Jeanette C. , Vandenbark Arthur A. , Offner Halina
Published: December 2022

Adoptive transfer of proteolipid protein 139–151-specific T cell lines was used to examine the role of androgens in regulating T cell cytokine secretion and the severity of experimental autoimmune encephalomyelitis (EAE) in the SJL mouse. In this study, we found that T cells from female mice transferred more severe EAE than T cells from male mice and that gender differences in clinical disease were due, at least in part, to differences in donor T cell cytokine secretion. T cell lines were selected from proteolipid protein 139–151-immunized female SJL mice in the presence or absence of exogenous androgens. Androgen-selected T cell lines secreted less IFN-γ and more IL-10 than untreated cell lines. Clinical disease induced by the adoptive transfer of androgen-selected T cell lines was less severe than disease induced with untreated T cell lines. Furthermore, androgen treatment of naive TCR transgenic T cells, during their first encounter with Ag, resulted in a shift in the balance of Th1/Th2 cytokines. This phenotype was maintained during subsequent stimulations in the absence of androgen. These results suggest that androgen present in the lymphoid microenvironment during the induction of an immune response can alter the development of effector T cells and may play an important role in governing gender differences in the immune response and susceptibility to autoimmune disorders.
μ-Surrogate Light Chain Physicochemical Interactions of the Human PreB Cell Receptor: Implications for VH Repertoire Selection and Cell Signaling at the PreB Cell Stage
Authors: Gauthier Laurent , Lemmers Bénédicte , Guelpa-Fonlupt Valérie , Fougereau Michel , Schiff Claudine
Published: December 2022

The surrogate light chain (SL) composed of the λ-like and VpreB polypeptides is organized as two Ig domains and an extra-loop structure. It associates to the μ-chain in preB cells. We have produced human VpreB, SL, two Fdμ (VH-CH1), and the two corresponding Fab-like (Fdμ-SL) recombinant proteins in baculovirus. The correctness of the general conformation of the proteins was assessed by epitope mapping and affinity measurements using a new batch of anti-VpreB mAbs. Plasmon resonance analysis showed that both VpreB and the entire SL associated with the Fdμ fragments, with Kd values of 3 × 10−8 M for VpreB-Fdμ and of 10−9 to 10−10 M, depending upon the VH, for SL-Fdμ. These results indicate that the λ-like chain, in addition to be covalently bound to the Cμ1 domain, also interacts with the VH domain. Therefore, a dual role of the SL emerges: 1) interaction of the C-domain of λ-like would release the μ-chain from its interaction with binding protein in the endoplasmic reticulum, and 2) interaction of a part of λ-like and most of VpreB would bind to VH, ensuring a “quality control” of the native heavy chain that represents the first step of selection of the B cell repertoire. We also demonstrated that two Fab-like fragments did not interact with each other, suggesting that activation of the cell surface preB receptor does not involve aggregation neither in cis nor in trans of the Fab-like structures.
Triggering of Chloride Ion Efflux from Human Neutrophils as a Novel Function of Leukocyte β2 Integrins: Relationship with Spreading and Activation of the Respiratory Burst
Authors: Menegazzi Renzo , Busetto Sara , Decleva Eva , Cramer Rita , Dri Pietro , Patriarca Pierluigi
Published: December 2022

PMN residing on immobilized fibronectin have been shown to respond to TNF with an intense and long lasting Cl− efflux that leads to a marked decrease of the unusually high basal Cl− content of these phagocytes. The finding that this Cl− efflux depends, at least in part, on β2 integrin engagement stimulated the present investigation, which addresses the question as to whether β2 integrins per se, in the absence of PMN agonists, are able to generate signals triggering Cl− efflux. We induced β2 integrin cross-linking by plating PMN onto surface-bound mAbs directed against either the common β-chain (CD18) or the individual α-chains (CD11a, CD11b, CD11c) of LFA-1, CR3, and gp150/95. Anti-CD18 mAbs triggered a marked release of Cl− ions, which was accompanied by spreading and activation of the respiratory burst. Cross-linking of gp150/95 and LFA-1 generated the most powerful signals for the activation of Cl− efflux. The results of three independent experimental approaches, i.e., kinetic studies, use of Cl− transport inhibitors, and modulation of Cl− efflux with different amounts of anti-β2 integrin mAbs, indicated that Cl− efflux regulates both spreading and respiratory burst triggered by β2 integrin cross-linking. Cl− efflux appears to be independent on either alterations of [Ca2+]i or changes in the plasma membrane potential and shows sensitivity to a raise in pHi. This study uncovers a new signaling ability of β2 integrins and contributes to highlight the role of Cl− efflux in the outside-in signal transduction pathway regulating adherence-dependent PMN responses.
Complement-Mediated Anti-HIV-1 Effect Induced by Human IgM Monoclonal Antibody Against Ganglioside GM2
Authors: Wu Xiaoshan , Okada Noriko , Momota Hiroshi , Irie Reiko F. , Okada Hidechika
Published: December 2022

HIV-infected cells aberrantly express a high level of antigenic glycosidic structures such as GM2 and Gg4. Some normal sera containing natural IgM Abs to GM2 and/or Gg4 cause C-mediated cytolysis of HIV-infected cells. In the present study we demonstrated that a human IgM anti-GM2 mAb (L55 Ab) can induce cytolysis of HIV-infected cells. Increased GM2 expression by HIV-1 infection of a human T cell line (MOLT4), a human monocyte cell line (U937), and human lymphoblastoid cells was confirmed by immunofluorescence staining with L55 Ab. These infected cells were readily lysed by L55 Ab in the presence of fresh human serum as a C source that alone did not cause cytolysis. L55 Ab also had the ability to destroy HIV-1 particles via C-mediated lysis. By adding L55 Ab together with human C to mixed culture of HIV-infected cells and naive cells, HIV-1 replication was significantly suppressed, and this effect was synergistic when L55 Ab was combined with a reverse transcriptase inhibitor and a proteinase inhibitor. Therefore, a human IgM anti-GM2 mAb may be effective in treating HIV-infected patients, especially when used together with chemotherapeutic agents.
Selective Induction of Protective MHC Class I-Restricted CTL in the Intestinal Lamina Propria of Rhesus Monkeys by Transient SIV Infection of the Colonic Mucosa
Authors: Murphey-Corb Michael , Wilson Lawrence A. , Trichel Anita M. , Roberts Donald E. , Xu Keyu , Ohkawa Susumu , Woodson Bruce , Bohm Rudolf , Blanchard James
Published: December 2022

The identification of mucosal immune responses required for protection against sexual transmission of HIV is essential for the development of an efficacious vaccine. To gain a better understanding of these responses, we have characterized the immune responses in the lamina propria (LP) and epithelium of the jejunum, the mesenteric lymph nodes, and peripheral blood (PBMC) of 11 rhesus monkeys following colonic exposure to two molecular clones of SIV. Two monkeys had no signs of infection. Three monkeys became persistently infected. Transient infections, characterized by the sporadic detection of virus in the periphery and/or detection of SIV-specific immune responses in either the gut-associated tissues or PBMC, were induced in six of the monkeys. One persistently infected and three transiently infected monkeys had high levels of SIV env-specific MHC class I restricted CTL in the jejunal LP. Another transiently infected monkey had SIV-specific IgA secreting B cells in the LP. Three or six months postexposure, these animals and four naive controls were challenged intracolonically with the heterologous primary isolate, SIV/DeltaB670. All four monkeys with strong SIV env-specific MHC-restricted CTL in the LP were protected, whereas none of the naive controls or the remaining seven monkeys with little or no CTL in the LP were protected. These experiments provide the first direct evidence that transient mucosal infection can induce SIV-specific immunity that remains localized to the gut-associated tissues. Furthermore, a strong correlation between SIV env-specific MHC-restricted CTL in the LP and protection against colonic mucosal challenge was observed.