bluuebunny/crossref_metadata_2025 huggingface dataset:
Intercellular adhesion molecule-1 deficiency protects MRL/MpJ-Fas(lpr) mice from early lethality.
Authors: Bullard D C , King P D , Hicks M J , Dupont B , Beaudet A L , Elkon K B
Published: January 2023

MRL/MpJ-Fas(lpr) (Fas(lpr)) mice develop a rapidly fatal form of systemic autoimmune disease characterized by glomerulonephritis and vasculitis similar to severe cases of systemic lupus erythematosus in humans. To evaluate the requirement for intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of tissue injury in this model, we created ICAM-1-deficient MRL/MpJ-Fas(lpr) (ICAM-1/Fas(lpr)) mice. ICAM-1 deficiency resulted in a striking improvement in the survival of Fas(lpr) mice (median +/- SEM survival of Fas(lpr) = 26 +/- 1.7 vs ICAM-1/Fas(lpr) = 47 +/- 2.4 wk, p < 0.0001) and the increased survival was associated with delayed elevations of blood urea nitrogen levels in the ICAM-1/Fas(lpr) mice. Histologic examination of the ICAM-1/Fas(lpr) mice revealed an overall reduction in glomerular disease and a significant reduction in vasculitis in the kidney, lung, skin, and salivary glands when compared with Fas(lpr). These findings indicate that ICAM-1 plays a major role in development of glomerular and vascular injury in Fas(lpr) mice.
Expression of Bright at Two Distinct Stages of B Lymphocyte Development
Authors: Webb Carol F. , Smith Elizabeth A. , Medina Kay L. , Buchanan Kent L. , Smithson Glennda , Dou Shenshen
Published: January 2023

The B cell regulator of Ig heavy chain transcription (Bright) is a DNA-binding protein that was originally discovered in a mature Ag-specific B cell line after stimulation with IL-5 and Ag. It binds to the intronic heavy chain enhancer and 5′ of the V1 S107 family VH promoter. Several studies suggested that Bright may increase transcription of the heavy chain locus, and expression in cell lines was limited to those representing mature B cells. We have now analyzed normal hemopoietic tissues for the expression of Bright during B lymphocyte differentiation. We expected to find Bright expression in a subset of mature spleen cells, but also observed Bright in a subset of normal B lymphocytic progenitors in both adult bone marrow (BM) and in fetal liver as early as day 12 of gestation. Bright was also expressed in the small percentage of CD4low cells in the thymus that are newly arrived from the BM and are not yet committed to the T lymphocyte lineage, but was not observed at later stages of T cell differentiation in either the spleen or thymus. Bright mRNA was not detected in the immature B lymphocytes that initially populate the spleen after migration from the BM. In addition, new splice variants of Bright were observed in fetal tissues. Thus, Bright expression is highly regulated in normal murine lymphocytes and occurs both early and late during B cell differentiation. These findings may have important implications for the function of Bright in regulating Ig transcription.
3′ IgH Enhancer Elements Shift Synergistic Interactions During B Cell Development
Authors: Ong Jane , Stevens Sean , Roeder Robert G. , Eckhardt Laurel A.
Published: January 2023

IgH gene expression is tightly controlled over the course of B cell development, B cell activation, and the subsequent differentiation of these cells into Ig-secreting plasmacytes. There are several transcriptional enhancers that map within and downstream of the IgH locus, and some of these have been clearly implicated in the developmental regulation of IgH gene assembly and expression. While some of the individual enhancers from this locus have been studied extensively, the functional interactions possible among this group of enhancers have been largely unexplored. In the present study, we have measured the transcriptional activities of combinations of enhancers introduced into B-lineage cell lines at several different developmental stages. We detected a developmental progression in which the 3′ enhancers are initially inactive, then become strongly active through synergistic interactions, and finally achieve a strong level of activity with little interdependency. The relative contributions of Eμ (the intron enhancer) and of the 3′ enhancers also change as a function of developmental stage. We discuss these results in light of parallel studies of developmental changes in transcription factor requirements.
IFN-γ-Deficient Mice Develop Severe Granulomatous Experimental Autoimmune Thyroiditis with Eosinophil Infiltration in Thyroids
Authors: Tang Haiwen , Sharp Gordon C. , Peterson Karin P. , Braley-Mullen Helen
Published: January 2023

To study the role of IFN-γ in the development of granulomatous experimental autoimmune thyroiditis (EAT), DBA1 mice with a disrupted IFN-γ gene were used for adoptive EAT induction. Effector cells from either IFN-γ+/+ or IFN-γ−/− donor mice activated with mouse thyroglobulin and anti-IL-2R mAb induced severe granulomatous EAT. A predominant infiltration of the thyroid by eosinophils was observed in recipients of IFN-γ−/− effector cells but not in recipients of IFN-γ+/+ cells. Compared with wild-type mice, thyroids of recipients of IFN-γ−/− effector cells had decreased expression of mRNA for Th1 cytokines and inducible nitric oxide synthetase. Expression of Th2 cytokine mRNA was comparable to that of IFN-γ+/+ mice, and expression of eotaxin was increased in the thyroids of recipients of IFN-γ−/− effector cells. Activation of cells from either IFN-γ+/+ or IFN-γ−/− donors in the presence of IL-12 also induced severe granulomatous EAT. Eosinophil infiltration in recipients of IFN-γ−/− cells was unaffected when effector cells were activated with IL-12, and thyroids expressed predominantly Th2 cytokines. The extent of fibrosis of recipient thyroids was generally greater when donor IFN-γ+/+ and IFN-γ−/− cells were activated with IL-12. Compared with IFN-γ+/+ mice, IFN-γ−/− mice produced lower levels of mouse thyroglobulin-specific autoantibodies after immunization with MTg and LPS. These results indicate that cells from both IFN-γ+/+ and IFN-γ−/− donors can induce severe granulomatous EAT. However, damage of thyroid follicles by IFN-γ−/− and that by IFN-γ+/+ cells appear to involve different mediators of inflammation.
The Role of Two Distinct Endothelial Molecules, Vascular Adhesion Protein-1 and Peripheral Lymph Node Addressin, in the Binding of Lymphocyte Subsets to Human Lymph Nodes
Authors: Salmi Marko , Hellman Jukka , Jalkanen Sirpa
Published: January 2023

Lymphocyte binding to high endothelial venules (HEV) in noninflamed peripheral lymph nodes (PLN) relies heavily on two endothelial adhesion molecules called vascular adhesion protein-1 (VAP-1) defined by mAb 1B2 and the peripheral lymph node addressins (PNAd) defined by mAb MECA-79. Data from several different groups indicate that these two molecules share several characteristics in expression, biochemical structure, and function, raising the possibility that VAP-1 may be identical to the 170- and 90-kDa species of PNAd glycoproteins. In this study, we show that many PLN HEV coexpress these two molecules. In parallel SDS-PAGE analyses, the m.w. of the 90- and 170-kDa forms of these molecules are indistinguishable. Nevertheless, we show by different metabolic labelings, by reciprocal cross-precipitations, and by immunofluorescence stainings of newly established VAP-1 transfectants that the 90- and 170-kDa species of PNAd and VAP-1 are distinct molecules. In functional terms, VAP-1 is strikingly selective in mediating PLN HEV adhesion of CD8-positive, but not of CD4-positive T cells. In contrast, PNAd contributes to the adhesion of both CD4-positive and CD8-positive cells to these vessels. Together, these data show that initial adhesion of CD8-positive lymphocytes to PLN HEV requires a PNAd- and a VAP-1-dependent step that are both essential and may occur simultaneously or sequentially.
Cutting Edge: Role of Macrophage Migration Inhibitory Factor in Inhibiting NK Cell Activity and Preserving Immune Privilege
Authors: Apte R. S. , Sinha D. , Mayhew E. , Wistow G. J. , Niederkorn J. Y.
Published: January 2023

The absence of MHC class I Ags on the corneal endothelium, which lines the anterior chamber of the eye, makes this cell layer potentially vulnerable to lysis by NK cells. However, aqueous humor (AH), which bathes the corneal endothelium, contains a 12-kDa protein which inhibits the NK-mediated lysis of corneal endothelial cells. An amino acid sequence analysis of AH revealed that this factor shared >90% homology with macrophage migration inhibitory factor (MIF). The NK inhibitory effect of AH was neutralized with anti-human MIF Ab. Moreover, mouse rMIF produced a similar inhibition of NK cell activity. However, neither rMIF nor AH inhibited the CTL-mediated lysis of allogeneic cells. rMIF prevented the release of perforin granules by NK cells but not CTLs. Although MIF displays proinflammatory properties, these results indicate that it can also inhibit at least one immune effector element, NK cells, and thereby contribute to immune privilege in the eye.
Inefficient Assembly and Intracellular Accumulation of Antibodies with Mutations in VH CDR2
Authors: Martin Tammy M. , Wiens Gregory D. , Rittenberg Marvin B.
Published: January 2023

We previously described secretion defects in four mutants of the murine anti-phosphocholine Ab, T15. The mutant heavy (H) chains had amino acid replacements in the VH complementarity-determining region 2 (HCDR2) and were expressed at normal intracellular levels. Here, the intracellular fate of the secretion-defective mutant heavy chains was investigated. Metabolic labeling demonstrated that the T15 wild-type Ab was secreted within a 4-h chase. In contrast, the mutant H chains accumulated with intracellular t1/2 values ranging from 10 to 24 h. The mutant H chains were associated with increased levels of the molecular chaperones BiP and GRP94, and remained endoglycosidase H sensitive, suggesting retention in the endoplasmic reticulum. Assembly of the mutant H chains with T15 light (L) chain was arrested at the H2 and H2L intermediate stages of the T15 wild-type pathway (H2 → H2L → H2L2). Even though some assembly with L chain occurred, it was not as a secretion-competent H2L2 Ig moiety. The T15 L chains coexpressed with mutant H chains were degraded efficiently except for a minor L chain population with a long t1/2 that was apparently protected at the H2L stage. To our knowledge, this is the first study demonstrating that intracellular half-lives of Ig H and L chains can be influenced by somatic mutations in HCDR2.
The α2 Domain of H-2Dd Restricts the Allelic Specificity of the Murine NK Cell Inhibitory Receptor Ly-49A
Authors: Sundbäck Jonas , Nakamura Mary C. , Waldenström Margareta , Niemi Eréne C. , Seaman William E. , Ryan James C. , Kärre Klas
Published: January 2023

Mouse NK lymphocytes express Ly-49 receptors, which inhibit cytotoxicity upon ligation by specific MHC I molecules on targets. Different members of the lectin-like mouse Ly-49 receptor family recognize distinct subsets of murine H-2 molecules, but the molecular basis for the allelic specificity of Ly-49 has not been defined. We analyzed inhibition of natural killing by chimeric MHC I molecules in which the α1, α2, or α3 domains of the Ly-49A-binding allele H-2Dd were exchanged for the corresponding domains of the nonbinding allele H-2Db. Using the Ly-49A-transfected rat NK cell line, RNK-mLy-49A.9, we demonstrated that the H-2Dd α2 domain alone accounts for allelic specificity in protection of rat YB2/0 targets in vitro. We also showed that the H-2Dd α2 domain is sufficient to account for the allele-specific in vivo protection of H-2b mouse RBL-5 tumors from NK cell-mediated rejection in D8 mice. Thus, in striking contrast to the α1 specificity of Ig-like killer inhibitory receptors for human HLA, the lectin-like mouse Ly-49A receptor is predominantly restricted by the H-2Dd α2 domain in vitro and in vivo.
Suppression of Immune Responses by CD8 Cells. I. Superantigen-Activated CD8 Cells Induce Unidirectional Fas-Mediated Apoptosis of Antigen-Activated CD4 Cells
Authors: Noble Alistair , Pestano Gary A. , Cantor Harvey
Published: January 2023

Stimulation of mature CD4 cells through the TCR induces cellular activation and expansion that are often followed by clonal elimination by a form of apoptosis3 termed activation-induced cell death. This process of CD4 cell apoptosis is generally thought to reflect clonal suicide and to be independent of other cell types. Here we show that during the response to the superantigen Staphylococcal enterotoxin A, activated CD8 cells, but not activated CD4 cells, suppress the CD4 proliferative response. Suppression by CD8 cells reflects their ability to induce CD4 cell apoptosis via ligation of Fas. Moreover, although activated CD8 cells that express Fas ligand and Fas eliminate CD4 cells through a Fas-dependent mechanism, they are themselves resistant to Fas-dependent apoptosis. These findings indicate a fundamental difference between the two major T cell subsets with regard to sensitivity to Fas-dependent apoptosis, expression of Fas ligand, and mediation of suppressive activity following immunization with superantigen.
Blockade of Chemokine Activity by a Soluble Chemokine Binding Protein from Vaccinia Virus
Authors: Alcamí Antonio , Symons Julian A. , Collins Paul D. , Williams Timothy J. , Smith Geoffrey L.
Published: January 2023

Chemokines direct migration of immune cells into sites of inflammation and infection. Chemokine receptors are seven-transmembrane domain proteins that, in contrast to other cytokine receptors, cannot be easily engineered as soluble chemokine inhibitors. Poxviruses encode several soluble cytokine receptors to evade immune surveillance, providing new strategies for immune modulation. Here we show that vaccinia virus and other orthopoxviruses (cowpox and camelpox) express a secreted 35-kDa chemokine binding protein (vCKBP) with no sequence similarity to known cellular chemokine receptors. The vCKBP binds CC, but not CXC or C, chemokines with high affinity (Kd = 0.1–15 nM for different CC chemokines), blocks the interaction of chemokines with cellular receptors, and inhibits chemokine-induced elevation of intracellular calcium levels and cell migration in vitro, thus representing a soluble inhibitor that binds and sequesters chemokines. The potential of vCKBP as a therapeutic agent in vivo was illustrated in a guinea pig skin model by the blockade of eotaxin-induced eosinophil infiltration, a feature of allergic inflammatory reactions. Furthermore, vCKBP may enable the rational design of antagonists to neutralize pathogens that use chemokine receptors to initiate infection, such as HIV or the malarial parasite.
NK Cells from Human MHC Class I (HLA-B) Transgenic Mice Do Not Mediate Hybrid Resistance Killing Against Parental Nontransgenic cells
Authors: Kung Sam K. P. , Su Ruey-Chyi , Graham Jeremy J. K. , Chamberlain John W. , Miller Richard G.
Published: January 2023

We have investigated the capacity of human MHC class I HLA-B gene products, HLA-B27, -B7 (fully human), and -B7Kb (human-mouse hybrid consisting of the α1 and α2 domains of HLA-B7, and the α3 and cytoplasmic domains of mouse H-2Kb), expressed on mouse NK cells during ontogeny to influence NK recognition of otherwise syngeneic mouse target cells. Despite a high level of surface expression of the transgene (comparable to that of endogeneous H-2DbKb molecules), the direct killing of YAC-1 targets, and the killing of P815 targets in a redirected lysis assay, the NK effectors of these transgenic mice could not mediate hybrid resistance-like killing of nontransgenic C57BL/6 target cells either in vitro or in vivo. Splenocytes from B6-B27 mice could be used to generate CTL lines against a B27-binding peptide, implying that T cells restricted by HLA-B27 developed during ontogeny. NK cells from B6-B27 could lyse B6-B27 Con A lymphoblasts pulsed with Db-binding peptide but not B27-binding peptides. Taken together, our results show that these human HLA-B transgene products cannot function as class I MHC “self” elements for mouse NK cells, even when present throughout ontogeny.
Nuclear Targeted Suppression of NF-κB Activity by the Novel Quinone Derivative E3330
Authors: Hiramoto Masaki , Shimizu Noriaki , Sugimoto Kotaro , Tang Jianwei , Kawakami Yutaka , Ito Masaharu , Aizawa Shin , Tanaka Hirotoshi , Makino Isao , Handa Hiroshi
Published: January 2023

The activation of NF-κB consists of at least three steps: degradation of IκBα, translocation of NF-κB into the nucleus, and post-translational modification of NF-κB (e.g., phosphorylation of p65). In the present study, we found that a novel quinone derivative E3330 selectively inhibited NF-κB-mediated gene expression without affecting any of these steps. E3330, when included in the culture medium, suppressed NF-κB DNA-binding activity in PMA-induced Jurkat cell nuclear extracts, suggesting that the inhibition by E3330 of NF-κB-mediated gene expression was due to its ability to suppress NF-κB DNA-binding activity. Fractionation of the nuclear extracts by column chromatography revealed that a nuclear factor enhanced NF-κB DNA-binding activity and that this enhancing activity was interrupted after treatment with E3330. Moreover, a major polypeptide with a molecular mass of 40 kDa was found to be in the highly purified fraction containing the NF-κB-enhancing activity and predominantly bind E3330. Taken together, these results suggest that the NF-κB activity, after dissociation from IκB, is enhanced by a nuclear factor that is active irrespective of PMA treatment, and the nuclear factor-mediated enhancement is selectively inhibited by E3330. Thus, we conclude that E3330 may belong to a novel class of anti-NF-κB drugs.
Neuropeptides, Via Specific Receptors, Regulate T Cell Adhesion to Fibronectin
Authors: Levite Mia , Cahalon Liora , Hershkoviz Rami , Steinman Lawrence , Lider Ofer
Published: January 2023

The ability of T cells to adhere to and interact with components of the blood vessel walls and the extracellular matrix is essential for their extravasation and migration into inflamed sites. We have found that the β1 integrin-mediated adhesion of resting human T cells to fibronectin, a major glycoprotein component of the extracellular matrix, is induced by physiologic concentrations of three neuropeptides: calcitonin gene-related protein (CGRP), neuropeptide Y, and somatostatin; each acts via its own specific receptor on the T cell membrane. In contrast, substance P (SP), which coexists with CGRP in the majority of peripheral endings of sensory nerves, including those innervating the lymphoid organs, blocks T cell adhesion to fibronectin when induced by CGRP, neuropeptide Y, somatostatin, macrophage inflammatory protein-1β, and PMA. Inhibition of T cell adhesion was obtained both by the intact SP peptide and by its 1–4 N-terminal and its 4–11, 5–11, and 6–11 C-terminal fragments, used at similar nanomolar concentrations. The inhibitory effects of the parent SP peptide and its fragments were abrogated by an SP NK-1 receptor antagonist, suggesting they all act through the same SP NK-1 receptor. These findings suggest that neuropeptides, by activating their specific T cell-expressed receptors, can provide the T cells with both positive (proadhesive) and negative (antiadhesive) signals and thereby regulate their function. Thus, neuropeptides may influence diverse physiologic processes involving integrins, including leukocyte-mediated migration and inflammation.
Generation of an Inhibitory Circuit Involving CD8+ T Cells, IL-2, and NK Cell-Derived TGF-β: Contrasting Effects of Anti-CD2 and Anti-CD3
Authors: Gray J. Dixon , Hirokawa Makoto , Ohtsuka Kazuo , Horwitz David A.
Published: January 2023

Although the phenomenon of immunosuppression is well established, the mechanisms involved in the generation of lymphocytes with down-regulatory activity are poorly understood. Unlike anti-CD3 antibodies, mitogenic combinations of anti-CD2 antibodies do not stimulate human PBL to produce IgM or IgG. In determining the reason for this difference, we have found that anti-CD2 triggers an inhibitory circuit facilitated by TGF-β provided by NK cells. Stimulation of PBL with anti-CD2, but not anti-CD3, generated substantial amounts of active TGF-β. NK cells were found to be a significant source of TGF-β and were the only lymphocyte population that constitutively produced this cytokine. Anti-CD2 enhanced the production of active TGF-β by purified NK cells. TGF-β. After the removal of NK cells or the addition of anti-TGF-β, anti-CD2 could stimulate Ig production. Anti-TGF-β had to be added within the first 24 h for a maximal effect. Moreover, a short, overnight exposure of CD8+ T cells to TGF-β could prime them for suppressor activity provided that IL-2 was also present. Thus, the presence of active TGF-β coincident with CD8+ T cell activation can condition these cells to mediate down-regulatory activity, and NK cells can serve as the source of this cytokine.
Decreased Production of TGF-β by Lymphocytes from Patients with Systemic Lupus Erythematosus
Authors: Ohtsuka Kazuo , Gray J. Dixon , Stimmler Mary M. , Toro Bricia , Horwitz David A.
Published: January 2023

TGF-β has marked inhibitory effects on the immune system but also serves as a costimulatory factor in the development of T cells with down-regulatory activities. This cytokine is secreted as a latent complex and converted extracellularly to its active form. We have recently learned that anti-CD2 is a potent inducer of lymphocyte-derived TGF-β and that NK cells are the predominant source. The objective of this study was to compare levels of constitutive, anti-CD2-induced and cytokine-regulated TGF-β produced by blood lymphocytes from patients with systemic lupus erythematosus (SLE) in comparison with healthy controls. Using a highly sensitive and specific bioassay to assess TGF-β, we report that unstimulated PBL from SLE patients, especially the NK cell subset, produced decreased levels of active TGF-β. In response to anti-CD2, concentrations of active and total TGF-β were also decreased in SLE. After learning that IL-2 and TNF-α enhance lymphocyte production of active TGF-β, we found that the addition of these cytokines was unable to increase active TGF-β to normal concentrations. Although we observed that IL-10 inhibited the production of active TGF-β, antagonism of this cytokine was unable to completely correct the defect. In two SLE patients with B cell hyperactivity, spontaneous IgG production was almost abolished by the combination of TGF-β and IL-2. Therefore, decreased production of each of these cytokines in SLE could be important in the perpetuation of B cell hyperactivity.
The Actin-Bundling Protein Fascin Is Involved in the Formation of Dendritic Processes in Maturing Epidermal Langerhans Cells
Authors: Ross Ralf , Ross Xiao-Lan , Schwing Jens , Längin Tina , Reske-Kunz Angelika B.
Published: January 2023

Dendritic cells (DC) are characterized by their unique potential to prime naive T cells. Epidermal Langerhans cells (LC), the DC resident in the epidermis, gain this immunostimulatory capacity following Ag contact in vivo or during in vitro culture of epidermal cell suspensions. To analyze differential gene expression in maturing LC, we constructed a highly representative cDNA library of cultivated LC (cLC) in λ ZAP II containing 18 × 106 independent clones. This library was screened with freshly isolated Langerhans cell (fLC)- and cLC-derived probes for cLC-specific cDNAs. The cDNAs identified were sequenced and analyzed by database searches. Two cDNA fragments were identified as fragments of fascin, indicating that fascin is differentially expressed in LC. By competitive RT-PCR, we confirmed that fascin is highly expressed in cLC cultivated for 1, 2, and 3 days, while no signals were obtained with fLC. Western blot and immunofluorescence analysis revealed cLC-specific expression of fascin on the protein level as well. Fascin is known to be involved in the organization of the actin cytoskeleton in cytoplasmatic extensions of nerve growth cones. Its differential expression in maturing LC coincides with the formation of numerous dendritic projections in LC. Their formation was inhibited by incubation of LC with fascin antisense oligonucleotides during cultivation. Therefore, we conclude that fascin is necessary for the formation of the dendritic processes of maturing Langerhans cells and may thus influence T cell-LC interaction.
Thymic Alterations Induced by 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Are Strictly Dependent on Aryl Hydrocarbon Receptor Activation in Hemopoietic Cells
Authors: Staples J. Erin , Murante Francis G. , Fiore Nancy C. , Gasiewicz Thomas A. , Silverstone Allen E.
Published: January 2023

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related congeners affect the immune system, causing immunosuppression and thymic atrophy in a variety of animal species. TCDD is believed to exert its effects primarily through the ligand-activated transcription factor, the aryl hydrocarbon receptor (AhR). Although the AhR is found at high levels in both thymocytes and thymic stroma, it is uncertain in which cells TCDD is activating the AhR to cause alterations in the thymus. Some investigators have suggested that stromal elements, primarily epithelial cells, within the thymus are the primary targets for TCDD. Others have suggested that atrophy is due to a direct effect on thymocytes, either by apoptosis or by altering the development of progenitor cells. By producing chimeric mice with TCDD-responsive (AhR+/+) stromal components and TCDD-unresponsive (AhR−/−) hemopoietic components, or the reverse, we have clarified the role of stromal vs hemopoietic elements in TCDD-induced thymic alterations. Our results show that the targets for TCDD-induced thymic atrophy and phenotypic alterations are strictly in the hemopoietic compartment and that TCDD activation of epithelial cells in the stroma is not required for thymic alterations. Furthermore, changes observed in the putative stem cell populations of these chimeric mice are also dependent on TCDD activation of the AhR in hemopoietic elements.
Extracellular HIV-1 Tat Protein Induces the Rapid Ser133 Phosphorylation and Activation of CREB Transcription Factor in Both Jurkat Lymphoblastoid T Cells and Primary Peripheral Blood Mononuclear Cells
Authors: Gibellini Davide , Bassini Alessandra , Pierpaoli Sabina , Bertolaso Lucia , Milani Daniela , Capitani Silvano , La Placa Michele , Zauli Giorgio
Published: January 2023

Extracellular HIV-1 Tat protein (0.1–100 ng/ml) induced a rapid (peak at 30 min) increase in the Ser133 phosphorylation levels of the transcription factor CREB in serum-starved Jurkat cells, as revealed by Western blot and indirect immunofluorescence analyses. Nuclear cAMP-responsive element (CRE) binding activity in electrophoretic mobility shift assays was constitutive in unstimulated Jurkat cells, showing only a small increase upon Tat treatment. However, transient transfection experiments performed with various chloramphenicol acetyl-transferase (CAT) constructs showed that Tat produced a fourfold induction of CAT activity only in the presence of a CRE-dependent CAT construct. Moreover, the use of plasmids encoding for GAL4-CREB fusion proteins demonstrated that Tat induction of pG4-CAT reporter gene required the CREB moiety of the GAL4-CREB fusion protein and that Ser133 CREB was essential for Tat activity. Extracellular Tat also stimulated Ser133 CREB phosphorylation in freshly isolated PBMC; this effect was completely blocked by either staurosporin, a broad-spectrum inhibitor of various protein kinases, or PD 98059, a specific inhibitor of mitogen-activated protein kinases (MAPK). Furthermore, extracellular Tat induced a rapid (peak at 5–15 min) stimulation of the MAPK catalytic activity in primary PBMC. Altogether, these findings suggest that HIV-1 Tat protein activates CREB in lymphoid cells through a signal cascade involving the MAPK pathway.
An Antagonistic IL-4 Mutant Prevents Type I Allergy in the Mouse: Inhibition of the IL-4/IL-13 Receptor System Completely Abrogates Humoral Immune Response to Allergen and Development of Allergic Symptoms In Vivo
Authors: Grunewald Susanne M. , Werthmann Antje , Schnarr Bernd , Klein C. Eberhard , Bröcker Eva B. , Mohrs Markus , Brombacher Frank , Sebald Walter , Duschl Albert
Published: January 2023

We have analyzed in vivo effects of the murine IL-4 mutant Q116D/Y119D (QY), which forms unproductive complexes with IL-4Rα and is an antagonist for IL-4 and IL-13 in vitro. Treatment of BALB/c mice with QY during immunization with OVA completely inhibited synthesis of OVA-specific IgE and IgG1. BALB/c-derived knockout mice lacking either IL-4 or IL-4Rα also did not develop specific IgE or IgG1, but mounted a much stronger IgG2a and IgG2b response than wild-type mice. In contrast, QY treatment of normal BALB/c mice suppressed specific IgG2a, IgG2b, and IgG3 synthesis, which may indicate the development of tolerance toward the allergen. Associated with the lack of IgE synthesis in QY-treated wild-type mice and in IL-4−/− mice used as a control was the failure to develop immediate cutaneous hypersensitivity or anaphylactic shock upon rechallenge. Interestingly, QY treatment also inhibited humoral immune responses and allergic reactivity in SJL/J mice, a strain that did not produce IgE, but displayed IgE-independent mast cell degranulation mediated by specific IgG1. We conclude that QY inhibits Ag-specific humoral immune responses and allergic symptoms mediated either by IgE or IgG1. It needs to be clarified how QY abrogates synthesis of IgG2a, IgG2b, and IgG3, but the induction of tolerance toward nonhazardous protein Ags should be advantageous for therapy of atopic disorders and other Th2-dominated diseases.
Only Selected Light Chains Combine with a Given Heavy Chain to Confer Specificity for a Model Glycopeptide Antigen
Authors: Czerwinski Marcin , Siemaszko Dorota , Siegel Don L. , Spitalnik Steven L.
Published: January 2023

The M and N human blood group glycopeptide Ags are carried on RBCs by glycophorin A. Previous results suggested that the murine humoral immune response against the N, but not the M, Ag is restricted. In addition, these results suggested that particular highly homologous heavy chains might be able to combine promiscuously with various light chains to yield anti-N specificity. To examine this, the current study used Fab phage methodology to couple an array of light chains, obtained from cDNA libraries isolated from immunized mice, to single Fd obtained from N61, N92, and 425/2B hybridomas. Interestingly, for the chimeric Fab to retain M or N specificity, the new light chains needed to belong to the same Vk gene family as the light chain from the parental, hybridoma-derived mAb. In some cases the new light chains modified the Fab affinity and fine specificity. For example, library-derived light chains coupled with the N92 Fd yielded chimeric Fab with increased affinity. In particular, the affinity of these univalent chimeric Fab for the N Ag was equivalent to that of the bivalent parental IgG mAb. Taken together, these results demonstrate that particular structures formed by the light chain V region are required to cooperate with a particular heavy chain V region to create a functional binding site for these glycopeptide Ags. They also demonstrate a lack of heavy chain promiscuity in the formation of murine anti-M and anti-N Abs.
Transient Gene Transfer of IL-12 Regulates Chemokine Expression and Disease Severity in Experimental Arthritis
Authors: Parks Elizabeth , Strieter Robert M. , Lukacs Nicholas W. , Gauldie Jack , Hitt Mary , Graham Frank L. , Kunkel Steven L.
Published: January 2023

Murine collagen-induced arthritis (CIA) is characterized by pannus formation, cell infiltration, and cartilage erosion, and shares histologic and immunologic features with rheumatoid arthritis. Numerous cytokines are reportedly associated with RA and/or CIA; however, their mechanistic role is not clear. To determine the role of IL-12 in CIA, DBA/1 LacJ mice were administered 3 × 108 plaque-forming units of mIL-12 i.p. in a nonreplicating adenoviral vector (AdIL-12) on day 25 following primary type II collagen immunization. Our studies demonstrated that systemic transient overexpression of IL-12 accelerated disease progression and augmented the arthritis severity relative to mice expressing a replication-deficient, E1-deleted Ad5 construct. A likely mechanism for this increase in pathology was the increase in the expression of cytokines and chemokines known to play a proinflammatory role in disease. In particular, levels of murine IFN-γ were significantly increased in mice overexpressing AdIL-12 relative to the replication-deficient, E1-deleted Ad5 construct. Interestingly, the C-X-C chemokine murine macrophage inflammatory protein-2, as well as the C-C chemokines murine monocyte chemoattractant protein-1 and murine macrophage inflammatory protein-1α were up-regulated by AdIL-12 relative to controls. In an additional set of studies, neutralization of endogenous IL-12 in CIA mice was shown to delay disease onset and attenuate disease severity. IFN-γ levels in the mice receiving anti-IL-12 were significantly decreased in joint homogenates. These studies demonstrate that IL-12 is an important cytokine involved in controlling the production of chemokines/cytokines leading to the evolution of experimental arthritis.
STAT6 Is Required for IL-4-Induced Germline Ig Gene Transcription and Switch Recombination
Authors: Linehan Leslie A. , Warren Wendy D. , Thompson Patricia A. , Grusby Michael J. , Berton Michael T.
Published: January 2023

Transcription of the germline Cγ1 and Cε Ig genes is believed to be a necessary prerequisite for isotype switching to IgG1 and IgE, respectively. IL-4 stimulation and ligation of CD40 can each independently induce low level germline γ1 and ε transcription in murine B cells. Together these signals act synergistically to promote high level germline transcription and are normally required for T-dependent isotype switching to IgG1 and IgE. The STAT6 transcription factor has been suggested to play a critical role in IL-4-induced activation of germline Cγ1 and Cε genes. To directly assess the role of STAT6 in IL-4R- and CD40-mediated germline transcription and switching, we have analyzed these events in splenic B cells from STAT6-deficient mice. Our results demonstrate that IL-4 does not induce detectable levels of germline γ1 or ε transcripts in STAT6-deficient B cells. Germline transcript expression induced by CD40 stimulation alone is unaffected, but synergism between CD40- and IL-4R-mediated signals is completely ablated. Switch recombination to Sγ1, as measured by digestion-circularization PCR, is dramatically reduced in STAT6-deficient B cells stimulated with CD40 ligand plus IL-4. Similarly, germline γ1 transcript expression and switch recombination to Sγ1 are also impaired in STAT6-deficient B cells stimulated with IL-4, IL-5, and anti-IgD Abs conjugated to dextran, a model for T-independent type II responses. These results directly demonstrate a critical role for STAT6 in the IL-4-mediated activation of germline Ig gene transcription and switch recombination in nontransformed B cells.
Differential Role of CTLA-4 in Regulation of Resting Memory Versus Naive CD4 T Cell Activation
Authors: Metz D. P. , Farber D. L. , Taylor T. , Bottomly K.
Published: January 2023

Regulation of peripheral T cell responses is critical for preserving self tolerance. Memory T cells have a lower threshold for activation through the TCR and are thought to be less dependent on costimulation than naive T cells, suggesting a requirement for more stringent regulation of memory T cells. We have recently shown that CD4 engagement apart from the TCR results in the inactivation of memory, but not naive, CD4 T cells. We show here that this inhibition requires ligation of CTLA-4, in that blocking CTLA-4-B7 interactions restores memory CD4 T cell responsiveness. Early signaling through CTLA-4 is possible because resting memory, but not naive, CD4 T cells contain intracellular stores of CTLA-4 that are continuously recycled between the cytoplasm and the cell surface. This mechanism ensures that low intensity TCR engagements, which are thought to be important for peripheral T cell longevity, do not cause memory T cell activation but instead raise their threshold for costimulatory signals. This may give memory T cells an extended lifespan with a reduced risk of inappropriate activation.
IL-7-Dependent Extrathymic Expansion of CD45RA+ T Cells Enables Preservation of a Naive Repertoire
Authors: Vieira Maria , Soares D. , Borthwick Nicola J. , Maini Mala K. , Janossy George , Salmon Mike , Akbar Arne N.
Published: January 2023

We have investigated the regulation of adult and cord blood CD45RA+ T cell proliferation and apoptosis to identify factors that may control the naive T cell pool. Cord CD45RA+ T cells were highly susceptible to spontaneous apoptosis as compared with CD45RA+ T cells from adults. Apoptosis was prevented by the addition of IL-2, IL-4, IL-7, and IL-15 which signal via the γ-chain of the IL-2 receptor. IL-7 prevented the decrease in Bcl-2 and Bcl-xL and induced cell cycling in up to 20% of cord T cells after 8 days, resulting in a threefold increase in cord T cell numbers. However, the expanded cells retained a CD45RA+CD45RO− phenotype. Similar results were obtained with adult CD45RA+ T cells. IL-7-expanded CD45RA+RO− T cells expressed CD45RO after stimulation through the TCR. Investigations into the regulation of replicative senescence showed that after 12 days in culture with IL-7, cord blood CD45RA+ T cell proliferation resulted in telomere shortening. Nevertheless, IL-7-expanded cord blood T cells still maintained longer telomeres than unstimulated adult T cells. IL-7 but not IL-2 could directly induce high telomerase activity which probably retarded the rate of telomere shortening in cord blood T cells. These results suggest that proliferation induced by IL-7 may be important for extrathymic expansion of neonatal CD45RA+ T cells and may also contribute to the maintenance of the adult CD45RA+ T cell pool.
Cutting Edge: The Tyrosine Phosphatase SHP-1 Regulates Thymocyte Positive Selection
Authors: Plas David R. , Williams Calvin B. , Kersh Gilbert J. , White Lynn S. , White J. Michael , Paust Silke , Ulyanova Tatiana , Allen Paul M. , Thomas Matthew L.
Published: January 2023

The binding kinetics of the TCR for its interacting ligand and the nature of the resulting signal transduction event determine the fate of a developing thymocyte. The intracellular tyrosine phosphatase SHP-1 is a potential regulator of the TCR signal transduction cascade and may affect thymocyte development. To assess the role of SHP-1 in thymocyte development, we generated T cell-transgenic mice that express a putative dominant negative form of SHP-1, in which a critical cysteine is mutated to serine (SHP-1 C453S). SHP-1 C453S mice that express the 3.L2 TCR transgene are increased in CD4 single positive cells in the thymus and are increased in cells that express the clonotypic TCR. These data suggest that the expression of SHP-1 C453S results in increased positive selection in 3.L2 TCR-transgenic mice and support a role for SHP-1 thymocyte development.
B Lymphocytes as Antigen-Presenting Cells for CD4+ T Cell Priming In Vivo
Authors: Constant Stephanie L.
Published: January 2023

The contribution of B lymphocytes as APCs for CD4+ T cell priming remains controversial, based on findings that B cells cannot provide the requisite ligating and costimulatory signals for naive T cells to be activated. In the current study, we have examined Ag-specific T:B cell collaboration under circumstances in which B cells take up Ag through Ig receptors in vivo. This results in their activation and an ability to effectively stimulate naive CD4+ T cells both in vitro and in vivo. The aim of this work was to establish some of the key molecular interactions, as well as kinetics, between Ag-specific T and B cells that enable this priming to take place. Our approach was to amplify the starting pools of both Ag-specific T and B cell populations in vivo to track directly the events during initial T:B cell collaborations. We show that the induction of optimal levels of T cell priming to a protein Ag requires the involvement of Ag-specific B cells. The interaction that results between Ag-specific T and B cells prevents the down-modulation of B7 costimulatory molecules usually observed in the absence of appropriate T cells. Moreover, this prevention in down-modulation is independent of CD40:CD40 ligand contact. Finally, we present data suggesting that once Ag-specific T and B cells interact, there is a rapid (1–2-h) down-regulation of antigenic complexes on the surface of the B lymphocytes, possibly to prevent them from engaging other T cells in the vicinity and therefore focus the initial interaction.
Annexin V Delays Apoptosis While Exerting an External Constraint Preventing the Release of CD4+ and PrPc+ Membrane Particles in a Human T Lymphocyte Model
Authors: Gidon-Jeangirard Carole , Hugel Bénédicte , Holl Vincent , Toti Florence , Laplanche Jean-Louis , Meyer Dominique , Freyssinet Jean-Marie
Published: January 2023

Phosphatidylserine exposure in the exoplasmic leaflet of the plasma membrane is one of the early hallmarks of cells undergoing apoptosis. The shedding of membrane particles carrying Ags testifying to their tissue origin is another characteristic feature. Annexin V, a protein of as yet unknown specific physiologic function, presents a high Ca2+-dependent affinity for phosphatidylserine and forms two-dimensional arrays at the membrane surface. In this study, we report the delaying action of annexin V on apoptosis in the CEM human T cell line expressing CD4 and the normal cellular prion protein (PrPc), two Ags of particular relevance to cell degeneration and with different attachments to the membrane. The effect of annexin V was additive to that of z-Val-Ala-Asp-fluoromethyl ketone, a potent caspase inhibitor. Annexin V significantly reduced the degree of proteolytic activation of caspase-3, and totally blocked the release of CD4+ and PrPc+ membrane particles. z-Val-Ala-Asp-fluoromethyl ketone was a more powerful antagonist of caspase-3 processing, but prevented the shedding of CD4+ vesicles only partially and had no effect on that of PrPc+ ones. These results suggest that an external membrane constraint, such as that exerted by annexin V, has important consequences on the course of programmed cell death and on the dissemination of particular Ags. In vivo, annexin V had a significant protective effect against spleen weight loss in mice treated by an alkylating agent previously shown to induce lymphocyte apoptosis.
Intraepithelial Lymphocytes Traffic to the Intestine and Enhance Resistance toToxoplasma gondiiOral Infection
Authors: Buzoni-Gatel Dominique , Debbabi Hajer , Moretto Magali , Dimier-Poisson Isabelle H. , Lepage Anne C. , Bout Daniel T. , Kasper Lloyd H.
Published: January 2023

Toxoplasma gondii Ag-primed intraepithelial lymphocytes (IEL) from the mouse intestine have been shown to be protective against an lethal parasite challenge when adoptively transferred into recipient mice. In the present study, we observed that Ag-primed IEL traffic to the intestine of naive mice following i.v. administration. Primed and CD8β+ IEL were the most efficient cells at homing to the host organ. In congenic mice, IEL migrated from intestine within several hours posttransfer. On Ag reexposure, the primed IEL return to the intestine where they enhance resistance as determined by reduction in the number of brain cysts. Treatment of recipient mice with anti-α4 and anti-αE Abs partially inhibited IEL intestinal homing. The Ab treatment dramatically impaired resistance to a subsequent oral infection. These finding indicate that lymphocyte homing is an important parameter in establishing long term immunity to recurrent infection with this parasite.
Involvement of Epitope Mimicry in Potentiation But Not Initiation of Autoimmune Disease
Authors: Rao Varada P. , Kajon Adriana E. , Spindler Katherine R. , Carayanniotis George
Published: January 2023

We have examined whether the peptide (368–381) from the murine adenovirus type 1 E1B sequence, exhibiting a high degree of homology with the known pathogenic thyroglobulin (Tg) T cell epitope (2695–2706), can induce experimental autoimmune thyroiditis (EAT) in SJL/J mice. The viral peptide was a poor immunogen at the T or B cell level and did not elicit EAT either directly or by adoptive transfer assays. Surprisingly, however, the viral peptide was highly antigenic in vitro, activating a Tg2695–2706-specific T cell clone and reacting with serum IgG from mice primed with the Tg homologue. The viral peptide also induced strong recall responses in Tg2695–2706-primed lymph node cells, and subsequent adoptive transfer of these cells into naive mice led to development of highly significant EAT. These data demonstrate that nonimmunogenic viral peptides can act as agonists for preactivated autoreactive T cells and suggest that epitope mimicry may at times play a potentiating rather than a precipitating role in the pathogenesis of autoimmune disease.
IL-18 Promotes Type 1 Cytokine Production from NK Cells and T Cells in Human Intracellular Infection
Authors: García Verónica E. , Uyemura Koichi , Sieling Peter A. , Ochoa María T. , Morita Craig T. , Okamura Haruki , Kurimoto Masashi , Rea Thomas H. , Modlin Robert L.
Published: January 2023

We investigated the role of IL-18 in leprosy, a disease characterized by polar cytokine responses that correlate with clinical disease. In vivo, IL-18 mRNA expression was higher in lesions from resistant tuberculoid as compared with susceptible lepromatous patients, and, in vitro, monocytes produced IL-18 in response to Mycobacterium leprae. rIL-18 augmented M. leprae-induced IFN-γ in tuberculoid patients, but not lepromatous patients, while IL-4 production was not induced by IL-18. Anti-IL-12 partially inhibited M. leprae-induced release of IFN-γ in the presence of IL-18, suggesting a combined effect of IL-12 and IL-18 in promoting M. leprae-specific type 1 responses. IL-18 enhanced M. leprae-induced IFN-γ production rapidly (24 h) by NK cells and in a more sustained manner (5 days) by T cells. Finally, IL-18 directly induced IFN-γ production from mycobacteria-reactive T cell clones. These results suggest that IL-18 induces type 1 cytokine responses in the host defense against intracellular infection.
CD38 Stimulation Lowers the Activation Threshold and Enhances the Alloreactivity of Cord Blood T Cells by Activating the Phosphatidylinositol 3-Kinase Pathway and Inducing CD73 Expression
Authors: Borrione Paolo , Peola Silvia , Mariani Sara , Besostri Barbara , Mallone Roberto , Malavasi Fabio , Pileri Alessandro , Massaia Massimo
Published: January 2023

We have recently described in cord blood T cells (CBTC) a novel pathway linking CD38 and CD73, two signal transducers with ecto-enzyme activity. The aim of this study was 2-fold: first, to characterize the mechanisms by which CD38 regulates CD73 expression; and second, to determine whether surface-induced CD73 modulates CBTC responses. A marked increase in CD73 expression was observed in CD38+ cells after incubation with the appropriate CD38 mAbs. The induction of CD73 was blocked by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3-K). CD38 stimulation induced tyrosine phosphorylation of the p85 regulatory subunit of PI3-K and its association with other tyrosine-phosphorylated proteins. Surface-induced CD73 was as efficient in delivering activatory signals as the CD73 constitutively expressed on adult T cells. Highly CBTC, totally unresponsive to mitogenic concentrations of plastic-immobilized CD3 mAb, proliferated vigorously when exposed to the combination of plastic-immobilized CD3 and CD73 mAbs. The reactivity to allogeneic irradiated PBMC was also significantly enhanced by CD38 stimulation and was dependent on CD73 expression. Thus, CD38 stimulation lowers the activation threshold of CBTC by the CD3/TCR complex and enhances their reactivity to allogeneic cells via activation of the PI3-kinase pathway and CD73 expression.