bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of ADP-Ribosyltransferase on Normal T Lymphocytes and Effects of Nicotinamide Adenine Dinucleotide on Their Function
Authors: Okamoto Shigefumi , Azhipa Olga , Yu Yajing , Russo Elizabeth , Dennert Gunther
Published: January 2023

ADP-ribosyltransferase (ADPRT) is a glycosylphosphatidylinositol-anchored cell surface enzyme on CTL. Expression of this enzyme correlates with suppression of CTL functions in the presence of its substrate β-nicotinamide adenine dinucleotide (NAD). To investigate the immunoregulatory importance of ADPRT on normal lymphocytes in vivo, NAD was injected into mice and the effects on cell-mediated and humoral immunity were assessed. Induction of both delayed-type hypersensitivity and CTL, but not Ab responses, are shown to be suppressed by NAD. Consistent with this, mature T cells, but not B cells or macrophages, express ADPRT and are able to ADP-ribosylate cell surface proteins. ADP-ribosylated molecules were identified as LFA-1, CD8, CD27, CD43, CD44, and CD45. Concomitant to ADP-ribosylation of these molecules, T cell trafficking to secondary lymphoid organs is suppressed by NAD. To examine whether this is due to effects of NAD on cell activation, Ag-stimulated responses were assayed in vitro. NAD is shown to inhibit induction of cell proliferation, cytotoxicity, and cytokine secretion. It is suggested that ADPRT regulates T cells on the level of transmembrane signaling via ADP-ribosylation of cell surface molecules. This effect is reported to be indirect, as it involves transmission of signals through TCRs, which are not ADP-ribosylated.
Type I IFNs Inhibit Human Dendritic Cell IL-12 Production and Th1 Cell Development
Authors: McRae Bradford L. , Semnani Roshanak Tolouei , Hayes Mark P. , van Seventer Gijs A.
Published: January 2023

We have investigated the role of type I IFNs (IFN-α and -β) in human T cell differentiation using anti-CD3 mAb and allogeneic, in vitro-derived dendritic cells (DC) as APCs. DC were very efficient activators of naive CD4+ T cells, providing necessary costimulation and soluble factors to support Th1 differentiation and expansion. Addition of IFN-αβ to DC/T cell cultures resulted in induction of T cell IL-10 production and inhibition of IFN-γ, TNF-α, and LT secretion. Diminished T cell IFN-γ production correlated with IFN-αβ-mediated inhibition of the p40 chain of the IL-12 heterodimer secreted by DC. Suppression of p40 IL-12 and IFN-γ was not due to increased levels of IL-10 in these cultures, and production of IFN-γ could be restored by exogenous IL-12. These data indicate that type I IFNs inhibit DC p40 IL-12 expression, which is required for development of IFN-γ-producing CD4+ T cells. Furthermore, when T cells were restimulated without IFN-β, these cells induced less p40 IL-12 from DC, suggesting that the functional properties of T cells may regulate DC function. Thus, IFN-αβ inhibits both IL-12-dependent and independent Th1 cytokine production and provides a mechanism for inhibition of IL-12-mediated immunity in viral infections.
TAP-Independent MHC Class I Peptide Antigen Presentation to Alloreactive CTL Is Enhanced by Target Cell Incubation at Subphysiologic Temperatures
Authors: Shi Yan , Smith Kelly D. , Lutz Charles T.
Published: January 2023

We investigated the peptide dependency of a group of CD8+ anti-HLA-B7 alloreactive CTL. The CTL killed target cells after acid denaturation of more than 98% of target cell surface peptide/MHC class I complexes. The CTL also killed TAP− HLA-B7-transfected T2 (T2B7) cells. The killing was enhanced by target cell incubation at 26°C. Despite these findings, which suggested peptide-independent allorecognition, CTL-mediated cytolysis was reduced or abolished by several point mutations affecting the HLA-B7 peptide-binding groove. Acid denaturation of HLA complexes on T2B7 cells prohibited CTL recognition. CTL recognition was restored by T2B7 cell incubation with β2-microglobulin and a single HPLC fraction containing peptides extracted from TAP+HLA-B7+ cells, but not by any of a panel of 17 synthetic HLA-B7-binding peptides. These findings indicated that CTL allorecognition was peptide specific. Sensitizing peptide was extracted from T2B7 cells only after incubation at 26°C. The amount of peptide detected in TAP+ cells was at least 10-fold and 100-fold greater than that detected in TAP− cells incubated at 26°C and at 37°C, respectively. TAP-independent peptide epitope presentation was sensitive to treatment with brefeldin A, but not sensitive to treatment with chloroquine, consistent with an endogenous peptide source. We propose that subphysiologic temperature incubation can enhance peptide/MHC class I presentation in the total absence of TAP function.
Analysis of the IFN-γ-Signaling Pathway in Macrophages at Different Stages of Maturation
Authors: Lucas David M. , Lokuta Mary A. , McDowell Mary Ann , Doan Joyce E. S. , Paulnock Donna M.
Published: January 2023

We previously demonstrated that the macrophage cell lines RAW 264.7 and WEHI-3 exhibit distinct patterns of gene expression in response to IFN-γ. This difference is controlled at the transcriptional level and results from a specific inability of the less mature WEHI-3 cells to utilize either the IFN-stimulated response element or the γ-activated sequence DNA regulatory element in response to stimulation with IFN-γ, while other aspects of IFN-γ gene induction remain intact. In the work described here, we examined the components of the IFN-γ signal transduction pathway in RAW 264.7 and WEHI-3 cells to determine whether differences in pathway components or activity exist in WEHI-3 cells that could give rise to this difference in transcriptional response. Reverse transcriptase-PCR (RT-PCR) and flow cytometric analyses indicated that the levels of IFN-γ receptor mRNA accumulation and protein expression are comparable for RAW 264.7 and WEHI-3 cells. RT-PCR and immunoblot analyses revealed that the principal components of this signaling pathway, including JAK1, JAK2, and STAT1, are present in both RAW 264.7 and WEHI-3 cells. However, analysis of STAT1 DNA-binding activity by electrophoretic mobility shift assay and of STAT1 phosphorylation by immunoblot revealed that this DNA-binding factor is active in RAW 264.7, but not in WEHI-3, cells after IFN-γ stimulation. These results demonstrate that the components of the IFN-γ signal transduction pathway are intact in WEHI-3 cells, but stimulation of these cells by IFN-γ does not result in STAT1 activation.
Interaction of HLA-E with Peptides and the Peptide Transporter In Vitro: Implications for its Function in Antigen Presentation
Authors: Ulbrecht Matthias , Modrow Susanne , Srivastava Rakesh , Peterson Per A. , Weiss Elisabeth H.
Published: January 2023

The assembly of MHC Ia molecules in the endoplasmic reticulum requires the presence of peptide ligands and β2m and is facilitated by chaperones in an ordered sequence of molecular interactions. A crucial step in this process is the interaction of the class I α-chain/β2m dimer with TAP, which is believed to ensure effective peptide loading of the empty class I molecule. We have previously demonstrated impaired intracellular transport of the class Ib molecule HLA-E in mouse myeloma cells cotransfected with the genes for HLA-E and human β2m, which is most likely attributable to inefficient intracellular peptide loading of the HLA-E molecule. We therefore analyzed the ability of HLA-E in the transfectant cell line to bind synthetic peptides by means of their ability to enhance cell surface expression of HLA-E. Peptide binding was confirmed by testing the effect on the thermostability of soluble empty HLA-E/human β2m dimers. Two viral peptides binding to HLA-E were thus identified, for which the exact positioning of the N terminus appeared critical for binding, whereas the contribution of the length of the C terminus seemed to be minor, allowing peptides as short as seven amino acids and up to 16 amino acids to exhibit considerable binding activity. Furthermore, we demonstrate that HLA-E interacts with TAP and that this interaction can be prolonged by the proteasome inhibitor N-acetyl-l-leucyl-l-leucyl-l-norleucinal, which reduces the intracellular peptide pool. The presented data indicate that HLA-E is capable of presenting peptide ligands similar to the repertoire of HLA class Ia molecules.
Transcriptional Repression of the IL-2 Gene in Th Cells by ZEB
Authors: Yasui Dag H. , Genetta Tom , Kadesch Tom , Williams Thomas M. , Swain Susan L. , Tsui Lisa V. , Huber Brigitte T.
Published: January 2023

Th1- and Th2-type cells mediate distinct effector functions via cytokine secretion in response to immunologic challenge. Precursor Th cells transcribe IFN-γ, IL-2, and IL-4 upon activation. Repeated stimulation of Th precursor cells in the presence of IL-4 leads to terminally differentiated Th2 cells that have lost the ability to transcribe the IL-2 gene. We provide evidence that repression of IL-2 gene expression in Th2 cells and partial repression in Th1 cells are mediated by ZEB, a zinc finger, E box-binding transcription factor. This factor binds to a negative regulatory element, NRE-A, in the IL-2 promoter, thereby acting as a potent repressor of IL-2 transcription.
In Situ Cancer Vaccination: An IL-12 Defective Vector/Replication-Competent Herpes Simplex Virus Combination Induces Local and Systemic Antitumor Activity
Authors: Toda Masahiro , Martuza Robert L. , Kojima Hidefumi , Rabkin Samuel D.
Published: January 2023

Intratumoral inoculation of replication-competent, attenuated herpes simplex virus (HSV) mutants inhibits tumor growth by direct cytotoxic viral replication and induction of a tumor-specific immune response. To boost the antitumor response, we describe a defective HSV vector encoding IL-12 as an adjuvant to in situ vaccination by the replication-competent HSV helper virus. The defective HSV vector system consists of defective particles containing tandem repeats of the cytokine genes (p40 and p35) in combination with a HSV helper virus. Heterodimeric IL-12 was expressed and secreted after IL-12 defective vector infection of tumor cells. In a syngeneic, bilateral established tumor model with CT26 murine colon carcinoma, unilateral intratumoral inoculation with an IL-12 defective/replication-competent HSV vector combination significantly reduced tumor growth of the inoculated and noninoculated contralateral tumors. This antitumor effect was significantly greater than with a lacZ-defective/replication-competent HSV vector combination, which itself was significantly greater than the mock inoculation. Efficacy is associated with enhancement of tumor-specific CTL activity, including specificity against the CT26 immunodominant MHC class I restricted Ag AH1, and IFN-γ production. There was no significant tumor growth inhibition after intratumoral inoculation of s.c. CT26 tumors in athymic mice. We conclude that this defective HSV vector system is an effective method for cytokine gene delivery to tumors in situ and IL-12 expression in tumors synergizes the antitumor activity mediated by the replication-competent HSV helper virus.
Cellular Mechanisms Involved in Protection Against Influenza Virus Infection in Transgenic Mice Expressing a TCR Receptor Specific for Class II Hemagglutinin Peptide in CD4+ and CD8+ T Cells
Authors: Bot Adrian , Casares Sofia , Bot Simona , von Boehmer Harald , Bona Constantin
Published: January 2023

Mice transgenic for a TCR that recognizes peptide110–120 of hemagglutinin of PR8 influenza virus in the context of MHC class II I-Ed molecules express the transgenes in both CD4+ and CD8+ T cells. We have found that these TCR-hemagglutinin (TCR-HA) transgenic mice display a significantly increased resistance to the primary infection with PR8 virus compared with the wild-type mice. The TCR-HA transgenic mice mounted significant MHC type II and enhanced MHC type I-restricted cytotoxicity as well as increased cytokine responses in both spleen and lungs after infection with PR8 virus. In contrast, the primary humoral response against PR8 virus was not significantly different from that of the wild-type mice. In vivo depletion and adoptive cell transfer experiments demonstrated that both CD4+ and CD8+ TCR-HA+ T cell subsets were required for the complete clearance of pulmonary virus following infection with a dose that is 100% lethal in wild-type mice. Whereas CD4+ TCR-HA+ T cells were necessary for effective activation and local recruitment of CD8+ T cells, CD8+ TCR-HA+ T cells showed a Th1-biased pattern and MHC type II-restricted cytotoxicity. However, in the absence of in vivo expression of MHC type I molecules on the infected cells, the protection conferred by the TCR-HA+ T cells was impaired, indicating that the enhanced MHC class I-restricted cytotoxicity due to TCR-HA+ CD4+ Th cells was a critical element for clearance of the pulmonary virus by the transgenic mice.
Analysis of Ligand-Induced and Ligand-Attenuated Epitopes on the Leukocyte Integrin α4β1: VCAM-1, Mucosal Addressin Cell Adhesion Molecule-1, and Fibronectin Induce Distinct Conformational Changes
Authors: Newham Peter , Craig Sue E. , Clark Katherine , Mould A. Paul , Humphries Martin J.
Published: January 2023

The leukocyte integrin α4β1 is a receptor for both cell surface ligands (VCAM-1 and mucosal addressin cell adhesion molecule-1 (MAdCAM-1)) and extracellular matrix components (fibronectin). Through regulated interactions with these molecules, α4β1 mediates leukocyte migration from the vasculature at sites of inflammation. Regulation of integrin activity plays a key role in controlling leukocyte-adhesive events and appears to be partly determined by changes in integrin conformation. Several mAbs that recognize ligand-induced binding site epitopes on integrins have been characterized, and a subset of these mAbs are capable of stimulating integrin-ligand binding. Conversely, some mAbs recognize epitopes that are attenuated by ligand engagement and allosterically inhibit ligand binding. To gain insight into ligand-specific effects on integrin conformation, we have examined the ability of different ligands to modulate the binding of four distinct classes (A, B1, B2, and C) of anti-α4 Abs to α4β1. VCAM-1 attenuated B (antifunctional) class epitopes via an allosteric mechanism and also allosterically inhibited the binding of the function-blocking anti-β1 mAb 13. Additional α4β1 ligands (fibronectin fragments, MAdCAM-1, and the CS1 peptide) also inhibited mAb 13-integrin binding; however, the epitopes of the class B anti-α4 mAbs were attenuated by the fibronectin fragments, but not by MAdCAM-1 or the CS1 peptide. Of the two anti-α4 class A mAbs examined, one recognized an epitope that was induced uniquely by VCAM-1. Taken together, these data suggest that overlapping but distinct binding mechanisms exist for different α4β1 ligands and that distinct conformational changes are induced upon integrin engagement by different ligands.
Leukotriene B4 Activates the NADPH Oxidase in Eosinophils by a Pertussis Toxin-Sensitive Mechanism That Is Largely Independent of Arachidonic Acid Mobilization
Authors: Lindsay Mark A. , Perkins Rosie S. , Barnes Peter J. , Giembycz Mark A.
Published: January 2023

Experiments were designed to investigate whether leukotriene (LTB4) receptors can couple directly to phospholipase A2 (PLA2) in guinea pig eosinophils and the role of endogenous arachidonic acid (AA) in LTB4-induced activation of the NADPH oxidase. LTB4 (EC50 ∼ 16 nM) and AA (EC50 ∼ 6 μM) generated hydrogen peroxide (H2O2) in a concentration-dependent manner and at an equivalent maximum rate (5–6 nmol/min/106 cells). LTB4 stimulated PLA2 over a similar concentration range that activated the NADPH oxidase, although kinetic studies revealed that the release of [3H]AA (t1/2 ∼ 2 s) preceded H2O2 generation (t1/2 > 30 s). Pretreatment of eosinophils with pertussis toxin abolished the increase in inositol(1,4,5)trisphosphate mass, [Ca2+]c, [3H]AA release, and H2O2 generation evoked by LTB4. Qualitatively identical results were obtained in eosinophils in which phospholipase C (PLC) was desensitized by 4β-phorbol 12,13-dibutyrate with the exception that [3H]AA release was largely unaffected. Additional studies performed with the protein kinase C inhibitor, Ro 31-8220, and under conditions in which Ca2+ mobilization was abolished, provided further evidence that LTB4 released [3H]AA independently of signal molecules derived from the hydrolysis of phosphatidylinositol(4,5)bisphosphate by PLC. Pretreatment of eosinophils with the PLA2 inhibitor, mepacrine, abolished LTB4-induced [3H]AA release at a concentration that inhibited H2O2 by only 36%. Collectively, the results of this study indicate that agonism of LTB4 receptors on guinea pig eosinophils mobilizes AA by a mechanism that does not involve the activation of PLC. In addition, although LTB4 effectively stimulated PLA2, a central role for AA in the activation of the NADPH oxidase was excluded.
Blockade of Antibody-Induced Glomerulonephritis with Crry-Ig, a Soluble Murine Complement Inhibitor
Authors: Quigg Richard J. , Kozono Yuko , Berthiaume Dawn , Lim Alice , Salant David J. , Weinfeld Andrew , Griffin Patricia , Kremmer Elisabeth , Holers V. Michael
Published: January 2023

A recombinant soluble form of the mouse membrane complement inhibitor Crry (complement receptor-related gene y) fused to IgG1 hinge, CH2, and CH3 domains has been created and designated Crry-Ig. Crry has been used because, similar to human soluble CR1, it demonstrates decay-accelerating activity for both the classical and alternative pathways of complement as well as cofactor activity for factor I-mediated cleavage of C3b and C4b. The mouse IgG1 isotype was incorporated because it is a noncomplement-activating isotype and, when fused to Crry, results in a complement inhibitor that should not be recognized as foreign when used chronically in murine models. Crry-Ig demonstrated complement-inhibitory activity in both the fluid phase and on target surfaces. Following in vivo injection, Crry-Ig manifested a two-phase serum elimination profile, a rapid initial loss most likely reflecting tissue redistribution and a second more prolonged decline with a t1/2 of 40 h. Inhibition of complement activation in mice following injection of Crry-Ig was demonstrated by a marked decrease in the ability of serum from treated mice to be activated by zymosan particles in vitro. Finally, in vivo efficacy of Crry-Ig was demonstrated by its ability to substantially diminish renal injury induced by complement-fixing nephrotoxic Ab. The use of Crry-Ig in vivo in murine models of chronic inflammatory and autoimmune disease should allow further insight into the potential therapeutic effects and possible untoward complications of continuous blockade of complement using inhibitors that act on activation products of C4 and C3.
Inducible Expression of the α1-Acid Glycoprotein by Rat and Human Type II Alveolar Epithelial Cells
Authors: Crestani Bruno , Rolland Corinne , Lardeux Bernard , Fournier Thierry , Bernuau Dominique , Poüs Christian , Vissuzaine Christiane , Li Lin , Aubier Michel
Published: January 2023

α1-Acid glycoprotein (AGP) is a major acute phase protein in rat and human. AGP has important immunomodulatory functions that are potentially important for pulmonary inflammatory response. The liver is the main tissue for AGP synthesis in the organism, but the expression of AGP in the rat lung has not been investigated. We show that AGP mRNA was induced in the lung of dexamethasone-, turpentine-, or LPS-treated rats, whereas AGP mRNA was not detected in the lung of control rats. In the lung of animals treated intratracheally with LPS, in situ hybridization showed that AGP gene expression was restricted to cells located in the corners of the alveolus, consistent with an alveolar type II (ATII) cell localization. The inducible expression of the AGP gene was confirmed in vitro with SV40 T2 cells and rat ATII cells in primary culture: maximal expression required the presence of dexamethasone. IL-1 and the conditioned medium of alveolar macrophages acted synergistically with dexamethasone. Rat ATII cells secreted immunoreactive AGP in vitro when stimulated with dexamethasone or with a combination of dexamethasone and the conditioned medium of alveolar macrophages. In vivo, in the human lung, we detected immunoreactive AGP in hyperplastic ATII cells, whereas we did not detect AGP in the normal lung. We conclude that AGP is expressed in the lung in cases of inflammation and that ATII cells are the main source of AGP in the lung.
TCR Agonist and Antagonist Exert In Vivo Cross-Regulation When Presented on Igs
Authors: Legge Kevin L. , Min Booki , Cestra Aimee E. , Pack Christopher D. , Zaghouani Habib
Published: January 2023

Ig-PLP1 and Ig-PLP-LR are chimeric Igs expressing proteolipid protein (PLP)-derived T cell agonist (PLP1) and antagonist (PLP-LR) peptides, respectively. Both chimeras, like free PLP1 and PLP-LR peptides, induce in vivo-specific T cell responses. However, the responses induced by Ig-PLP1 and Ig-PLP-LR were cross-reactive with both PLP1 and PLP-LR peptides, while those induced by free peptides were not. Surprisingly, despite the cross-reactivity of the responses, when Ig-PLP1 and Ig-PLP-LR were administered together into mice, a dose-dependent down-regulation of both T cell responses and a reduction of IL-2 production to background levels was observed. In contrast, when T cells induced by either Ig chimera were stimulated in vitro with mixtures of free PLP1 and PLP-LR peptides, there was no down-regulation of proliferation or decrease in IL-2 production. These data indicate that Ig-PLP1 and Ig-PLP-LR exert adverse reactions on one another at the level of naive T cells, resulting in an opposite antagonism. However, naive T cells experiencing either chimera develop into cross-reactive cells, acquire resistance to TCR triggering by closely related but different peptides, and support responsiveness.
Tissue-Specific Up-Regulation of B7-1 Expression and Function During the Course of Murine Relapsing Experimental Autoimmune Encephalomyelitis
Authors: Karandikar Nitin J. , Vanderlugt Carol L. , Eagar Todd , Tan Litjen , Bluestone Jeffrey A. , Miller Stephen D.
Published: January 2023

B7/CD28-mediated costimulation is a promising target for therapeutic intervention in autoimmune diseases. However, studies addressing the differential functional roles of B7-1 and B7-2 in several autoimmune models have resulted in conflicting data, perhaps due to the temporal dynamics of B7-1 and B7-2 surface expression on different cell types and/or at different sites during an autoimmune response. We examined the temporal expression of B7 costimulatory molecules in the CNS and in various lymphoid organs during the course of murine relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE). Following immunization of SJL mice with the immunodominant proteolipid protein epitope, PLP139–151, surface expression of B7-1 was up-regulated on B cells, T cells, and macrophages, relative to B7-2, on CNS-infiltrating cells and on splenocytes. Similar enhancement in splenic B7-1 expression could be induced in SJL mice by the adoptive transfer of PLP139–151-specific cells or by immunization with CFA alone. These changes were not observed on lymph node cells, including those isolated from lymph nodes draining the immunization site, which maintained the predominant B7-2 expression pattern seen in naive mice. These phenotypic expression patterns correlated with the functional predominance of B7-1 in costimulating T cell activation when employing APCs from the spleen or CNS of mice with ongoing R-EAE, while B7-2 remained functionally predominant on lymph node APCs. Variation of phenotypic expression and functional dominance of costimulatory molecule expression in different lymphoid compartments during an active inflammatory autoimmune response has important implications in immune regulation, autoimmune pathogenesis, and therapeutic strategies.
The Role of Estrogen Receptors and Androgen Receptors in Sex Steroid Regulation of B Lymphopoiesis
Authors: Smithson Glennda , Couse John F. , Lubahn Dennis B. , Korach Kenneth S. , Kincade Paul W.
Published: January 2023

Several observations suggest that sex steroids might participate in steady state regulation of B lymphopoiesis. B cell precursors decline dramatically in bone marrow of pregnant or estrogen-treated mice. Reciprocally, the same cell populations are increased in hypogonadal mice or male castrates. Estrogen treatment of hypogonadal mice reduced precursors to normal. However, questions remain about which hormones and receptors are the most important. Furthermore, these observations need to be reconciled with advances regarding new sex steroid receptors. We have now characterized B lymphopoiesis in androgen receptor-deficient testicular feminization (Tfm) mice. Testicular feminization mice had substantially elevated numbers of B cell precursors in the bone marrow and B cells in the spleen as compared with wild-type mice. The importance of one estrogen receptor (ERα) was evaluated in gene-targeted mice, and B cell precursors were found to be within the normal range. Our previous studies indicated that hormone receptors in stromal cells may be important for estrogen-mediated suppression of B lymphopoiesis. We now show that estrogen-mediated inhibition of B cell precursor expansion in culture was blocked by a specific estrogen receptor antagonist (ICI 182,780). Stromal cells derived from ERα-targeted bone marrow were fully estrogen responsive. RT-PCR analyses of these stromal cells revealed splice-variant transcripts of ERα, as well as message for a recently discovered estrogen-binding receptor, ERβ. Thus, androgens may normally inhibit B lymphopoiesis through the androgen receptor, whereas estrogens might utilize one or more receptors to achieve the same physiologic response.
An NFAT-Dependent Enhancer Is Necessary for Anti-IgM-Mediated Induction of Murine CD5 Expression in Primary Splenic B Cells
Authors: Berland Robert , Wortis Henry H.
Published: January 2023

CD5 is a 67-kDa membrane glycoprotein the expression of which in murine splenic B cells is induced by surface IgM cross-linking. To analyze this induction, we transiently transfected primary splenic B cells with luciferase reporter constructs driven by various wild-type and mutated CD5 5′-flanking sequences. The transfected cells were subsequently cultured in medium with or without F(ab′)2 anti-IgM (anti-IgM), and luciferase expression was assayed. Using this approach, we identified a 122-bp enhancer element necessary for anti-IgM-mediated induction of the CD5 promoter. Electrophoretic mobility shift assays indicated that four inducible and four constitutive complexes form on the enhancer fragment in nuclear extracts of primary B cells. Supershift assays revealed that two of the inducible complexes contained NFATc. Point mutations that abolished NFAT binding severely impaired enhancer function. Thus, CD5 is a target of NFAT in B cells. A third inducible complex required an intact H4TF-1 site. One of several constitutive complexes required an intact Ebox site while a second required an intact putative ets binding site. Mutation of the H4TF-1, Ebox, and Ets sites, in the presence of wild-type NFAT sites, significantly reduced the activity of the enhancer. Therefore, the induction of B cell CD5 expression requires NFAT binding and binding to at least one of three additional sites in the CD5 enhancer.
Reduced Tumorigenicity and Augmented Leukocyte Infiltration After Monocyte Chemotactic Protein-3 (MCP-3) Gene Transfer: Perivascular Accumulation of Dendritic Cells in Peritumoral Tissue and Neutrophil Recruitment Within the Tumor
Authors: Fioretti Francesca , Fradelizi Didier , Stoppacciaro Antonella , Ramponi Simona , Ruco Luigi , Minty Adrian , Sozzani Silvano , Garlanda Cecilia , Vecchi Annunciata , Mantovani Alberto
Published: January 2023

Monocyte chemotactic protein-3 (MCP-3) is a C-C chemokine that interacts with the CCR1, CCR2, and CCR3 receptors and has a spectrum of action encompassing T cells, NK cells, eosinophils, and dendritic cells (DC), in addition to mononuclear phagocytes. This broad spectrum of action prompted the present study aimed at assessing the antitumor activity of MCP-3 in a gene transfer approach and at providing information as to the actual in vivo leukocyte recruiting capacity of MCP-3. P815 mastocytoma cells transfected with the gene coding MCP-3 (P815/MCP-3) grew in syngeneic hosts and underwent rejection. Rejection was associated with profound alterations of leukocyte infiltration and resistance to subsequent challenge with P815 cells. Tumor-associated macrophages, already present in copious numbers, T cells, eosinophils, and neutrophils, increased in tumor tissues after gene transfer. DC, identified as DEC205+, high MHC class II+, CD11c+ cells, did not increase substantially in the tumor mass. However, in peritumoral tissues, DC accumulated in perivascular areas. P815/MCP-3-transfected tumor cells grew normally in nude mice. Increased accumulation of macrophages and polymorphonuclear neutrophils was evident also in nude mice. mAb against CD4, CD8, and IFN-γ, but not against IL-4, inhibited rejection of MCP-3-producing cells. An anti-polymorphonuclear mAb caused only a retardation of MCP-3-elicited tumor rejection. Thus, MCP-3 gene transfer elicits tumor rejection by activating type I T cell-dependent immunity. It is tempting to speculate that altered trafficking of APCs, which express receptors and respond to MCP-3, together with recruitment of activated T cells, underlies activation of specific immunity by MCP-3-transfected cells.
Proteasome Activation Occurs at an Early, Premitochondrial Step of Thymocyte Apoptosis
Authors: Hirsch Tamara , Dallaporta Bruno , Zamzami Naoufal , Susin Santos A. , Ravagnan Luigi , Marzo Isabel , Brenner Catherine , Kroemer Guido
Published: January 2023

Proteasomes and mitochondrial membrane changes are involved in thymocyte apoptosis. The hierarchical relationship between protease activation and mitochondrial alterations has been elusive. Here we show that inhibition of proteasomes by two specific agents, lactacystin or MG132, prevents all manifestations of thymocyte apoptosis induced by the glucocorticoid receptor agonist dexamethasone or by the topoisomerase II inhibitor etoposide. Lactacystin and MG132 prevent the early disruption of the mitochondrial transmembrane potential (ΔΨm), which precedes caspase activation, exposure of phosphatidylserine, and nuclear DNA fragmentation. In contrast, stabilization of the ΔΨm using the permeability transition pore inhibitor bongkrekic acid or inhibition of caspases by N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone does not prevent the activation of proteasomes, as determined with the fluorogenic substrate N-succinyl-l-leucyl-l-leucyl-l-valyl-l-tyrosine-7-amido-4-methylcoumarin. Thus, proteasome activation occurs upstream from mitochondrial changes and caspase activation. Whereas the proteasome-specific agents lactacystin and MG132 truly maintain thymocyte viability, a number of protease inhibitors that inhibit nuclear DNA fragmentation (acetyl-Asp-Glu-Val-Asp-fluoromethylketone; N-Boc-Asp(OMe)-fluoromethylketone; N-tosyl-l-Phe-chloromethylketone) do not prevent the cytolysis induced by DEX or etoposide. These latter agents fail to interfere with the preapoptotic ΔΨm disruption. Altogether, our data indicate that different proteases may be involved in the pre- or postmitochondrial phase of apoptosis. Only those protease inhibitors that interrupt the apoptotic process at the premitochondrial stage can actually preserve cell viability.
Antigen-Specific Cytolysis by Neutrophils and NK Cells Expressing Chimeric Immune Receptors Bearing ζ or γ Signaling Domains
Authors: Roberts Margo R. , Cooke Keegan S. , Tran Annie-Chen , Smith Kent A. , Lin Wei Yu , Wang Martin , Dull Thomas J. , Farson Deborah , Zsebo Krisztina M. , Finer Mitchell H.
Published: January 2023

TCR- and IgG-binding Fc receptors (FcγR) mediate a variety of critical biologic activities including cytolysis via the structurally related ζ- and γ-chains. In previous studies, we have described chimeric immune receptors (CIR) in which the ligand-binding domain of a heterologous receptor or Ab is fused directly to the cytoplasmic domain of the TCR ζ-chain. Such ζ-CIRs efficiently trigger cytotoxic function of both T and NK cells in a target-specific manner. In this report, we compared the ability of both ζ- and γ-CIRs to activate the cytolytic function of two distinct classes of FcγR-bearing effectors, NK cells and neutrophils. Mature neutrophils expressing ζ- and γ-CIR were generated in vivo from murine hemopoietic stem cells following transplantation of syngeneic mice with retrovirally transduced bone marrow or in vitro from transduced human CD34+ progenitors following differentiation. Both ζ- and γ-based CIRs were capable of activating target-specific cytolysis by both NK cells and neutrophils, although the ζ-CIR was consistently more efficient. The experimental approach described is a powerful one with which to study the role of nonlymphoid effector cells in the host immune system and permits the rational design of immunotherapeutic strategies that rely on harnessing multiple immune cell functions via CIR-modified hemopoietic stem cells or progenitors.
In Vitro Effects of IL-12 on HIV-1-Specific CTL Lines from HIV-1-Infected Children
Authors: McFarland Elizabeth J. , Harding Paul A. , MaWhinney Samantha , Schooley Robert T. , Kuritzkes Daniel R.
Published: January 2023

We studied the in vitro effects of IL-12 on HIV-1-specific CTL lines derived from PBMC of HIV-1-infected children. HIV-1-specific CTL lines were derived by limiting dilution following Ag-specific stimulation of PBMC from HIV-1-infected children and were maintained with repeated anti-CD3 stimulation. Following incubation with IL-12 for 5 to 7 days, HIV-1-specific cytotoxicity was augmented in a dose-dependent fashion (mean increase, 94 ± 83 lytic units; p = 0.0006). Experiments performed with CD3-blocking Abs and MHC-mismatched targets demonstrated that the IL-12-enhanced activity was MHC restricted and dependent on cells bearing CD3. The effect of IL-12 on proliferation of the CTL lines as tested by [3H]TdR uptake was minimal, with stimulation indexes ranging from 1.25 to 4.9. The effects of IL-12 on cytotoxicity were not significantly altered by addition of Ab to the IL-2R (anti-Tac) in quantities sufficient to block exogenous IL-2 (p = 0.15), demonstrating that endogenous IL-2 activity is not required for IL-12-enhanced cytolytic activity. Likewise, addition of neutralizing Ab specific for IFN-γ did not change IL-12-enhanced cytotoxicity (p = 0.61). The in vivo role of IL-12 in the generation and the stimulation of CTL remains to be determined; however, its ability to augment HIV-1-specific CTL in vitro adds additional support for IL-12 as a candidate for immune-based therapy of HIV-1.
Transcription of a Broad Range of Self-Antigens in Human Thymus Suggests a Role for Central Mechanisms in Tolerance Toward Peripheral Antigens
Authors: Sospedra Mireia , Ferrer-Francesch Xavier , Domínguez Orlando , Juan Manel , Foz-Sala Màrius , Pujol-Borrell Ricardo
Published: January 2023

The role of the thymus in the induction of tolerance to peripheral antigens is not yet well defined. One impending question involves how the thymus can acquire the diversity of peripheral nonthymic self-Ags for the process of negative selection. To investigate whether peripheral Ags are synthesized in the thymus itself, we have determined the expression of a panel of circulating and cell-bound peripheral Ags, some of which are targets of autoimmune diseases, at the mRNA level in total thymic tissue and in its main cellular fractions. Normalized and calibrated RT-PCR experiments demonstrated the presence of transcripts of nonthymic self-Ags in human thymi from 8 days to 13-yr-old donors. Out of 12 glands, albumin transcripts were found in 12; insulin, glucagon, thyroid peroxidase, and glutamic acid decarboxylase (GAD)-67 in six, thyroglobulin in five, myelin basic protein and retinal S Ag in three, and GAD-65 in one. The levels of peripheral Ag transcripts detected were age-related but also showed marked interindividual differences. Cytokeratin-positive stromal epithelial cells, which are a likely cellular source for these, contained up to 200 transcript copies of the most expressed peripheral Ags per cell. These results implicate the human thymus in the expression of wide representation of peripheral self-Ags and support the view that the thymus is involved in the establishment of tolerance to peripheral Ags. The existence of such central mechanism of tolerance is crucial for the understanding of organ-specific autoimmune diseases.
Human Muscle Cells Express a Functional Costimulatory Molecule Distinct from B7.1 (CD80) and B7.2 (CD86) In Vitro and in Inflammatory Lesions
Authors: Behrens Lüder , Kerschensteiner Martin , Misgeld Thomas , Goebels Norbert , Wekerle Hartmut , Hohlfeld Reinhard
Published: January 2023

The B7 family of costimulatory molecules likely includes members distinct from B7.1 (CD80) and B7.2 (CD86). After stimulation with IFN-γ or TNF-α, human myoblasts selectively express BB-1, but not B7.1 or B7.2. BB-1 is detected by anti-BB-1, a mAb cross-reacting with B7.1 (but not B7.2) and an as yet undefined costimulatory molecule. The absence of B7.1 and B7.2 in BB-1-positive myoblasts was confirmed by RT-PCR. The molecule detected by anti-BB-1 is functional, because anti-BB-1 mAb and CTLA4Ig (but not anti-B7.1- or anti-B7.2-specific mAbs) completely inhibit Ag presentation by cytokine-induced myoblasts to HLA-DR-matched Ag-specific CD4+ T cell lines. Stimulation of myoblasts with IL-4 induces B7.1 and B7.2, as well as BB-1, but with different time kinetics. Stimulation of CD40-positive myoblasts with anti-CD40 mAb selectively induces BB-1, whereas stimulation with CD40L-transfected mouse L cells induces BB-1 and B7.1, with different kinetics. To assess whether BB-1 is expressed in muscle tissue, we investigated 23 muscle biopsy specimens from patients with polymyositis, dermatomyositis, inclusion body myositis, Duchenne muscular dystrophy, and nonmyopathic controls by immunohistochemistry and confocal laser microscopy. We found that, in all inflammatory myopathy cases, but not in normal muscle, many muscle fibers strongly react with anti-BB-1. In contrast, muscle fibers did not react with B7.1- or B7.2-monospecific mAbs in any of the pathologic specimens or in normal muscle. Our results demonstrate that human muscle cells can be induced to selectively express BB-1, a functional costimulatory molecule distinct from B7.1 and B7.2. This molecule may play an important role in the immunobiology of muscle.
Nasal Immunization of Nonhuman Primates with Simian Immunodeficiency Virus p55gagand Cholera Toxin Adjuvant Induces Th1/Th2 Help for Virus-Specific Immune Responses in Reproductive Tissues
Authors: Imaoka Koichi , Miller Christopher J. , Kubota Mitsuru , McChesney Michael B. , Lohman Barbara , Yamamoto Masafumi , Fujihashi Kohtaro , Someya Kenji , Honda Mitsuo , McGhee Jerry R. , Kiyono Hiroshi
Published: January 2023

Female rhesus macaques were nasally immunized with p55gag (p55) of SIV and cholera toxin as a mucosal adjuvant. Nasal immunization induced Ag-specific IgA and IgG Abs in mucosal secretions (e.g., cervicovaginal secretions, rectal washes, and saliva) and serum. Furthermore, high numbers of p55-specific IgA and IgG Ab-forming cells were induced in mucosal effector sites, i.e., uterine cervix, intestinal lamina propria, and nasal passage. p55-specific CD4+ T cells in both systemic and mucosal compartments expressed IFN-γ and IL-2 (Th1-type)- as well as IL-5, IL-6, and IL-10 (Th2-type)-specific mRNA. Moreover, p55-specific CTL activity was demonstrated in lymphocytes from blood, tonsils, and other lymphoid tissues. These results show that nasal immunization with SIV p55 with cholera toxin elicits both Th1- and selective Th2-type cytokine responses associated with the induction of SIV-specific mucosal and serum Abs, and CTL activity. These results offer a promise for the development of protective mucosal immunity to SIV.
Signaling Pathways Mediated by the TNF- and Cytokine-Receptor Families Target a Commoncis-Element of the IFN Regulatory Factor 1 Promoter
Authors: Gupta Sanjay , Xia Dengfeng , Jiang Man , Lee Sangjin , Pernis Alessandra B.
Published: January 2023

CD40 activation of B cells is strongly influenced by the presence of cytokines. However, the molecular basis for the interplay between these distinct stimuli is not clearly delineated. IFN regulatory factor 1 (IRF-1) is a transcription factor activated by either CD40 or cytokines. We have found that these different sets of signals target a common cis-acting element in the promoter of this gene, the IRF-1 gamma-activated site (GAS). Targeting of the IRF-1 GAS is not confined to activation via CD40 but extends to other stimuli that mimic the CD40 signaling cascade, like TNF-α and EBV. In contrast to induction of STATs by cytokines, the IRF-1 GAS-binding complex activated by CD40, TNF-α, or EBV contains Rel proteins, specifically p50 and p65. In this system, simultaneous exposure to CD40L together with either IL-4 or IFN-γ does not lead to the activation of novel Rel/STAT complexes. Given the importance of IRF-1 in a variety of biologic functions from proliferation to apoptosis, our findings support the notion that modulation of IRF-1 levels may be a critical control point in B cell activation.
Human CD5 Signaling and Constitutive Phosphorylation of C-Terminal Serine Residues by Casein Kinase II
Authors: Calvo Javier , Vildà Josep M. , Places Lourdes , Simarro María , Padilla Olga , Andreu David , Campbell Kerry S. , Aussel Claude , Lozano Francisco
Published: January 2023

CD5 is a lymphocyte surface glycoprotein with a long cytoplasmic domain suitable for phosphorylation and signal transduction, which is involved in the modulation of Ag-specific receptor-mediated activation and differentiation signals. In this study, we use Jurkat T cell transfectants of CD5 cytoplasmic tail mutants to reveal phosphorylation sites relevant to signal transduction. Our results show that casein kinase II (CKII) is responsible for the constitutive phosphorylation of CD5 molecules at a cluster of three serine residues located at the extreme C terminus (S458, S459, and S461). Furthermore, the yeast two-hybrid system demonstrates the specific association between the C-terminal regions of the CD5 cytoplasmic tail and the regulatory β subunit of CKII. We demonstrate that CKII associates with and phosphorylates the C-terminal region of CD5, a conserved domain known to be relevant for the generation of second lipid messengers, and thereby enables at least one component of its signaling funcion.
Regulation of the Activity of IFN-γ Promoter Elements During Th Cell Differentiation
Authors: Zhang Feng , Wang Ding Zhe , Boothby Mark , Penix Laurie , Flavell Richard A. , Aune Thomas M.
Published: January 2023

Before they can deliver their effector functions, CD4+ Th cells must differentiate into Th1 or Th2 subsets. We have prepared reporter transgenic mice that express the luciferase gene under the control of proximal (prox.IFN-γ) and distal (dist.IFN-γ) regulatory elements from the IFN-γ promoter to permit investigation of mechanisms that regulate IFN-γ gene transcription during Th cell differentiation. Precursor Th cells (pTh) contain high levels of cAMP response element binding protein-activation transcription factor-1 (CREB-ATF1) proteins that bind these promoter elements from the IFN-γ gene, and these cells fail to express promoter activity. Restimulated effector Th (eTh) cells have reduced levels of CREB-ATF1 proteins, their nuclear extracts exhibit reduced CREB-ATF1 binding and greater Jun and Jun-ATF2 binding to dist.IFN-γ, and eTh cells express promoter activity. CREB directly competes with effector T cell nuclear proteins for dist.IFN-γ binding, and overexpression of CREB inhibits both prox.IFN-γ- and dist.IFN-γ-directed transcription in Jurkat T cells. IL-12-stimulated Th1 differentiation increases dist.IFN-γ activity in restimulated eTh1 cells; eTh1 nuclear extracts form increased levels of Jun-ATF2-dist.IFN-γ complexes. Taken together, these data suggest that both de-repression and trans-activation contribute to the induction of IFN-γ gene transcription during Th1 differentiation.
CD36 Is Required for Phagocytosis of Apoptotic Cells by Human Macrophages That Use Either a Phosphatidylserine Receptor or the Vitronectin Receptor (αvβ3)
Authors: Fadok Valerie A. , Warner Mary L. , Bratton Donna L. , Henson Peter M.
Published: January 2023

In vivo, apoptotic cells are efficiently removed by professional or nonprofessional phagocytes, a process thought to be essential for tissue remodeling and resolution of inflammation. Macrophages recognize apoptotic cells by several mechanisms, including recognition of exposed phosphatidylserine (PS); however, PS recognition on apoptotic cells has not been identified as a feature of human macrophages. The purpose of this study was to determine whether human monocyte-derived macrophages could be stimulated to recognize PS, defined as inhibition of phagocytosis by PS-containing liposomes. We also assessed the potential roles for scavenger receptors, CD14, and lectins. Uptake of apoptotic neutrophils into unstimulated macrophages was blocked about 50% by Arg-Gly-Asp-Ser and anti-αv, and up to 20% by oxidized low density lipoprotein and N-acetylglucosamine, implying a major role for integrin and minor roles for scavenger and lectin receptors. Uptake into macrophages stimulated with β-1,3-glucan was blocked 50% by PS liposomes and 40% by oxidized low density lipoprotein, suggesting that the macrophages had switched from using integrin to recognition of PS. MEM-18 and 61D3 (anti-CD14 mAbs) were poor inhibitors of apoptotic neutrophil uptake, but good inhibitors of apoptotic lymphocyte uptake. The switch to PS recognition was accompanied by down-regulation of αvβ3 expression and function. Anti-CD36 blocked uptake into unstimulated or stimulated macrophages, suggesting CD36 involvement not only with the αvβ3 integrin mechanism (as previously reported) but also with PS recognition. A maximum of 70% inhibition was achieved by combining anti-CD36 with either anti-av or PS liposomes.
Blood Monocyte Migration to Acute Lung Inflammation Involves Both CD11/CD18 and Very Late Activation Antigen-4-Dependent and Independent Pathways
Authors: Li Xian Chang , Miyasaka Masayuki , Issekutz Thomas B.
Published: January 2023

In acute lung inflammation, blood neutrophils and monocytes migrate into the lung parenchyma and bronchoalveolar space. The infiltration of the inflamed lung by monocytes is poorly understood because of difficulties in quantifying these cells in the presence of resident macrophages. Radiolabeled monocytes were used to study monocyte migration into the inflamed rat lung. Monocytes and neutrophils were purified from blood, labeled with 51Cr and 111In, respectively, and injected i.v. into rats given an intratracheal injection of LPS. The accumulation of 51Cr-labeled monocytes increased >10-fold in the lung parenchyma and 170-fold in the bronchoalveolar lavage (BAL) 18 h after LPS. 111In-labeled neutrophils increased >30-fold in the lung tissue and 500-fold in the BAL. Treatment of rats with a blocking anti-CD18 mAb inhibited monocyte accumulation in the lung and BAL by about 30%, whereas blocking very late activation Ag-4 (VLA-4) had no effect. Combined blockade of VLA-4 and CD18 inhibited approximately 30% of the migration to the lung parenchyma, but decreased the BAL by 80%. Monocyte migration to cutaneous inflammation was completely abolished by the combined mAb treatment. Neutrophil accumulation in the lung and BAL was not decreased by blocking either CD18 or VLA-4 and was only partially reduced by blocking CD18 plus VLA-4. Thus, monocyte migration to the LPS inflamed lung is substantially CD11/CD18 and VLA-4 independent, but accumulation in BAL is mediated by CD18 and VLA-4. Monocytes as well as neutrophils may use a previously unrecognized endothelial adhesion and migration pathway in the lung.