bluuebunny/crossref_metadata_2025 huggingface dataset:
IFN-gamma and TNF-alpha induce redistribution of PECAM-1 (CD31) on human endothelial cells.
Authors: Romer L H , McLean N V , Yan H C , Daise M , Sun J , DeLisser H M
Published: December 2022

Platelet endothelial adhesion molecule-1 (PECAM-1/CD31) is a glycoprotein adhesion molecule of the Ig superfamily that is constitutively expressed on leukocytes, platelets, and endothelial cells where it concentrates at the intercellular borders of adjacent cells. Recent studies have confirmed that endothelial PECAM-1 is involved in the recruitment of neutrophils into inflammatory sites. However, the effects of inflammatory cytokines such as TNF-alpha and IFN-gamma on endothelial PECAM-1 expression are not known. We studied the effect of several inflammatory cytokines on human umbilical vein endothelial cell PECAM-1 expression. We found that TNF-alpha and IFN-gamma produced dose-dependent changes in the surface distribution of PECAM-1, with a loss of the typical staining of PECAM-1 at intercellular junctions. Because TNF-alpha and IFN-gamma did not alter PECAM-1 transcription or total surface PECAM-1, these changes in PECAM-1 localization are most consistent with a redistribution of the protein away from intercellular junctions. This cytokine-induced surface redistribution of PECAM-1 was associated with changes in PECAM-1 cytoskeletal association but did not involve the expression of different alternatively spliced variants of the molecule. Given the involvement of endothelial PECAM-1 in neutrophil recruitment, redistribution of PECAM-1 may serve as a mechanism for regulating the transmigration of leukocytes across the vascular endothelium.
Differences in the degree of depletion, rate of recovery, and the preferential elimination of naive CD4+ T cells by anti-CD4 monoclonal antibody (GK1.5) in young and aged mice.
Authors: Rice J C , Bucy R P
Published: December 2022

We compared the degrees of depletion and the rates of recovery of CD4+ T cells in young and old mice given a single injection of an anti-CD4 (GK1.5) mAb. We found that in young mice the CD4+ population of T cells was completely depleted from the peripheral blood and, subsequently, the lymph nodes and spleen. Young mice recovered 80% of CD4+ T cells within 100 days after the injection. Although aged mice have less CD4+ T cells, CD4+ T cells were depleted by only 60% after mAb injection. This finding was associated with prolonged circulation of Ab-coated T cells. The rate of repopulation of peripheral CD4+ T cells was also affected by aging, because 5 times less CD4+ T cells appear in aged than in young mice between days 14 and 100 post injection. Further, we found that the CD4+ T cells that are depleted in both young and old mice by injection of anti-CD4 mAb are CD45RBhigh, suggesting a preferential depletion of immature cells. Finally, the thymus appears to be required to repopulate the T cell population post injection, because thymectomized young mice failed to recover CD3+CD4+ cells.
Presentation of endogenous and exogenous antigens is not affected by inactivation of E1 ubiquitin-activating enzyme in temperature-sensitive cell lines.
Authors: Cox J H , Galardy P , Bennink J R , Yewdell J W
Published: December 2022

Little is known regarding the mechanism by which MHC class I-associated peptides are generated. Proteins can be targeted for degradation by the covalent attachment of ubiquitin. The first step in ubiquitin conjugation to proteins is its binding to E1 ubiquitin-activating enzyme. To study the role of ubiquitin-targeted protein degradation in Ag processing, we used two mutant cell lines with temperature-sensitive E1 proteins, and a recombinant vaccinia virus expressing wild-type human E1. One of the cell lines examined (hamster ts20 cells) was previously reported to have a minimal capacity after a 1-h incubation at 41 degrees C to present osmotically loaded OVA to a T cell hybridoma, as assessed by IL-2 release. Even after incubating the same cells for 1 h at 43 degrees C, we failed to detect an E1-related decrease in the presentation of biosynthesized or osmotically loaded OVA to splenic T cells, as measured by target cell lysis. We introduce the use of mouse tsA1S9 cells to Ag-processing studies and provide the initial biochemical characterization of their defect in protein ubiquitination. Relative to parental L929 cells, after thermal inactivation of E1, these cells actually demonstrate enhanced presentation of endogenous or exogenous viral Ags to T cells. Our findings do not support a role for protein ubiquitination in Ag processing, and indicate that either the temperature-sensitive cell lines examined do not exhibit a sufficient reduction in ubiquitin-conjugating activity to affect the generation of antigenic peptides, or that ubiquitin-targeted proteolysis is not essential for processing the two exogenous and six endogenous Ags examined.
Nonresponders to hepatitis B vaccine can present envelope particles to T lymphocytes.
Authors: Desombere I , Hauser P , Rossau R , Paradijs J , Leroux-Roels G
Published: December 2022

The mechanisms causing nonresponsiveness to hepatitis B surface Ag (HBsAg) vaccines in humans remain largely unknown. The increased incidence of nonresponsiveness in subjects with HLA-DR3 or -DR7 haplotype suggests that immune response mechanisms governed by genes of the MHC are involved. It is conceivable that APC of nonresponders are defective in the presentation of HBsAg because they are unable to adequately take up, process, or present this Ag. To examine this hypothesis we have used PBMC from nonresponders to present recombinant particles containing S or PreS2-S sequences to HBsAg-specific T cell lines from haplo-identical responder vaccinees. The proliferative response of these lines was used to evaluate the efficacy of Ag presentation. Unfractionated PBMC from five DR2+ and six DR7+ nonresponders did not proliferate to HBsAg in vitro, whereas they vigorously proliferated upon stimulation with tetanus toxoid, thus ruling out the presence of a generalized immunodeficiency. All DR2(15)+ nonresponders were able to present hepatitis B envelope Ag to HBsAg-specific, DR1501-restricted T cells. PBMC from six DR7+ nonresponders were all able to present HBsAg to DR07-restricted T cell lines and PBMC from three DPw4+ nonresponders were able to present HBsAg to DP0402-restricted T cell lines. Additional experiments showed that PBMC from two nonresponders presented HBsAg equally well and sometimes better than PBMC from two partially HLA-matched high responders. We conclude that HLA-DR2+, -DR7+, and -DPw4+ nonresponder vaccinees are able to take up, process and present HBsAg to allogeneic, haplo-identical T cell lines in vitro.
Murine T and B cell responses to natural and recombinant hornet venom allergen Dol m 5.02 and its recombinant fragments.
Authors: King T P , Kochoumian L , Lu G
Published: December 2022

White-face hornet venom allergen, Dol m 5.02, is a protein of 204 amino acid residues. This protein and its overlapping fragments, of 53-114 residues in size, containing the N-terminal, middle, and C-terminal regions of the molecule, can be expressed in high yield in bacteria by using the plasmid vector pQE12. Natural (n) and recombinant (r) Dol m 5.02s and the r-fragments are about equally immunogenic for IgG Ab response in BALB/c mice. n-Dol m 5.02 induces mainly murine IgG Abs specific for its discontinuous B cell epitopes and, to a lesser extent, Abs specific for its continuous epitopes. r-Dol m 5.02 and the r-fragments induce only Abs specific for continuous B cell epitopes that are common with those of the n-protein. Abs specific for the discontinuous epitopes show higher affinity than those specific for the continuous epitopes. r-Dol m 5.02 and the r-fragments are as efficient as n-Dol m 5.02 in inducing murine T cell responses specific for the n-protein. The differences in the immunogenicity of n- and r-proteins or r-peptide fragments for B and T cell responses are related to their conformations, inasmuch as only the n-protein is cross-linked by four disulfide bonds. These findings are relevant to the potential use of r-fragments as immunotherapeutic reagents in humans.
In vivo state of antiviral CTL precursors. Characterization of a cycling cell population containing CTL precursors in immune mice.
Authors: Razvi E S , Welsh R M , McFarland H I
Published: December 2022

The in vivo state of CD8+ mouse memory CTL specific to lymphocytic choriomeningitis virus (LCMV) was characterized. During acute LCMV infection, the majority of the LCMV-specific CTL activity tested immediately ex vivo was mediated by CD8+ L-selectin- Mac-1+ CTL. The L-selectin- population of CD8+ cells elicited during acute infection also carried > 99% of the restimulatable CD8+ CTL precursors (CTLp) to LCMV, and these required added IL-2 for development into effectors in vitro. In contrast with the acute infection, most of the virus-specific CTLp in immune mice were L-selectin+. Examination of CD8+ T cells in LCMV-immune mice revealed that a L-selectin+ blast-size population of cycling CD8+ cells contained CTLp, which developed into effector CTL in the absence of added IL-2. These cells also expressed Mac-1 and IL-2R. Flow cytometric sorting for IL-2R+ and IL-2R- CD8+ cells in the immune animal revealed, by limiting dilution analysis, similar frequencies of CTLp in both populations. In bulk restimulation assays, the CD25+ CTLp did not require added IL-2 for their in vitro development into effectors, whereas the CD25- CTLp did. Hence, the different requirements for CTLp to effector development in vitro reflect qualitative differences in the in vivo state of the CTLp in the various subpopulations. LCMV-specific memory CTLp that did not require added IL-2 for differentiation were also found in the small-size, noncycling, CD8+L-selectin- cells. In contrast, the small-size, noncycling, CD8+L-selectin+, and CD8+IL-2R- populations also carried CTLp, but these required added IL-2 for development into effector CTL. Hence, T cell memory to LCMV is distributed among various lymphocyte subpopulations in immune animals, and the presence of an activated cycling cell component may account for the long-term perpetuation of antiviral immunologic memory.
Diversity of V gamma gene segments rearranged to the J gamma 4 gene in mice.
Authors: Atsuta N , Nishimura H , Nakamura N , Emoto M , Iwatsuki T , Yoshikai Y
Published: December 2022

Because there are limited numbers of V gamma gene segments and most V gamma rearrangements occur within clusters of the J gamma-C gamma genes in mice, gamma-chains display limited diversity compared with other TCR chains. In this study, we examined the nucleotide sequences of the V gamma-J gamma genes expressed in the gamma delta T cells appearing at the inflamed sites after Salmonella infection in DBA/2 mice. Most of the productive gamma gene rearrangements were V gamma 1-J gamma 4, whereas V gamma 2 and a unique V gamma, the 5' region of which was identical with sequences of the V gamma 2 gene, and the 3' region of which was identical with that of the V gamma 1 gene, were found to be rearranged to J gamma 4 gene, albeit at low frequency. Analysis of the ontogenic appearance of the rearrangements in the J gamma 4-C gamma 4 locus revealed that V gamma 2-J gamma 4 gene rearrangement was frequent in fetal thymocytes at the early stage of gestation. Most of the early fetal V gamma 2-J gamma 4 rearrangements exhibited the identical junction, a nonfunctional canonical sequence. The sequence analysis of the coding joint and the reciprocal recombination signal joint suggests that short homology-mediated direct recombination and chromosomal inversion mechanism are involved in fetal V gamma 2-J gamma 4 gene rearrangement. Taken together, our data suggest that the recombination of multiple V gamma segments with J gamma 4 can diversify the V gamma repertoire.
Melanoma patients immunized with melanoma cell vaccine induce antibody responses to recombinant MAGE-1 antigen.
Authors: Hoon D S , Yuzuki D , Hayashida M , Morton D L
Published: December 2022

The MAGE-1 gene was recently characterized to encode an immunogenic tumor Ag on several types of human tumors, including melanoma. This Ag is expressed in a wide variety of human tumors and not in normal cells, except testicular tissue, as assessed through specific mRNA analysis. In this study we cloned the MAGE-1 gene exon 3 region from a colon carcinoma cell line and expressed it in Escherichia coli. The recombinant MAGE-1 protein was affinity purified. By using Western blot analysis, IgG and IgM anti-MAGE-1 Abs were detected in the sera of melanoma patients. Fifty-three patients immunized with a melanoma cell vaccine (MCV) were assessed for anti-MAGE-1 IgG responses by using a MAGE-1 Ag-specific ELISA. The MCV consisted of three melanoma cell lines that expressed MAGE-1. Comparisons of anti-MAGE-1 IgG response pre-MCV treatment with 12- to 16-wk post-MCV treatment were made. Fifty-seven percent of the patients immunized with the MCV showed significant enhancement of IgG response to recombinant MAGE-1 protein. Patients who responded had no particular HLA-A or -B allele expression pattern. Melanoma patients immunized with whole cell MCV containing MAGE-1 can enhance anti-MAGE-1 IgG Abs. Recombinant MAGE-1 protein can be used to assess patient response to MAGE-1 and will be investigated as a potential cancer vaccine against a wide variety of human tumors that express MAGE-1.
Mechanisms underlying acute mast cell-induced leukocyte rolling and adhesion in vivo.
Authors: Gaboury J P , Johnston B , Niu X F , Kubes P
Published: December 2022

It has been proposed that a primary detector mechanism for tissue infection or injury may be the mast cell that releases agents that recruit leukocytes to the appropriate site at risk. The objective of this study was to evaluate the early mechanisms involved in mast cell-induced leukocyte recruitment. We used intravital microscopy to visualize leukocyte-rolling flux and adhesion in single 25 to 40 microns venules in mesenteric preparations that were treated with the mast cell-degranulating agent, compound 48/80 (CMP 48/80). Superfusion of the rat mesentery with CMP 48/80 caused a dose-dependent rise in the number of rolling and adherent cells, events significantly reduced by: 1) mast cell stabilizers, ketotifen, or cromolyn, and 2) chronic treatment with CMP 48/80 to deplete mast cell constituents. The increase in leukocyte flux associated with CMP 48/80 was blocked by diphenhydramine (H1-receptor antagonist) and an anti-P-selectin Ab (PB1.3), but not by the 5-lipoxygenase inhibitor, MK 886. The reduction in the flux of rolling leukocytes translated into fewer adherent leukocytes with diphenhydramine or PB1.3. The CMP 48/80-induced rise in leukocyte adhesion, but not leukocyte flux, was reduced by the platelet-activating factor (PAF)-receptor antagonist (WEB 2086) and an anti-CD18 Ab (CL26). MK 886 did not prevent the increased leukocyte adhesion. In vitro data revealed that mast cell-derived PAF induced essentially all of the leukocyte adhesion to endothelium or protein-coated plastic. These data suggest that mast cell degranulation induces P-selectin-dependent leukocyte rolling and CD18-dependent leukocyte adhesion via histamine and PAF, respectively.
Antitumoral properties of aged human monocytes.
Authors: McLachlan J A , Serkin C D , Morrey K M , Bakouche O
Published: December 2022

It is known that older people are more sensitive to cancer and infectious agents and need more time to recover from such disorders. Can this difference in sensitivity to cancer and infections between elderly and younger people be a result of a difference in their immune systems and, more specifically, in the way monocytes react to infectious agents and cancer cells? To determine what happens after cells have aged, human monocytes were purified from young donors (approximately 25 years of age) and from older donors (65 years of age or older) and tested for their ability to respond to the polyclonal activator LPS. Our results showed that monocytes from aged donors (aged monocytes), when compared with monocytes from younger donors (young monocytes) did lose part of their cytotoxicity against tumor cells (A375 human melanoma cells and L929 murine fibroblast cells). In addition, aged monocytes displayed a sharp decrease in IL-1 secretion, but did display the intracellular 31 kDa IL-1 precursor. Moreover, aged monocytes displayed a decrease in the production of reactive oxygen intermediates such as NO2 and H2O2. Finally, aged monocytes stimulated by LPS displayed an increase in intracellular cyclic AMP and have lost their protein kinase C translocation from the cytosol to the plasma membranes. These results suggest that age affects the immunologic and antitumoral properties of human monocytes.
Balance between IL-1 beta, TNF-alpha, and their specific inhibitors in chronic renal failure and maintenance dialysis. Relationships with activation markers of T cells, B cells, and monocytes.
Authors: Descamps-Latscha B , Herbelin A , Nguyen A T , Roux-Lombard P , Zingraff J , Moynot A , Verger C , Dahmane D , de Groote D , Jungers P
Published: December 2022

Patients with end-stage renal disease present an immunodeficiency that paradoxically coexists with activation of most immunocompetent cells, and the roles of chronic uremia and maintenance dialysis are poorly understood. We determined circulating levels of IL-1 beta and IL-1Ra, TNF-alpha and its soluble receptors (TNF-sR55 and TNF-sR75), and activation markers of T cells (soluble CD25), B cells (soluble CD23), and monocytes (neopterin) in a large cohort of undialyzed patients at various stages of chronic renal failure and in dialyzed patients on maintenance hemodialysis or chronic peritoneal dialysis. The progression of uremia was associated with a gradual increase in soluble CD25, CD23, and especially neopterin levels. Although IL-1 beta could not be detected, IL-1Ra levels were significantly increased from the earliest stage of renal failure. Plasma levels of TNF-alpha, TNF-sR55, and TNF-sR75 progressed with the severity of renal failure and correlated with soluble CD25, CD23, and neopterin levels, whereas IL-1Ra levels correlated exclusively with TNF-sR55 levels. Compared with undialyzed patients, levels of IL-1 beta were higher in patients on maintenance hemodialysis, whereas those of IL-1Ra were lower and decreased further at the end of dialysis sessions. In contrast, both TNF-sR55 and TNF-sR75 levels were significantly higher than in undialyzed patients and increased further at the end of dialysis sessions in the absence of an increase of TNF-alpha. Such an imbalance between cytokines and their inhibitors may play a pivotal role in the multifaceted process of immune dysfunction.
TNF-alpha expression by resident microglia and infiltrating leukocytes in the central nervous system of mice with experimental allergic encephalomyelitis. Regulation by Th1 cytokines.
Authors: Renno T , Krakowski M , Piccirillo C , Lin J Y , Owens T
Published: December 2022

The inflammatory cytokines IFN-gamma and TNF-alpha have been demonstrated in various autoimmune diseases, and are thought to participate in the induction and pathogenesis of disease. TFN-alpha is a cytopathic cytokine that is cytotoxic for oligodendrocytes in vitro and has been implicated in the pathology of multiple sclerosis and its animal model experimental allergic encephalomyelitis (EAE). We used reverse transcriptase (RT)-PCR to study the kinetics, cellular source, and regulation of cytokine gene expression in the central nervous system (CNS) of SJL/J mice with myelin basic protein-induced EAE at different stages of the disease. The expression of CD3, IL-2, IFN-gamma, and TNF-alpha mRNA was barely detectable in the CNS of unmanipulated mice or mice that were immunized with adjuvant but showed no symptoms. These mRNAs were readily detectable in the CNS of mice during peak disease, then coordinately dropped to background levels during remission. Analysis of cells isolated from the CNS of mice with acute EAE showed that the Th1 cytokines, IL-2 and IFN-gamma, were produced by infiltrating CD4+ T cells. In contrast, TNF-alpha was predominantly transcribed by non-T mononuclear CNS cells, the majority of which were identified as microglia and macrophages by their Mac-1 phenotype. Microglia could be discriminated by their low expression of CD45. Incubation of freshly derived, adult microglia from normal, uninfiltrated, CNS with activated Th1 supernatant induced the production of TNF-alpha mRNA. Therefore, TNF-alpha is made by both CNS-resident microglia and infiltrating macrophages during EAE, and this production is tightly controlled by cytokines secreted by infiltrating CD4+ T cells.
Analysis of the autoantibody response to fibrillarin in human disease and murine models of autoimmunity.
Authors: Takeuchi K , Turley S J , Tan E M , Pollard K M
Published: December 2022

Fibrillarin, a component of the U3 RNP particle, is a target for the spontaneously arising autoantibodies in human scleroderma and a monoclonal autoantibody (72B9) derived from the autoimmune mouse strain (NZB x NZW) F1. Autoantibodies against fibrillarin can also be induced in H-2s mice by treatment with mercuric chloride (HgCl2). The objective of this study was to compare the spontaneously occurring anti-fibrillarin autoantibody response with the autoantibody response induced by HgCl2 treatment. Immunofluorescence microscopy on human HEp2, mouse 3T3, and Xenopus XIK-2 cells, immunoblotting with use of nuclear extract from human MOLT-4, mouse 3T3, and Xenopus XIK-2 cells, and immunoprecipitation with use of in vitro translation products of RNA transcripts from yeast fibrillarin cDNA were used in this analysis. Both spontaneous and induced autoantibodies displayed common reactivity in that, irrespective of the antigenic source, they gave the same nucleolar immunofluorescence pattern and a restricted immunoblotting reactivity targeting predominantly the 34-kDa protein fibrillarin. Immunoprecipitation of N- and C-terminal truncated fibrillarin constructs also demonstrated a common pattern of reactivity. All Abs precipitated a fragment comprising amino acids 1-312 but not a smaller fragment containing amino acids 1-257. The majority of sera could not precipitate an N-terminal truncated molecule consisting of amino acids 157-327. These immunoprecipitation experiments support recognition of a common epitope requiring both N- and C-regions, which may be exemplified by the reactivity of murine monoclonal anti-fibrillarin autoantibody 72B9. Our results indicate that spontaneous human and toxin-induced murine autoantibodies to fibrillarin share common reactivity against this highly conserved nucleolar protein.
The NK1.1 antigen in NK-mediated F1 antiparent killing in vitro.
Authors: Kung S K , Miller R G
Published: December 2022

NK cells in lethally irradiated F1(A x B) hybrid mice can reject parental A or B strain bone marrow cells, a phenomenon called "hybrid resistance." The recognition mechanism used by the NK cells remains unknown. Our laboratory has previously described an in vitro model for hybrid resistance, and we have used it here to test whether the NK surface marker, NK1.1, is involved in such recognition. We found that 1) an anti-NK1.1 mAb (PK136) inhibited F1 lymphokine-activated killer (LAK) antiparent lysis if the LAK expressed NK1.1. Other mAb, even a mAb such as 2B4 that recognizes the same LAK as PK136, did not produce inhibition. 2) The F(ab')2 fragment of PK136 also inhibited lysis. 3) F1 LAK generated from athymic nude mice were as effective antiparent killers as LAK from normal mice and were equally inhibitable by anti-NK1.1 mAb, strengthening the conclusion that killing is mediated by NK cells and not T cells. 4) As previously shown by others, addition of anti-NK1.1 mAb to a mixture of NK1.1+ LAK cells and NK-resistant FcR+ cells allowed lysis of the FcR+ cells via "redirected lysis," in which the anti-NK1.1 mAb binds to NK1.1 on the NK cells and FcR on the target cell. The ability of anti-NK1.1 mAb to inhibit direct lysis and enhance redirected lysis is most consistent with NK1.1 being a receptor involved in NK activation.
Regulation of the production of the RANTES chemokine by endothelial cells. Synergistic induction by IFN-gamma plus TNF-alpha and inhibition by IL-4 and IL-13.
Authors: Marfaing-Koka A , Devergne O , Gorgone G , Portier A , Schall T J , Galanaud P , Emilie D
Published: December 2022

Production by endothelial cells of the regulated on activation normal T expressed and secreted chemokine (RANTES) has recently been evidenced during delayed-type hypersensitivity (DTH) reactions and may contribute to the local accumulation of macrophages and CD4+ memory T lymphocytes. To document the mechanism inducing RANTES production in this condition, we analyzed the effect of cytokines known to influence the formation of DTH granulomas. Little or no RANTES was produced after stimulation of HUVEC with IFN-gamma, IL-1 beta, or TNF-alpha. However, the combination TNF-alpha+IFN-gamma induced a strong RANTES production. In situ hybridization experiments with a RANTES probe showed that this synergy was also observed at the mRNA level and that the effect of the combination was mainly to increase the amount of RANTES mRNA per cell. The expression of the luciferase gene under the control of the RANTES gene regulatory elements was analyzed; TNF-alpha and the combination TNF-alpha+IFN-gamma activated the regulatory elements. Sequential treatment of HUVEC with TNF-alpha and IFN-gamma showed that IFN-gamma sensitized HUVEC to the stimulating effect of TNF-alpha. The production of RANTES induced by TNF-alpha+IFN-gamma was partly but significantly inhibited by the Th2-type cytokines IL-4 and IL-13. In contrast, IL-10 had no effect. These results indicate that the microenvironment of DTH granulomas, containing high levels of both TNF-alpha and IFN-gamma, may be responsible for RANTES production by perigranulomatous endothelial cells. Inhibition of this production by Th2-type cytokines may be a mechanism by which these cytokines interfere with the formation of DTH granulomas.
Ligand binding by the IL-2 receptor is modulated by intracellular determinants of the IL-2 receptor beta-chain.
Authors: Goldsmith M A , Amaral M C , Greene W C
Published: December 2022

The biologic actions of IL-2 are mediated by the IL-2R, a multisubunit receptor complex displayed on the surface of lymphocytes and select other hematopoietic lineages. The IL-2R exhibits multiple affinities for IL-2 that result from the monomeric (alpha), heterodimeric (alpha beta and beta gamma), and heterotrimeric (alpha beta gamma) assembly of different receptor subunits. In the present studies, we have used a series of IL-2R mutants in a transient mammalian expression system to investigate the potential role of intracellular receptor regions in the ligand-binding functions of the IL-2R. Analyses of chimeric and deletion mutants of the IL-2R beta subunit have revealed that its intracellular domain critically and selectively influences high affinity ligand binding mediated through the extracellular domains of the alpha beta-heterodimeric receptor. In contrast, intermediate affinity binding of IL-2 by beta gamma-heterodimeric receptors exhibits no dependence on the cytoplasmic domain of IL-2 R beta. Further, co-expression of either a full-length or severely truncated form of IL-2 R gamma to generate an alpha beta gamma-heterotrimeric complex also overcomes the functional dependence upon the cytoplasmic tail of IL-2 R beta. Collectively, our findings suggest that the cytoplasmic domain of IL-2R beta produces intrasubunit transmembrane conformational changes in this receptor subunit that promote extracellular IL-2 binding in combination with IL-2R alpha. These findings have important implications for the receptor dynamics involved in both ligand binding and signal transduction as well as for clinical applications pertaining to altering IL-2R function.
B cell superstimulatory influenza virus (H2-subtype) induces B cell proliferation by a PKC-activating, Ca(2+)-independent mechanism.
Authors: Rott O , Charreire J , Semichon M , Bismuth G , Cash E
Published: December 2022

The influenza virus hemagglutinin glycoprotein (HA) induces a vigorous B cell proliferation and Ig-synthesis by an unknown activation mechanism, which is susceptible to the inhibitory effects of anti-Ig and anti-class II mAbs. To gain further insight into the activation mode of this T cell-independent, B cell "superstimulatory" virus, we analyzed the sensitivity of H2-subtype virus-mediated B cell activation to the inhibitory effects of various signal transduction-blocking agents and compared it to the well characterized anti-mu-mediated and the LPS-employed pathway. Cyclic-AMP agonists (cAMP-analogues, pentoxifylline, cholera toxin, and forskolin) blocked HA-mediated activation of B cells only at concentrations at least 50-fold higher than required for blocking of anti-mu-induced activation. However, HA-treatment failed to induce an increase in intracellular cAMP levels in responding B cells. The B cell response to HA was highly resistant to calcineurin-inhibitory cyclosporin-A treatment and did not result in a measurable Ca2+ influx. Similarly, HA failed to induce an increase in tyrosine phosphorylations, including phosphorylation of phospholipase C gamma 2. HA-activated B cells showed an increase in membrane-associated protein kinase C activity, and depletion of protein kinase C by pretreatment of B cells with phorbol esters inhibited a subsequent activation by HA. Collectively, our results provide a new example of B cell stimulation by multivalent type-2 Ags, which seems to be mediated by a phosphatidylinositol- and Ca(2+)-independent signaling pathway.
Regulation of lymphocyte pseudopodia formation by triggering the integrin alpha 4/beta 1.
Authors: Szabo M C , Teague T K , McIntyre B W
Published: December 2022

Video microscopy and digital imaging were used to quantitatively analyze lymphocyte adhesion and formation of pseudopodia on the extracellular matrix protein fibronectin (FN). A morphology kinetics assay comparing pseudopodial extension values over a 24-h period showed that HPB-ALL T leukemic cells undergo a wave of morphologic change, returning to a round shape after 8 h. Using anti-alpha 4 and anti-alpha 5 mAbs and a panel of cell types that are single or double positive for expression of the alpha 4/beta 1 and alpha 5/beta 1 FN binding integrins, it was determined that cell adhesion to FN was influenced by both beta 1-integrins, whereas alpha 4/beta 1 was found to be the major FN receptor mediating pseudopodia extension. The protein kinase inhibitor staurosporine, the protein kinase C inhibitors calphostin C and chelerythrine, and the protein tyrosine kinase inhibitor herbimycin A blocked pseudopodial extension in HPB-ALL cells. In contrast, two cAMP-dependent protein kinase inhibitors H8 and H89 did not inhibit. Inhibitors of phospholipase A2, lipoxygenases, and cyclooxygenases could block formation of pseudopodia, yet had little or no effect on cell adhesion to FN. The preincubation of cells with arachidonic acid could prevent the inhibition mediated by the reversible phospholipase A2 inhibitor cibacron blue. We conclude that the formation of lymphocyte pseudopodia in response to FN can utilize the adhesive and signaling activities of the alpha 4/beta 1-integrin and the enzymatic activities of protein kinases and phospholipases.
Transgenic control of perforin gene expression. Functional evidence for two separate control regions.
Authors: Lichtenheld M G , Podack E R , Levy R B
Published: December 2022

Perforin is a pore-forming effector molecule of CTL and NK cells. To characterize perforin gene expression and its transcriptional control mechanisms in vivo, expression of a cell surface tag, i.e., human CD4, was driven by 5.1 kb of the murine perforin 5' flanking and promoter region in transgenic mice. Six out of seven transgenic lines expressed the perforin-tag hybrid gene at low to intermediate levels, depending on the integration site. Tissues not yet reported to contain perforin-expressing lymphocytes were identified. Transgene expression occurred in all cells that physiologically are able to express perforin, i.e., in T cells and NK cells, and in some T cells that normally may express little or no perforin. At the whole organ level, significant amounts of transgenic mRNA and endogenous perforin mRNA were co-expressed in the lymphoid organs, as well as in the lung, the ileum, the oviduct/uterus, and the bone marrow. At the single cell level, the perforin tag was present on NK cells and on CD8+, as well as on CD4+ T cells. Also targeted were Thy-1.2+ gamma delta T cells, but not Thy1.2- gamma delta T cells, B cells, nor monocytes. During thymic T cell development, transgene expression occurred in double negative (CD4-CD8-) thymocytes and was detected at all subsequent stages, but exceeded the expression levels of the endogenous gene in the thymus. In conclusion, the analyzed perforin 5' flanking and promoter region contains important cis-acting sequences that restrict perforin expression to T cells and NK cells, and therefore provides a unique tool for manipulating T cell and/or NK cell-mediated immune responses in transgenic mice. On the other hand, the normal control of perforin gene expression involves at least one additional negative control mechanism that was not mediated by the transgenic promoter and upstream region. This control restricts perforin gene expression in thymically developing T cells and in most resting peripheral T cells, but can be released upon T cell activation.
Binding analysis of antibodies produced by precursor and branchpoint intermediates of an anti-influenza hemagglutinin B cell clone. Parallel replacement mutations do not confer increased avidity for hemagglutinin.
Authors: Rickert R C , Wloch M K , Hahn R L , Clarke S H
Published: December 2022

We have previously described a set of secondary (2 degrees) hybridomas that are specific for the Sb site of influenza hemagglutinin (HA(Sb)) and share the H37-68 ld (68ld). Cells producing 68ld Abs dominated the 2 degree response of one BALB/c mouse, but were virtually absent from the primary (1 degree) and 2 degree responses of other BALB/c mice. To understand the basis for the idiosyncratic nature of this response, we have assessed the functional importance of a conserved DJ junctional sequence. We find that substitutions of conserved residues within this sequence drastically reduced HA(Sb) binding, providing an explanation for the absence of this ld from 1 degree responses. We have also examined intraclonal affinity maturation by generating Abs inferred to have been produced by the unmutated precursors and branchpoint intermediates of the largest 68ld clone. Interestingly, unmutated Abs of this clone bound HA(Sb) well, comparable to that of some 2 degree 68ld hybridoma Abs. Abs produced by branchpoint intermediates of this clone have a lower affinity for HA(Sb) than their unmutated precursor, despite containing parallel amino acid replacement mutations. Thus, expansion of this clone did not follow the paradigm of stepwise increases in affinity. Together, these data underscore the interconnective nature of precursor frequency and somatic mutation in the formation of the 2 degree response to a given Ag.
Addition of a mu-tailpiece to IgG results in polymeric antibodies with enhanced effector functions including complement-mediated cytolysis by IgG4.
Authors: Smith R I , Coloma M J , Morrison S L
Published: December 2022

The 18-amino acid carboxyl-terminal tailpiece from IgM (mutp) has now been added to the carboxyl-termini of IgG1, IgG2, IgG3, and IgG4 constant regions to produce recombinant IgM-like IgGs. Polymeric IgGs obtained by this approach possess up to six Fcs and 12 antigen-combining sites, greatly increasing the avidity of their interactions with other molecules. Not surprisingly, the C activity of normally active IgG1 and IgG3 and somewhat less active IgG2 Abs is shown to be dramatically enhanced upon polymerization. The multiple Fcs present in a single molecule apparently allow for more efficient interactions with the multiple C1q heads present in C1, the first component of the classical C cascade. An unexpected result however, is that IgG4, normally devoid of C activity, when polymerized in the same fashion directs C-mediated lysis of target cells almost as effectively as the other polymers. Interestingly though, IgG4mutp does not deplete C activity in a standard consumption assay using soluble Ag. The other gamma mutp isotypes are capable of depleting 100% of the serum lytic ability even in the absence of Ag, whereas IgG4mutp shows no evidence of activity in this assay under any of the conditions tested. Additionally, we show that, in contrast to monomeric IgG, polymeric IgGs bind with very high affinity to Fc gamma receptor II (Fc gamma RII), a low affinity receptor for wild-type antibodies; however, binding to Fc gamma Rl, the high affinity receptor, appears to be unaltered. Finally, the in vivo t1/2 of the gamma mutp proteins is decreased relative to wild-type IgG, apparently because of rapid clearance of the polymeric fraction.
Secondary deletional recombination of rearranged switch region in Ig isotype-switched B cells. A mechanism for isotype stabilization.
Authors: Zhang K , Cheah H K , Saxon A
Published: December 2022

Analysis of the Ig switch (S) region structure from many stable isotype-switched B cells reveals that the majority of the ultimate switch recombination sites are located at the 5' end, or even upstream of the 5' end of the Smu region. These findings strongly contrast with results from switch circle analysis which reveal that the primary switch recombination sites are preferentially distributed in the middle or at the 3' end of the Smu region. Secondary deletion within recombined chimeric S regions has been proposed as a mechanism to account for the different results. We directly tested whether secondary deletion of rearranged chimeric S regions indeed occurs in human cells. Circular DNAs representing secondary deletion events in gamma switched cells were isolated and characterized by using a specially designed PCR-based approach. Nucleotide sequence analysis revealed that all clones had the S gamma 1-Smu-S gamma 1 structure. Thus, these cloned fragments resulted from secondary deletion/recombination events within a chimeric Smu-S gamma 1 switch region, i.e., rearrangement between its 5' Smu and 3' S gamma 1. Analysis of the Smu region from stimulated B cells also revealed that some Smu regions undergo an internal deletion/rearrangement between the 5' and 3' ends. These results definitively demonstrate that secondary deletion/recombination of chimeric S regions of isotype-switched B cells occurs. Such secondary deletion/recombination events potentially can be responsible for isotype stabilization of switched B cells, as the active Ig gene may have an insufficient amount of retained chimeric switch sequence to serve as a substrate for further S-S recombination.
Induction of tumor-reactive CTL from peripheral blood and tumor-infiltrating lymphocytes of melanoma patients by in vitro stimulation with an immunodominant peptide of the human melanoma antigen MART-1.
Authors: Rivoltini L , Kawakami Y , Sakaguchi K , Southwood S , Sette A , Robbins P F , Marincola F M , Salgaller M L , Yannelli J R , Appella E
Published: December 2022

MART-1 is an Ag expressed on melanomas and melanocytes, and is recognized by the majority of HLA-A2-restricted tumor-specific tumor-infiltrating lymphocytes (TIL) from melanoma patients. In the present study we have analyzed 10 potential 9-mer epitopes containing the HLA-A2.1 binding motifs for their ability to induce melanoma-specific T cell lines. Antimelanoma CTL could be generated only with MART-1(27-35) peptide, which has been previously shown to be recognized by a majority of HLA-A2-restricted TIL. Anti-MART-1(35-43)-specific CTL could also be induced, but these T cells did not recognize melanoma cells. MART-1(27-35)-specific CTL could be effectively generated from a total of 11 of 12 PBL and from 3 of 3 TIL derived from HLA-A2+ melanoma patients, as well as from 2 of 4 PBL from HLA-A2+ healthy donors by in vitro stimulation with autologous PBMC pulsed with the synthetic MART-1(27-35) peptide. These CTL lines specifically lysed and release cytokines (TNF-alpha, IFN-gamma, and GM-CSF) in response to T2 cells pulsed with MART-1(27-35), as well as to HLA-A2+ MART-1+ melanoma cells. CTL generated with MART-1(27-35) also lysed uncultured HLA-A2+ melanoma cells derived from tumor biopsies, indicating that this MART-1 epitope is likely to be expressed in association with HLA-A2 on the surface of tumor cells in vivo. CTL lines generated with MART-1(27-35) mediated 25- to 100-fold higher lytic activity than MART-1-reactive CTL grown from TIL in the presence of high dose IL-2. These results demonstrate that MART-1(27-35) peptide may represent an ideal candidate for Ag-specific immunotherapy in melanoma patients.
Human IL-1 beta processing and secretion in recombinant baculovirus-infected Sf9 cells is blocked by the cowpox virus serpin crmA.
Authors: Howard A D , Palyha O C , Griffin P R , Peterson E P , Lenny A B , Ding G J , Pickup D J , Thornberry N A , Schmidt J A , Tocci M J
Published: December 2022

Biologically active, mature IL-1 beta (mIL-1 beta) is released from activated monocytes after proteolytic processing from an inactive precursor (pIL-1 beta). IL-1 beta converting enzyme (ICE), the first member of a newly discovered family of cysteine proteinases, is required for this processing event. The cleaved cytokine is released from monocytes by an unknown mechanism which does not employ a standard hydrophobic signal sequence. As in mammalian fibroblasts, insect Sf9 cells do not normally process or secrete human IL-1 beta. The expression of active ICE enables Sf9 cells to process 31-kDa pIL-1 beta correctly at Asp27 and Asp116, and to export 17.5-kDa mIL-1 beta. The recombinant heterodimeric human enzyme purified from Sf9 cells possesses a sp. act. of 2.9 +/- 0.5 x 10(6) U/mg and is indistinguishable from native ICE with regard to its subunit composition and catalytic properties. In this system, co-expression of the cowpox virus crmA gene, an extremely potent serpin inhibitor of ICE (Ki < 7 pM), inhibits ICE activation completely and blocks pIL-1 beta processing and mIL-1 beta secretion by approximately 95%. The results indicate that ICE, in addition to its processing function, facilitates the transport of IL-1 beta across the plasma membrane.
Proinflammatory cytokines potentiate thrombospondin-mediated phagocytosis of neutrophils undergoing apoptosis.
Authors: Ren Y , Savill J
Published: December 2022

Apoptosis leads to swift recognition, ingestion, and degradation of intact senescent neutrophils by macrophages. This protects tissues from leakage of noxious contents from dying cells and may promote resolution of inflammation. However, little has been known of the mechanisms that regulate macrophage capacity for apoptotic cells during an inflammatory response. We examined whether proinflammatory cytokines modulated phagocytosis of senescent neutrophils undergoing apoptosis by human monocyte-derived macrophages at 4 days maturity (4d M phi), an in vitro model of neutrophil "disposal" by apoptosis. Pretreatment of 4d M phi with granulocyte-macrophage-CSF increased the proportion of 4d M phi taking up apoptotic PMN in a concentration-dependent fashion by up to approximately 240%. This was by a rapid effect detectable by 4 h and exerted on the M phi, not the PMN. Granulocyte-macrophage-CSF also increased the number of apoptotic PMN taken up by each M phi. IFN-gamma, IL-1 beta, TNF-alpha, and TGF-beta 1 also enhanced phagocytosis, but IL-4 and IL-6 had no effect. In each case, the cytokine-expanded phagocytic subpopulation employed the thrombospondin (TSP)-dependent recognition mechanism defined for mature M phi, in which M phi vitronectin receptor and CD36 cooperate. However, commensurate increases in M phi expression of VnR, TSP, or CD36 were not detectable, indicating that TSP-mediated recognition can be recruited by other mechanisms. Cytokines did not recruit phosphatidylserine-dependent recognition, the other major mechanism by which some macrophage populations ingest apoptotic cells. Thus, M phi phagocytosis of apoptotic neutrophils can be potentiated by proinflammatory cytokines, suggesting a mechanism for negative feedback control of neutrophil number at inflamed sites.
IL-8 specifically binds to endothelial but not to smooth muscle cells.
Authors: Schönbeck U , Brandt E , Petersen F , Flad H D , Loppnow H
Published: December 2022

The proinflammatory cytokine and potent chemoattractant IL-8 is involved in regulation of infectious or inflammatory processes. Human vascular endothelial cells (EC) and smooth muscle cells (SMC) probably contribute to these responses by recognition and/or production of rIL-8. We demonstrate here in competitive binding studies with radiolabeled rIL-8 that EC and fibroblasts, but not SMC, specifically bind IL-8 with low affinity. The binding was not saturated by ligand concentrations up to 80 nM 125I-rIL-8. Unlabeled neutrophil-activating peptide-2 competed the binding of 125I-rIL-8, although less potently than unlabeled rIL-8, as reported previously for polymorphonuclear neutrophils. In contrast, connective tissue-activating peptide III, platelet factor 4, or lysozyme did not reduce binding of 125I-rIL-8 to EC or fibroblasts. In accordance with these binding studies, EC and fibroblasts, but not SMC, expressed human IL-8 receptor type I mRNA. Neither cell type expressed mRNA for IL-8 receptor type II. Stimulation with IL-1 alpha or LPS did not alter the results obtained in PCR or binding studies. Although SMC did not express specific binding sites for IL-8, Western blot experiments showed that IL-1 alpha-, TNF-, or LPS-stimulated SMC released two major immunoreactive isoforms of IL-8 in a time- and dose-dependent manner. The m.w. were similar to IL-8 isoforms released by EC or mononuclear cells. The differential capacity of EC and SMC to produce IL-8 and express IL-8 binding sites indicates that vascular cell-derived IL-8 may contribute to differential regulation of infectious and inflammatory responses in the vessel wall.
Endogenous IL-6 and IL-10 contribute to the differentiation of CD40-activated human B lymphocytes.
Authors: Burdin N , Van Kooten C , Galibert L , Abrams J S , Wijdenes J , Banchereau J , Rousset F
Published: December 2022

This study was initiated to explore the contribution of endogenous cytokines to CD40-induced B cell proliferation and differentiation. First, both CD40 and Ag receptor (AgR) cross-linking induced purified tonsillar human B lymphocytes to secrete the same pattern of cytokines, including IL-1 beta, IL-6, IL-10, granulocyte-macrophage-CSF, and TNF-alpha, whereas IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-7, granulocyte (G)-CSF, or IFN-gamma were not detected. Second, cotriggering of CD40 and AgR resulted in additive secretion of both IL-6 and IL-10. Addition of IL-4 to CD40-activated B cells increased IL-6 levels but decreased IL-10 levels. In contrast, exogenous IL-10 diminished IL-6 levels. Neutralization of IL-6 and IL-10 using blocking Abs did not alter CD40-induced B cell growth. In contrast, IL-6 neutralization markedly inhibited the IL-4-induced IgE secretion (57 +/- 10%) as well as the IgG and IgM production resulting from AgR and CD40 cotriggering (49 +/- 16.5 and 29.5 +/- 4.5%, respectively). Blocking IL-10 inhibited the IgA secretion (25 +/- 2.7%) obtained after CD40 activation and the production of IgG, IgA, and IgM (24.1 +/- 5.6, 25 +/- 8, and 42 +/- 6.5%, respectively) by B lymphocytes undergoing dual ligation of CD40 Ag and AgR. Simultaneous neutralization of both endogenous IL-6 and IL-10 resulted in an increased inhibition of Ig secretion for B cells cotriggered by CD40 Ag and AgR (65-75%). Thus, endogenously produced IL-6 and IL-10 are involved in the differentiation of CD40-activated B cell.