bluuebunny/crossref_metadata_2025 huggingface dataset:
The adhesion molecules used by monocytes for migration across endothelium include CD11a/CD18, CD11b/CD18, and VLA-4 on monocytes and ICAM-1, VCAM-1, and other ligands on endothelium.
Authors: Meerschaert J , Furie M B
Published: December 2022

CD11/CD18 and VLA-4 integrins mediate interactions of monocytes with HUVEC cultured on human amniotic tissue. In the present study, the roles of individual CD11/CD18 integrins and endothelial adhesion molecules were examined using blocking mAbs and peptides. After 20 min of incubation, monocyte adhesion to and migration across unstimulated endothelium was dependent primarily on CD11a/CD18. When incubation was extended to 2 h to allow for completion of migration, either CD11a/CD18 or CD11b/CD18 could be used. Similarly, either CD11a/CD18 or CD11b/CD18 could be used by monocytes to bind to and traverse IL-1 beta-stimulated endothelium. Although both CD11a/CD18 and CD11b/CD18 are known to bind to ICAM-1, results of Ab-mixing experiments suggest that alternative ligands on HUVEC for CD11/CD18 integrins also may be used during transendothelial migration of monocytes. Our previous studies indicate that VLA-4 on monocytes interacts primarily with VCAM-1 on unstimulated endothelium. In contrast, migration of monocytes across IL-1 beta-stimulated endothelium was less dependent on VCAM-1. mAbs directed against binding sites for VLA-4 in domain 1 and domain 4 of VCAM-1 did not, by themselves, inhibit interactions of monocytes with stimulated HUVEC. VLA-4-dependent migration across IL-1 beta-stimulated endothelium was markedly inhibited only when mAbs to VCAM-1 were added in combination with peptides of fibronectin. Therefore, VLA-4 can interact with either VCAM-1 or alternative ligands on IL-1 beta-stimulated HUVEC-amnion cultures to mediate transendothelial migration of monocytes.
TNF-induced superoxide anion production in adherent human neutrophils involves both the p55 and p75 TNF receptor.
Authors: Richter J , Gullberg U , Lantz M
Published: December 2022

TNF, a potent activator of neutrophil granulocytes, acts via two cell-surface receptors: the p55-TNF receptor (TNF-R55) and the p75-TNF receptor (TNF-R75), which can be cleaved from the cell surface and thus form soluble TNF-binding proteins (TNF-BP). The role of the two receptors in activation of the neutrophil respiratory burst was investigated. Two mAbs reacting with TNF-R55 (H398 and TBP2) induced O2 release in a similar manner but to a lesser extent than TNF. TBP2, however, required preincubation at 4 degrees C to exert its effect. Preincubation of neutrophils (both at 4 and 37 degrees C) with mAb to TNF-R75 decreased TNF-induced superoxide anion production by 67 and 64%, respectively, indicating the essential role also for TNF-R75 in neutrophil activation. This inhibitory effect could not be explained by cross-down-regulation of TNF-R55 because the TNF-R75 mAb had no effect on TNF binding to TNF-R55 as determined by binding of 125I-labeled TNF or release of TNF-R55-BP as measured by ELISA. Furthermore, the TNF-R75 mAb did not decrease superoxide anion generation induced by the TNF-R55 mAb H398, thus ruling out that the inhibitory effect of the TNF-R75 mAb is due to inhibition of the signaling pathway downstream of TNF-R55. In contrast to the TNF-R75 mAb, TNF-R55 mAbs induced down-regulation of TNF-R75 and shedding of both TNF-R55-BP and TNF-R75-BP. We conclude that both TNF-R55 and TNF-R75 are involved in TNF-induced activation of the neutrophil respiratory burst.
Cross-linking of ICAM-1 on T cells induces transient tyrosine phosphorylation and inactivation of cdc2 kinase.
Authors: Chirathaworn C , Tibbetts S A , Chan M A , Benedict S H
Published: December 2022

Intercellular adhesion molecules (ICAM)-1 and -3 coexist on T lymphocytes and are counter-receptors for the integrin LFA-1. Signaling through ICAM-3 stimulates a number of T cell functions and involves phosphorylation of Fyn, Lck, CD45, and other proteins. In contrast, this type of specific signaling event has not been described for signaling through ICAM-1. Here, tyrosine phosphorylation of cellular proteins was examined after cross-linking of ICAM-1. Tyrosine phosphorylation of the 34-kDa cdc2 protein kinase was induced transiently after stimulation of the leukemic T cell line, Molt-3, or peripheral blood T cells. Stimulation through ICAM-1 had no effect on constitutive presence of cdc2 or phosphorylation of cdc2 on threonine. cdc2 kinase activity was constitutive in peripheral blood T cells, and transient inhibition of kinase activity after ICAM-1 stimulation correlated kinetically with phosphorylation of cdc2 on tyrosine.
Expression of sialyl Lewis(x) and Lewis(x) defines distinct stages of chicken B cell maturation.
Authors: Masteller E L , Lee K P , Carlson L M , Thompson C B
Published: December 2022

Commitment of cells to the B lineage in chickens occurs only during a brief period of embryogenesis. B cell progenitors then progress through discrete developmental stages resulting in the production of mature B cells that are competent to form a functioning humoral immune system in the adult bird. During embryogenesis, chicken B cell precursors undergo tissue and developmental stage-specific changes in cell-surface glycosylation. Immature B cell progenitors that migrate to the bursa of Fabricius express the carbohydrate epitope sialyl Lewis(x). Such cells undergo initial clonal expansion within the bursa without undergoing gene conversion. Beginning between days 15 and 17 of embryogenesis, B cells in the bursa undergo a tissue specific change in surface glycosylation that results in the loss of sialyl Lewis(x) expression and the acquisition of the related carbohydrate structure Lewis(x). Expression of Lewis(x) identifies B lymphocytes that have begun to undergo gene conversion. Before emigration from the bursa, bursal lymphocytes undergo another phenotypic switch in glycosylation and down-regulate Lewis(x) expression. Therefore, developmental switches in glycosylation allow us to distinguish three populations of B cells in the bursa of Fabricius at defined stages of development and suggest that regulation of cell-surface glycosylation plays a role in B cell development in the chicken.
Mouse surfactant protein-D. cDNA cloning, characterization, and gene localization to chromosome 14.
Authors: Motwani M , White R A , Guo N , Dowler L L , Tauber A I , Sastry K N
Published: December 2022

Surfactant protein-D (SP-D) is a collectin found associated with surfactant in the lung. SP-D has also been functionally characterized as an opsonin for diverse microorganisms and a chemoattractant for phagocytic cells. To determine the structure of mouse SP-D, we isolated and characterized clones from a B6/CBAF1J strain lung cDNA library using a PCR-derived genomic probe. The deduced sequence predicts a 19-amino acid signal sequence, a 25-amino acid long NH2 terminus with two cysteines, followed by an uninterrupted collagen domain with 59 Gly-X-Y repeats. Next, a short "neck" domain of 28 amino acids, with a potential to form trimeric alpha-helical coiled coil is found ending in a COOH-terminal 125-amino acid carbohydrate recognition domain. The mature mouse SP-D protein of 355 amino acids shows strong homology to rat (92% identity), human (76%), and bovine (72%) SP-D amino acid sequences. Northern blot and RT-PCR analysis revealed that the mouse SP-D gene is expressed predominantly in lung and, surprisingly, also in heart, stomach, and kidney but not in brain. In contrast, mouse surfactant protein-A (SP-A) mRNA expression was found to be restricted to lung. Human lung and stomach, but not heart or liver, were found to express SP-D mRNA, as determined by PCR. The mouse SP-D gene (Sftp4) has been localized to chromosome 14 (to a region syntenic to human chromosome 10), closely linked to the genes for other collagenous lectins, mannose-binding protein-A (MbI1), and SP-A (Sftp1).
An ospA frame shift, identified from DNA in Lyme arthritis synovial fluid, results in an outer surface protein A that does not bind protective antibodies.
Authors: Fikrig E , Liu B , Fu L L , Das S , Smallwood J I , Flavell R A , Persing D H , Schoen R T , Barthold S W , Malawista S E
Published: December 2022

Passive immunization with murine or human Abs to outer surface protein A (OspA) can protect mice against Borrelia burgdorferi, but OspA Abs elicited during natural infection in mice or humans are unable to clear the spirochete from the infected host. To examine Ab binding by OspA during the course of human infection, we amplified the operon encoding full-length ospA and ospB from synovial fluids of a patient with chronic Lyme arthritis, the first such recoveries from human material, at four separate time points over 4.5 mo, and expressed OspA in Escherichia coli. OspA mAbs that passively protected mice from infection did not bind one of the expressed OspAs, because of a deletion in ospA that resulted in a frame shift and premature stop codon near the carboxyl terminus. However, expressed OspA from a later synovial fluid sample did not contain this deletion. Thus, although altered forms of OspA, which potentially can influence host immune effectiveness, do occur in the human host, they cannot be the only factors responsible for microbial persistence.
Controlled recruitment of monocytes and macrophages to specific organs through transgenic expression of monocyte chemoattractant protein-1.
Authors: Fuentes M E , Durham S K , Swerdel M R , Lewin A C , Barton D S , Megill J R , Bravo R , Lira S A
Published: December 2022

Transgenic mice overexpressing the chemokine monocyte chemoattractant protein-1 (MCP-1) in the thymus and central nervous system have a higher number of mononuclear cells in those tissues than do control littermates. In the thymus, there is a modest increase in the number of Mac-1 and F4/80 positive cells, but no apparent change in the number of lymphoid cells. A more pronounced mononuclear infiltrate is detected in transgenic mice expressing MCP-1 in the brain. The vast majority of the recruited cells in the brain are monocytes and macrophages, as defined by light microscopy, and ultrastructural and immunohistochemical criteria. Such cells are found in a perivascular orientation with minimal parenchymal infiltration, possibly as a consequence of the accumulation of MCP-1 in the vessels, as shown by immunohistochemistry. The mononuclear cell infiltrate in the brain can be significantly amplified by LPS treatment, suggesting that the recruitment properties of MCP-1 can be potentiated by additional factors.
Protein kinase C activation precedes arachidonic acid release during IgG-mediated phagocytosis.
Authors: Karimi K , Lennartz M R
Published: December 2022

Previous studies demonstrated that both protein kinase C (PKC) and arachidonic acid (AA) are required for IgG-mediated phagocytosis by human monocytes. We have characterized a calcium-independent "phagocytic" phospholipase A2 (designated pPL) that mediates arachidonic acid release. The present studies were designed to order PKC and pPL in the phagocytic signaling pathway. The PKC inhibitors staurosporine and calphostin C caused a coordinated decrease in phagocytosis of IgG-opsonized erythrocytes and arachidonic acid release. The PLA2 activators mastoparan and melittin restored phagocytosis to PKC-inhibited cells, but were ineffective in monocytes pretreated with the pPL inhibitor bromoenol lactone. Similarly, PKC activation with PMA and diacylglycerol enhanced phagocytosis in the absence, but not in the presence, of bromoenol lactone. These results indicate that pPL may be regulated by an upstream phosphorylation event. Thus, we examined the effects of Ab-opsonized glass bead ingestion, okadaic acid-mediated inhibition of phosphatases, and PMA treatment on the activity of pPL and on its distribution between the cytosolic and membrane-associated compartments. IgG-opsonized erythrocytes and okadaic acid caused an overall increase in pPL activity, with a twofold increase in membrane-associated pPL. PMA treatment caused a 1.8-fold increase in membrane-associated pPL activity. Okadaic acid and PMA mimic IgG-opsonized erythrocytes with respect to membrane activation of pPL, suggesting that pPL activity may be regulated by PKC. Collectively, these results indicate that pPL activity is modulated by PKC during IgG-mediated phagocytosis, and that the PKC requirement can be bypassed by direct activation of pPL.
Oligoclonality of lung T lymphocytes following exposure to allergen in asthma.
Authors: Burastero S E , Crimi E , Balbo A , Vavassori M , Borgonovo B , Gaffi D , Frittoli E , Casorati G , Rossi G A
Published: December 2022

We were interested in studying the lung allergen-specific T cell repertoire in different conditions of allergen exposure in subjects with atopic asthma. Twenty-one allergic individuals were studied: 17 subjects suffering mainly from asthma and 4 from rhinitis. They all performed spirometry and methacholine challenge. All patients were subjected to bronchoalveolar lavage (BAL), either at base line (no challenge) or after allergen or histamine challenge, and the TCR repertoire of their lung T cells was studied with heteroduplex analysis. Expansion of single T cell clones was observed in one (of seven) asthmatic subject that reported a recent exposure to allergen and had high bronchial hypersensitivity to methacholine, and in seven/seven asthmatic subjects who underwent BAL after they suffered an early asthmatic reaction to experimental allergen inhalation. Remarkably, ex vivo expanded clones included allergen-specific T cells. In two of the seven subjects who underwent BAL after allergen challenge, two different lung segments were lavaged. A strikingly symmetrical distribution of the expanded clones was found in these samples. Control subjects and six of seven asthmatic patients studied at base line showed polyclonality of lung T cells. In conclusion, T lymphocytes are clonally expanded in the lower respiratory tract only in asthmatic subjects exposed to allergen. These results suggest that in allergic asthma, the inhalation of sensitizing allergens can recruit to the lung T lymphocytes that include allergen-specific T cell clones.
Primary hormonogenic sites as conserved autoepitopes on thyroglobulin in murine autoimmune thyroiditis. Secondary role of iodination.
Authors: Kong Y C , McCormick D J , Wan Q , Motte R W , Fuller B E , Giraldo A A , David C S
Published: December 2022

We hypothesized earlier that conserved T cell epitopes and those unique to mouse thyroglobulin (MTg) contributed to its total thyroiditogenicity in murine autoimmune thyroiditis. Recent studies of synthetic peptides from human Tg (HTg) revealed no immunodominant epitopes. The role of iodine residues, considered by some to render Tg immunogenic, became unclear, since only one 12-mer peptide contained thyroxine (T4) positioned at hormonogenic site 2553. It primed T cells for thyroiditis transfer, but noniodinated peptide containing thyronine (T0) was not compared. To determine 1) whether other primary hormonogenic sites were likewise immunogenic and 2) whether iodination was requisite for this and other sites to be an autoepitope, we derivatized three pairs of 12-mer peptides, 1-12, 2549-2560, 2559-2570, containing T0 or T4 at positions 5, 2553, or 2567, respectively. The six peptides were used to stimulate MTg-primed cells in vitro and to immunize mice. None directly induced thyroiditis; peptide Abs were the lowest in mice given hT0(2567) or hT4(2567). Of the three T4-containing peptides, hT4(5) and hT4(2553), but not hT4(2567), stimulated MTg-primed or HTg-primed T cells in vitro, with hT4(2553) being the stronger. Comparing hT0(2553) with hT4(2553), both activated MTg-primed, or peptide-primed, T cells to transfer thyroiditis. The marked immunogenicity of noniodinated hT0(2553) and the poor antigenicity of hT4(5) and hT4(2567) demonstrate that immunogenicity of a conserved hormonogenic site is dependent more on its amino acid sequence than on T4 substitution. Iodination may enhance antigenicity and/or binding affinity, but it is not required for a Tg hormonogenic site to be an autoepitope.
Antiviral cytotoxic T lymphocytes in vaginal mucosa of simian immunodeficiency virus-infected rhesus macaques.
Authors: Lohman B L , Miller C J , McChesney M B
Published: December 2022

The mucosal immune system of the female reproductive tract is of central importance for protection against sexually transmitted diseases, including HIV; however, this arm of the immune system remains poorly understood. Antiviral CTL responses never have been documented in the genital tract and the role of CTL in this anatomic site is unknown. In this study, CD8+ intraepithelial lymphocytes (IEL) in the vaginas of six simian immunodeficiency virus (SIV)-infected female rhesus macaques were identified by immunohistochemistry to be CD2+ and TCR beta-chain+. In addition, the majority of CD8+ IEL contained TIA-1+ cytoplasmic granules that are associated with CTL activity. CD8+ T cells were isolated from the vaginal epithelium and submucosa and amplified by limiting dilution in the presence of feeder cells. SIV p55gag and/or gp160env-specific lysis was detected in cultures of vaginal epithelial but not submucosal CD8+ T lymphocytes. Estimated SIV-specific precursor CTL frequencies were higher in the vaginal CD8+ IEL population of chronically infected monkeys than in the same cells from acutely infected monkeys or a naive control monkey. These results provide the first demonstration that antiviral CTL are present in the vaginal epithelium, and suggest that a vaccine may be able to generate anti-HIV CTL in the genital mucosa.
Myelin antigen-coupled splenocytes suppress experimental autoimmune encephalomyelitis in Lewis rats through a partially reversible anergy mechanism.
Authors: Vandenbark A A , Celnik B , Vainiene M , Miller S D , Offner H
Published: December 2022

Mechanisms of adult tolerance induced by injecting myelin Ag/ECDI (ethyl carbodiimide)-coupled splenocytes (Ag-SPL) were evaluated in Lewis rat experimental autoimmune encephalomyelitis (EAE). Rats could be tolerized against the major encephalitogenic epitope of guinea pig basic protein (Gp-BP), residues 72-89, using either S72-89-SPL or crude spinal cord homogenate (SCH)-SPL. In contrast to lymph node responses that were not affected significantly, the proliferation responses of blood T cells were markedly inhibited at the peak of EAE and during the recovery period to both Gp-BP and S72-89, but not to purified protein derivative (PPD), demonstrating Ag-specific tolerance. Tolerance induction reduced the number of infiltrating spinal cord (SC) cells, especially recruited CD45RC+ cells, as well as SC proliferation responses to S72-89 throughout the course of EAE. In contrast, SC response to PPD was increased at onset of EAE, but later during recovery the PPD response was also decreased compared with control rats. Tolerance induced by S72-89-SPL in blood and SC T cells could be reversed by incubation in IL-2, in accordance with an anergy model. BP-specific T cells preincubated in vitro with Gp-BP-SPL were rendered unresponsive to Gp-BP or S72-89, compared with the same T cells preincubated with histone (Hist)-SPL that remained Ag responsive. Consistent with an anergy model, preincubation with BP-SPL+IL-2 partially prevented tolerance induction to BP. T cells tolerized in vitro to BP-SPL induced milder EAE with delayed onset compared with control-tolerized T cells that produced lethal disease. These results demonstrate the efficacy of myelin Ag-coupled SPL in preventing EAE by selective tolerization of encephalitogenic T cells through a partially reversible anergy-induction mechanism.
CD38 signal transduction in human B cell precursors. Rapid induction of tyrosine phosphorylation, activation of syk tyrosine kinase, and phosphorylation of phospholipase C-gamma and phosphatidylinositol 3-kinase.
Authors: Silvennoinen O , Nishigaki H , Kitanaka A , Kumagai M , Ito C , Malavasi F , Lin Q , Conley M E , Campana D
Published: December 2022

Ligation of CD38 inhibits proliferation and induces apoptosis of human immature B cells, but the molecular mechanisms underlying this function are unknown. We found that CD38 dimerization with the specific mAbs T16 and IB4 induces rapid and transient tyrosine phosphorylation of several intracellular proteins in the immature B cell lines RS4;11, REH, 380, Nalm6, and OP-1. This effect could be markedly reduced by incubating cells with the tyrosine kinase inhibitors genistein, staurosporine, and herbimycin A. CD38 dimerization induced tyrosine phosphorylation of the protein kinase syk and increased syk kinase activity. CD38 dimerization also induced tyrosine phosphorylation of phospholipase C-gamma and of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-K). The latter was accompanied by a distinct increase in PI 3-kinase activity in the immunoprecipitates obtained with an anti-phosphotyrosine Ab. In contrast to the signaling triggered by surface Ig engagement in B lymphocytes, CD38 ligation did not appear to induce tyrosine phosphorylation of the src-like protein tyrosine kinases lyn, fyn, and btk, or of vav- and ras-GTPase-activating protein, nor did it induce detectable changes in cytosolic CA2+ concentrations. CD38 signaling also differed from cytokine-induced signaling in that it did not cause tyrosine phosphorylation of Jak1 and Jak2. Finally, CD38 ligation did not inhibit IL-3-induced tyrosine phosphorylation of Jak2. These results identify CD38 as a cell surface receptor with signal transduction properties activated by dimerization. Induction of signal transduction by CD38 ligation implies the existence of a yet unidentified natural ligand of CD38.
Identification of a population of CD4+ CTL that utilizes a perforin- rather than a Fas ligand-dependent cytotoxic mechanism.
Authors: Williams N S , Engelhard V H
Published: December 2022

Although cytotoxic activity has generally been shown to reside in the CD8+ population of T lymphocytes, there are numerous examples of cytotoxic CD4+ T cells. In the present study, we found that after depletion of CD8+ T cells, CD4+ T cells cultured in short-term in vitro mixed lymphocyte cultures developed strong Ag-specific cytotoxic activity. Such CD4+ CTL lysed both Fas+ and Fas- target cells and developed in Fas ligand-deficient gld mice. Thus, in contrast to several recent reports involving long-term T cell clones or short-term mitogen-activated splenocytes, the cytotoxic activity of these CD4+ CTL is not dependent on Fas-Fas ligand interactions. However, CD4+ cells derived in a similar manner from perforin knockout mice showed greatly decreased target cell lysis, indicating that their cytotoxic activity is primarily dependent on a perforin-based mechanism. The Fas-Fas ligand pathway has been shown to be important in the maintenance of peripheral tolerance, but to have a minimal role in protection against viral and bacterial pathogens. Thus, the existence of a population of CD4+ CTL that utilizes perforin re-emphasizes the likely importance of these cells in a classical defensive role against foreign pathogens, in addition to their already implied role as modulators of the immune response.
Therapeutic antitumor response after immunization with a recombinant adenovirus encoding a model tumor-associated antigen.
Authors: Chen P W , Wang M , Bronte V , Zhai Y , Rosenberg S A , Restifo N P
Published: December 2022

Recombinant adenovirus (rAd), deleted of critical genes that enable viral replication and replaced with genes encoding heterologous proteins, has been shown to be a safe and effective vector in gene therapy studies. To evaluate a potential role for rAd as an immunogen, we used two different replication-defective type 2 rAds encoding the model Ag, beta-galactosidase (beta-gal). To determine whether rAd elicited the kind of immune responses therapeutic in an anti-tumor setting, the beta-gal-expressing adenocarcinoma, CT26.CL25, was used. Splenocytes from BALB/c mice immunized with 1 x 10(7) infectious units (iu) of rAd demonstrated anti-beta-gal activity after in vitro culture with the relevant L(d) beta-gal peptide. Adoptive transfer of these same splenocytes produced dramatic regression of established pulmonary metastases. However, when tumor-bearing mice were treated with 1 x 10(7) iu of rAd, no reduction in established disease was observed even when rAd was given with exogenous IL-2. To increase the viral dose delivered to each animal, we used an E1-E4-deleted rAd that could be grown to much higher titers. Significant reduction occurred with 10-fold more rAd (1 x10(8) iu) was administered. Exogenous IL-2 administration with 1 x 10(8) iu of rAd resulted in augmentation of this anti-tumor effect. These findings demonstrate that when using a nonreplicating virus, the viral dose is directly related to the immune response generated. These data constitute the first reported use of rAd in the treatment of an established experimental cancer and may have implication for the treatment of human cancer.
Secretion of IL-16 (lymphocyte chemoattractant factor) from serotonin-stimulated CD8+ T cells in vitro.
Authors: Laberge S , Cruikshank W W , Beer D J , Center D M
Published: December 2022

At sites of inflammation, mononuclear cells are in close contact with aggregated platelets. Although the physiologic role of this association is not clear, this proximity suggests that platelet-derived mediators may play a role in chemoattraction of T lymphocytes. In the current study we investigated serotonin receptor-bearing lymphocyte modulation of T cell migration. Serotonin-stimulated human blood mononuclear cells secrete lymphocyte chemoattractant activity with selective activity for CD4+ T cells. This chemoattractant activity was observed within 2 h of exposure to serotonin and was blocked by serotonin type 2 receptor antagonists. Molecular sieve chromatography of supernatant from serotonin-stimulated PBMCs revealed a single peak of T cell chemoattractant activity with an apparent molecular mass of 56 kDa and a pl of 9.1. Neutralizing experiments with specific mAbs indicated that the serotonin-induced chemotactic factor was the previously characterized lymphocyte chemoattractant factor (LCF), recently designated IL-16. Serotonin induced secretion of IL-16 from CD8+, not CD4+, T cells which did not require the de novo protein synthesis. These studies suggest that serotonin, via serotonin type 2 receptors, may promote the recruitment of CD4+ T lymphocytes into an inflammatory focus.
Activation of NK cells by CC chemokines. Chemotaxis, Ca2+ mobilization, and enzyme release.
Authors: Loetscher P , Seitz M , Clark-Lewis I , Baggiolini M , Moser B
Published: December 2022

The responses of cloned human NK cells (ERNK57) to seven CC chemokines (monocyte chemotactic protein-1 (MCP-1), MCP-2, MCP-3, RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein-1 alpha (MIP-1 alpha), MIP-1 beta, and 1309) and two CXC chemokines (IL-8 and IP-10) were studied. Except for 1309, all CC chemokines induced chemotaxis of the NK cells in vitro, whereas the CXC chemokines were inactive. Maximal activity was obtained at 1 nM for MCP-1 and 10 to 100 nM for the other CC chemokines. The response showed a typically bimodal concentration dependence in all cases, except for RANTES, which induced a linear increase of migration over the concentration range of 0.1 to 1000 nM. A transient rise of the cytosolic-free Ca2+ concentration ([Ca2+]i), which is characteristic for chemokine-stimulated leukocytes, was observed in NK cells after stimulation with all six active chemokines. Since granule exocytosis is required for NK cell-dependent target killing, the effect of CC chemokines on exocytosis was tested. All CC chemokines that induced chemotaxis and [Ca2+]i changes also induced the release of granzyme A and N-acetyl-beta-D-glucosaminidase from cloned and blood NK cells, as well as CD8+ T cells after pretreatment with cytochalasin B. Maximum release was obtained from NK cells, and amounted to 35% and 13% of the total content of granzyme A and N-acetyl-beta-D-glucosaminidase, respectively. The capacity of cloned NK cells and CD8+ T cells to respond to chemokines depended on the time in culture after stimulation with PHA in the presence of irradiated feeder cells, and maximum responses were observed after 10 to 16 days. Our results demonstrate that CC chemokines activate NK cells, and are, therefore, not only attractants for monocytes, T lymphocytes, and eosinophil and basophil granulocytes.
Activated alpha beta-CD8+, but not alpha alpha-CD8+, TCR-alpha beta+ murine intestinal intraepithelial lymphocytes can mediate perforin-based cytotoxicity, whereas both subsets are active in Fas-based cytotoxicity.
Authors: Gelfanov V , Gelfanova V , Lai Y G , Liao N S
Published: December 2022

CD8 single-positive (CD8+) T cells in murine intestinal intraepithelial lymphocytes (iIEL) consist of alpha alpha-CD8+ and alpha beta-CD8+ subpopulations. Cytotoxicity represents an important function of peripheral CD8+ T cells, so we examined perforin-granzymebased and Fas-based cytotoxicity of activated CD8+ TCR-alpha beta+ iIEL subsets. We found that allospecific CTL activity was induced from alpha beta-CD8+ iIEL but not from alpha alpha-CD8+ iIEL even when allospecific TCR were present on the iIEL, as demonstrated by using 2C TCR transgenic mice. On the other hand, both CD8+ iIEL subsets proliferated upon allostimulation with a lower responder frequency than CD8+ LN cells. The alpha alpha-CD8+ TCR-alpha beta+ iIEL appeared to lose their ability to perform perforin-based killing after activation through TCR because fresh cells lysed P815 cells coated with anti-TCR beta-chain (TCR-beta) mAb, whereas cells activated by plate-bound anti-TCR mAb did not. Of interest, both activated CD8+ TCR-alpha beta+ iIEL subsets, but not fresh cells, were able to mediate Fas-based killing when triggered with PMA and CA2+ ionophore.
Specific Th1 cytokine down-regulation associated with primary clinically derived human immunodeficiency virus type 1 Nef gene-induced expression.
Authors: Collette Y , Chang H L , Cerdan C , Chambost H , Algarte M , Mawas C , Imbert J , Burny A , Olive D
Published: December 2022

HIV-1 infection is associated with a progressive and functional decline in the CD4+ lymphoid Th1 subset. Here, we propose that the HIV nef gene product may function as a specific regulator of Th1 cytokine production. By use of a T cell-specific inducible expression system, we show that upon T cell activation, induced nef expression down-regulated both IL-2 and IFN-gamma production in a dose-dependent manner, whereas IL-4, IL-9, IL-13, IL-8, and TNF-alpha production remained unaffected. In addition to this, independent transfected clones expressing various nef genes, including nef sequences amplified directly from an HIV-1 primary clinical isolate, displayed a similar pattern of cytokine expression. The specific Th1 impairment induced by nef, therefore, seems to be an important and conserved feature of HIV-1 infection and may represent a significant function of this viral gene in AIDS pathogenesis.
TCR in Fas-sensitive T cells from labial salivary glands of patients with Sjögren's syndrome.
Authors: Sumida T , Matsumoto I , Murata H , Namekawa T , Matsumura R , Tomioka H , Iwamoto I , Saito Y , Mizushima Y , Hasunuma T , Maeda T , Nishioka K
Published: December 2022

Apoptosis is found in labial salivary glands of patients with Sjogren's syndrome (SS). To analyze the pathogenesis of apoptosis in labial salivary glands of SS patients, we examined the expression of Fas Ag and Fas ligand (FasL) and TCR on T cells susceptible to anti-Fas mAbs (CH-11). Fas Ag is expressed on epithelial cells and mononuclear cells in the salivary glands as observed by an immunohistochemical method. FasL is over-expressed specifically on T cells infiltrating into the labial salivary glands as seen by an reverse transcription-PCR method. These results suggest that apoptosis in SS lips is mediated by a Fas/FasL pathway. PCR single-strand conformation polymorphism (SSCP) clearly demonstrated that more than 40% of the T cells accumulated in labial salivary glands are deleted by incubation with CH-11 for 24 h in vitro, indicating that these expanded cells are Fas sensitive. junctional sequence analysis showed that the same conserved amino acid motifs (LAGG, RLA, SLG, QGPG, PGG, GGE, RGR, KPG, AGD, and MLG) in complementarity determining region 3 (CDR3) are found in Fas-sensitive T cell clones, whereas they are not detected in Fas-resistant clones, suggesting that Fas-sensitive T cells recognize restricted T cell epitopes on autoantigens. In conclusion, the findings suggest that Fas-sensitive T cells in labial salivary glands of SS patients are generated by Ag stimulation and might function as autoreactive T cells.
Are transporter associated with antigen processing (TAP) and tapasin class I MHC chaperones?
Authors: Solheim J C , Carreno B M , Hansen T H
Published: December 2022

Class I MHC heavy chains associate with many proteins in the endoplasmic reticulum, including TAP, calnexin, calreticulin, and the newly defined tapasin molecule. Recent studies have begun to resolve the nature of how these proteins interact with class I as well as the functional significance of each of these interactions. We propose here that TAP and tapasin are leading candidates to be highly specific chaperones for the class I molecule.
Costimulation of CD28- T lymphocytes by 4-1BB ligand.
Authors: DeBenedette M A , Shahinian A , Mak T W , Watts T H
Published: December 2022

The interaction of the T cell surface protein CD28 with its ligand, B7-1 or B7-2, provides a critical costimulatory signal for T cell activation. T cells from CD28- mice are deficient in a variety of responses, including those to lectins and allogeneic spleen cells. However, some immune responses do occur in CD28- mice, suggesting the existence of alternate costimulatory pathways. In this work, we show that T cells purified from CD28- mice respond to B lymphomas expressing 4-1BB ligand (4-1BBL), a member of the TNF gene family. This response is inhibited by a soluble form of 4-1BB, the T cell surface receptor for 4-1BBL. Thus, 4-1BBL/4-1BB interaction provides costimulatory signals to T cells independent of signaling through the CD28 receptor. We find that 4-1BBL is inducible on splenic B cells by CD40 ligand/CD40 interaction or by culturing of splenic dendritic cells, treatments that also induce B7 family molecules. CD28- T cells fail to respond in an MLR to resting allogeneic spleen cells. However, treatment of spleen cells with CD40 ligand renders them competent in activation of CD28- T cells. In contrast to results using B lymphomas as APC, soluble 4-1BB fails to inhibit the T cell response to activated spleen cells. This failure of soluble 4-1BB to block an MLR between CD28+ or CD28- T cells and allogeneic spleen cells is in contrast to a previous report with CD28+ cells.
Induction and prevention of colonic inflammation in IL-2-deficient mice.
Authors: Ehrhardt R O , Lúdvíksson B R , Gray B , Neurath M , Strober W
Published: December 2022

Gene-targeted mice deficient for IL-2 (IL-2 -/- mice) are free of apparent disease when maintained under germfree conditions but develop colitis and autoimmunity in a conventional environment. Here we show that colitis can be reproducibly induced in IL-2 -/- mice, but not in IL-2 +/+ mice, by i.p. immunization with Ag in CFA; thus enabling the systematic study of the immunopathogenesis of the colitis. We found that TNP-KLH or TNP-OVA had the most significant effect in inducing colitis, and while TNP-KLH immunization leads to the early appearance of activated T cells in the colons of both IL-2 -/- and IL-2 +/+ mice, only lamina propria cells of IL-2 -/- mice produced high amounts of INF-gamma. Moreover, both infiltrating colon CD4+ (69%) and CD8+ (6%) T cells secrete large amounts of IFN-gamma; however, only the depletion of CD4+ T cells leads to abrogation of the inflammation. In further analysis, we showed that the high IFN-gamma production is IL-12 driven, since colonic tissues of IL-2 -/- mice but not IL-2 +/+ mice show the presence of heterodimeric IL-12 and co-administration of anti-IL-12 with TNP-KLH completely prevented colitis and significantly reduced IFN-gamma production. Finally, we demonstrate that IL-2 -/- mice are deficient in their ability to induce Th2 responses after TNP-KLH challenge and that such immunization also leads to autoimmune-like phenomena in other organs of IL-2 -/- mice. These findings suggest that in the absence of IL-2 systemic administration of Ag induces primarily Th1 cells driven by overexpression of heterodimeric IL-12.
The EBV IL-10 homologue is a selective agonist with impaired binding to the IL-10 receptor.
Authors: Liu Y , de Waal Malefyt R , Briere F , Parham C , Bridon J M , Banchereau J , Moore K W , Xu J
Published: December 2022

BCRF1 is an EBV homologue of human IL-10 (hIL-10) and is known as viral IL-10 (vIL-10). As found earlier for the effects of vIL-10 on mouse mast cells and CD4+ T cells, the efficiency of inhibition by vIL-10 of IL-2 production by human CD4+ T cell clones is approximately 1000-fold diminished compared with hIL-10. We studied the interaction of vIL-10 and an epitope-tagged homologue, vIL-10His6, with recombinant mouse and human IL-10 receptors (mIL-10R, hIL-10R). vIL-10His6 has approximately 1000-fold lower affinity for recombinant IL-10R than does hIL-10, yet stimulates proliferation of mouse Ba/F3 (BaF)-mIL-10R- and human TF1-hIL-10R-transfected cells with a sp. act. comparable to or greater than that of the cellular cytokine. In contrast, BaF-hIL-10R cells are approximately 1000-fold less sensitive to vIL-10His6 than are BaF-mIL-10R cells. An anti-hIL-10R mAb (3F9) blocks responses to both hIL-10 and vIL-10His6, while a soluble form of hIL-10R effectively neutralizes biologic responses only to hIL-10 by both BaF-IL-10R transfectants and normal human peripheral blood cells. The results indicate that biologic responses to both hIL-10 and vIL-10 require the known IL-10R, and suggest the existence of at least one additional IL-10R subunit. We suggest that vIL-10 is a selective agonist that is impaired in its ability to bind the defined IL-10R, which we now designate as IL-10R alpha.
The role of B7-1 and LFA-3 in costimulation of CD8+ T cells.
Authors: Parra E , Wingren A G , Hedlund G , Kalland T , Dohlsten M
Published: December 2022

This study compares the ability of LFA-3 (CD58) and B7-1 (CD80) ligands to provide costimulatory signals for superantigen (SAg)-stimulated CD8+ and CD4+ T cells. We show that B7-1 and LFA-3 costimulation activate CD8+ T cells to proliferation, cytokine production (IL-2, TNF, and IFN-gamma), and cytotoxicity. A long-lasting proliferative response was observed after combined DR/B7-1/LFA-3 costimulation. Detailed analysis of SEA-activated CD8+ T cells revealed that maximal production of IFN-gamma was seen in LFA-3-costimulated cells, while production of IL-2 was mainly induced after B7-1 costimulation. A fivefold increase in the IFN-gamma production was observed when activated CD8+ T cells were costimulated with Chinese hamster ovary (CHO)-DR/LFA-3 cells compared with the secretion induced by CHO-DR/B7-1. In contrast, SEA-treated CD4+ T cells costimulated with B7-1 or LFA-3 gave rise to a similar production of IFN-gamma, suggesting a preferential function for the CD2/LFA-3 pathway in the regulation of IFN-gamma in CD8+ T cells. Moreover, the generation of CTL was supported similarly by B7-1 and LFA-3 costimulation, but not by CHO-DR cells. We conclude that ligation of the CD28 and CD2 receptors mediate distinct effect on CD8+ and CD4+ T cell effector functions.
Elimination of N-linked glycosylation sites from the human IgA1 constant region: effects on structure and function.
Authors: Chuang P D , Morrison S L
Published: December 2022

IgA1 Abs possess conserved N-linked glycosylation sites in the second C region and secreted tailpiece domains. To understand the role of these carbohydrates in the structure and function of human IgA1, site-directed mutants that produce human IgA1 lacking either one or both of the N-linked carbohydrate sites have been produced. When the mutant heavy chains are expressed in myeloma lines producing the relevant kappa-light chain, efficient secretion of the monomer and dimer forms of IgA1 is seen. In addition, higher polymer forms of the IgA molecules lacking the third domain carbohydrate, either singly or in the double mutant, are present. Functional analysis of the IgA1 proteins has shown significant differences between the various mutants and wild-type IgA. The carbohydrate mutants show a reduced affinity for their target Ag, dansyl. All of the IgA1 molecules retained the ability to bind to the polymeric Ig receptor. C3 binding was observed for all of the IgA molecules, with the IgA mutants lacking the third domain carbohydrate showing a reduced ability to bind C3; however, IgA did not effectively activate the alternative pathway, as determined by factor B cleavage and terminal complex binding. These studies demonstrate that N-linked glycosylation in the constant domain of human IgA1 plays an important role in the biologic properties of IgA1.
Mapping of the catalytic and epitopic sites of human CD38/NAD+ glycohydrolase to a functional domain in the carboxyl terminus.
Authors: Hoshino S , Kukimoto I , Kontani K , Inoue S , Kanda Y , Malavasi F , Katada T
Published: December 2022

We reported that 1) ecto-NAD+ glycohydrolase (NADase) activity induced upon differentiation of HL-60 cells is localized on the extracellular carboxyl-terminal side of CD38 and that 2) CD38 ligation by specific mAbs is followed by protein tyrosine phosphorylation in the cells. The strategy selected for identifying the relevant catalytic domains of the molecule relies upon the production in COS-7 cells of carboxyl-terminal deletion mutants of CD38. The mutants with fewer than 15 amino acids deleted at the carboxyl terminus of the 300-amino acid wild-type molecule maintained NADase activity, whereas those with more than 27 amino acids deleted did not. The general inference is that the carboxyl-terminal 273-285 sequence bears the site of enzyme activity. Introduction of site-directed mutation of a conserved cysteine residue (Cys275), located in the 273-285 sequence, completely abolished NADase activity. The second issue resolved in this work is the definition of an epitope of the agonistic anti-CD38 mAbs. To this aim, a panel of selected anti-CD38 mAbs was tested using these mutants and various CD38 fragments as the target in immunoblot analyses. All of the epitopes recognized by mAbs inducing protein tyrosine phosphorylation were mapped on an identical site containing the carboxyl-terminal sequence of 273-285. The conclusion is that the discrete carboxyl-terminal sequence identified in the present study not only plays a key role in its ecto-NADase activity, but actually constitutes the epitopes exploited by the agonistic anti-CD38 mAbs for transmembrane signaling.
Intracellular IL-1 receptor antagonist promoter: cell type-specific and inducible regulatory regions.
Authors: Jenkins J K , Drong R F , Shuck M E , Bienkowski M J , Slightom J L , Arend W P , Smith M F
Published: December 2022

The objective of these studies was to examine the molecular mechanisms involved in transcriptional regulation of the gene for the intracellular structural variant of the IL-1 receptor antagonist (icIL-1Ra) molecule. By reverse transcription-PCR analysis, constitutive expression of endogenous icIL-1Ra mRNA was observed in the epithelial cell lines A431 and HT-29, but not in the macrophage cell lines RAW 264.7 and U937, or in the lymphocyte cell lines Raji and Jurkat. However, icIL-1Ra mRNA expression was observed in response to stimulation with LPS in RAW 264.7 cells and to PMA and LPS in U937 cells. To examine the mechanisms of transcriptional regulation, 4.5 kb of the 5' flanking sequence was isolated from the human icIL-1Ra gene, sequenced, cloned into a luciferase expression vector (pIC4525.Luc), and examined in transfection studies. The pIC4525.Luc construct exhibited a pattern of expression in epithelial and macrophage cell lines similar to that of the endogenous icIL-1Ra gene. To obtain a generalized map of cell type-specific and inducible cis-acting DNA elements, nested 5' deletional mutants of the icIL-1Ra promoter were constructed. Results from transfection studies with these icIL-1Ra promoter/luciferase fusion constructs indicated that constitutive expression in epithelial cells was under the control of three positively acting regions located between bases -4525 to -1438, -288 to -156, and -156 to -49. In contrast, basal expression of pIC4525.Luc in transfected but unstimulated RAW 264.7 cells was under the control of a weak inhibitory region located between bases -4525 to -1438 and a strong positive element between -156 and -49. LPS induction of icIL-1Ra transcription in RAW 264.7 cells was regulated by strong positively acting DNA regions between bases -1438 to -909 and -156 to -49. In summary, the proximal region of the icIL-1Ra promoter, between bases -156 to -49, contains positive cis-acting elements that are needed for expression in both epithelial and monocyte cell lines. However, our results indicate that the ability of this proximal promoter region to control expression is strongly influenced, both positively and negatively, by other upstream cis-acting elements in a cell type-specific manner.
Bispecific monoclonal antibody complexes bound to primate erythrocyte complement receptor 1 facilitate virus clearance in a monkey model.
Authors: Taylor R P , Sutherland W M , Martin E N , Ferguson P J , Reinagel M L , Gilbert E , Lopez K , Incardona N L , Ochs H D
Published: December 2022

We investigated the feasibility of using bispecific mAb complexes to redirect and improve the efficiency of the primate E complement receptor 1-based clearance reaction to remove a virus from the circulation. As an initial approach, we used bacteriophage phiX174 as an immunologic model for mammalian viruses. Bispecific complexes were prepared by chemically cross-linking a mAb specific for complement receptor 1 with a mAb specific for the bacteriophage phiX174. In a monkey model these complexes facilitate rapid and quantitative binding of the target bacteriophage to E in vitro and in vivo. Moreover, after in vivo binding to E, the complexes containing mAb and prototype virus are rapidly cleared from the circulation of rhesus and cynomolgus monkeys without loss of E. Our findings suggest that bispecific mAb complexes, in concert with primate E complement receptor 1, may have therapeutic utility in the treatment of diseases associated with blood-borne pathogens.
Cytolytic and cytostatic properties of an anti-human Fc gammaRI (CD64) x epidermal growth factor bispecific fusion protein.
Authors: Goldstein J , Graziano R F , Sundarapandiyan K , Somasundaram C , Deo Y M
Published: December 2022

A bispecific fusion protein (H22-EGF) that binds simultaneously to the epidermal growth factor receptor (EGF-R) and to the high affinity receptor for the Fc portion of human IgG, Fc gammaRI (CD64), has been successfully constructed and expressed. For this construction, genomic DNA encoding the Fd fragment of humanized anti-Fc gammaRI mAb, H22, which binds Fc gammaRI at an epitope that is distinct from the Fc binding site, was fused to cDNA encoding human epidermal growth factor (EGF), a natural ligand for EGF-R. The resulting H22Fd-EGF-expressing vector was transfected into a myeloma cell line that was transfected previously with a vector containing DNA encoding the H22 kappa-light chain. SDS-PAGE analysis of purified H22-EGF demonstrated that the fusion protein was secreted predominantly as H22Fab'-EGF monomer (approximately 55 kDa), even though a free Cys residue exists in the hinge region of the H22 Fab' component. Using a novel bispecific flow cytometry-binding assay, we demonstrated that the purified bispecific fusion protein, H22-EGF, was able to bind simultaneously to soluble Fc gammaRI and EGF-R-expressing cells. H22-EGF inhibited the growth of EGF-R-overexpressing tumor cells and mediated dose-dependent cytotoxicity of these cells in the presence of Fc gammaRI-bearing cytotoxic effector cells. These results suggest that this fusion protein may have therapeutic utility for EGF-R-overexpressing malignancies.