bluuebunny/crossref_metadata_2025 huggingface dataset:
Histamine augments cytokine-stimulated IL-11 production by human lung fibroblasts.
Authors: Zheng T , Nathanson M H , Elias J A
Published: December 2022

Histamine mediates its effects via histamine receptors and by participating in a multicellular cytokine cascade. IL-11 is a stromal cell-derived cytokine with biologic activities that overlap with IL-6. To further understand the biology of histamine and IL-11, we determined whether histamine regulates the production of IL-11 by human lung fibroblasts. Histamine was a weak stimulator of IL-11 production. Importantly, it also interacted in a synergistic fashion with TGF-beta 1 to further augment IL-11 protein production and mRNA accumulation. This synergistic interaction was not altered by the H2 receptor antagonist cimetidine and could not be reproduced with the H2 receptor agonist 4-methylhistamine. In addition, it was not abrogated by the cyclic nucleotide-dependent protein kinase inhibitor N-(2-1-guanidinoethyl)-5 isoquinolinesulfonamide hydrochloride), and histamine and TGF-beta 1 did not stimulate intracellular cAMP. In contrast, the synergy was abrogated by the H1 histamine receptor antagonists diphenhydramine and pyrilamine, could be reproduced when histamine was replaced with the H1 agonist 2-methylhistamine, and was abrogated by the calmodulin antagonists N-(6-aminohexyl)-1-napthalenesulfonamide), N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide), and trifluoperazine dichloride and by the intracellular calcium chelator 1,2-bis-(2-amino-5-bromo-phenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester. In addition, although TGF-beta 1 did not alter cytosolic Ca2+, histamine caused a biphasic increase in cytosolic Ca2+, and the majority of cells incubated with TGF-beta 1 plus histamine exhibited sustained Ca2+ oscillations. These studies demonstrate that histamine is an important regulator of fibroblast IL-11 production, that histamine interacts with TGF-beta 1 in the induction of this cytokine, and that this interaction is mediated, to a great extent, by a pretranslational mechanism that is dependent on H1 receptors and a calcium/calmodulin-dependent activation pathway.
Binding of IL-1 alpha, IL-1 beta, and IL-1 receptor antagonist by soluble IL-1 receptors and levels of soluble IL-1 receptors in synovial fluids.
Authors: Arend W P , Malyak M , Smith M F , Whisenand T D , Slack J L , Sims J E , Giri J G , Dower S K
Published: December 2022

These studies have examined the binding of the three IL-1 ligands, IL-1 alpha, IL-1 beta, and IL-1 receptor antagonist (IL-1 ra), to soluble forms of types I and II IL-1Rs (sIL-1RI and sIL-1RII). This interaction was measured in direct binding experiments, in which the ligands bound to immobilized sIL-1R, and in inhibition experiments, in which sIL-1R in solution inhibited the binding of IL-1 ligands to immobilized sIL-1R. In addition, the effects of sIL-1R on the detection of IL-1 ligands by ELISA were examined. Finally, levels of sIL-1R in synovial fluid samples were determined, and their effects on measurement of IL-1 in these samples were estimated. IL-1 beta bound more avidly to sIL-1RII than IL-1 alpha or IL-1ra, primarily because of a slow dissociation rate. In contrast, IL-1 ra bound more avidly than IL-1 alpha or IL-1 beta to sIL-1RI, again because of a slow dissociation rate. sIL-1RII and sIL-1RI inhibited the detection of IL-1 beta and IL-1ra, respectively, by ELISA. Low levels of sIL-1RI (approximately 1.0-2.5 ng/ml) were present in all synovial fluids, irrespective of the degree of inflammation, and were correlated inversely with the levels of measured IL-1ra. In contrast, higher levels of sIL-1RII (approximately 10-20 ng/ml) were found in inflammatory synovial fluids and were not correlated with IL-1ra levels. IL-1 beta could not be detected in any synovial fluid. These results suggest that some IL-1 beta and IL-1ra may be bound in vivo to sIL-1RII and sIL-1RI, respectively, leading to underestimations of cytokine concentrations in body fluids when measured by ELISA.
Developmental ages of the thymic epithelium and of the T cell precursors together determine the proportions of peripheral CD4+ cells.
Authors: Adkins B , Hamilton K
Published: December 2022

In earlier studies on chimeric animals, we found that fetal thymocytes produced peripheral T lymphocyte populations depleted of CD4+ cells. This occurred whether the fetal thymocytes matured in the presence of adult or fetal thymic stromal cells. In contrast, fetal liver cells that differentiated in the adult thymus generated normal proportions of peripheral CD4+ cells. Because fetal liver cells are thought to be the immediate precursors to fetal thymocytes, we proposed that fetal thymic stroma would modulate the differentiation of fetal liver cells; specifically, that fetal liver cells maturing in the fetal thymus would resemble fetal thymocytes and produce low levels of peripheral CD4+ cells. To test this hypothesis, fetal thymic lobes were colonized in vitro with fetal liver cells and subsequently transplanted in vivo to Thy-1 congenic hosts. Under these conditions, fetal liver cells produced reduced proportions of CD4+ peripheral progeny. The under-representation of CD4+ peripheral T cells was apparently governed by the thymic epithelium because similar results were obtained with 2-deoxyguanosine-treated fetal thymuses colonized by fetal liver cells. In contrast, adult bone marrow cells made normal levels of CD4+ peripheral T cells whether maturation occurred in the fetal or the adult thymus. Thus, pre-T cells (fetal liver or adult bone marrow) lose the capacity to respond to fetal thymic stromal cells during development. These results indicate that the proportions of CD4+ cells in peripheral tissues are regulated by a combination of the developmental ages of the T cell precursors and the thymic stromal environment.
Antigen-presenting function of human endothelial cells. Direct activation of resting CD8 T cells.
Authors: Epperson D E , Pober J S
Published: December 2022

We characterized the response of resting human CD8 T cells to allogeneic endothelial cells (EC). Both resting and IFN-gamma-pretreated EC stimulate similar CD8 T cell proliferative responses (peak, day 5 to 6), whereas only IFN-gamma-pretreated EC stimulate CD4 T cells. The response increases with increasing numbers of CD8 T cells from 25,000 to 400,000/well. The proliferation of CD8 T cells is inhibited by mAbs reactive with CD8 or HLA-A and -B molecules but not with CD4 or HLA-DR. mAb blocking studies show a role for CD2, LFA-3, and CD59, but not for intercellular adhesion molecule-1, intercellular adhesion molecule-2, very late activation Ag-4, vascular cell adhesion molecule-1, CD28, or CD28 ligand, as costimulatory molecules. The stimulation of resting CD8 T cells by EC causes secretion of IL-2 and IFN-gamma but not IL-4. Both proliferation and IFN-gamma secretion are inhibited by mAb to the IL-2R alpha subunit (CD25). Limiting dilution analysis suggests that approximately 1 in 20,000 resting CD8 T cells secrete IL-2 in response to allogeneic EC. EC stimulate greater than 1 in 10,000 CD8/CD45RO+ cells but fewer than 1 in 40,000 CD8/CD45RA+ cells, which indicates that primarily memory CD8 T cells respond to EC. Coculturing CD8 cells with EC stimulates a sufficient level of endothelial class II MHC expression to subsequently support a CD4 T cell proliferative response. The ability of memory CD8 T cells to proliferate against allogeneic EC, a nonclassical APC, and their ability to stimulate EC may contribute to the initiation of vascularized organ graft rejection.
Platelet-activating factor induces the tyrosine phosphorylation and activation of phospholipase C-gamma 1, Fyn and Lyn kinases, and phosphatidylinositol 3-kinase in a human B cell line.
Authors: Kuruvilla A , Pielop C , Shearer W T
Published: December 2022

Platelet-activating factor (PAF) is a versatile lipid mediator of inflammation in a variety of biologic systems. We have previously reported that one of the earliest events in the signal transduction pathway of PAF in a human B lymphoblastoid cell line was the induction of tyrosine kinase activity concomitant with the activation of phospholipase C (PLC). We now demonstrate the occurrence of multiple tyrosine phosphorylation-dependent events which follow the interaction of PAF with its receptor on B cells. Anti-phosphotyrosine immunoprecipitates from lysates of PAF-stimulated cells, when fractionated by SDS-PAGE and analyzed by Western blotting with anti-PLC-gamma 1, showed that maximal tyrosine phosphorylation of this enzyme occurred within 2 min of stimulation. This phenomenon was verified by immunoprecipitating with anti-PLC-gamma 1 and subsequently probing with anti-phosphotyrosine. Immunoprecipitation of the tyrosine kinases, Fyn and Lyn, from PAF-stimulated cells, and use of these immunoprecipitates in kinase assays established that the activation of both kinases also occurred within the first 2 min of stimulation with phosphorylation occurring on their tyrosine residues. Additionally, we also provide evidence for the tyrosine phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase (PtdIns 3-kinase) and activation of this kinase by PAF in a dose-dependent manner, maximal activation occurring within 10 min post-stimulation. We have thus demonstrated that the activation of tyrosine kinases is an important proximate step in PAF-mediated signal transduction in B cells, leading to tyrosine phosphorylation and activation of PLC-gamma 1, Fyn and Lyn kinases, and PtdIns 3-kinase.
Outcome of Kupffer cell antigen presentation to a cloned murine Th1 lymphocyte depends on the inducibility of nitric oxide synthase by IFN-gamma.
Authors: Roland C R , Walp L , Stack R M , Flye M W
Published: December 2022

The interaction between lymphocytes and the resident hepatic macrophage, the Kupffer cell (KC), is relevant to the phenomenon of immune tolerance to Ags entering the liver. Tolerance to Ag administered via the portal vein can be prevented by the rare earth lanthanide metal, gadolinium (Gd). Therefore, we studied the ability of OVA-responsive, H-2d-restricted Th1 clones to proliferate in response to KCs from DBA/2J (H-2d) mice that had been injected with either saline (control) or a Gd solution. Whereas control KCs functioned as effective APCs, KCs from Gd-injected mice (GdKC) were incapable of sustaining the proliferative response of the Th1 clone to the 16 mer of OVA (323-339). This lack of proliferation was determined not to be caused by impaired Ag processing, but rather was the result of IFN-gamma-stimulated nitric oxide (NO) release by the APC: 1) In vitro addition of the nitric oxide synthase (NOS) inhibitor NG-methyl-L-arginine (NMMA) restored the ability of the Gd-treated KC to stimulate clone proliferation. 2) Additional of anti-IFN-gamma, but not anti-IL-2 or anti-IL-4, prevented the induction of NOS in the Gd-exposed KC and was associated with clone proliferation. 3) IFN-gamma levels from clone-GdKC-OVA cocultures closely paralleled the nitrite released by GdKCs. 4) Only the addition of rIFN-gamma, and not IL-2 or IL-4, to cultures of purified GdKCs resulted in the release of nitrite. The results of the study suggest an autocrine loop initiated by the interaction of the clone's TCR with the class II MHC molecule presenting processed OVA on the surface of KC. This interaction stimulates the Th1 lymphocyte to release IFN-gamma, which in turn induces NO release by KCs isolated from Gd-injected mice. This release of NO blocks Th1 proliferation. Such a feedback loop may have particular relevance to Ag-specific tolerance, which is not only induced by the administration of Ag into the portal vein, but is also prevented by Gd pretreatment of the recipient animal.
Tyrosine phosphorylation of VCP, the mammalian homologue of the Saccharomyces cerevisiae CDC48 protein, is unusually sensitive to stimulation by sodium vanadate and hydrogen peroxide.
Authors: Schulte R J , Campbell M A , Fischer W H , Sefton B M
Published: December 2022

A mAb produced by immunization of mice with tyrosine-phosphorylated proteins from activated B lymphocytes was found to recognize valosin-containing protein (VCP). VCP is the mammalian homologue of the Saccharomyces cerevisiae CDC48 protein and has localized regions of sequence identity with the yeast Sec18 and Pas1 proteins and the mammalian NSF protein, all of which are important in intracellular vesicular traffic or formation. VCP was found to be constitutively phosphorylated on tyrosine in Rous sarcoma virus-transformed fibroblasts. Phosphorylation of VCP on tyrosine was stimulated only modestly during activation of B lymphocytes by ligation of membrane Ig. In contrast, treatment of B cells with either H2O2 or a combination of H2O2 and Na3VO4 greatly increased tyrosine phosphorylation of VCP. These results may suggest that under normal conditions tyrosine phosphorylation of VCP has a rapid turnover and that it can be detected easily only when dephosphorylation is inhibited by artificial means.
An Ig heavy chain enhancer of the channel catfish Ictalurus punctatus: evolutionary conservation of function but not structure.
Authors: Magor B G , Wilson M R , Miller N W , Clem L W , Middleton D L , Warr G W
Published: December 2022

The teleost fishes are among the earliest evolutionary lineages to have an Ig heavy chain (IgH) locus whose organization approximates that of mammals. To understand transcriptional control of the IgH locus in a teleost fish and to gain insight into the evolution of the control elements, the enhancer activity in the IgH locus of the channel catfish, Ictalurus punctatus, was investigated. Segments of the locus extending from upstream of the proximal JH gene to 2.5 kb downstream of the second transmembrane (TM2) exon of the mu gene were tested in transient transfection expression assays in murine myeloma and T cell lines, and in catfish B lymphoblastoid, monocyte-like, and putative T cell lines. In marked contrast to mammals, no enhancer activity was observed in the catfish JH to C mu intron, but strong enhancer activity (approaching that of the murine IgH intronic enhancer) was identified in a 1.8-kb segment that included the TM2 exon. This catfish enhancer was active in a B lineage-specific manner in both catfish and murine cells. It was not localized in a small core region, but appeared to contain multiple, dispersed cooperative elements rich in octamer- and mu E5-related motifs. Although the catfish IgH enhancer shares functional characteristics with the mammalian IgH intronic and 3' enhancers, its unusual organization does not permit any obvious inferences concerning evolutionary relationships between the catfish enhancer and any one of the murine IgH enhancers.
Individual effects of the DR11-variable beta-chain residues 67, 71, and 86 upon DR(alpha,beta 1*1101)-restricted, peptide-specific T cell proliferation.
Authors: McKinney J S , Fu X T , Swearingen C , Klohe E , Karr R W
Published: December 2022

The four members of the HLA-DR11 family of class II molecules vary only by three or fewer amino acids via dimorphisms among DR beta-chain residues 67, 71, and 86. However, they differ markedly in their abilities to induce proliferation of DR(alpha,beta 1*1101)-restricted, peptide-specific T cell clones. To dissect which DR11-variable residues, individually and in combination, mediate these functional differences, we used as APC transfectants expressing DR molecules with one of all possible permutations of DR11-variable sequences, including the four DR11 family members, and four additional DR11 variant mutants. The abilities of the wild-type or mutant molecules to present two distinct influenza peptide Ags, HA307-19 and HA128-45, to T cells was assessed in in vitro T cell proliferation assays. Of the naturally dimorphic DR11 positions, residue beta 71 variation significantly influenced the ability of DR11 molecules to present both peptides to DR(alpha,beta 1*1101)-restricted T cells. Residue beta 86 variation had relatively less influence than reported in several other DR and peptide systems. Residue beta 67 variation usually appeared irrelevant to T cell proliferation, but in two mutants led to unexpected T cell proliferation independent of nominal peptide Ag. Peptide binding, assessed by flow cytometry, was not found to be altered by any mutations that disrupted DR(alpha,beta 1*1101)-like presentation. These data indicate that residue beta 71 exerts a central role in influencing the functional differences among DR11 molecules, whereas the widely studied dimorphism of residue beta 86 is not as generally influential in DR11 as in other alleles.
Genomic organization of mouse and human Bruton's agammaglobulinemia tyrosine kinase (Btk) loci.
Authors: Sideras P , Müller S , Shiels H , Jin H , Khan W N , Nilsson L , Parkinson E , Thomas J D , Brandén L , Larsson I
Published: December 2022

Btk is a cytoplasmic protein tyrosine kinase (PTK) that has been directly implicated in the pathogenesis of X-linked agammaglobulinaemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. We have isolated phage and cosmid clones that allowed us to deduce the genomic structure of mouse and human Btk loci. The mouse and human genes are contained within genomic regions that span approximately 43.5 kb and 37.5 kb, respectively. Both loci contain 18 coding exons ranging between 55 and 560 bp in size with introns ranging in size from 164 bp to approximately 9 kb. The 5'-untranslated regions are encoded by single exons located approximately 9 kb upstream of the first coding exon. Exon 18 encodes for the last 23 carboxyl-terminal amino acids and the entire 3'-untranslated region. The location of intron/exon boundaries in the catalytic domains of the mouse and human Btk loci differs from that found in other described sub-families of intracellular PTKs, namely that of Src, Fes/Fer, Csk, and Abl/Arg. This observation is consistent with the classification of Btk together with the recently characterized kinases, Tec and Itk, into a separate sub-family of cytoplasmic PTKs. Putative transcription initiation sites in the mouse and human Btk loci have been determined by using the rapid amplification of cDNA ends assay. Similar to many other PTK specific genes, the putative Btk promoters lack obvious TATAA and CAAAT motifs. Putative initiator elements and potential binding sites for Ets (PEA-3), zeste, and PuF transcription factors are located within the 300 bp which are located upstream of the major transcription start site in both species. These sequences can mediate promoter activity when placed upstream of a promotorless chloramphenicol acetyl transferase reporter gene in an orientation-dependent manner. The present analysis will significantly facilitate the mutational analyses of patients with XLA and the further characterization of the function and regulation of the Btk molecule.
HLA-G polymorphisms in African Americans.
Authors: van der Ven K , Ober C
Published: December 2022

HLA-G is a class I MHC gene most notable for its restricted tissue distribution. The unique expression of this gene in extravillous cytotrophoblast at the maternal-fetal interface suggests that HLA-G plays a critical role in human pregnancy. Previous studies have reported that HLA-G has a limited repertoire of rare alleles, unlike the classical class I alleles, which are among the most polymorphic human genes. To determine the full extent of sequence variation in this gene, we examined the nucleotide variation in the first six exons of this gene in 45 healthy African American persons. By using sensitive techniques that detect > 95% of nucleotide substitutions, we detected two sequence variations in the alpha-1 domain (exon 2) and 24 sequence variations in the alpha-2 domain (exon 3) that result in amino acid substitutions. These data indicate that HLA-G is potentially capable of presenting a wide variety of peptides and of eliciting an allogeneic response, similar to the classical class I HLA genes.
Connective tissue-activating peptide III desensitizes chemokine receptors on neutrophils. Requirement for proteolytic formation of the neutrophil-activating peptide 2.
Authors: Härter L , Petersen F , Flad H D , Brandt E
Published: December 2022

The connective tissue-activating peptide III (CTAP-III), which is released from activated platelets, represents an inactive precursor of the chemokine neutrophil-activating peptide 2 (NAP-2). Leukocytes and leukocyte-derived proteases have been found to convert CTAP-III into NAP-2 by proteolytic cleavage at the N terminus. We demonstrate here that rapid and efficient formation of NAP-2 is mediated by neutrophil granulocytes (PMN) but not by monocytes or lymphocytes. However, as seen in a degranulation assay, neutrophils processing CTAP-III did not become activated by the generated NAP-2 and even exhibited decreased responsiveness to high doses of NAP-2 or IL-8, but not to FMLP. The desensitizing effect, being maximal already after 5 min of preincubation with CTAP-III, was not mediated through binding of the precursor to specific receptors but correlated with the rapid down-modulation of common NAP-2/IL-8 high affinity binding sites. A similar functional and receptor desensitization was observed in PMN pre-exposed to nonstimulatory doses of NAP-2. Specific inhibition of the CTAP-III-cleaving enzyme by the serine protease inhibitor aprotinin abrogated the CTAP-III, but not the NAP-2-mediated effects. Desensitization of PMN by CTAP-III was due to NAP-2 generated by proteolytic truncation of CTAP-III. Our results suggest that CTAP-III may regulate PMN activation by protecting processing cells from premature activation.
Macrophage function in simian AIDS. Killing defects in vivo are independent of macrophage infection, associated with alterations in Th phenotype, and reversible with IFN-gamma.
Authors: Brodie S J , Sasseville V G , Reimann K A , Simon M A , Sehgal P K , Ringler D J
Published: December 2022

Infection of macrophages (M phi) in vitro with M phi-tropic isolates of simian immunodeficiency virus (SIV) did not affect killing of Cryptococcus neoformans up to 16 days after inoculation (p < 0.05). Conversely, alveolar M phi from animals with SIV-induced AIDS killed C. neoformans less efficiently (10.4 +/- 2.8% killing) and, when stimulated with phorbol myristate, produced less superoxide anion (O2-; 0.15 +/- 0.02 O2-/h/mg M phi protein) than M phi from uninfected monkeys (21.8 +/- 1.6% killing and 0.29 +/- 0.02 O2-/h/mg M phi protein). In contrast, killing and O2- release were accentuated in SIV+ asymptomatic animals (25.8 +/- 2.3% killing and 0.40 +/- 0.04 O2-/h/mg M phi protein; p < 0.05). M phi-mediated killing and O2- production could be restored by culturing the affected cells in supernatants derived from Con A-stimulated PBMC of uninfected or SIV+ asymptomatic monkeys. Supernatants with restorative properties had high IFN-gamma bioactivity (63.4 +/- 11.0 U/ml) and elevated IL-10 concentrations (75.3 +/- 10.4 pg/ml) as compared with PBMC supernatants derived from animals with AIDS (IFN-gamma, 9.7 +/- 4.9 U/ml; IL-10, 24.0 +/- 10.1 pg/ml). Functional restoration was found to be dependent, in part, on the presence of IFN-gamma, as neutralizing Abs to IFN-gamma significantly inhibited functional restoration in active supernatants. Moreover, the inactivity of supernatants from mitogen-stimulated PBMC cultures derived from animals with AIDS was not solely dependent upon the loss of CD4+ lymphocytes, inasmuch as purified pulmonary alveolar and peripheral blood CD4+ T cells from only uninfected and SIV+ asymptomatic animals, and not those from animals with AIDS, produced IFN-gamma upon mitogen stimulation. Collectively, these findings suggest that functional aberrations in alveolar M phi from animals with AIDS are not directly due to virus infection but likely result from changes in the pulmonary microenvironment in association with the multisystemic loss and dysfunction of CD4+ T cells.
Induction of T cell tolerance by pretreatment with anti-ICAM-1 and anti-lymphocyte function-associated antigen-1 antibodies prevents antigen-induced eosinophil recruitment into the mouse airways.
Authors: Nakao A , Nakajima H , Tomioka H , Nishimura T , Iwamoto I
Published: December 2022

To determine whether the induction of Th2 cell tolerance down-regulates Ag-induced eosinophil recruitment into the tissue, we studied the effect of the in vivo pretreatment with anti-ICAM-1 mAb, anti-LFA-1 mAb, and a soluble Ag on Ag-induced eosinophil infiltration in the trachea of sensitized mice. The in vivo pretreatment with anti-ICAM-1 mAb, anti-LFA-1 mAb, and a soluble Ag inhibited Ag-induced eosinophil infiltration in the mouse trachea in an Ag-specific manner. The pretreatment with anti-LFA-1 mAb and the Ag also had a weak inhibitory effect on the Ag-induced eosinophil infiltration. In contrast, the pretreatment with Ag alone, the combination of anti-ICAM-1 mAb and anti-LFA-1 mAb alone, or the combination of anti-ICAM-1 mAb and the Ag had no significant effect. In addition, the pretreatment with the two mAb and the Ag and, to a lesser degree, the pretreatment with anti-LFA-1 mAb and the Ag also prevented Ag-specific IgE production in mice. In vitro Ag-induced IL-2, IL-4, and IL-5 production was decreased in spleen cells of the mice pretreated with anti-ICAM-1 mAb, anti-LFA-1 mAb, and the Ag, but not with Ag alone or with the combination of anti-ICAM-1 mAb and anti-LFA-1 mAb alone, indicating the induction of both Th1 and Th2 cell tolerance in vivo. These results indicate that the induction of Th2 cell tolerance prevents Ag-induced eosinophil recruitment into the tissue and IgE Ab production and that ICAM-1/LFA-1 interaction is involved as a costimulatory signal in inducing T cell tolerance to a soluble Ag.
Primary sequence of an alternatively spliced form of CR1. Candidate for the 75,000 M(r) complement receptor expressed on chimpanzee erythrocytes.
Authors: Birmingham D J , Shen X P , Hourcade D , Nickells M W , Atkinson J P
Published: December 2022

Chimpanzee erythrocytes express a 75,000 M(r) complement receptor (E-CR) that binds C3b bearing immune complexes and is recognized by an anti-CR1 mAb (E11). Human erythrocytes express the type 1 CR (CR1), the most common form being 220,000 M(r) and consisting of 30 short consensus repeats (SCRs) for its entire extracellular region. The purpose of this investigation was to determine the structure of the 75,000 M(r) chimpanzee E-CR. A chimpanzee cell line was identified that expressed a 220,000 M(r) CR1, and a 75,000 M(r) molecule that was recognized by E11 and could bind human C3i. Utilizing this cell line, chimpanzee CR1 cDNA was amplified in overlapping segments by the PCR, using primer pairs specific for various regions of human CR1 cDNA. Direct sequencing of the PCR-amplified products revealed 6044 nucleotides encoding the entire 220,000 M(r) chimpanzee CR1. This nucleotide sequence was 98.8% homologous to that of the human 220,000 M(r) CR1. Amplification using a CR1 primer from the signal peptide and from the cytoplasmic region yielded a 1985-bp PCR product, termed CR1a. The CR1a sequence was identical with the sequence encoding SCRs 1 to 6, SCRs 28 to 30, and the transmembrane and cytoplasmic regions of chimpanzee CR1. This alternatively spliced product of chimpanzee CR1 would encode a protein of 71,000 peptide m.w. with six potential N-glycosylation sites. Amplification employing a CR1 primer from SCR 1 and from the 3' untranslated region yielded a second PCR product of 1731 bp. This sequence, termed CR1b, encoded eight SCRs, followed by a hydrophobic region that ended in a stop codon. The first six SCRs of CR1b were closer in homology to the first six SCRs of a human CR1-like genomic sequence (97.4%) than to those of the chimpanzee CR1 (94.8%). Taken together, these sequence data suggest that the 75,000 M(r) chimpanzee E-CR is encoded by CR1a, an alternative splice variant of chimpanzee CR1. The CR1b is presumably derived from an RNA species related to the CR1-like genomic sequence previously described only in humans.
Interference with the binding of a naturally processed peptide to class II alters the immunodominance of T cell epitopes in vivo.
Authors: Nikcevich K M , Kopielski D , Finnegan A
Published: December 2022

T lymphocytes elicited in response to an immunizing Ag usually recognize only one or a few immunodominant peptides. The mechanisms governing this process are poorly understood. This study examines the consequences of peptide competition on immunodominance. Immunization of B10.A mice with the native Staphylococcus aureus nuclease protein primes T cells to the dominant 86-100 peptide presented in association with I-Ek class II molecules. To render the 86-100 peptide incapable of binding to the class II molecule, single amino acid substitutions were introduced in the native Staphylococcus aureus nuclease protein within a putative I-Ek class II binding motif. Introduction of residue changes at positions 89 and 91 in the protein prevents 86-100-specific T cell clone recognition of the protein in vitro. Competition studies demonstrate that substitutions at residues 89 or 91 decreased the I-Ek binding affinity of the 86-100 peptide. Immunization of B10.A mice with the L89F or Y91S mutant proteins does not prime T cells to the dominant 86-100 peptide; T cells are primed instead to I-Ek-restricted subdominant peptide(s) encompassed by the residues 111-135. In vitro binding studies demonstrate that both the 111-130 and 116-135 synthetic peptides compete with a labeled I-Ek-binding peptide 20-fold less efficiently than the dominant 86-100 peptide, suggesting that these subdominant peptides may be of lower binding affinity than the dominant 86-100 peptide. These results support the hypothesis that dominance is dependent on peptide binding affinity for the appropriate class II molecule and the ability to compete with other peptides, derived from the same Ag, for class II binding.
Structural analysis of the rat homologue of CD1. Evidence for evolutionary conservation of the CD1D class and widespread transcription by rat cells.
Authors: Ichimiya S , Kikuchi K , Matsuura A
Published: December 2022

The cDNA encoding the rat homologue of CD1 was isolated and the complete nucleotide sequence was determined. It contained an open reading frame of 1008 bp that was capable of encoding a polypeptide with 336 amino acids composed of hydrophobic leader and transmembrane sequences, three extracellular domains, and 5' and 3' untranslated sequences. Comparison of the amino acid sequence of rat CD1 with those of other species revealed that it showed the highest similarity to mouse CD1, which belongs to the CD1D class of the CD1 system and is distinct from the classic CD1 class including CD1a, CD1b, and CD1c expressed primarily on human thymocytes and some dendritic cells. Widespread transcription of rat CD1 was readily detected by Northern blot analysis in nonlymphoid organs, including the liver, kidney, and heart, as well as in lymphoid organs, including the thymus, lymph node, and spleen. Intestinal expression was also demonstrated by the more sensitive reverse transcription-PCR method. Immunoprecipitation with a rabbit anti-rat CD1 Ab showed that rat CD1 was expressed on the cell surface as a beta 2-microglobulin-associated heterodimer. Southern blot analysis of inbred rat strains suggested that rat CD1 shows limited polymorphism and that only one CD1 gene is detectable in the F344 rat genome. These results provide evidence for the conservation of CD1D class through mammalian evolution and an apparent lack of the classic CD1 class genes in rodents. Functional similarity of rodent CD1 is implied.
Analysis of allele-specific contact sites of natural HLA-DR17 ligands.
Authors: Malcherek G , Gnau V , Stevanovic S , Rammensee H G , Jung G , Melms A
Published: December 2022

The sequence motif of peptide ligands naturally associated with DR17 has indicated conserved residues at the relative positions P1-P4-P6-P 8.9 or 10. Eight naturally processed DR17 ligands were synthesized to study the role of conserved residues in DR17 binding. In their majority, they showed an excellent ability to bind to purified DR17 molecules. Binding experiments with variant peptides confirmed aspartate as the DR17-specific contact site at P4. In addition, hydrophobic or aromatic residues at P1 and P9, probably interacting with the NH2- and COOH-terminal pockets, and lysine or chemically related amino acids at P6 were important for binding. A core peptide of 10 amino acids, bordered by the terminal contact sites, is sufficient, although the ability to bind is reduced approximately 10-fold compared with the binding capacity of the natural ligand. Ala substitution of flanking stretches at either end completely restores the binding capacity to that of the natural ligand. This suggests that regions flanking the peptide core contribute to the binding strength nonspecifically, i.e., by forming H-bonds to MHC molecules. Natural DR1 and DR12 ligands like HLA-A2 (103-117) and transferrin receptor (140-156) failed to bind to DR17 molecules. However, substituting leucine for aspartate at P4 transformed DR1 and DR12 ligands into excellent DR17 binders. This conversion, enabled by a single amino acid substitution, emphasizes the importance of aspartate as the DR17-specific contact site and suggests that terminal contact residues are shared among DR1, DR12, and DR17 ligands. In contrast, additional aspartates introduced next to the contact site at P4 impaired the binding capacity. Regarding this specific role of asparate we expect that DR17-specific ligands will be rarely found among "promiscuous" peptides binding to several different DR molecules.
Partial purification of murine tumor-associated peptide epitopes common to histologically distinct tumors, melanoma and sarcoma, that are presented by H-2Kb molecules and recognized by CD8+ tumor-infiltrating lymphocytes.
Authors: Itoh T , Storkus W J , Gorelik E , Lotze M T
Published: December 2022

The B16/BL6 melanoma is a relatively nonimmunogenic tumor expressing low levels of MHC class I molecules. BL6 clones expressing transfected H-2Kb class I molecules were, by contrast, highly immunogenic in immunocompetent mice. Tumor-infiltrating lymphocytes (TILs) generated from the H-2Kb+ BL6 lesions (Thy 1.2+, CD8+, CD4-) efficiently lysed H-2Kb+ melanoma (CL8-1 and 2Kb38) and the H-2Kb+ nonrelated 3-methylcholanthrene (MCA)-105 sarcoma, but not the H-2Kb- parental melanoma BL6-8. This strongly suggests that CL8-1, 2Kb38, and MCA-105 express identical or cross-reactive T cell epitopes recognized by CL8-1 TILs in the context of the H-2Kb class I allele. To identify the T cell epitopes, peptides were acid-eluted from various cells, and fractionated by HPLC. Five HPLC fractions (F1mel-F5mel) of 70 tested contained peptides derived from H-2Kb+ CL8-1 melanoma (but not H-2Kb- melanomas) that were capable of conferring susceptibility to CL8-1 TIL lysis on H-2b-expressing target cells (EL4, C1R.Kb), but not on H-2d-expressing P815 target cells. CL8-1 TILs failed to recognize peptides derived from H-2Kb+ nonmelanoma targets such as EL4 or normal B6 splenocytes. Interestingly, CL8-1 TILs appeared to recognize peptide species contained in two HPLC fractions derived from the MCA-105 sarcoma (F1sar and F5sar). Conversely, TILs derived from MCA-105 lesions recognized MCA-105 and CL8-1 tumor cells, as well as F5mel and F5sar peptides presented by EL4 targets. These data support common murine tumor-associated peptide epitopes presented by H-2Kb and recognized by CD8+ CTLs derived from histologically distinct tumors, melanoma and sarcoma.
Neutrophil apoptosis is associated with a reduction in CD16 (Fc gamma RIII) expression.
Authors: Dransfield I , Buckle A M , Savill J S , McDowall A , Haslett C , Hogg N
Published: December 2022

Resolution of inflammation involves removal of recruited neutrophils from inflamed sites via a noninflammatory mechanism, possibly involving neutrophil apoptosis and engulfment/phagocytosis by macrophages. In this study, we describe the reduction in surface expression (> 90%) of the neutrophil molecule Fc gamma RIII (CD16) during in vitro culture at 37 degrees C, which was found to be temporally associated with the appearance of neutrophils with apoptotic morphology during in vitro culture and inhibitable by granulocyte-macrophage colony-stimulating factor (GM-CSF), which postpones apoptosis in the neutrophil. By using dual fluorescence analysis, CD16 "low" expressing neutrophils showed reduced staining with the DNA-binding dye propidium iodide, suggesting that CD16 low expressing neutrophils were apoptotic. Separation of CD16 "high" and CD16 "low" expressing neutrophils by fluorescence-activated cell sorting revealed that morphologically apoptotic cells exhibited the CD16 low phenotype. We did not observe similar marked changes in expression of other neutrophil surface molecules (including other phosphatidylinositol (PI)-linked molecules), indicating that generalized loss of surface molecules does not occur during apoptosis. We believe this to be the first reported cell type-specific membrane alteration in a surface glycoprotein associated with apoptosis, suggesting that the program of cell death in the neutrophil, in addition to morphologic and nuclear changes, includes alterations in expression of surface receptors.
Impairment of ligand binding and growth signaling of mutant IL-2 receptor gamma-chains in patients with X-linked severe combined immunodeficiency.
Authors: Ishii N , Asao H , Kimura Y , Takeshita T , Nakamura M , Tsuchiya S , Konno T , Maeda M , Uchiyama T , Sugamura K
Published: December 2022

The IL-2R gamma-chain is an indispensable subunit for the functional IL-2R. Recently, mutations of the gamma-chain have been reported to be closely associated with X-linked severe combined immunodeficiency (XSCID). The present study reveals that three patients with XSCID have three different mutations in the gamma-chain; a point mutation, a two consecutive-base deletion, and lack of the second exon in mRNA. The point mutation that we have detected is C to T, which results in one amino acid substitution of valine for alanine in the extracellular domain of the IL-2R gamma-chain (named AV mutant). The two-base deletion detected causes a frame shift of the coding region in the SH2 subdomain in the cytoplasmic domain (named tSH mutant). Transfection studies performed with the mutant gamma-chains demonstrated that the AV mutant and tSH mutant failed to bind to IL-2 and to transduce growth signals, respectively. These findings indicate that the gamma-chain gene mutations that accompany XSCID induce loss of the gamma-chain function, possibly resulting in stagnation of the differentiation and development of T cells.
Impaired T and B cell subpopulations involved in a chronic disease induced by mouse hepatitis virus type 3.
Authors: Jolicoeur P , Lamontagne L
Published: December 2022

A chronic viral infection can occur when the host immune system fails to detect the viruses. Mouse hepatitis virus type 3 (MHV3) seems to be an excellent model for the study of the relationship between viral-induced immunodeficiency and the development of chronic disease. Animals that survive acute hepatitis develop a chronic disease characterized by viral persistency in various organs, including the brain, spleen, and thymus, and they eventually die within the next 3 mo postinfection (p.i.). To verify whether T and B cell immunodeficiency occurs either in the acute or chronic phase of the disease, the percentage and absolute number of splenic T and B lymphocytes, thymic T, or bone marrow B-lineage cell subpopulations were recorded at various times p.i. in pathogenic L2-MHV3-infected and nonpathogenic YAC-MHV3-infected (C57BL/6 x A/J) F1 mice. Splenic T and B cells were depleted as early as 48 h p.i., and maintained at low levels for up to 3 mo until death of mice. Such depletions resulted from thymic depletion in all T cell subpopulations, and in B (cytoplasmic micro-chain+ and surface micro-chain+) lymphocytes only in the bone marrow of pathogenic L2-MHV3-infected mice. In vitro studies of purified thymic stromal cells have produced a nonproductive L2-MHV3 replication with a low viral transmission to complexed thymocytes. However, pre-B and B cells have supported a productive viral replication, generating abnormal forms, which leads to cell lysis. These results are discussed in relation to viral persistency in the brain and lymphoid cells, which results from a chronic impairment of cellular and humoral immune mechanisms that are involved in the viral elimination process.
Human monoclonal autoantibodies specific for the bullous pemphigoid antigen 1 (BPAg 1).
Authors: Peyron E , Nicolas J F , Réano A , Roche P , Thivolet J , Haftek M , Schmitt D , Péronne C , Banchereau J , Rousset F
Published: December 2022

Bullous pemphigoid (BP) is an acquired blistering skin disease associated with the production of IgG autoantibodies to the 230-kDa BP Ag (BPAg1). To better characterize the epitopes of BPAg1, we generated immortalized B cell lines secreting human mAbs (HumAbs) to BPAg1 from two BP patients whose sera reacted with native BPAg1 but not with a recombinant BP55 carboxyl-terminal peptide. Ab-producing B cell lines were established by EBV infection of CD40-activated PBMCs. Three independent clonal lines were obtained that secreted IgG HumAbs, including one IgG1 kappa (BP3) and two distinct IgG4 kappa (BP1 and BP2). These three HumAbs immunoprecipitated BPAg1. Blocking immunofluorescence experiments and phylogenetic studies showed that these Abs recognize different epitopes of BPAg1. This analysis with HumAbs further extends the serologic demonstration of the wide variety of epitopes recognized by BPAg1 autoantibodies which contrasts with the limited number of epitopes recognized by thyroid peroxidase monoclonal autoantibodies.
Evidence for polyclonal T cell activation in murine models of systemic lupus erythematosus.
Authors: Rozzo S J , Drake C G , Chiang B L , Gershwin M E , Kotzin B L
Published: December 2022

CD4+ T cells have been shown to be important in the development of disease in murine models of SLE. We compared the TCR V beta repertoires of young (healthy) and older (diseased) New Zealand hybrid mice as well as non-autoimmune strains to characterize changes in TCR usage associated with the development of disease. Despite large increases in the total number of splenic CD4+ T cells with age in diseased mice, we noted little skewing of the V beta repertoire. For example, diseased NZB.H-2bm12 mice failed to exhibit a significant change in the percentage of any V beta subset despite a fivefold increase in the number of CD4+ T cells. Strains without lupus-like disease, including NZB.H-2b mice, demonstrated no increase in CD4+ T cell numbers with age. Similar to NZB.H-2bm12 mice, (NZB x SWR)F, and (NZB x NZW)F1 mice showed disease-related increases in CD4+ T cell numbers, but no changes in V beta repertoire that could be linked to disease development. Differences in V beta usage between young autoimmune and non-autoimmune strains of mice matched for either MHC or background genes were consistent with genetic influences unrelated to disease. Overall, the heterogeneous repertoire of proliferating T cells provides evidence for polyclonal T cell expansion in murine models of lupus and suggests that activation either involves a multitude of conventional self-antigens or may be independent of the TCR. However, the requirement for specific class II MHC molecules suggests that this polyclonal T cell expansion is dependent on a much smaller and specific autoreactive response.
Generation of antigen-specific CD8+ CTLs from naive precursors.
Authors: Mehta-Damani A , Markowicz S , Engleman E G
Published: December 2022

Class I MHC-restricted CTLs are an important component of the host immune response against viral infections, and CTL effectors can often be isolated from infected individuals. However, the mechanism responsible for the induction of CTLs is incompletely understood because, in part, of the difficulty in generating such cells in vitro from naive precursors. In the present study we have used human peripheral blood dendritic cells (DCs), devoid of CD4+ T cells, to sensitize naive CD8+ T cells to exogenous Ags, resulting in the generation of Ag specific CTL effectors. With this system, Ag-specific CTL lines were generated to a complex glycoprotein, keyhole limpet hemocyanin, and to multiple small (9-15 amino acids) synthetic peptides derived from conserved regions of the HIV-1 gag and envelope proteins. The HIV-1-specific CTLs demonstrated potent HLA class I restricted killing of both Ag pulsed and virally infected target cells. In contrast to Ag-pulsed DCs, Ag-pulsed monocytes failed to sensitize CTL precursors although they could be used as feeders for purposes of CTL expansion and as target cells in cytolytic assays. With the use of the system described herein, a detailed analysis of the primary human T cell response to foreign Ags is now feasible, and CTL of desired specificity can be generated for potential clinical use in adoptive immunotherapy protocols.
Allelic differences in TCR gamma-chains alter gamma delta T cell antigen reactivity.
Authors: Kalataradi H , Eyster C L , Fry A , Vollmer M K , Fu Y X , Born W K , O'Brien R L
Published: December 2022

Mouse gamma delta T cell hybridomas from various strains that express a TCR-V gamma 1/V delta 6 respond weakly to an autologous Ag and more strongly to a short segment of the mycobacterial heat shock protein-60 (HSP-60). However, V gamma 1/V delta 6 hybridomas derived from AKR mice show greatly reduced or absent responses to these stimuli. To determine whether the lack of response in these AKR hybridomas is caused by polymorphisms found in the expressed AKR gamma and TCR-delta genes or, instead, stems from other genes in AKR, we crossed an AKR mouse with a responder mouse, C57BL/10 (B10), and prepared hybridomas from F1 progeny. Expression of an AKR V gamma 1-J gamma 4-C gamma 4 gene correlated with nonresponsiveness, whereas expression of a B10 V gamma 1-J gamma 4-C gamma 4 gene in most hybridomas ensured responses to both self Ag and the HSP-60 peptide. An allelic difference in the expressed V gamma 6 gene was irrelevant to these responses. Moreover, transfection of a functional B10 V gamma 1-J gamma 4-C gamma 4 gene into an F1 hybridoma variant that had lost the AKR version of this gene restored responses. The allelic gamma gene products differ at nine amino acids in the V region, and three amino acids in the C region. In addition, the AKR C gamma 4 region contains a 16-amino acid insertion. We propose that amino acid differences among those encoded by the AKR V gamma 1-J gamma 4-C gamma 4 gene are responsible for the lack of response, and reduce the ability of the TCR-gamma delta to bind the relevant Ag.