bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional and phenotypic analysis of thymocytes in SCID mice. Evidence for functional response transitions before and after the SCID arrest point.
Authors: Rothenberg E V , Chen D , Diamond R A
Published: December 2022

Thymocytes from severe combined immune deficient (SCID) mice undergo developmental arrest at an early stage, before most TCR gene rearrangement. They therefore represent a natural test case to assess those aspects of T cell development that are TCR independent. Multiparameter flow cytometry was used to analyze the array of immature phenotypes present in the SCID thymus at steady state, as defined by the markers CD4, CD5, Sca-1, NK1.1, CD44, heat-stable antigen (HSA), and IL-2R alpha. The results suggest a simple developmental block in SCID mice rather than a program of aberrant differentiation. SCID thymocytes displayed efficient, developmentally regulated functional responses. Approximately 20-25% of the cells, mostly within the IL-2R alpha +HSA+CD44low fraction, could be induced to express IL-2. This IL-2 inducibility was highly dependent on IL-1 costimulation, in agreement with the behavior of normal immature thymocytes. These results formally demonstrate that competence to express IL-2 is developed independently of TCR expression or gene rearrangement. Comparison of the response properties of various SCID thymocyte subsets indicated that IL-2 inducibility is first likely to be acquired at an early (Sca-1++CD44++HSAlow) stage. A later functional transition was revealed by comparing patterns of IL-2R alpha regulation in normal and SCID IL-2R alpha +HSA+CD44low thymocytes. The SCID thymocytes uniformly maintained IL-2R alpha expression on in vitro stimulation, whereas only a minority of the normal cells in the corresponding subset could do so unless IL-1 was also added. The SCID arrest point thus appears to separate the IL-2R alpha +HSA+CD44low stage into distinct early (TCR independent) and late phases. Normal cells that progress beyond the SCID arrest point appear to lose, rather than gain, competence to make various responses, even before they leave the IL-2R alpha +HSA+CD44low stage. A model is proposed in which discrete changes in functional competence define novel transitions in early thymocyte development, at least some of which may be linked to TCR-beta gene rearrangement before positive or negative selection.
Probing the human receptor for C5a anaphylatoxin with site-directed antibodies. Identification of a potential ligand binding site on the NH2-terminal domain.
Authors: Oppermann M , Raedt U , Hebell T , Schmidt B , Zimmermann B , Götze O
Published: December 2022

Molecular cloning has revealed the DNA sequence of the human receptor for the C5a anaphylatoxin (C5aR). In this study, mAb and polyclonal antibodies with specificities for deduced hydrophilic sequences of the receptor protein were employed to determine the expression and the topography of C5aR on PBL. Evidence was obtained that an antigenically homogenous receptor exists on human neutrophils, eosinophils, and monocytes that binds C5a and C5a(desArg). The assignment of epitopes to extra- or intracellular receptor domains as determined by FACS analysis of intact or permeabilized cells confirmed the general topography of the C5aR that had been predicted by hydropathy analysis. Competitive binding studies were performed to examine whether extracellular receptor domains participate in ligand binding. The occupation of the receptor by C5a inhibited only the binding of antibodies that were specific for the receptor's aminoterminal domain (C5aR-EX1). The anti-EX1 mAb S5/1 effectively antagonized C5a/C5a(desArg) biological activity. A heptameric peptide (D15DKDTLD21) was identified as the smallest receptor fragment that was recognized by the mAb S5/1 or by polyclonal rabbit anti-EX1 lg. These results imply that an amino acid sequence rich in aspartate within the receptor aminoterminus represents both an immunodominant epitope and a ligand binding site on the C5aR.
Peptide-induced changes in class I heavy chains alter allorecognition.
Authors: Bluestone J A , Kaliyaperumal A , Jameson S , Miller S , Dick R
Published: December 2022

Class I molecules of the MHC are intimately involved in the development and function of CD8+ T cells. Small peptides, derived from endogenous proteins, bind within the Ag binding groove created by the beta-pleated sheets and alpha-helices of the alpha 1 and alpha 2 domains of the class I molecule. This peptide-MHC complex has been shown to influence allorecognition by CD8+ T cells. However, the precise role of peptide in alloantigen recognition remains unclear. We have previously shown that conformational changes induced in the class I molecules can be identified as specific alterations in serologic epitopes. These results suggested that alloreactive T cells may detect structural changes in MHC based on the nature of the peptide binding to the class I protein. Here, we have shown that, in at least some instances, alloreactivity may not depend on the recognition of a precise self-peptide but on an epitope on the class I molecule influenced by the peptide. The nature of specific peptides expressed by class I-bearing cells may, therefore, have a dramatic effect on T cell development, self-tolerance, and alloreactivity.
Multiple pathways are involved in the extracellular processing of MHC class I-restricted peptides.
Authors: Kozlowski S , Corr M , Shirai M , Boyd L F , Pendleton C D , Berzofsky J A , Margulies D H
Published: December 2022

T cell stimulation by certain class I-restricted antigenic peptides, such as the HIV 1 gp160-derived peptide, P18, requires peptide processing by angiotensin-1 converting enzyme (ACE) in FCS. We observed that longer versions of P18 and the murine cytomegalovirus pp89-derived core peptide, pMCMV, which could stimulate T cell hybridomas in FCS, were not as sensitive to the ACE inhibitor captopril as P18. Using cell-free soluble murine class I MHC molecules and protease inhibitors, we found that there are pathways of differing efficiency that use enzymes other than ACE for the proteolytic processing of peptides in serum. The kinetics of the generation of T cell stimulatory activity among P18 variant peptides in serum differed with peptide length, and with the nature of amino and COOH-terminal extensions. Such processing occurs in human plasma as well as in FCS. The understanding of this processing, its kinetics, and its inhibitors can lead to better design of peptide-based therapies, including vaccines.
The TAPA-1 molecule is associated on the surface of B cells with HLA-DR molecules.
Authors: Schick M R , Levy S
Published: December 2022

TAPA-1 is a transmembrane protein that has been shown to be involved in cell growth and cellular adhesion. Our studies were aimed at determining the mechanisms of the biologic phenomena mediated by TAPA-1, which include the identification of proteins that are associated with it on the surface of lymphocytes. We and others have previously shown that Leu-13, a leukocyte Ag, is one such molecule and that in B cells TAPA-1 is associated with the CD19 Ag. Herein we identify an additional molecule, HLA-DR, that is noncovalently associated on the surface of B cells with TAPA-1. This association was first detected by immunoprecipitation by anti-TAPA-1 and by anti-HLA-DR antibodies in the presence of mild detergents. The initial observation was confirmed by 2-dimensional SDS-PAGE and by direct identification of TAPA-1 in anti-HLA-DR immunoprecipitates by Western blot analysis. The association of the two molecules on the surface of a human B cell line was shown by cocapping experiments. In addition, antibodies to both molecules can induce cellular adhesion and an antiproliferative effect. Because the tissue distribution of these two molecules only partially overlaps, with TAPA-1 being expressed on most cell types and MHC class II expressed on a more restricted group of tissue, it is possible that the TAPA-1 molecule provides a basic function that can augment a cell type specific activity. In B cells the association of TAPA-1 with CD19 and HLA-DR may increase cellular interaction and play a supporting role in the transmission of specific signals.
Distinct divalent cation requirements for integrin-mediated CD4+ T lymphocyte adhesion to ICAM-1, fibronectin, VCAM-1, and invasin.
Authors: Shimizu Y , Mobley J L
Published: December 2022

Integrins are a large family of cell surface receptors that mediate the adhesion of cells to other cells and to components of the extracellular matrix. Various divalent cations, particularly Ca2+ and Mn2+, have been shown to modulate the functional activity of many different integrins expressed on a wide variety of cell types. In this study, we have characterized the divalent cation requirements for the adhesion of human peripheral CD4+ T cells to four distinct integrin ligands: the alpha 4 beta 1 and alpha 5 beta 1 ligand fibronectin, the alpha 4 beta 1 ligand VCAM-1, the LFA-1 ligand ICAM-1, and the alpha 4 beta 1 bacterial ligand invasin. We find that there are distinct divalent cation requirements for T cell adhesion to each of these ligands: 1) Mg2+/EGTA treatment selectively up-regulates T cell adhesion to ICAM-1; 2) Mn2+ coordinately up-regulates adhesion to ICAM-1, fibronectin, and VCAM-1, with a peak response at 100 microM Mn2+; 3) Ca2+ can selectively support adhesion to VCAM-1 induced by activation and inhibit Mn(2+)-dependent adhesion to ICAM-1; and 4) binding to invasin is maximal in the presence of Ca2+, Mg2+, or Mn2+. Furthermore, divalent cation modifications do not fully up-regulate T cell adhesion to fibronectin, VCAM-1, and ICAM-1, because additional cell activation with phorbol ester treatment can further enhance adhesion in the presence of Mn2+. These results suggest that modification of divalent cations may provide a mechanism by which an individual integrin receptor/ligand interaction can be specifically and selectively regulated.
Structural analysis of proteolytic products of MHC class II-invariant chain complexes generated in vivo.
Authors: Newcomb J R , Cresswell P
Published: December 2022

The MHC class II alpha beta heterodimer associates with invariant (I) chain in the endoplasmic reticulum and remains associated until the complex reaches a post-Golgi compartment. During early stages of transport, I chain blocks peptide binding to alpha beta dimers. I chain is proteolytically cleaved in a post-Golgi compartment releasing alpha beta dimers that can bind antigenic peptides and transport them to the cell surface. Human B lymphoblastoid cell lines grown in leupeptin, a sulfhydryl protease inhibitor, accumulate a partial proteolytic product of the I chain called leupeptin-induced protein (LIP). LIP remains associated with alpha beta dimers. We find, using chemical cross-linking, sucrose gradient sedimentation, and size exclusion chromatography, that the alpha beta LIP complex retains the nine-subunit structure described for alpha beta I complexes. Unlike the alpha beta I complex, in certain detergents the alpha beta LIP nonamer is unstable and dissociates into trimers containing one alpha, beta, and LIP molecule. This finding emphasizes the reported stoichiometry of the alpha beta I complex as a nine-subunit structure comprised of three alpha beta I trimers. Also, these data indicate that the region(s) of I chain necessary for retaining the nonameric structure lie within the LIP fragment, but that domains to the C-terminus of the LIP cleavage site act to further stabilize the nine chain structure. In addition, alpha beta I complexes containing forms of human I chain encoding the p35/p43 N-terminal cytoplasmic extension responsible for endoplasmic reticulum retention can transport to post-Golgi proteolytic compartments where LIP is formed.
Monocytes do not make mast cells when cultured in the presence of SCF. Characterization of the circulating mast cell progenitor as a c-kit+, CD34+, Ly-, CD14-, CD17-, colony-forming cell.
Authors: Agis H , Willheim M , Sperr W R , Wilfing A , Krömer E , Kabrna E , Spanblöchl E , Strobl H , Geissler K , Spittler A , Boltz-Nitulescu G , Majdic O , Lechner K , Valent P
Published: December 2022

Mast cells (MC3) belong to the hemopoietic system and arise from hemopoietic precursor cells. Human MC progenitors can be detected in the bone marrow as well as in the peripheral blood (pb) and are responsive to the mast cell growth factor SCF, the ligand of the c-kit tyrosine kinase receptor. However, little is known about the subsets of cells that become committed to and differentiate into mature human MC. In this study, the identity of the circulating MC progenitor, previously felt to be a monocyte (Mo) or basophil (Ba), was investigated. For this purpose, CD14+ pb monocytes, CD17+ pb basophils and CD34+ cord blood cells were purified to homogeneity (> 95%) from mononuclear cells (normal adult donors, n = 17, cord blood, n = 2) by counter-flow centrifugation followed by cell sorting with mAb. In the presence of rhSCF, MC developed in long term suspension culture from pure CD34+ cells but not from pure Mo, pure Ba, or Ly (MC-tryptase levels on day 42: CD14+ Mo: 3.7 +/- 0.8 vs CD17+ Ba: 3.2 +/- 0.5 vs Ly: 2.0 +/- 1.5 vs control: 196.5 +/- 92.5 ng/ml, p < 0.001). Depletion of CD34+ cells from MNC resulted in a loss of MC in long term suspension culture, whereas depletion of either Mo, Ba, or Ly did not. In methyl-cellulose cultures in the presence of rhSCF, MC and tryptase could be detected in pure (CFU-mast) and mixed (CFU-myeloid/mast) MC colonies. Together, MC do not originate from circulating Mo, Ba, or Ly. The circulating MC progenitor is a CD34+, c-kit+, Ly-, CD14-, CD17- colony-forming cell. This is the first definitive demonstration that mast cells are replenished directly from early hemopoietic progenitors and thus form a unique cell lineage within the hemopoietic system.
Differential cytokine regulation of complement C3, C4, and factor B synthesis in human intestinal epithelial cell line, Caco-2.
Authors: Andoh A , Fujiyama Y , Bamba T , Hosoda S
Published: December 2022

In the intestinal tract, the local synthesis of C3 and components of both the classical (C4) and alternative (factor B) C activation pathway has previously been demonstrated in vivo. However, the cellular source of this local C synthesis has not been identified. In this study, we demonstrated the syntheses of C3, C4, and factor B in the human colonic adenocarcinoma cell line Caco-2, which is regarded as a good experimental model of normal human intestinal epithelial cells. The results of metabolic labeling experiments indicated that the intra- and extracellular molecular sizes and subunit structures of Caco-2-derived C3, C4, and factor B were compatible with previously reported values for these components in other cells. The functional activities of C3 and C4 in the supernatants were also demonstrated by hemolytic titration assay. Furthermore, C syntheses in this line were independently upregulated by several human cytokines: C3 synthesis was dose-dependently enhanced by the addition of IL-1 beta or TNF-alpha; C4 synthesis was enhanced by the addition of IL-6 or IFN-gamma in the same manner; and the addition of IL-1 beta or IL-6 also induced a dose-dependent increase in factor B synthesis. These enhancing effects were confirmed to be specific for individual cytokines by experiments using anti-human cytokine antibodies. It is likely that intestinal epithelial cells are local production sites of C3, C4, and factor B, and that local C syntheses in the intestine are independently regulated by several cytokines, derived from monocytes/macrophages and T cells resident in the mucosal microenvironment.
Induction by IL-9 and suppression by IL-3 and IL-4 of the levels of chromosome 14-derived transcripts that encode late-expressed mouse mast cell proteases.
Authors: Eklund K K , Ghildyal N , Austen K F , Stevens R L
Published: December 2022

Immature, rIL-3-dependent mouse bone marrow-derived mast cells (BMMC) contain high steady-state levels of the mouse mast cell protease (mMCP) 5 transcript but undetectable levels of the mMCP-1, mMCP-2, or mMCP-4 transcripts even though all four of their genes reside at a locus on chromosome 14. These mast cells can be induced by recombinant c-kit ligand (rKL) to obtain high steady-state levels of the mMCP-4 transcript and by rIL-10 to obtain high steady-state levels of the mMCP-1 and mMCP-2 transcripts. rIL-3 and rKL both elicit the differentiation of progenitor cells into immature BMMC and then stimulate their proliferation. We now report that although rIL-9 alone has no effect on BMMC proliferation as assessed by their incorporation of [3H]thymidine, rIL-9 in combination with rKL enhances the long term viability of BMMC. Furthermore, rIL-9 in the presence of rKL stimulates mouse BMMC to undergo a phenotypic change by inducing accumulation of high steady-state levels of the mMCP-1 and mMCP-2 transcripts. In contrast, in BMMC, the presence of rIL-4 suppresses the rIL-9-induced accumulation of the mMCP-1 and mMCP-2 transcripts, the rIL-10-induced accumulation of the mMCP-1 and mMCP-2 transcripts, and the rKL-induced accumulation of the mMCP-4 transcript, but not the rIL-3-induced accumulation of the mMCP-5 transcript. The presence of rIL-3 also suppresses the rIL-9-induced accumulation of the mMCP-1 and mMCP-2 transcripts. Because of their counter-regulatory actions on the steady-state levels of transcripts that encode three late-expressed serine proteases in BALB/cJ mice, rIL-4 and rIL-3 both inhibit the final stages of differentiation and maturation of mast cells. Because rIL-4, unlike rIL-3, is neither an inducer of early-expressed proteases nor alone a proliferative factor for BMMC, the counterregulatory actions of rIL-3 and rIL-4 on differentiation and maturation of these mouse mast cells are independent of their other functions.
Particle digestibility is required for induction of the phosphatidylserine recognition mechanism used by murine macrophages to phagocytose apoptotic cells.
Authors: Fadok V A , Laszlo D J , Noble P W , Weinstein L , Riches D W , Henson P M
Published: December 2022

One of the characteristic features of programmed cell death in vivo is the rapid recognition and removal of apoptotic cells by macrophages. Although there are several potential mechanisms by which the macrophage can identify a cell as apoptotic, it has been shown recently that murine-elicited macrophages stereospecifically recognize phosphatidylserine (PS) exposed on the surface of apoptotic cells. The particulate stimulus, beta-1, 3-glucan, stimulates bone marrow-derived macrophages to express several characteristics of inflammatory macrophages, and induced these cells to recognize PS on apoptotic cells; this activity was correlated with the ability to form rosettes with PS-expressing RBC. Induction of PS recognition in bone marrow-derived macrophages was associated with digestibility of the stimulus, because L, but not D amino acid particles or latex, were able to stimulate macrophage recognition of PS. The requirement for digestibility could be bypassed by the addition of exogenous TGF-beta, which induced macrophage recognition of PS after stimulation with either latex or D amino acid particles. That endogenously produced TGF-beta played a role in the glucan-stimulated response was indicated by the ability of anti-TGF-beta antibodies to inhibit digestible particle-induced recognition of PS. The induction of the PS recognition mechanism correlated well with the expression of other markers for the inflammatory phenotype. These studies indicate that the PS receptor may be a marker for the inflammatory phenotype, which appears to be induced by the phagocytosis of particulate digestible stimuli. Endogenously produced TGF-beta is suggested to play an autocrine or paracrine priming role in the induction of the PS receptor.
Lipocortin-1 fragments inhibit neutrophil accumulation and neutrophil-dependent edema in the mouse. A qualitative comparison with an anti-CD11b monoclonal antibody.
Authors: Perretti M , Ahluwalia A , Harris J G , Goulding N J , Flower R J
Published: December 2022

The activity of the steroid-inducible protein lipocortin-1 (LC1; with a primary sequence of 346 amino acids; also called annexin 1), a fragment corresponding to amino acids 1-188 and a short peptide from the N-terminus (amino acid 2-26) were tested for anti-inflammatory actions in three models of acute inflammation in the mouse in comparison with a mAb anti-CD11b (alpha CD11b). In the mouse air-pouch model LC1, fragment 1-188 and peptide Ac2-26 exhibited powerful inhibitory effects (ED50 approximately 5.2, 38 and 88 micrograms/mouse, respectively) on leukocyte migration elicited by IL-1. LC1 was approximately 200 times more potent than Ac2-26 on a molar basis although both gave maximal inhibitions, in contrast fragment 1-188 only produced a partial dose-response curve. LC1 was approximately 20 times more potent on a molar basis in this assay than the alpha CD11b mAb. Peptide Ac2-26 and the mAb alpha CD11b also blocked cell migration into the air-pouch induced by IL-8 with approximately the same potency. In the mouse skin edema and zymosan peritonitis assays Ac2-26 was inhibitory (ED50 of 200 micrograms/mouse) but less so than the alpha CD11b antibody (ED50 approximately 0.5 mg/mouse). Both LC1 (10 micrograms) and Ac2-26 (200 micrograms) completely blocked FMLP-induced neutropenia in the mouse. Studies using an inactivated LC1 preparation, which binds to the same high affinity binding sites as the biologically active material, indicated that the short peptide acts on the same sites as the native LC1. This study confirms the activity of LC1 in another model of experimental inflammation and suggests that it acts partly through inhibition of leukocyte activation with an overall effect qualitatively comparable to the blocking of CD11b portion of a beta 2-integrin complex. It also shows that peptides derived from the N-terminal domain of LC1 may mimic the activity of the full length molecule and points the way for a new family of anti-inflammatory substances that inhibit leukocyte trafficking.
Visualization of the endogenous NF-kappa B p50 subunit in the nucleus of follicular dendritic cells in germinal centers.
Authors: Feuillard J , Körner M , Fourcade C , Costa A , Binet J L , Debré P , Raphael M
Published: December 2022

NF-kappa B, a 50 kDa/65 kDa (p50/p65) heterodimer, is a ubiquitous transcription factor involved in the positive regulation of various immune genes. The aim of this study was to determine whether NF-kappa B is related to a particular cell type and/or differentiation step during immunopoiesis. Using in situ hybridization on sections from non HIV hyperplastic lymph nodes, we found that the gene of the 105 kDa precursor of p50 was overexpressed in the light zone of germinal centers, with a network aspect, which suggested the involvement of follicular dendritic cells (FDC). By immunohistochemistry, p50 protein was detected in the cytoplasm and nucleus of FDC, confirming the involvement of FDC. Furthermore, p50 protein was detected in the cytoplasm of all lymphocytes. Thus, we focused our study on isolated FDC clusters from normal tonsils. As showed on tissue sections, we detected the p50 in both cytoplasm and nucleus of FDC. Nuclei of lymphocytes from FDC clusters were negative. We next studied p65 and c-Rel protein expression in FDC clusters. p65 was detected in the cytoplasm of FDC, whereas nuclei were negative. Furthermore, p65 was detected in the nuclei of some lymphocytes. c-Rel protein was detected only in the cytoplasm of lymphocytes and not in the nucleus and cytoplasm of FDC. Our results indicated that, in the context of T cell-dependent B cell immunopoiesis occurring in FDC clusters, p50 is mainly related to FDC with a massive overexpression in the nuclei, whereas p65 is expressed in a scattered manner in the nuclei of lymphocytes and c-Rel protein exclusively in the cytoplasm of lymphocytes from FDC clusters. These results suggested that the two subunits of NF-kappa B and the c-Rel protein have different roles in different cell types during B cell immunopoiesis.
Structural and immunologic analysis of gene triplications in the Ig heavy chain constant region locus.
Authors: Brusco A , Cariota U , Bottaro A , Boccazzi C , Plebani A , Ugazio A G , Galanello R , van Leeuwen A M , DeLange G G , Depelchin S
Published: December 2022

The Ig H chain C region is a multigene family often involved in genomic rearrangements leading to deleted and duplicated haplotypes, most probably through unequal crossing over between homologous regions within the locus. The frequency of these haplotypes in Italy is around 2.7% each. Using PFGE analysis in two unrelated Italian families we found an abnormal high m.w. band, inherited in a Mendelian fashion. To assess the extension of the haplotype we performed Southern blot analysis using several specific Ig H chain C probes. In both cases, the haplotype turned out to be triplicated, with three copies of the genes from A1 to E. In one family segregation of a duplication from EP to G4 was also observed. Analysis of polymorphic loci suggests that the two triplications are of independent origin. Serological detection of IgA2 allotypes demonstrated the functional activity of the genes at the 3' end of the triplicated locus, ruling out any major effect of these large genomic rearrangements on Ig class switching. Furthermore, the triplicated haplotype does not seem to give rise to any clinically significant immunological impairment or increase in Ig serum concentrations.
A single engineered amino acid substitution changes antibody fine specificity.
Authors: Kussie P H , Parhami-Seren B , Wysocki L J , Margolies M N
Published: December 2022

During the acquisition of humoral immunity, the process of somatic hypermutation introduces nucleotide substitutions into expressed antibody (Ab) V region genes. Studies employing in vitro mutagenesis have shown that recurrent mutations observed in vivo often enhance the affinity of the target Ab for Ag. Here we show that a single amino acid replacement at position 35 in the H chain of an unmutated Ab with specificity for p-azophenylarsonate (Ars) confers specificity for the structurally related hapten p-azophenylsulfonate (Sulf) while abolishing specificity for Ars. The mutant Ab binds Sulf with an affinity characteristic of Ab produced by memory B cells. The same mutation in the somatically mutated anti-Ars Ab 36-71, for which the Fab crystal structure is known, resulted in a significant shift in fine specificity from Ars to Sulf. Examination of the crystal structure suggests that the specificity change is caused by a decrease in binding site size and/or new hydrogen bond geometry. Because the mutation at position 35 had been observed in somatically mutated Ab elicited by immunization with Ars followed by Sulf, the results confirm that somatic mutation in vivo can alter Ab specificity. The results also support the potential of Ab engineering to alter antigenic specificity.
Normal V(D)J coding junction formation in DNA ligase I deficiency syndromes.
Authors: Petrini J H , Donovan J W , Dimare C , Weaver D T
Published: December 2022

Bloom syndrome and a clinically related syndrome represented by the cell line 46BR have been associated with reduction in DNA ligase I activity. In these syndromes, DNA ligase I deficiency severely impairs the development and function of the immune system. We undertook analysis of DNA ligase I-deficient cells to determine whether the observed immune deficiency is attributable to a perturbation in the process of V(D)J recombination. V(D)J recombination in Bloom syndrome cell lines and 46BR was examined by a transient transfection assay. No effect on the fidelity of coding and signal junction formation in DNA ligase I-deficient cells was observed. The frequency of V(D)J recombination in DNA ligase I-deficient cells was also examined using recombination substrates modified to function in human cells. Similar recombination frequencies were observed in normal and DNA ligase I-deficient cells, demonstrating that the efficiency of the V(D)J recombination process is unaffected by alterations in DNA ligase I activity. Rearranged immunoglobulin loci from Bloom syndrome cell lines and patient material were molecularly cloned by an inverse polymerase chain reaction strategy which should be applicable to a variety of human immunodeficiency syndromes and were indistinguishable from those found in normal bone marrow samples. Our data argue that the immune system defects associated with DNA ligase I deficiency do not result from perturbation of the V(D)J recombination pathway.
T and B epitope determination and analysis of multiple antigenic peptides for the Schistosoma mansoni experimental vaccine triose-phosphate isomerase.
Authors: Reynolds S R , Dahl C E , Harn D A
Published: December 2022

Schistosoma mansoni triose-phosphate isomerase (TPI), a glycolytic enzyme, was originally identified as the target of the mAb M.1 that conferred protection when the antibody was administered in vivo. In this study we increase the evidence that schistosome TPI is a potential vaccine Ag by showing that it also a potent inducer of IL-2 and IFN-gamma production (Th1 responses), driving production of these cytokines in the same cell populations of infected animals that have high Th2 responses directed at other parasite egg Ag. With the goal of synthetic peptide vaccine design, rTPI was used to determine specific T and B cell epitopes recognized by two strains of mice representing high and moderate responders (C57Bl/6J and CBA/J). All selected epitopes were from nonconserved regions of TPI and thus parasite-specific. We then defined minimal size immunoreactive epitopes and synthesized four-armed multiple antigenic peptides (MAP) consisting of T and B cell epitopes that could be recognized by both strains of mice in the same molecule. Characterization of the immunoreactivity of the MAP showed that higher antibody recognition of the MAP was attained when the B cell epitope was placed on the amino termini relative to the T cell epitope, whereas equivalent immunoreactivity occurred for the T cell epitopes when located at either position. Most interesting was the finding that one of the minimal T cell epitopes, when incorporated into the MAP, required enlarging to retain immunoreactivity. Finally we showed that both the full-length TPI molecule and the final version of the MAP were immunogenic to T cells in naive animals and induced cross-recognition in the form of IL-2 and IFN-gamma production.
CD40-activated surface IgD-positive lymphocytes constitute the long term IL-4-dependent proliferating B cell pool.
Authors: Galibert L , Durand I , Banchereau J , Rousset F
Published: December 2022

In vitro, B cells undergo long term proliferation when triggered through their CD40 surface molecule and in the presence of IL-4. Here, we show that cells that proliferate in this culture system lose their germinal center (GC) features and acquire or maintain non-GC markers. When separated by the magnetic cell separation system, both sIgD+ and sIgD- B cells can proliferate in this culture system, sIgD+ B cells exhibiting a higher rate of growth than sIgD- cells. Simultaneous flow cytometric measurement of sIgD and DNA content revealed that B lymphocytes can keep their sIgD after entry into cell cycle. Experiments using G8 Id-positive B lymphocytes allowed us to follow the evolution of sIgD+ and sIgD- cells in a reconstituted B cell population. Long term proliferating cells are sIgD+/sIgM(+)-derived B lymphocytes whereas the initial sIgD-/sIgM- cells are lost. Taken together, these data show that anti-CD40 + IL-4 activated sIgD+ B blasts express non-GC characteristics and that sIgD+ B cells preferentially proliferate in the CD40 system. The possible in vivo role of IL-4 + CD40 signaling is discussed.
CD11b/CD18 integrin and a beta-glucan receptor act in concert to induce the synthesis of platelet-activating factor by monocytes.
Authors: Elstad M R , Parker C J , Cowley F S , Wilcox L A , McIntyre T M , Prescott S M , Zimmerman G A
Published: December 2022

We determined the mechanism by which opsonized zymosan particles, which are derived from yeast and composed of carbohydrate polymers, stimulate platelet-activating factor (PAF) synthesis by monocytes. A role for CD11b/CD18 was demonstrated because antibodies to this integrin decreased PAF synthesis, zymosan bearing only a ligand for CD11b/CD18 (iC3b) induced the synthesis of PAF, and monocytes that did not express CD11b/CD18 produced much less PAF than control monocytes. Ligation of CD11b/CD18 was not sufficient for PAF synthesis suggesting that an additional receptor was involved. Monocytes are known to bind beta-glucan which is a major component of zymosan. Opsonized beta-glucan particles stimulated the synthesis of PAF, and a soluble form of beta-glucan partially inhibited PAF synthesis in response to opsonized zymosan. Two lines of evidence suggested that the beta-glucan receptor mediating this response was distinct from CD11b/CD18. First, CD11b/CD18-deficient monocytes produced PAF when stimulated by zymosan opsonized with isolated C3b, a molecule that binds to complement receptor type 1 (CD35). Second, inducing contact of monocytes with zymosan by centrifugation resulted in PAF synthesis that was not inhibited by antibodies to CD11b/CD18. The combination of soluble beta-glucan and antibodies to CD11b/CD18 completely blocked PAF synthesis in response to opsonized zymosan. Together, these results demonstrate that induction of maximal PAF synthesis by serum-opsonized zymosan requires the concerted interactions of monocyte receptors for iC3b and beta-glucan. Additionally, they suggest that CD11b/CD18 facilitates binding of the particle and that a beta-glucan receptor transduces the activation signal.
Production of IL-8 and monocyte chemotactic peptide-1 by peripheral blood monocytes. Disparate responses to phytohemagglutinin and lipopolysaccharide.
Authors: Liebler J M , Kunkel S L , Burdick M D , Standiford T J , Rolfe M W , Strieter R M
Published: December 2022

The temporal recruitment of leukocytes to a site of inflammation is dependent on a complex interplay of a number of soluble mediators. Recently, two families of chemotactic cytokines have been discovered. The -C-X-C-family, which includes IL-8, appears to recruit neutrophils and lymphocytes. In contrast, the -C-C-family, which includes monocyte chemotactic peptide-1 (MCP-1), appears to recruit predominantly monocytes. Monocytes, after their arrival at a site of inflammation, could further amplify the immune response by secreting IL-8 and MCP-1. We sought to define conditions under which human peripheral blood monocytes produce IL-8 and MCP-1. Using serum-free media, we found that PHA-stimulated monocytes expressed MCP-1 and IL-8 protein and mRNA in a dose-dependent manner. However, the onset of mRNA expression for MCP-1 occurred at least 3 h later than did the onset of IL-8 mRNA expression. IL-8 and MCP-1 gene expression by monocytes appeared to require de novo protein synthesis, in that cycloheximide blocked the expression of mRNA for both IL-8 and MCP-1 in PHA-stimulated cells. However, treatment of monocytes with cycloheximide resulted in the superinduction of IL-8 compared with control monocytes. Monocytes costimulated with PHA and LPS demonstrated enhanced amounts of IL-8 mRNA and protein, but sharply decreased amounts of MCP-1 mRNA and protein. The addition of serum to culture media increased both the constitutive and PHA-induced production of monocyte-derived MCP-1 and IL-8, but had no effect on the inhibition of PHA-stimulated MCP-1 production by LPS. These findings suggest that distinct pathways of activation exist for the production of monocyte-derived IL-8 and MCP-1. The differential expression of these different but related polypeptides may offer a means of control of the type of immune cells that are recruited to a site of inflammation.
Characterization of a high molecular weight tumor necrosis factor-alpha mRNA in influenza A virus-infected macrophages.
Authors: Sprenger H , Bacher M , Rischkowsky E , Bender A , Nain M , Gemsa D
Published: December 2022

Infection by influenza A virus has previously been shown to prime macrophages for a high TNF-alpha production. Influenza A virus induced a TNF-alpha mRNA accumulation that consisted of two types: a regular 1.7 kb and an additional high m.w. 2.4 kb species in murine macrophages, and a high m.w. 3.6 kb species in human monocytes. In this study, we further characterized this virus-induced, novel high m.w. TNF-alpha mRNA. The additional high m.w. TNF-alpha mRNA represented a true polyadenylated mRNA and its induction required exposure to infectious viruses. The regular and the high m.w. TNF-alpha mRNA were both found in the nuclear fraction and the cytoplasm. We excluded that the novel high m.w. TNF-alpha mRNA was an intron-containing precursor TNF-alpha mRNA that could have persisted in virus-infected macrophages. When TNF-alpha exons 1 to 4 and TNF-alpha exons 2 to 4 were amplified by polymerase chain reaction, only regular and no high m.w. bands were detected. By use of specific TNF-alpha intron I and intron III cDNA we could definitely demonstrate the absence of introns in the high m.w. TNF-alpha mRNA. The high m.w. TNF-alpha mRNA was free of TNF-beta and TNF intergenic region elements but contained the 5' and 3' untranslated region of TNF-alpha. Influenza A virus infection also induced a double band of IL-1 beta and IL-6 mRNA. Whether this novel high m.w. TNF-alpha mRNA represents a virus-induced abnormality or a superinduction of an otherwise normal but minor TNF-alpha transcript, and whether this high m.w. TNF-alpha mRNA species codes for a biologically active product, remains to be examined.
Exocytosis of a subpopulation of specific granules coincides with H2O2 production in adherent human neutrophils.
Authors: Suchard S J , Boxer L A
Published: December 2022

Unlike polymorphonuclear leukocytes (PMN) that are activated in suspension, PMN plated onto extracellular matrix (ECM) proteins exhibited a prolonged delay in the onset of the respiratory burst in response to PMA, FMLP, A23187, or GM-CSF. The present study was focused on examining the events leading to H2O2 release in adherent PMN. The time course of stimulated H2O2 release from PMN plated on fibrinogen (FG) kinetically paralleled cell spreading and lactoferrin release. In contrast, the release of another specific granule component, vitamin B12-binding protein, preceded H2O2 generation by at least 20 min, suggesting the differential mobilization of subpopulations of specific granules. FMLP-stimulated PMN from a patient with leukocyte adhesion deficiency (LAD), lacking CD11/CD18 integrins and unable to adhere to FG, attached and spread on thrombospondin (TSP) and generated substantial amounts of H2O2. However, PMN from a second LAD patient, able to attach but not spread on TSP, failed to generate oxidant. These data indicated that PMN spreading might be a prerequisite for H2O2 generation and that CD11/CD18 integrins are likely not the only surface receptors involved in this response. Exposing PMN to the intracellular Ca2+ chelators MAPTAM or BAPTA significantly reduced H2O2 generation in response to the receptor-mediated agonists, FMLP or GM-CSF, but did not affect PMA-stimulated H2O2 generation. Under conditions that resulted in reduced H2O2 generation (i.e., MAPTAM or BAPTA plus FMLP or GM-CSF), lactoferrin, but not vitamin B12-binding protein, release was also reduced, suggesting a link between the secretion of lactoferrin-containing specific granules and H2O2 production. Since neither MAPTAM or BAPTA blocked PMN spreading, it appeared that cell spreading alone was not sufficient for H2O2 production. Thus, the major requirement for H2O2 generation appeared to be the exocytosis of a distinct population of lactoferrin-containing specific granules. In support of this observation, PMN-like HL-60 cells, which lack specific granules, attached and spread on FG but failed to release H2O2. Thus, we postulate that the delivery of cytochrome b from lactoferrin-containing specific granules to the plasma membrane during activation contributes to oxidant production in PMN adherent to ECM proteins.
The suppressor factor of T suppressor cells induced by tolerogenic conjugates of ovalbumin and monomethoxypolyethylene glycol is serologically and physicochemically related to the alpha beta heterodimer of the T cell receptor.
Authors: Chen Y , Takata M , Maiti P K , Mohapatra S , Mohapatra S S , Sehon A H
Published: December 2022

Ovalbumin-specific, H-2Kd restricted, CD8+ Ts cells of clone 17.2 were shown to produce an OVA-specific Ts cell factor (TsF17.2) possessing the same Ag specificity and MHC restriction as those of the intact Ts cells. The Ts cell clone was generated from a single cell of the spleen of a mouse which had been immunosuppressed by injection of tolerogenic OVA(mPEG)12 conjugate. For the elucidation of the nature of TsF17.2, it was characterized by serologic, physicochemical, and Western blot analyses. It was found that 1) the OVA-specific suppression of in vitro antibody production by TsF17.2 could be blocked by mAb H28-710 which binds to an epitope of the constant region of the alpha-chain of TCR; 2) the TsF17.2 could be sequestered by, and eluted from, immunosorbents prepared by coupling to Affi-Gel Hz the H28-710 mAb or the mAb H57-597 and F23.1 which are specific, respectively, for an epitope of the constant region of the beta-chain and an epitope of the V beta 8 region of the TCR; and 3) the TsF17.2 had a pl of 7.0, m.w. of 84,000, and consisted of two disulfide-linked subunits of 42,000 each. After electroelution from the SDS-PAGE gel, the m.w. 84,000 molecule retained its capacity to suppress in vitro antibody production in an OVA-specific manner. From all these results it was concluded that this Ts cell factor may represent a soluble form of the alpha beta heterodimer of TCR of cloned Ts cells.
T cell receptor V beta gene expression in monozygotic twins. Discordance in CD8 subset and in disease states.
Authors: Davey M P , Meyer M M , Bakke A C
Published: December 2022

The peripheral T cell repertoire is shaped by positive and negative selection. These intrathymic events are dependent on the direct interaction of MHC and TCR molecules. Inasmuch as one possible mechanism for HLA-linked disease involves the role that these molecules play in shaping the peripheral T cell repertoire, an understanding of how stable the repertoire remains is an important question that will influence future studies. The purpose of this study was to analyze the stability of the T cell repertoire in monozygotic twins. To investigate this question the percentage of CD4 and CD8 T cells expressing TCR V beta gene products was determined for seven sets of healthy monozygotic twins ages 2 through 44. V beta expression was determined by three-color flow cytometric analysis using antibodies to V beta-5.1, -5.2, -5.3, -6.7, -8, and -12. The percentage of CD4 cells expressing each V beta gene was highly concordant between twins. In contrast, differences were noted for V beta expression within the CD8 subset. This was especially marked when sets of twins were studied (n = 3) where one individual had an underlying disease. Although expression in the CD4 subset was again concordant, significant differences were noted within the CD8 subset compared to the healthy twin. These data indicate that in both health and disease, the CD4 T cell repertoire is tightly regulated although often sizable differences have developed in the CD8 compartment.
Cytolysin gene expression in the islets of diabetic BioBreeding/Worcester rats.
Authors: Jiang Z , Makowiecki J , Mordes J P , Woda B A
Published: December 2022

Diabetes prone (DP) BB/Wor rats develop spontaneous autoimmune diabetes mellitus caused by a T cell-dependent process that destroys pancreatic beta cells. Neither the inciting immune system defect nor the mechanism by which beta cells are destroyed is known with certainty. DP rats are severely deficient in certain T cell subsets including CD8+ cytotoxic T cells (Tc) and RT6+ T cells. Diabetes-resistant (DR) BB/Wor rats can be rendered diabetic if depleted of RT6+ T cells. To investigate the mechanisms of beta cell destruction in BB rat diabetes, we determined: 1) the relative abundance of Tc and NK cells in the islets of acutely diabetic DP and RT6-depleted DR rats and 2) expression of mRNA encoding cytolysin, a cytolytic pore-forming protein produced by both Tc and NK cells. We found that in the islets of acutely diabetic DP rats NK cells were about three times more abundant than in diabetic RT6-depleted DR rats. Conversely, in the islets of diabetic DR rats, Tc were three times more abundant than NK cells. In addition, cytolysin gene expression was detected in about 60% of the islets of both DP and DR rats. These data suggest that cytolysin may be a mechanism by which Tc and NK cells damage B cells in vivo.
Regulation of adhesion molecule expression in Kaposi's sarcoma cells.
Authors: Yang J , Xu Y , Zhu C , Hagan M K , Lawley T , Offermann M K
Published: December 2022

Kaposi's sarcoma (KS) is a neoplasm with multifocal vascular lesions that is often seen in homosexual HIV-infected individuals. Infiltrates of leukocytes are characteristic components of KS lesions, and the products of leukocytes have been shown to enhance the proliferation of KS cells in vitro and most likely are crucial for the development of KS lesions in vivo. It is therefore likely that the expression of cellular adhesion molecules (CAM) is a critical determinant in the pathogenesis of KS by dictating the numbers and types of leukocytes that accumulate in areas predisposed to KS. We report that in the absence of inducers, KS cells in culture expressed low levels of ICAM-1 and undetectable VCAM-1 and E-selectin. ICAM-1, VCAM-1, and E-selectin were all induced by dsRNA (poly (I:C)), IL-1 beta, TNF-alpha, and LPS in KS cells. All of these agents increased NF-kappa B binding activity in nuclear extracts from KS cells. Neither human dermal fibroblasts nor human aortic smooth muscle cells had detectable VCAM-1 protein expression in response to conditions that led to high levels of VCAM-1 expression in KS cells. Although E-selectin expression was induced in KS cells, the peak cell surface protein levels were less than 25% the levels achieved on HUVEC or human dermal microvascular endothelial cells (HMEC). These low levels resembled the levels that were induced in HMEC immortalized with SV 40 large T Ag. These data indicate that multiple proinflammatory agents can induce NF-kappa B binding activity and can enhance ICAM-1, VCAM-1, and E-selectin expression in KS cells. The increased CAM expression enhances leukocyte binding to KS cells. Thus, the induction of CAM expression could be an early event in the development of KS by recruiting leukocytes into KS lesions, thereby providing factors that could potentiate the development of KS.
Identification of two V beta 7-specific viral superantigens.
Authors: Ignatowicz L , Kappler J W , Marrack P , Scherer M T
Published: December 2022

The commonly used strains of laboratory mice have mouse mammary tumor viruses (MTV) integrated at various locations in their DNA. The number and position of these integrants varies from one strain of mouse to another. It has recently been shown that the genomes of many of the MTV code for superantigens. The predicted amino acid sequences of these superantigens and their specificity for TCR V beta differs for each MTV integrant. This study contains the predicted amino acid sequence and V beta specificity of two MTV superantigens that had not previously been analyzed. The results show that both of these MTV superantigens are specific for TCR that bear V beta 7, but unlike the MTV7 superantigen not for receptors bearing V beta 6 or V beta 8.1. The data also support the conclusion of previous studies that the COOH-terminal sequence of these proteins is a major factor in controlling their V beta reactivity.