bluuebunny/crossref_metadata_2025 huggingface dataset:
A peripheral mechanism preserves self-tolerance to a secreted protein in transgenic mice.
Authors: Whiteley P J , Poindexter N J , Landon C , Kapp J A
Published: December 2022

We have examined mechanisms of tolerance to circulating self-proteins in mice that are transgenic for human insulin. Normal, nontransgenic mice develop serum antibody responses when injected with human insulin in CFA; syngeneic transgenic mice do not. B cell responsiveness was assessed by immunizing with human insulin coupled to a T-independent Ag, Brucella abortus. No differences were found in the numbers of insulin-specific splenic plaque-forming cells between transgenic and nontransgenic mice suggesting that insulin-specific B cells are not tolerant in transgenic mice. Similarly, APC from transgenic and nontransgenic mice display no differences in their ability to process and present human insulin to human insulin-specific T cells in vitro. However, marked differences were detected between transgenic and nontransgenic T cells. Lymph node T cells from transgenic mice primed with human insulin provided no detectable helper activity for secondary antibody responses to human insulin whereas, lymph node T cells from nontransgenic mice did. Nevertheless, lymph node T cells from transgenic mice developed significant proliferative responses to human insulin. Lymph node T cells obtained from transgenic and nontransgenic mice were fused to BW5147 and human insulin-specific T cell hybridomas were generated. The fact that human insulin-specific T cell hybridomas were obtained from the transgenic mice suggests that these T cells were not clonally deleted. In addition, APC from transgenic mice did not stimulate human insulin-specific hybridomas from normal mice in the absence of exogenous insulin. We suggest that T cells specific for human insulin are not deleted in the thymus of transgenic mice because APC in the thymus do not bear the requisite levels of endogenous human insulin/Ia complexes. Therefore, we conclude that tolerance in the transgenic mice is preserved by peripheral mechanisms.
Lymphokine-regulated differential expression of mRNA for p75kDa-IL-2R and p55kDa-IL-2R in a cloned B lymphoma line (BLC1-CL-3 cells).
Authors: Nakanishi K , Matsui K , Hirose S , Yoshimoto T , Hiroishi K , Kono T , Hada T , Hamaoka T , Higashino K
Published: December 2022

IL-5 renders BCL1-CL-3 (CL-3) cells responsive to IL-2 by increasing the number of high affinity IL-2R, whereas IL-4 prohibits such action of IL-5 to prepare CL-3 cells responsive to IL-2. Here we have found that genes for p75kDa-IL-2R and p55kDa-IL-2R are differentially regulated by IL-4 and IL-5. Nonstimulated CL-3 cells constitutively express mRNA for p75kDa-IL-2R and p55kDa-IL-2R. IL-5 stimulation principally augments the expression of p75kDa-IL-2R mRNA (4- to 8-fold), although modestly increasing the expression of p55kDa-IL-2R mRNA. Kinetic studies have revealed a maximal increase in p75kDa-IL-2R mRNA expression at 12 h and a decline thereafter, substantiating our previous kinetic study of the expression of high affinity IL-2R after the IL-5 stimulation. By contrast, IL-4 stimulation modestly increases the expression of p75kDa-IL-2R mRNA, whereas markedly reducing the expression of p55kDa-IL-2R mRNA, irrespective of whether CL-3 cells were stimulated with IL-4 alone or together with IL-5 and IL-2. Moreover, addition of IL-4 into the culture containing IL-5 and IL-2 causes striking reduction in the level of J-chain mRNA, which otherwise is markedly induced by stimulation with IL-5 and IL-2. These results clearly illustrate the differential regulation of p75kDa- and p55kDa-IL-2R-gene expression by IL-5 and IL-4, and reinforce our notion that increased expression of high affinity IL-2R induced by IL-5 is responsible for the IL-2 competent state, and decreased expression of p55kDa-IL-2R by IL-4 is responsible for IL-2 unresponsive state.
Structural basis of Kbm8 alloreactivity. Amino acid substitutions on the beta-pleated floor of the antigen recognition site.
Authors: Hunt H D , Pullen J K , Dick R F , Bluestone J A , Pease L R
Published: December 2022

We have analyzed the functional significance of the four amino acid differences between the parental H-2Kb and mutant H-2Kbm8 glycoproteins. Six bm8 variants including single substitutions at residues 22, 23, 24, and 30 as well as paired substitutions at residues 23, 30 and 22, 24 were generated and transfected into L cells. Surface expression of these H-2Kb variants was analyzed using monoclonal antibodies which bind to well-defined H-2Kb epitopes. No alterations introduced into the conformational structure of H-2Kb by the amino acid substitutions were detected. The effect of these substitutions on CTL recognition was initially analyzed using the following bulk CTL: either H-2Kb anti-H-2Kbm8, H-2Kbm8 anti-H-2Kb, or third party anti-H-2Kb. The alloreactivity between H-2Kb and H-2Kbm8 is dominated by the amino acid substitution at residue 24 (Glu----Ser). The complete bm8 phenotype, however, also requires the additional substitution at residue 22 (Tyr----Phe). The H-2Kbm8 anti-Kb bulk CTL reacted with both variant H-2Kbm8 molecules containing single substitutions at amino acid positions 22 or 24 but not the variant molecule containing both substitutions. Further analysis using three individual H-2Kbm8 anti-Kb CTL clones indicated the complexity of the self Kbm8 phenotype. Clone 8B1.20 did not react to changes in residues 22 or 24. The 8B1.32 clone reacted with the change at residue 22 but not with the change at residue 24, although the 8B1.54 clone reacted with the change at residue 24 but not with the change at residue 22. The changes in residues 23 (Met----Ile) and/or 30 (Asp----Asn) did not impact significantly on the alloantigenic properties of Kbm8 as determined by both the bulk and cloned CTL populations. According to the three-dimensional class I structure the substitution at amino acid 24 is inaccessible to the TCR. The location of this substitution within the Ag recognition site implies that altered peptide binding, and not a disruption of MHC residues that interact with the TCR, is responsible for the alloreactivity between H-2Kb and H-2Kbm8.
Biosynthesis pathway of gp90MEL-14, the mouse lymph node-specific homing receptor.
Authors: van de Rijn M , Weissman I L , Siegelman M
Published: December 2022

The mouse lymph node specific homing receptor gp90MEL-14 is a 95-kDa molecular mass ubiquitinated cell surface molecule involved in the binding of lymphocytes to high endothelial venules in peripheral lymph nodes. The molecule is thought to consist of a core protein to which ubiquitin side chains are covalently bound. Recently we cloned the cDNA encoding the core protein; this cDNA clone encodes for a polypeptide with an estimated molecular mass of 37 kDa. We have studied the biosynthesis of gp90MEL-14 in an effort to explain the difference in molecular mass between the core protein and the 95-kDa mature molecule. Pulse labeling experiments show a rapid synthesis of a 70-kDa precursor form that contains high-mannose N-linked oligosaccharides. On processing of the high-mannose oligosaccharides into complex N-linked oligosaccharides, the precursor matures in a single step into the 95-kDa form. Experiments using deglycosylating enzymes and inhibitors of N-linked glycosylation demonstrate that the molecular mass of deglycosylated gp90MEL-14 is 45 kDa; extensive N-linked glycosylation is responsible for the difference in molecular mass with the mature 95-kDa form. The core protein molecular weight of in vitro transcribed and translated gp90MEL-14 cDNA is consistent with the estimated molecular mass of 37 kDa, calculated from the cDNA sequence of the core protein, and 8 to 10 kDa less than the protein molecular mass of gp90MEL-14 translated in vivo in the presence of tunicamycin (45 kDa). Inasmuch as we have ruled out glycosylation as accounting for this discrepancy, this is consistent with the addition of one ubiquitin moiety to the core protein during biosynthesis. Limited proteolysis confirms the similarity between in vitro transcribed gp90MEL-14 cDNA and the tunicamycin form of gp90MEL-14.
Activation of C3H/HeJ macrophage tumoricidal activity and cytokine release by R-chemotype lipopolysaccharide preparations. Differential effects of IFN-gamma.
Authors: Flebbe L M , Chapes S K , Morrison D C
Published: December 2022

We have investigated the relative immunostimulatory activities of S-chemotype LPS and R-chemotype LPS preparations on C3H/HeJ peritoneal macrophages in vitro. As assessed by either secretion of TNF-alpha or IL-1, some of the R-chemotype LPS manifest significant activity on these normally LPS-unresponsive cells. The expression of IL-1 activity by R-LPS-stimulated C3H/HeJ macrophages was unaffected by IFN-gamma; however, this cytokine significantly enhanced TNF-alpha production by the same cells. The R-chemotype LPS preparations alone were not able to activate C3H/HeJ macrophages to become tumoricidal but activity could readily be demonstrated in the presence of IFN-gamma. Of potential importance is the observation that the profile of relative activity of the various R-chemotype LPS preparations for macrophage activation does not parallel that previously obtained by us for the C3H/HeJ B-lymphocyte activation.
Secondary structure and immunogenicity of hybrid synthetic peptides derived from two Plasmodium falciparum pre-erythrocytic antigens.
Authors: Londoño J A , Gras-Masse H , Dubeaux C , Tartar A , Druilhe P
Published: December 2022

Multicomponent synthetic vaccines containing both B and T cell epitopes belonging to two different pre-erythrocytic Ag of Plasmodium falciparum are presented. In a di-component hybrid, a circumsporozoite T cell epitope and a peptide representing a liver stage-specific Ag were connected to obtain a reciprocal reinforcement of helical potentials. In a tri-component hybrid, a sequence corresponding to the circumsporozoite repeat tetrapeptide (NPNA) was tandemly synthesized on the N-terminal end of the di-component hybrid. Both hybrid molecules were able to adopt a partial helical conformation in water as determined by circular dichroism studies. To analyze if the different components were immunologically functional in these vaccines, mich bearing genetic backgrounds known to respond or not to the individual components were immunized with the hybrids. The tri-hybrid peptide showed high immunogenic capacity as it elicited, in both H-2b and H-2k mice, high antibody responses against every separate individual sequence. Moreover, the antibodies induced by these conformationally restricted peptides were able to recognize the corresponding native proteins in the liver schizont and the sporozoite surface. H-2d mice, in which the immune response to the individual components was genetically restricted, did respond against the di-hybrid peptide. The tri-hybrid peptide, in which NPNA repeats were present, lacked this H-2d-priming capacity but it triggered antibody production in H-2d mice previously primed with the di-hybrid peptide. These results indicate that multivalent vaccines can provide positive (potentiating) effects by carefully combining structurally well defined epitopes; however, negative (suppressive) effects are also possible suggesting that selection of multivalent vaccine components will require testing of combined molecules to optimize specific immune responses and avoid undesirable effects which may result from negative molecular interactions.
Recombinant granulocyte-macrophage colony-stimulating factor restores deficient immune responses in mice with chronic Trypanosoma cruzi infections.
Authors: Reed S G , Grabstein K H , Pihl D L , Morrissey P J
Published: December 2022

Spleen cells from mice with chronic Trypanosoma cruzi infection generate a minimal plaque-forming response to SRBC in vitro. Addition of granulocyte-macrophage (GM)-CSF to cultures of spleen cells from chronically infected mice restored the plaque-forming cells (PFC) response to normal levels. Splenic adherent cells from chronically infected mice were deficient in their ability to reconstitute the PFC response of accessory cell-depleted normal spleen cells. Preincubation of splenic adherent cells from infected mice with GM-CSF restored their ability to reconstitute the PFC response of adherent cell depleted cultures. Ia Ag expression by splenic adherent cells from chronically infected mice was significantly lower compared to Ia Ag expression of cells from normal mice. Incubation of splenic adherent cells from chronically infected mice for 48 h with GM-CSF increased levels of Ia Ag expression to approximately those of uninfected mice. Peritoneal macrophages from infected mice produced IL-1 after incubation with GM-CSF at levels equivalent to those produced by similarly treated control macrophages. Spleen cells from chronically infected mice showed significant induction of IL-2 mRNA after GM-CSF treatment, and the addition of the anti-IL-2 mAb to GM-CSF supplemented cultures of spleen cells from infected mice blocked the restoration of the anti-SRBC PFC response. Thus, the ability of GM-CSF to restore the anti-PFC response to SRBC appears to involve the up-regulation of accessory cell function that includes increased Ia Ag expression and the induction of IL-1 production. These events also involve increased IL-2 production with resultant up-regulation of the response to SRBC by spleen cells from infected mice. Finally, it was shown that treatment of infected mice with rGM-CSF completely restored their depressed PFC production in vivo.
Rate and mechanism of generation of beta 2-microglobulin mutants from a heterozygous murine cell line.
Authors: Rajan T V , Moffat L F , Frankel W N
Published: December 2022

Mutants that fail to express the B2mb allele can be readily isolated from a B2ma/b heterozygous cell line called R8 by a single step immunoselection protocol using a mAb against beta 2 microglobulin protein in the presence of complement. We present data that demonstrate that the mutants preexist the selection and arise at a rate of about 10(-6)/cell per generation. Southern blot analyses suggest that the mutants have lost the B2mb gene and the linked allele at the Emv-15 proviral integration site telomeric to the B2m locus. Surprisingly, the deletion of the first exon of the B2mb gene that is observed in other cell lines is not found in R8. Possible reasons for this observation are discussed.
CD8 functions as an inhibitory ligand in mediating the immunoregulatory activity of CD8+ cells.
Authors: Hambor J E , Kaplan D R , Tykocinski M L
Published: December 2022

Antisense and sense transfection technologies were employed in cellular coculture systems to investigate the physiologic requirements for CD8-dependent immunoregulation. Our data indicate that cells bearing genetically engineered CD8 molecules incorporating a glycoinositolphospholipid membrane anchor, as well as fixed cells bearing natural CD8 molecules, retain specific, CD8-dependent immunoregulatory activity. These findings together support the novel concept that CD8, a molecule traditionally thought of as a receptor, can function as an inhibitory ligand. CD8-dependent inhibition was shown to induce nonresponsiveness, persisting for at least 24 h, in Ag-specific responders. Moreover, only cells undergoing primary, but not secondary, antigenic stimulation through their TCR were found to be susceptible to CD8-dependent inhibition. Both CD4+ and CD8+ responding T cells were inhibited by CD8+ modulatory cells. These functional analyses of inhibitory and responder cells in CD8-dependent inhibition lay the groundwork for developing artificial CD8-based immunomodulatory peptides and deciphering CD8's role in natural immunoregulation.
Frequency analysis of human peripheral blood B cells producing IgM-rheumatoid factor. Differential effects of stimulation with monoclonal antibodies to CD3 and Staphylococcus aureus.
Authors: Hirohata S , Inoue T , Miyamoto T
Published: December 2022

The extent and nature of IgM-rheumatoid factor (RF) precursors within normal human B cells were examined by utilizing two different polyclonal B cell stimulators, Staphylococcus aureus Cowan I (SA) and immobilized mAb to the CD3 molecular complex (64.1). In cultures stimulated with SA, B cells produced IgM-RF in the presence of T4 cells, factors generated from mitogen-activated T cells (TF), or IL-2. Similarly, in cultures stimulated with immobilized anti-CD3, T4 cells that had been treated with mitomycin C (T4 mito) induced the production of large amounts of IgM-RF. Limiting dilution analyses revealed that the precursor frequencies of IgM-RF-producing cells induced by SA + TF and by immobilized anti-CD3-activated T4 mito were 0.008 +/- 0.001/100 B cells (n = 7) and 0.043 +/- 0.004/100 B cells (n = 6) (mean +/- SEM), respectively. Of note, the proportion of IgM-secreting cells that produced IgM-RF was much greater in cultures stimulated with SA + TF (30 to 61%) than that noted in cultures containing immobilized anti-CD3-stimulated T4 mito (1.0 to 3.9%). When B cells were co-stimulated with both SA and immobilized anti-CD3-activated T4 mito, the frequency of IgM-RF producing cells increased further to 0.12 to 0.27/100 B cells (4.6 to 21.2% of IgM-producing cells). These results indicate that both SA and immobilized anti-CD3 are potent stimulators of IgM-RF precursors. Moreover, the combination of SA and immobilized anti-CD3 provides a very potent in vitro signal for IgM-RF elaboration, inducing the production of this autoantibody from 1 to 3 in 1000 circulating normal B cells.
Unusual expression of IgG Fc receptors on peripheral granulocytes from patients with leukocyte adhesion deficiency (CD11/CD18 deficiency).
Authors: Majima T , Minegishi N , Nagatomi R , Ohashi Y , Tsuchiya S , Kobayashi K , Konno T
Published: December 2022

Leukocyte adhesion deficiency (LAD) is a hereditary disease characterized by defective expression of leukocyte adhesion glycoproteins; lymphocyte function-associated Ag-1 (CD11a/CD18), CR3 (CD11b/CD18) and p150,95 (CD11c/CD18). Granulocytes, monocytes, and lymphocytes of patients with LAD show profoundly defective in vivo and in vitro adherence-dependent immune functions. We investigated the expression of FcR for IgG on polymorphonuclear cells (PMN) and monocytes from patients with LAD, and their luminol- and lucigenin-enhanced chemiluminescence production in response to SRBC sensitized with murine (m) IgG2a and IgG2b. Unstimulated patient PMN showed an enhanced chemiluminescence in response to mIgG2a-SRBC and an increased phagocytosis of mIgG2a-SRBC. The up-regulated functions were inhibited by monomeric human IgG in a dose-dependent manner, which was attributed to an increase in expression of FcRI on patient PMN, as shown by flow cytometry using monoclonal antibody, 32.2, specific for human FcRI. In contrast, neither the expression of FcR on the monocytes of LAD patients nor their FcR-mediated functions were different from those of controls.
Demonstration of cross-reactive antibodies able to elicit lysis of both HIV-1- and HIV-2-infected cells.
Authors: Norley S G , Mikschy U , Werner A , Staszewski S , Helm E B , Kurth R
Published: December 2022

A total of 100% of sera from a large number of HIV-1-infected patients contained antibodies able to elicit Antibody-dependent cellular cytotoxicity lysis of cells infected with the HIV-1 isolates IIIB or RF. Levels of activity could not be correlated with activities in ELISA or neutralizing antibody assays nor with the clinical status of the patients. Surprisingly, 8 of 156 patients sera could additionally elicit lysis of HIV-2-infected cells, and cold target competition assays demonstrated that the cross-reactivity was apparently mediated via recognition of common epitope(s) expressed on the surface of cells infected with either group of HIV. The ADCC mechanism was shown to be mediated by a CD16+ lymphocyte. This demonstration of an effector mechanism able to attack and eliminate cells infected with a wide range of HIV strains has obvious implications for development of putative vaccines.
Association of the human CD3-zeta chain with the alpha beta-T cell receptor/CD3 complex. Clues from a T cell variant with a mutated T cell receptor-alpha chain.
Authors: Geisler C , Schøller J , Wahi M A , Rubin B , Weiss A
Published: December 2022

The TCR for Ag, on the majority of human T cells, is a disulfide-linked heterodimer composed of TCR-alpha and -beta chains noncovalently associated with the monomorphic CD3 complex composed of the CD3-gamma, -delta, -epsilon, and -zeta chains. The interactions involved in the assembly of the various components of this multimeric protein complex are not fully understood. In this report, a variant of the human leukemic T cell line Jurkat that synthesized all of the known components of the TCR/CD3 complex but fails to express the TCR/CD3 complex at the cell surface is further characterized. This variant, J79, has a mutated TCR-alpha chain that does not affect the assembly of the pentameric form (TCR-alpha beta-CD3-gamma delta epsilon) of the TCR/CD3 complex but inhibits the assembly of the CD3-zeta homodimer with the rest of the complex (TCR-alpha beta-CD3-gamma delta epsilon----TCR-alpha beta-CD3-gamma delta epsilon zeta 2). Transfecting a wild-type TCR-alpha gene into J79 reconstituted expression of a complete functionally competent TCR/CD3 complex at the cell surface. The results indicate that the TCR-alpha chain plays a crucial role in the assembly of the CD3-zeta homodimer with the pentameric form of the TCR/CD3 complex.
An analysis of insulin receptor expression and binding on MHC class I positive and negative human lymphoblastoid cells.
Authors: Liegler T , Alexander J , Cresswell P , Goldfine I , Goodenow R S
Published: December 2022

Recent studies have demonstrated a specific membrane association between the polymorphic class I antigens of the MHC and the insulin receptor (IR). In some reports, variation in IR binding is attributed to the class I phenotype. To extend these observations, we have examined whether MHC class I products influence IR function, in particular, high affinity ligand-specific binding. Using two independent methods, we have compared IR affinity and density on a human B-lymphoblastoid cell line expressing HLA-A, -B products vs a null mutant and a set of transfectants where HLA-A3, -B7, or -Bw58 expression has been restored through gene transfer. The results from radioreceptor assay and RIA measuring IR binding and expression indicate that although there is a decrease in high affinity insulin binding sites in the HLA-A, -B null mutant, ligand binding cannot be restored in the transfectants expressing HLA-A or -B alleles. We conclude that the MHC class I products do not determine the insulin-binding phenotype of the cells examined in this study. Alternatively, we propose that insulin receptor heterogeneity in affinity and density may be influenced by other undetermined factors inherent to clonally expanded cells, thereby complicating dissection of IR/MHC interactions.
Mast cells and pulmonary fibrosis. Identification of a histamine releasing factor in bronchoalveolar lavage fluid.
Authors: Broide D H , Smith C M , Wasserman S I
Published: December 2022

As elevated bronchoalveolar lavage (BAL) fluid histamine levels are noted in patients with pulmonary fibrosis (PF), we assayed BAL fluid from 16 patients with PF for the presence of a histamine releasing factor (HRF). HRF activity was assayed by measuring release of the preformed mast cell-derived mediators, histamine, or beta-hexosaminidase (beta-hex) from a purified population of IL-3 dependent mouse bone marrow derived mast cells (MBMMC) or human blood basophils. Mean BAL cell free histamine levels in the patients with PF was 1226 +/- 1349 pg/ml, whereas BAL histamine levels in a comparison group of six non-PF patients was 118 +/- 60 pg/ml. HRF was significantly elevated in BAL fluid of patients with PF (mean beta-hex release 24.5 +/- 12.9%; range 6.8 to 52.4%) compared to the non-PF group of patients (mean beta-hex release 7.9 +/- 7.7%; range 1.8 to 20.7%). The PF HRF not only degranulated MBMMC, but also induced the generation of the arachidonic acid metabolite leukotriene C4 from MBMMC (24.6 +/- 4.2 ng leukotriene C4/10(6) MBMMC). The PF HRF did not appear to be a cytokine previously identified in BAL fluid of patients with PF (i.e., platelet derived growth factor or insulin growth factor-1) or a human cytokine able to degranulate human basophils (i.e., IL-1, or granulocyte-macrophage-CSF) as these recombinant human cytokines did not induce MBMMC beta-hex release. Physicochemical characterization of the HRF revealed that it was relatively heat stable, pronase sensitive and on Sephadex G-75 and G-200 column chromatography had an apparent molecular mass of 30 to 50 kDa. The ability of PF BAL to induce beta-hex release from MBMMC was not dependent on IgE as unsensitized or lactic acid treated MBMMC release similar amounts of beta-hex compared to MBMMC sensitized with IgE. Thus, BAL fluid of patients with PF contains an HRF that induces beta-hex release from MBMMC via an IgE-independent mechanism. The presence of the HRF could explain elevated BAL histamine levels in patients with PF.
Anaphylatoxin binding and degradation by rat peritoneal mast cells. Mechanisms of degranulation and control.
Authors: Fukuoka Y , Hugli T E
Published: December 2022

Incubation of radiolabeled human C3a with rat peritoneal mast cells resulted in high levels of uptake and extensive degradation of the ligand. Both cell-bound and free radiolabeled human C3a underwent extensive degradation by rat mast cells even at 0 degrees C. We examined several protease inhibitors for their ability to prevent degradation of radiolabeled human C3a by the rat mast cells. The inhibitors PMSF, chymostatin, and soybean trypsin inhibitor were most effective in preventing radiolabeled human C3a degradation. Degradation of the cell-bound ligand was totally inhibited only by PMSF. These compounds are effective inhibitors of a chymotrypsin-like enzyme (chymase) extracted from rat mast cells. Chemical cross-linking of radiolabeled human C3a to surface components on the rat mast cells, in the presence of PMSF, revealed one major and two minor bands. The mast cell component in both the major and minor bands proved to be chymase-associated based on a direct comparison with purified chymase isolated from rat mast cells. However, neither antichymase antibody nor chymase inhibitors influenced the degranulating activity of C3a on rat mast cells that occur independently of the C3a-chymase interactions. We conclude that there are neither specific C3a-binding sites on rat mast cells nor specific receptors whose occupancy leads to cellular activation. Although human C3ades Arg is inactive on guinea pig ileal and lung tissue, it binds to and induces degranulation of rat mast cells, as well as enhances vascular permeability in rat skin, at concentrations nearly identical to that of intact C3a. The fact that both C3a and C3ades Arg stimulated mast cell activation, at concentrations in excess of 10(-6) M, argues against specific binding sites for the anaphylatoxin on rat mast cells. It is proposed that the cationic C3a molecule activates rat mast cells in a secretory and nonlytic manner by a nonspecific mechanism similar to that of other polybasic compounds.
Two ultraviolet tumor-specific suppressor cell clones. One expresses Ig RNA and the other expresses T cell receptor-alpha and -beta RNA.
Authors: Kraig E , Kannapell C C , Fischbach K , Zellmer V , Trial J
Published: December 2022

Two splenic cell clones were derived from mice bearing UV-induced skin tumors; both clones are I-J+, Thy-1+, and J11D+. Cellular extracts, but not culture supernatants, from the two clones, designated T4 and T5, specifically suppress primary cytotoxic and proliferative responses to UV-induced tumor targets. Therefore, clones T4 and T5 would appear to function as putative I-J+ suppressor cells with specificity for an antigenic epitope common to several UV-induced tumors. To derive more information concerning the status of Ag-receptor genes in the suppressive clones, we analyzed the rearrangement and expression of Ig and TCR genes. The first clone, T4, expressed Thy-1, a T cell-surface marker, yet it had not rearranged its TCR-beta, -gamma, and -delta gene families, nor were TCR-alpha, -beta, -gamma, or -delta transcripts detectable. Instead, clone T4 contained rearranged Ig H chain genes and expressed mu RNA. Because neither lambda nor kappa L chain transcripts were detected, this clone could not utilize a complete Ig molecule as its Ag receptor. On the other hand, clone T5 contained exclusively TCR gene rearrangements; it expressed alpha and beta transcripts of the correct sizes. However, the level of RNA that could encode the delta-chain of the CD3 accessory molecule was below detection, so it is unlikely that this TCR was expressed on the cell surface. The rearrangement of Ig genes in one clone and TCR genes in the other clone does not necessarily indicate that these genes encode the Ag receptors used by these clones. However, it suggests either that suppressor-like cells can derive from different lymphocyte precursor lineages or that rearrangements of the known TCR genes or the Ig genes are irrelevant to the function of this cell lineage.
CD4-CD8- T cells from mice with collagen arthritis display aberrant expression of type l K+ channels.
Authors: Grissmer S , Hanson D C , Natoli E J , Cahalan M D , Chandy K G
Published: December 2022

Expression of voltage-gated K+ channels in mAb-defined T cell subsets from normal mice and mice with experimental autoimmune arthritis was studied with the patch-clamp whole-cell recording technique in combination with fluorescence microscopy. CD4+CD8- Th cells from DBA/1 LacJ mice with type II collagen arthritis expressed low levels of type n K+ channels, and CD4-CD8+ T cells (cytotoxic) showed small numbers of type l or n' K+ channels, like their phenotypic counterparts in normal mice. CD4-CD8-Thy-1.2+ (double negative or DN) T cells from the diseased mice, however, displayed an abundance of type l K+ channels compared to DN T cells in normal mice, or mice immunized with CFA. Furthermore, the aberrant expression of type l K+ channels correlated with the presence of active disease. DN T cells from mice with SLE, type-1 diabetes mellitus, and experimental allergic encephalomyelitis, also exhibited a high number of type l K+ channels. These results suggest that expression of numerous type l K+ channels may be a useful marker for DN T cells associated with these autoimmune disorders.
Graft-versus-host mortality induced by noncytolytic CD4+ T cell clones specific for non-H-2 antigens.
Authors: Miconnet I , Huchet R , Bonardelle D , Motta R , Canon C , Garay-Rojas E , Kress M , Reynes M , Halle-Pannenko O , Bruley-Rosset M
Published: December 2022

The relative contribution of individual non-H-2 Ag and of T cell subsets that initiate graft-vs-host reaction (GVHR) as well as the mechanism responsible for histopathologic lesions are still a matter of debate. To address these questions and to favor the selection of T cells primed in vivo against non-H-2 Ag important in GVHR we derived T cell clones from spleens of (DBA/2 x B10.D2)F1 (H-2d) mice developing this reaction after the graft of B10.D2 (H-2d) cells incompatible for numerous non-H-2 Ag plus Mlsa. The pattern of reactivity of eight selected clones against cells from different strains of mice including (BXD)RI strains indicated that one CD4+ clone is specific for Mlsa and seven additional clones (six CD4+ and one CD8+) are specific for four different non-H-2 Ag (Ag.I-IV) and proliferate in an H-2-restricted manner. The same series of experiments suggested that Ag.I and II are poorly polymorphic and allowed to propose the localisation of the genes controlling Ag.I (chromosome 1) and Ag.III (chromosome 4). All the clones show a triple (alpha, beta, gamma) mRNA transcript for TCR but at their surface they express the alpha/beta-heterodimer. The clone specific for Mlsa expresses V beta 6 and that specific for Ag.IV expresses V beta 8.1. Rapid mortality accompanied by clinical and histologic signs of severe GVHR was observed after administration of CD4+ clones (together with host-syngeneic bone marrow) derived early after grafting and specific for Ag.I and II but not after administration of: 1) CD8+ cytolytic clone derived early after grafting and specific for Ag.IV; 2) CD4+ clones derived late after grafting and specific for Ag.III; and 3) CD4+ clone specific for Mlsa. A clear correlation was established between the capacity of CD4+ clones to induce GVHR mortality, to mediate host-specific DTH and to release a high level of TNF. In conclusion: 1) the reaction against a single non-H-2 Ag is sufficient to provoke lethal GVHR; 2) the capacity to provoke GVHR mortality depends on antigenic specificity and functional properties of the responding clones; 3) the inflammatory process mediated by CD4+ clones may play a major role whereas the specific CD8+ T cell-mediated cytolytic activity is not necessarily lethal.
The effects of differentiating agents on IL-1 beta production in cultured human monocytes.
Authors: Janson R W , Joslin F G , Arend W P
Published: December 2022

Human monocytes aged in medium exhibit a decrease in LPS-induced IL-1 beta production in comparison with fresh cells. The objective of these experiments was to evaluate the effects of differentiation for 1 or 6 days in IFN-gamma, 1,25-(OH)2-vitamin D3, granulocyte-macrophage CSF, or in various combinations of these agents on both steady state IL-1 beta mRNA levels and protein production in LPS-stimulated monocytes. Monocytes preincubated in IFN-gamma for 1 day, then cultured for 24 h with LPS, exhibited similar kinetics of IL-1 beta mRNA production, but a higher peak mRNA level at 8 h after LPS stimulation, in comparison with cells cultured in medium before LPS stimulation. An increase in IL-1 beta protein production with variable secretion was noted in monocytes preincubated in IFN-gamma before stimulation with LPS. Monocytes preincubated for 1 day in 1,25-(OH)2-D3 alone or in both IFN-gamma and 1,25-(OH)2-D3 exhibited similar kinetics and peak expression of LPS-induced IL-1 beta mRNA levels as cells cultured in medium. However, monocytes preincubated for 1 day in both agents displayed a 50% or greater restoration in LPS-induced IL-1 beta synthesis and secretion in comparison with fresh cells. Granulocyte-macrophage-CSF did not augment the effects of the other differentiating agents on IL-1 beta production. Monocytes cultured over 6 days in differentiating agents failed to exhibit any restoration in LPS-induced IL-1 beta production. These in vitro-derived macrophages exhibited very low levels of both IL-1 beta mRNA and protein production under any culture condition. These results suggest that the effects of differentiating agents on LPS-induced IL-1 beta production may in part be related to the state of maturation of the monocyte. Furthermore, a repression in IL-1 beta transcription that is not reversed by exposure to differentiating agents may be acquired by monocytes as they mature into macrophages in vitro.
The regulation of resistance to Schistosoma mansoni by auto-anti-idiotypic immunity. III. An analysis of effects on epitopic recognition, idiotypic expression, and anti-idiotypic reactivity at the clonal level.
Authors: Phillips S M , Lin J J , Galal N , Linette G P , Walker D J , Perrin P J
Published: December 2022

Auto-anti-idiotypic mechanisms can regulate the protective immune response against Schistosoma mansoni. Anti-idiotypic responses were stimulated by immunization of mice either with nonspecifically induced lymphoblasts, produced with Con A, or with Ag-induced lymphoblasts bearing specific idiotypic receptors. The effect of the induced anti-idiotypic response upon clonotypic cellular reactivity was assessed in vitro through the suppression of antigen-mediated blast transformation by cloned T cells and in vivo by suppression of resistance to S. mansoni and delayed-type hypersensitivity responses against specific Ag. Differential regulation of humoral immune responses was studied at the levels of specific epitopic recognition, the expression of specific Id, and the production of anti-idiotypic responses directed against mAb bearing specific Id. Anti-idiotypic sensitization resulted in variable (10 to 90%) suppression of the immune response to discrete antigenic epitopes, the expression of specific idiotypic phenotypes, and anti-idiotypic, antiparatopic responses against T cell clonotypes and antibody idiotypic phenotypes. In vitro admixture and in vivo challenge studies resulted in consonant differential suppression. Thus idiotypic regulation can mold the fine specificities of the protective immune response to S. mansoni at the clonal level and may provide an approach to optimize the expression and assessment of resistance.
Transmembrane ion conductance in human B lymphocyte activation.
Authors: Brent L H , Butler J L , Woods W T , Bubien J K
Published: December 2022

Human B lymphocytes were examined to determine whether transmembrane ion conductance plays a role in cell activation. Mitogens (anti-human IgM F(ab')2 fragment (anti-mu) and PMA) were used to stimulate B lymphocytes. Mitogen-induced DNA synthesis was inhibited by tetraethylammonium-Cl (TEA), 4-aminopyridine (4AP), verapamil, and diltiazem in a dose-dependent manner. This inhibition was not due to reduction in cell viability as determined by trypan blue exclusion. Mitogen-induced increases in RNA synthesis were partially inhibited by TEA and 4AP and were more completely inhibited by verapamil and diltiazem. Mitogen-induced cell volume increases were not affected by TEA or 4AP but were completely inhibited by verapamil and diltiazem. B lymphocytes stimulated with anti-mu expressed G1 phase cell surface antigens in the presence of TEA or 4AP, but failed to do so in the presence of verapamil or diltiazem. Substitution of PMA for anti-mu as the mitogen did not alter the effects of TEA or 4AP. However, verapamil inhibited PMA-induced expression of G1 phase cell surface markers although diltiazem did not. The patch clamp technique was used to directly examine plasma membrane ionic currents in whole-cell, cell-attached, and inside-out patch configurations. Activation of B lymphocytes with either anti-mu or the Ca2+ ionophore, A23187, inhibited opening of one type of channel in cell-attached patches. In inside-out patches, this channel type conducted current when the bath [Ca2+] was low (6 X 10(-8) M) but failed to conduct current when the bath [Ca2+] was increased above 1 X 10(-6) M. The results of these experiments are consistent with the hypothesis that activation of B lymphocytes induces alterations in plasma membrane ion conductance. Single channel studies suggest that activation induced increases in [Ca2+]i may directly inhibit a specific set of plasma membrane ion channels as one mechanism by which transmembrane ion flux is altered.
T lymphocytes that express CD4 and the alpha beta-T cell receptor but lack Thy-1. Preferential localization in Peyer's patches.
Authors: Harriman G R , Lycke N Y , Elwood L J , Strober W
Published: December 2022

Thy-1- T cells expressing CD4 and the alpha beta-TCR have been identified in murine lymphoid tissues. These cells are particularly prevalent in Peyer's patches (PP), representing 17 +/- 3% of PP CD4 T cells, whereas they are much less prevalent in spleen, lymph nodes, lamina propria, or peritoneum. Phenotypic studies of fresh-isolated PP T cells demonstrate that all PP CD4 T cells (both Thy-1- and Thy-1+) express CD3, alpha beta-TCR, and CD5 (Lyt-1), whereas none coexpress CD8 (Lyt-2). Thy-1- and Thy-1+ CD4 T cell lines generated from PP also coexpress CD3 and alpha beta-TCR, but are heterogeneous in expression of CD5 and again do not coexpress CD8. Further studies revealed that Thy-1- CD4+ T cells were not present in nude mice. Short term stimulation of Thy-1+ CD4+ PP T cells with anti-CD3 resulted in loss of Thy-1 in a substantial fraction of these cells. Functional studies of Thy-1- and Thy-1+ CD4+ PP T cells indicate that fresh-isolated Thy-1- CD4+ cells do not proliferate in response to insoluble anti-CD3 but do proliferate when stimulated with soluble anti-CD3 in the presence of feeder cells. In contrast, Thy-1+ CD4+ cells proliferate well to both stimuli. However, Thy-1- CD4+ PP T cells adapted to in vitro culture exhibit vigorous proliferative responses when stimulated with either form of anti-CD3. Evaluation of lymphokine secretion by fresh-isolated Thy-1- and Thy-1+ CD4+ PP T cells revealed that both make substantial amounts of IL-2; however, Thy-1- T cells made less IL-4 than their Thy-1+ counterparts. Neither population made IL-5 or IFN-gamma. Similarly, Thy-1- and Thy-1+ CD4 T cell lines made similar amounts of IL-2; again Thy-1- T cells made less IL-4; and in this case Thy-1- T cells made IL-5 albeit significantly less than the Thy-1+ cells. Finally, immunohistochemical studies suggested that many of the CD4+ T cells in PP germinal centers were Thy-1-, indicating that Thy-1- and Thy-1+ CD4 T cells differ in their distribution within the PP. These studies thus define a phenotypically and functionally distinct T cell population which is most prevalent in murine Peyer's patches.
Regulation of human cytotoxic T lymphocyte development by IL-7.
Authors: Hickman C J , Crim J A , Mostowski H S , Siegel J P
Published: December 2022

The effects of IL-7 on the generation of human CTL in alloantigen-, virus-, and lectin-stimulated systems were examined. Addition of IL-7 at the onset of cultures resulted in marked (up to 80-fold) augmentation of cytotoxicity accompanied by smaller (1.5- to 4-fold) increases in total lymphocyte number. Studies of CTL development in purified lectin-stimulated CD8+ T cell populations demonstrated that IL-7 could act directly on the CD8+ lymphocyte subset to augment cytotoxicity. In MLC, the IL-7-induced enhancement of cytotoxicity was found to be mediated primarily by the CD8+ subpopulation of lymphocytes. Late addition of IL-7 (day 5 of 7) resulted in an increase in cytolytic activity that was associated with little or no increase in total or activated CD8+ lymphocyte number indicating that IL-7 may act as a differentiation factor for human CTL. A role for endogenous IL-7 in CTL development was suggested by the observation that addition of neutralizing antiserum to IL-7 to MLC at initiation (or 5 days thereafter) resulted in decreased levels of cytotoxicity. These results indicate that IL-7 can exert major up-regulatory effects on human CTL development and suggest that these effects are both proliferative and differentiative.
CD8+ suppressor T cell clone capable of inhibiting the antigen- and anti-T cell receptor-induced proliferation of Th clones without cytolytic activity.
Authors: Hisatsune T , Enomoto A , Nishijima K , Minai Y , Asano Y , Tada T , Kaminogawa S
Published: December 2022

A CD8+ Ts clone 13G2 was established from lymph node cells of bovine alpha s1-casein-primed C57BL/6 mice by in vitro antigenic stimulation followed by maintenance with IL-2-containing medium. The clone suppressed the Ag-induced proliferative responses of CD4+ Th cell clones without detectable cytotoxicity for both APC and responding T cells. The clone was able to suppress the in vitro proliferative response and antibody formation of Ag-primed lymph node cells. The suppression was Ag-nonspecific and not restricted to the MHC. The clone was able to suppress the proliferation of Th clones induced by an immobilized anti-TCR antibody in which APC was absent. The clone was, however, unable to suppress the proliferation of Th clones induced by anti-CD3 or IL-2. Thus, the mechanism of suppression by 13G2 was found to be due to a direct action on Th by inhibiting a consequence of signal transduction initiated through the TCR.
Germinal center cells are a major IL-5-responsive B cell population in peripheral lymph nodes engaged in the immune response.
Authors: Rabinowitz J L , Tsiagbe V K , Nicknam M H , Thorbecke G J
Published: December 2022

Germinal center formation and the development of B cell memory in lymphoid tissue is a T cell-dependent process. The specific B cell-T cell interactions, and/or cytokines, resulting in germinal center cell growth have not yet been identified. Germinal center B cells were separated from other lymph node (LN) B cells by panning on peanut agglutinin (PNA)-coated dishes. Resulting fractions enriched for PNA+ (germinal center) B cells, and the PNA- (other) LN B cells from immune SJL mice were assayed for proliferation in the presence of cytokines. PNA+ and PNA- B cells responded equally to IL-4 in the anti-mu co-stimulator assay. In contrast, PNA+ B cells responded to murine (r)IL-5 or human B cell growth factor in the dextran sulfate (DxS) co-stimulator assay, to a much greater degree than did PNA- B cells. The same results were obtained with PNA+ and PNA- cells from LAF1 mice. Unfractionated LN B cells from nonimmunized SJL or BALB/c mice did not respond to IL-5 with or without DxS. B cell populations from BALB/c mice such as from spleen and peritoneal cavity, which are known to be high in Ly-1+B cells, responded to IL-5 alone, and more dramatically, to IL-5 as a co-stimulator with DxS. Such populations of cells from SJL mice, which are known to contain low numbers of Ly-1+B cells, responded markedly less. These results are consistent with those of others which show that in nonimmunized mice, Ly-1+B cells are a major IL-5 responsive subpopulation. IL-1 enhanced the proliferation of PNA+ cells in response to rIL-5 and had no effect on PNA- cells. IL-4 and IL-5 did not enhance each other's effects as co-stimulators of proliferation. In contrast to PNA+ B cells from immune LN, B cells activated by Escherichia coli endotoxin exhibited no responses to rIL-5. The present results indicate that in immune LN, PNA+, germinal center B cells constitute a prominent IL-5-responsive population.
Antigen-specific damage to brain vascular endothelial cells mediated by encephalitogenic and nonencephalitogenic CD4+ T cell lines in vitro.
Authors: Sedgwick J D , Hughes C C , Male D K , MacPhee I A , ter Meulen V
Published: December 2022

Experimentally induced and naturally occurring inflammatory diseases of the central nervous system (CNS) are often associated with a breakdown of the blood-brain barrier and edema within the CNS itself. CD4+ T cells are now clearly implicated in the pathogenesis of the induced CNS disease, experimental autoimmune encephalomyelitis, and previous in vivo experiments had indicated that these cells may be capable of directly damaging the CNS vasculature. To assess the capacity of CD4+ T cells to damage brain vascular endothelial cells (EC) in vitro, two lines with specificity for myelin basic protein and OVA were prepared and added to cultures of EC. We show here that both lines, when added in a resting state, severely disrupt the EC monolayers in an Ag-specific manner. The interaction is dependent on the recognition of Ag in the context of MHC class II and is blocked in the presence of mAb specific for CD4. Addition of T cell lines preactivated on irradiated thymocyte APC caused a high level of Ag nonspecific damage to the EC, which was not blocked by the addition of anti-CD4 mAb. Supernatants derived from these latter cells did not alone damage the EC monolayers despite the presence of TNF activity suggesting that T cell-EC contact may be required for these cell lines to mediate their effector function. Both resting and preactivated lines adhered strongly to the EC in the absence of Ag. The capacity of CD4+ T cells to strongly adhere to, and disrupt the integrity of, brain vascular EC may be important in the early stages of CNS disease mediated by this cell type.