bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of identical V alpha V beta gene pairs by IE-alloreactive and IE-restricted, antigen-specific T cells from MHC disparate mice. Evidence for thymic selection of V(D)J combinations.
Authors: Nakajima P B , Betz C J , Hansburg D
Published: December 2022

Alloreactive T cell hybridomas specific for IEk and/or IEb MHC Ag were obtained from IE-nonexpressor (IE alpha b) mice. The TCR V alpha and V beta gene segments used were identified by Northern blot and RNase protection. A large proportion (24 of 80 hybridomas tested) employed the same V alpha genes (V alpha 11.1 or V alpha 11.2) as are utilized in the IEk and IEb restricted response to the Ag cytochrome c. Of these 24 alloreactive hybridomas, 10 also expressed V beta genes utilized in the IE plus cytochrome c repertoire. Structural similarity between the two related sets of TCR indicates that V alpha segments can play a determining role in MHC specificity. These data also suggest that thymic selection based on TCR reactivity with self-MHC products acts on particular V(D)J combinations rather than on V alpha V beta pairings alone.
Frequency of B cells committed to the production of antibodies to insulin in newly diagnosed patients with insulin-dependent diabetes mellitus and generation of high affinity human monoclonal IgG to insulin.
Authors: Casali P , Nakamura M , Ginsberg-Fellner F , Notkins A L
Published: December 2022

Circulating autoantibodies to insulin can be detected in patients with insulin-dependent (type I) diabetes mellitus (IDDM) at the onset of the clinical disease. To characterize the autoantibody response in IDDM patients, we determined the frequency of circulating B cells committed to the production of IgM, IgG, and IgA to insulin in 12 newly diagnosed IDDM patients and, for comparison, in 9 healthy subjects and 17 insulin-treated IDDM patients. We found that B cells committed to the production of anti-insulin IgG, but not IgM, autoantibodies are present at much higher frequency in the circulation of newly diagnosed IDDM patients before insulin treatment (0.209 +/- 0.142%, mean value +/- SD of total IgG-producing cell precursors) as compared with age-matched healthy controls (0.032 +/- 0.030% of total IgG-producing cell precursors). In IDDM patients who had been treated with insulin, cells producing IgG antibody to insulin were 0.177 +/- 0.139% of total IgG-producing cell precursors. Generation of IgG mAb from B cells of IDDM patients revealed that they were monoreactive, i.e., they bound to insulin, but to none of the other Ag tested, and displayed a high affinity for insulin (Kd approximately 10(-7) moles/liter). In contrast, the IgG mAb derived from healthy subjects were polyreactive, i.e., they bound to all Ag tested, and displayed a low to moderate affinity for insulin (Kd approximately 10(-5) to 10(-6) moles/liter). These findings show that lymphocytes committed to the production of high affinity IgG autoantibodies to insulin are common in the B cell repertoire at the onset of IDDM.
Muramyl dipeptide induces production of hemopoietic growth factors in vivo by a mechanism independent of tumor necrosis factor.
Authors: Broudy V C , Kaushansky K , Shoemaker S G , Aggarwal B B , Adamson J W
Published: December 2022

Monocyte products including TNF and IL-1 can stimulate hemopoietic growth factor production in vitro and in vivo. Endotoxin-resistant C3H/HeJ mice have a cellular defect in the regulation of TNF production and provide a model in which to examine the relative importance of TNF and IL-1. To assess the relative role of TNF and IL-1, we injected the synthetic bacterial cell wall derivative muramyl dipeptide (MDP) or LPS into C3H/HeJ mice. Both MDP and LPS stimulated hemopoietic growth factor production in a dose-dependent manner. Northern blot analysis of splenic and lung mRNA revealed that MDP treatment induced granulocyte-macrophage CSF and macrophage-CSF transcripts, whereas LPS treatment induced macrophage-CSF transcripts. Messenger RNA for granulocyte-CSF, IL-3, IL-4, and IL-5 was not detected in control or treated mice. MDP treatment induced IL-1 mRNA but not TNF mRNA, and TNF bioactivity was not detected in the serum of MDP-treated mice. In contrast, LPS treatment induced TNF production. These results identify the hemopoietic growth factors induced by MDP and LPS in vivo, and suggest that MDP stimulates growth factor production by a mechanism independent of TNF.
Cyclosporin a modulation of tumor necrosis factor gene expression and effects in vitro and in vivo.
Authors: Nguyen D T , Eskandai M K , DeForge L E , Raiford C L , Strieter R M , Kunkel S L , Remick D G
Published: December 2022

We investigated, in vitro and in vivo, the cyclosporin A (CsA) regulation of LPS-induced TNF gene expression and subsequent pathophysiologic changes. In vitro dose-response kinetics data showed that CsA inhibited TNF bioactivity in the supernatant without delaying its production, whereas Northern blot and in situ hybridization analysis demonstrated that CsA did not inhibit TNF mRNA expression. We then sought to examine the in vivo effects of CsA (75 mg/kg) in CBA/J mice that were primed with CFA, and injected 2 wk later with LPS. CsA demonstrated suppression of local levels (ascites) of TNF as measured by either bioactivity or an anti-murine TNF ELISA. However, CsA did not decrease mRNA for TNF, or cell-associated TNF. In vivo kinetics studies were performed to show that CsA blocked both local (ascites) and systemic (plasma) LPS-induced TNF production without delaying these effects. CsA inhibited the neutrophilia and lymphopenia that developed after the LPS challenge, but did not block the lung neutrophilic infiltrate. These observations are helpful in understanding the role of the macrophage in CsA immunosuppression, particularly with regard to the ability of CsA to block LPS-induced TNF secretion.
Phosphatidylcholine is a quantitatively more important source of increased 1,2-diacylglycerol than is phosphatidylinositol in mast cells.
Authors: Kennerly D A
Published: December 2022

The widely accepted hypothesis that the increased 1,2-diacylglycerol (DAG) in stimulated cells is derived from phosphoinositides was tested by comparing the pattern of molecular species of phosphatidylinositol (PI) to that of DAG in mast cells. For any glycerol-based lipid, molecular species are defined by unique combinations of the two fatty acids esterified to glycerol. The quantitative frequency distribution of these molecular species represent a "fingerprint" that provides a sensitive approach to assessing precursor/product relationships. Based on mass, the molecular species fingerprints PI, phosphatidylcholine (PC), phosphatidylethanolamine, and phosphatidylserine were determined in unstimulated mast cells and compared to that of the DAG found after stimulation by IgE R bridging, compound 48/80 and the Ca+2 ionophore A23187. The molecular species fingerprint of DAG before stimulation was quite different from that of PI.IgE R cross-linking caused a 1.5 to 2-fold increase in DAG mass 1 to 3 min after stimulation with a concomitant shift in the pattern of DAG molecular species to one that bore only a partial resemblance to that of PI suggesting that considerably less than half of the incremental DAG is likely derived from PI. Ten to 20 min after Ag challenge, DAG levels became maximal (3.2- and 2.9-fold, respectively), but its molecular species pattern returned toward that seen in unstimulated cells suggesting that only perhaps 25% of the incremental DAG might be derived from PI. The molecular species fingerprint of DAG much more closely resembled that PC suggesting that as much as 75% of the incremental DAG might be derived from PC. Similar observations were made when 48/80 and A23187 were used as secretory agonists. These experiments indicate that the DAG participating in the "phosphoinositide cycle" represents a quantitatively modest fraction of the DAG accumulating in stimulated mast cells and suggest that mechanisms other than PI hydrolysis, including perhaps a "PC cycle," are more important than previously assumed in causing the rise in DAG during activation.
Endocytosis, intracellular trafficking, and processing of membrane IgG and monovalent antigen/membrane IgG complexes in B lymphocytes.
Authors: Davidson H W , West M A , Watts C
Published: December 2022

Human B lymphoblastoid cell lines specific for tetanus toxin/toxoid were used in our earlier studies to demonstrate the rapid endocytosis of monovalent Ag and its processing, as a complex with mIgG. Here we show that the mIgGR for Ag is endocytosed in the presence or absence of Ag and that at any given time about 60% of this recycling pool of membrane (m) Ig is inside the cell. During the earliest detectable stages of Ag processing a high proportion of Ag fragments resolved on SDS gels were bound to intact mIg. However, at later times, as fragmented Ag accumulated, the fragments were precipitable only with antibodies against the Fab region of mIgG indicating proteolytic fragmentation of this receptor. Fractionation of cell homogenates on self-forming Percoll gradients revealed that at least two compartments are involved in Ag processing: a low density endosome compartment and a dense "late endosome"/lysosomal compartment. The spectrum of Ag fragments observed in each fraction differed: fragments produced at later times during processing were detected only in the late endosome/lysomal fraction whereas the earliest observed fragments were found both in this fraction and in the low density fractions. Monovalent Ag/mIgG complexes appear to have an increased probability, compared to unoccupied mIgG, of targeting to proteolytically active compartments leading to processing of the Ag/mIg complex and to accelerated degradation of the mIgG.
The role of physical forces on cytotoxic T cell-target cell conjugate stability.
Authors: Hubbard B B , Glacken M W , Rodgers J R , Rich R R
Published: December 2022

Theoretical considerations suggest that external forces play a significant role in cell-cell conjugate formation and may lead to the misinterpretation of adhesion data. To test this, the stability of conjugates formed between CTL and fibroblast target cells (TC) was examined in the controlled shear environment of a parallel plate flow chamber. Murine fibroblast targets expressing class I maternally transmitted Ag Mtaa or Mtab were grown on a glass slide that formed one wall of the flow chamber and were used in conjunction with anti-Mtaa and anti-Mtab specific mouse CTL clones to establish a panel of Ag-reciprocal targets and lymphocytes. Although cytolysis assays indicated that lymphocytes recognized and destroyed appropriate but not inappropriate targets, the stability of some CTL/TC conjugates was Ag independent. In all cases, the conjugate stability was shear dependent over a 100-fold range (0.04 to 4.0 dynes/cm2). For some clones, the ratio of the stabilities of Ag-specific CTL/TC conjugates to nonspecific conjugates was significantly enhanced with increasing shear. This implies that the role of Ag specificity in CTL/TC adhesion may be misinterpreted if the shear environment of CTL/TC conjugates is unknown or uncontrolled. Kinetic analysis revealed that conjugate stability was dependent on the exposure time to external forces and that there existed two populations of conjugates; weak associations that disengaged within the first 30 s of flow, and strong associations that remained attached even after a 5-min exposure to a steady shear stress. The stability of Ag-specific CTL/TC conjugates at 0.04 dynes/cm2 was enhanced by 50% as the temperature was increased from 25 to 37 degrees C, whereas the stability of nonspecific CTL/TC associations was not affected. This result indicates that significant Ag-specific strengthening may occur at physiologic temperatures. This work suggests the importance of attention to role of fluid mechanical shear stress in standard adhesion assays.
Cytotoxic activity and lymphokine production of T cell receptor (TCR)-alpha beta+ and TCR-gamma delta+ cytotoxic T lymphocyte (CTL) clones recognizing HLA-A2 and HLA-A2 mutants. Recognition of TCR-gamma delta+ CTL clones is affected by mutations at positions 152 and 156.
Authors: Spits H , Paliard X , Engelhard V H , de Vries J E
Published: December 2022

TCR-gamma delta+ CTL clones were generated from CD4-CD8- T cells that were stimulated twice with the cell line JY. Either IL-2 or IL-4 was used as growth factor. A number of TCR-gamma delta+ clones were found to lyse the stimulator cell line JY. Two of these clones secreted N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase activity after stimulation with JY cells. The cytotoxic activity of these two clones was blocked by a mAb specific for HLA-A2. Moreover, these two TCR-gamma delta+ clones selectively lysed human fibroblast line M1 and murine P815 cells transfected with DNA fragments encoding HLA-A2 but not those transfected with HLA-B7 encoding DNA, indicating that these clones recognize HLA-A2. Analysis of the recognition of HLA-A2 by using target cells transfected with mutated HLA-A2 encoding genes revealed that the nature of the amino acid at position 152 of the molecule is critical for recognition of the TCR-alpha beta+ as well as the TCR-gamma delta+ CTL clones since replacement of Val for Ala at that position resulted in abrogation of recognition of one TCR-gamma delta+ and one TCR-alpha beta+ clone and substitution of Val for Glu affected recognition of all clones. Substitution of Leu for Trp at position 156 abrogated recognition by one TCR-gamma delta+ and one TCR-alpha beta+ T cell clone, but recognition by the other clones was not changed. All clones were able to secrete IL-2, IFN-gamma, and GM-CSF but not IL-4 after activation.
Relative inefficiency of soluble recombinant CD4 for inhibition of infection by monocyte-tropic HIV in monocytes and T cells.
Authors: Gomatos P J , Stamatos N M , Gendelman H E , Fowler A , Hoover D L , Kalter D C , Burke D S , Tramont E C , Meltzer M S
Published: December 2022

Macrophages are major viral reservoirs in the brain, lungs, and lymph nodes of HIV-infected patients. But not all HIV isolates infect macrophages. The molecular basis for this restrictive target cell tropism and the mechanisms by which HIV infects macrophages are not well understood: virus uptake by CD4-dependent and -independent pathways have both been proposed. Soluble rCD4 (sCD4) binds with high affinity to gp 120, the envelope glycoprotein of HIV, and at relatively low concentrations (less than 1 microgram/ml) completely inhibits infection of many HIV strains in T cells or T cell lines. HTLV-IIIB infection of the H9 T cell line was completely inhibited by prior treatment of virus with 10 micrograms/ml sCD4: no p24 Ag or HIV-induced T cell syncytia were detected in cultures of H9 cells exposed to 1 x 10(4) TCID50 HTLV-IIIB in the presence of sCD4. Under identical conditions and at a 100-fold lower viral inoculum, 10 micrograms/ml sCD4 had little or no effect on infection of monocytes by any of six different HIV isolates by three different criteria: p24 Ag release, virus-induced cytopathic effects, and the frequency of infected cells that express HIV-specific mRNA. At 10- to 100-fold higher concentrations of sCD4, however, infection was completely inhibited. Monoclonal anti-CD4 also prevented infection of these same viral isolates in monocytes. The relative inefficiency of sCD4 for inhibition of HIV infection in monocytes was a property of the virion, not the target cell: HIV isolates that infect both monocytes and T cells required similarly high levels of sCD4 (100 to 200 micrograms/ml) for inhibition of infection. These data suggest that the gp120 of progeny HIV derived from macrophages interacts with sCD4 differently than that of virions derived from T cells. For both variants of HIV, however, the predominant mechanism of virus entry for infection is CD4-dependent.
V region sequences of an idiotypically connected family of pathogenic anti-DNA autoantibodies.
Authors: O'Keefe T L , Bandyopadhyay S , Datta S K , Imanishi-Kari T
Published: December 2022

The H and L chain V region sequences of nine anti-DNA mAb that are representative of a pathogenic population of autoantibodies produced by the nephritis prone (SWR x NZB)F1 (SNF1) mice, were determined. These nine anti-DNA autoantibodies were idiotypically connected members of a cross-reactive Id family called the Id564 cluster. Moreover, these autoantibodies were all cationic in charge, IgG2b in isotype, and their H chain C regions had the normal SWR parent's allotype. Although derived from two different SNF1 animals, these pathogenic autoantibodies possessed highly homologous Leader-VH sequences that could account for their idiotypic cross-reactivity. Furthermore, the VH region sequences of these anti-DNA antibodies contained numerous basic residues that could impart their cationic charge. The Leader-VH sequences of these autoantibodies were also highly homologous to that of an anti-NP antibody-related germ-line gene of C57BL/6 mice, called VH-23. Among these nine pathogenic autoantibodies, three sets of clonally related anti-DNA antibodies could be identified. Thus the Id564 cluster of cationic anti-DNA autoantibodies of SNF1 mice are encoded by highly related VH genes, and this idiotypically connected population of pathogenic autoantibodies are selected to undergo an oligoclonal expansion in the lupus-prone SNF1 mice.
Human epidermal Langerhans cells express only the 40-kilodalton Fc gamma receptor (FcRII).
Authors: Schmitt D A , Hanau D , Bieber T , Dezutter-Dambuyant C , Schmitt D , Fabre M , Pauly G , Cazenave J P
Published: December 2022

In man, three distinct classes of receptors for the Fc fragments of IgG (FcRI, II, III) have been defined. The FcRI has a Mr of about 72 kDa, binds human IgG-coated E, and is recognized by mAb such as 32. The FcRII has a Mr of 40 kDa, binds murine IgG1-coated E, and reacts with the mAb IV.3 and CIKM5, which recognize CDw32 moieties. Lastly, the FcRIII has a Mr of 50 to 70 kDa and is recognized by anti-CD16 mAb. In the present study we have shown that i) only murine IgG1-coated E form rosettes with 49 +/- 1.5% (mean +/- SEM, n = 9) of CD1a+ epidermal cells (EC) (which represent Langerhans and indeterminate cells) and that ii) the mAb anti-FcRII CIKM5 prevents this rosette formation. Among the mAb reacting with the three different types of FcR, only those recognizing FcRII i) stain about 55 +/- 1.5% (mean +/- SEM, n = 9) of the CD1a+ EC and ii) reveal the presence of dendritic cells in epidermal sheets obtained by suction blister. Under the electron microscope i) apparently all the cells forming rosettes or reacting with the gold-labeled anti-FcRII mAb (CIKM5 or the F(ab) fragment of IV.3) contained Birbeck granules and ii) the gold-labeled mAb were internalized in unfixed Langerhans cells by receptor-mediated endocytosis and accumulated in lysosomes. Labeling by the anti-FcRII mAb of the CD1a+ cells in suspension disappears after 48 h of culture. All these observations strongly suggest that CD1a+ EC express only the FcRII. This conclusion was confirmed by immunoprecipitation experiments, whereas no specific immunoprecipitate was noted with the anti-FcRI or anti-FcRIII mAb, the anti-FcRII mAb immunoprecipitated a protein of Mr 40 kDa.
Inducible and tissue-specific expression of rat alpha-1-acid glycoprotein in transgenic mice.
Authors: Dewey M J , Rheaume C , Berger F G , Baumann H
Published: December 2022

alpha-1-Acid glycoprotein (AGP), which is produced in the mammalian liver and secreted into the blood-stream, is regulated by steroid hormones and by mediators of the acute phase response. In vitro transfection studies have shown that the response to glucocorticoids requires a cis-acting regulatory element, termed the glucocorticoid response element, that is located within 120 bp of the transcriptional start-site of the gene; induction by the acute phase mediators requires a different element, termed the distal regulatory element (DRE), that is located about 5 kb upstream of the start-site. To determine if these elements function in vivo, we have produced and characterized transgenic mice containing rat AGP gene constructs with and without the DRE. Five transgenic lines were produced from a 9.5-kb genomic AGP containing 4.7 kb of the 5' flanking region; this construct lacks the DRE. Another transgenic line was derived from a 10.7-kb clone that contains 5.3 kb of 5' flanking sequences including the DRE. All transgenic mice produced high levels of immunologically detectable rat AGP in the circulation, comparable to or in excess of that found in normal rats. There were correspondingly high concentrations of rat AGP transcripts in the liver. Transgene expression in all lines was induced in response to dexamethasone and during acute inflammation resulting from LPS treatment. The DRE-containing transgene underwent a greater induction in response to LPS than to dexamethasone; the transgene lacking the DRE responded similarly to both treatments. In cultured primary hepatocytes, the DRE-containing transgene was induced by the acute phase cytokines IL-1 and IL-6, and by dexamethasone, administered individually or in combination; the transgene lacking the DRE responded only to dexamethasone, and was not affected by the peptide hormones. Together, these results provide in vivo evidence supporting the notion that a minimum of two upstream sequences are responsible for the inflammatory induction of rat AGP. One element, which is located within the smaller 9.7-kb restriction fragment, is responsive to glucocorticoids and is likely to be the glucocorticoid response element located close to the transcriptional start site. The other element, the DRE, is located much further upstream and is responsible for imparting responsiveness to the acute phase cytokines.
Identification of class I MHC mRNA in human first trimester trophoblast cells by in situ hybridization.
Authors: Hunt J S , Fishback J L , Chumbley G , Loke Y W
Published: December 2022

Special gestation-related regulatory mechanisms for the expression of class I Ag by trophoblast cells directly exposed to maternal blood and tissues may be required for semiallogeneic pregnancy to be successful. Analysis of class I MHC mRNA by in situ hybridization and class I MHC Ag by immunohistology has revealed two phenotypically distinct subpopulations of trophoblast cells in term placentas and extraplacental membranes. Trophoblast cells external to the placenta are mRNA +/Ag+. They contain class I mRNA and express class I Ag that differ serologically from HLA-A,B,C. In contrast, trophoblast cells forming the syncytial layer of placental villi are mRNA-/Ag-. By immunohistology, trophoblast cells in 1st trimester placental tissues are similar to those in term tissues. In our study, in situ hybridization was used to determine if patterns of trophoblast cell class I mRNA were the same or different. Trophoblast cells external to the placental villi in 1st trimester tissues contained class I mRNA as would be predicted from the results with term tissues. Unexpectedly, class I mRNA was found in villous trophoblast cells. Thus, these studies identified an mRNA+/Ag- trophoblast cell subpopulation. The results suggest that tissue-specific mechanisms may interfere with translation of class I mRNA in 1st trimester villous trophoblast cells and/or that the protein products of the mRNA are not identified by available mAb.
Structural characterization of H chain-associated idiotopes of anti-p-azophenylarsonate monoclonal antibodies.
Authors: Parhami-Seren B , Sharon J , Margolies M N
Published: December 2022

The majority of antibodies directed against p-azophenylarsonate (Ars) protein conjugates elicited during secondary immune responses of A/J mice bear a heritable cross-reactive Id (CRIa or IdCR) which corresponds to the utilization of a unique combination of variable region gene segments that can differ by somatic mutations. One such monoclonal anti-Ars antibody, 44-10, bears IdCR as defined by rabbit antisera but does not react with two anti-idiotypic mAb, 5Ci and AD8, which react with all primary (unmutated) IdCR+ antibodies and some secondary response IdCR+ antibodies. We therefore determined the complete sequence of antibody 44-10, which differs from the germline encoded (unmutated) IdCR+ antibody 36-65 at four positions in the H chain V region (VH): position 55 in the second complementarity determining region, 100 and 107 (D-gene junctions) and 110 (in JH2). The 44-10 L chain is unmutated. Sequence analyses of five other secondary immune response anti-Ars IdCR+ antibodies chosen on the basis of sharing one or more of the amino acid substitutions found in 44-10, were correlated with idiotypic expression of this set of antibodies. The results suggest that the mutation at VH position 55 (Asn----Lys) is responsible for loss of the 5Ci idiotope. To substantiate this hypothesis, oligonucleotide-directed mutagenesis of the germline encoded (unmutated) IdCR+ antibody was used to produce two mutants, one with VH Lys 55 and the other containing residues at positions 100, 107 and 110 identical to those found in 44-10. Id binding studies on these mutants confirm that 5Ci idiotope loss is due to conformational changes resulting from a mutation at VH position 55. This mutation also results in loss of the AD8 idiotope in the structural context of antibody 44-10.
IL-4 and granulocyte-macrophage colony-stimulating factor selectively increase HLA-DR and HLA-DP antigens but not HLA-DQ antigens on human monocytes.
Authors: Gerrard T L , Dyer D R , Mostowski H S
Published: December 2022

A number of cytokines were tested for their ability to modulate HLA-DR Ag expression on normal human monocytes. IL-4, granulocyte-macrophage (GM)-CSF as well as IFN-gamma were able to increase HLA-DR Ag expression on monocytes. IFN-alpha was also able to augment HLA-DR Ag expression, but to a lesser degree. Macrophage-CSF, granulocyte-CSF, TNF-alpha, TNF-beta, and IL-6 were not able to augment HLA-DR Ag expression. There were distinct patterns in the ability of different cytokines to augment class II histocompatibility Ag expression. IL-4 and GM-CSF selectively increased only HLA-DR and HLA-DP, but did not increase HLA-DQ antigens on monocytes. IFN-gamma, however, was able to augment the expression of HLA-DR, HLA-DP, and HLA-DQ Ag. Combinations of IFN-gamma with either IL-4 or GM-CSF did not show any synergy for the augmentation of any of the class II antigens on monocytes.
IL-6 is an accessory signal in the alternative CD2-mediated pathway of T cell activation.
Authors: Lorré K , Van Damme J , Verwilghen J , Baroja M L , Ceuppens J L
Published: December 2022

Recent studies have demonstrated that IL-1 and IL-6 are synergistic accessory signals for activation of T cells. In this study, highly purified human T cells were cultured with either a stimulating pair of anti-CD2 mAb or with immobilized anti-CD3 mAb. Monocytes, a cellfree monocyte culture supernatant or IL-1 were required for anti-CD2-stimulated T cell proliferation, and they each strongly enhanced anti-CD3-induced T cell growth. IL-6 was synergistic with IL-1 as a helper factor for T cell growth after activation via CD2, but we could not demonstrate any effect of IL-6 in the CD3 pathway. The mechanism of the synergistic helper activity of IL-1 and IL-6 on T cell activation in the CD2 pathway was further examined. IL-1 (but not IL-6) was required for induction of IL-2 production. Both IL-1 and IL-6 enhanced IL-2R (p55) expression and the proliferative response to IL-2. T cell proliferation after stimulation with anti-CD2 and IL-1 or IL-1/IL-6 proceeded through an autocrine IL-2-dependent pathway. Moreover we found that, in the absence of IL-1, IL-6 still supported a transient and limited proliferation of anti-CD2- (but not of anti-CD3-) stimulated T cells, which apparently was independent of the autocrine growth factors IL-2 or IL-4. Our data suggest that IL-6 is important as an accessory signal for T cell growth in the CD2 pathway of T cell activation.
Monoclonal anti-idiotype induces protection against the cytotoxicity of the trichothecene mycotoxin T-2.
Authors: Chanh T C , Rappocciolo G , Hewetson J F
Published: December 2022

An IgG1 mAb, designated HD11, specific for the trichothecene mycotoxin T-2 and capable of neutralizing its cytotoxicity was used to generate a syngeneic monoclonal anti-Id antibody. The generated anti-Id mAb, designated DE8, specifically bound to HD11 anti-T-2 mAb, and not to IgG1 mAb of irrelevant specificity or to normal mouse Ig. DE8 inhibited the binding of HD11 anti-T-2 to T-2-BSA-coated plates, whereas a control anti-Id mAb did not, suggesting recognition of an Id determinant associated with the T-2 binding site of HD11. Moreover, the binding of HD11 to DE8 and that of DE8 to HD11 were specifically inhibited by free T-2 mycotoxin. DE8 mAb was efficient in abrogating the protective effect of HD11 in the cytotoxicity of T-2 on the human epidermoid carcinoma cell line Hep-2. In vivo immunization of BALB/c mice with DE8 conjugated to KLH induced an anti-T-2 antibody titer comparable to that obtained with T-2-OVA immunization, whereas immunization with unconjugated DE8 resulted in a lower titered anti-T-2 response. Immunization with DE8-keyhole limpet or with unconjugated DE8 induced anti-T-2 antibody responses characterized by expression of "HD11-like" Id and by protection against T-2 cytotoxicity. However, the T-2-OVA-induced anti-T-2 response lacked the HD11+ Id and was only partially protective against T-2 cytotoxicity. This represents the first demonstration of the use of an anti-Id based vaccine in the in vivo induction of a protective antibody response against the cytotoxicity of a nonproteinaceous, small m.w. biologic toxin, whose very toxic nature precludes its use as the immunogen.
Expression of murine Fc receptors for IgG.
Authors: Schreiber R E , Buku A , Unkeless J C
Published: December 2022

There are two distinct genes that encode murine low affinity Fc gamma RII, murine Fc gamma RII alpha, and murine Fc gamma RII beta, which are transcribed in specific cell lineages. Fc gamma RII alpha transcripts are present in macrophages, NK cells, and mesangial cells; Fc gamma RII beta transcripts are expressed in Fc gamma R-bearing B cells, T cells, and macrophages. We have devised a sandwich ELISA to quantify the expression of Fc gamma RII alpha protein. The ELISA is specific for Fc gamma RII alpha, and does not detect the closely related Fc gamma RII beta protein. Upon stimulation with IFN-gamma the Fc gamma RII beta- macrophage cell line J774a expressed a twelvefold enhanced level of Fc gamma RII alpha protein. Peritoneal macrophages synthesized varying amounts of Fc gamma RII alpha. High levels of Fc gamma RII alpha were observed in resident and thioglycollate-elicited peritoneal macrophages, but no Fc gamma RII alpha was detected in Bacillus Calmette Guérin-elicited macrophages. J774a cells stimulated with rIL-6 bound approximately twice as much anti-Fc gamma RII mAb 2.4G2 IgG as did unstimulated controls. However, the Fc gamma RII alpha-specific ELISA showed no change in the amount of Fc gamma RII alpha expressed. A probe encompassing the extracellular coding sequence of Fc gamma RII beta hybridized to two distinct transcripts that were elevated in rIL-6-stimulated J774a cells. One of these transcripts had the same mobility in electrophoresis as Fc gamma RII alpha mRNA and hybridized to an Fc gamma RII alpha-specific probe, whereas the other transcript was larger and did not hybridize to probes specific for either Fc gamma RII alpha or Fc gamma RII beta. Moreover, we confirmed, with an Fc gamma RII beta-specific probe, that J774a cells do not make Fc gamma RII beta mRNA. Thus, the larger transcript appears to encode a novel Fc gamma RII. We suggest that the increased level of binding of the anti-Fc gamma RII mAb 2.4G2 to rIL-6-induced cells represents translation of a Fc gamma R distinct from Fc gamma RII alpha or Fc gamma RII beta.
Production by K 562 cells of an inhibitor of adherence-related functions of human neutrophils.
Authors: Amar M , Amit N , Huu T P , Chollet-Martin S , Labro M T , Gougerot-Pocidalo M A , Hakim J
Published: December 2022

Certain tumor cells generate factors that inhibit neutrophil chemotaxis. Our study was designed to explore whether such factors are produced by K 562 malignant cells and whether these have a broader effect in altering neutrophil functions. After 48 h of in vitro culture of K 562 cells, the culture medium and the cells were separated, lyophilized, and extracted with ethanol. These K 562 products, i.e., either the cell or supernatant extract, inhibited both nonstimulated locomotion and locomotion induced either by FMLP or activated serum. Furthermore, K 562 products inhibited neutrophil adherence and oxidative burst induced by opsonized zymosan, whereas oxidative burst induced by PMA or FMLP was not altered. K 562 products had an inhibitory effect on the PMN binding to iC3b-coated particles. They did not modify Mo1 expression of resting cells, did not alter the up-regulation of the receptor induced by FMLP but inhibited the FMLP-induced capping of Mo1 Ag. Con A capping was also inhibited. Actin polymerization in FMLP-stimulated PMN, as measured by flow cytometry and phalloidin binding to F-actin, was inhibited by K 562 products. The inhibitory factor present in K 562 products (cell and culture supernatant) was purified in three steps including gel filtration, ion-exchange chromatography, and IEF. The eluted active fraction corresponded to single band of about 8 kDa on SDS-PAGE. From these experiments, it is concluded that K 562 malignant cells in culture contain and release a low molecular mass factor (congruent to 8 kDa) that inhibits all adherence-related functions of neutrophils, whereas it does not alter FMLP- or PMA-induced oxidative burst. Further studies are needed to assess whether products of other tumor cells also act on the neutrophil by inhibiting adherence-related functions, Mo1 function and capping, and actin polymerization.
Cellular and humoral immune responses to Plasmodium falciparum gametocyte antigens in malaria-immune individuals. Limited response to the 48/45-kilodalton surface antigen does not appear to be due to MHC restriction.
Authors: Riley E M , Ong C S , Olerup O , Eida S , Allen S J , Bennett S , Andersson G , Targett G A
Published: December 2022

We have examined immune responses to a cultured Plasmodium falciparum gametocyte lysate and to an affinity-purified preparation of the 48/45-kDa gamete surface Ag in a group of 30 malaria immune individuals and in 24 Europeans with no previous exposure to malaria. Cellular responses were assessed in vitro by lymphoproliferation and production of IFN-gamma; antigamete antibodies were detected by immunofluorescence, Western blotting, and competitive ELISA. Cells from all the malaria immune donors responded to the gametocyte lysate in both assays while cells from nonimmune donors gave only weak proliferative responses. Antigamete antibodies were detected in the serum of all the immune donors but not in serum from nonimmunes. Nonimmune donors were completely unresponsive to the purified 48/45-kDa surface Ag while cells from 40% of immune donors responded by either proliferation or IFN-gamma production. Only 3 of 30 immune donors had detectable antibodies to the 48/45-kDa Ag. Class II HLA type was determined for 27 of the immune donors but no relationship between HLA-DR or -DQ and responsiveness to the 48/45-kDa Ag was discerned. The possible reasons for limited recognition of this gamete surface Ag are discussed.
Mechanisms of deoxyguanosine lymphotoxicity. Human thymocytes, but not peripheral blood lymphocytes accumulate deoxy-GTP in conditions simulating purine nucleoside phosphorylase deficiency.
Authors: Fairbanks L D , Taddeo A , Duley J A , Simmonds H A
Published: December 2022

This study was designed to simulate purine nucleoside phosphorylase (PNP) deficiency by preincubating with guanosine (Guo) to minimize PNP activity while investigating the metabolism of [14C] deoxyguanosine (dGuo) at physiologic concentrations (10 microM) by unstimulated thymocytes, tonsil-derived T and B lymphocytes, and peripheral blood cells over short time periods. GTP was the principal metabolite formed from dGuo by all cell types with functional PNP and hypoxanthine-guanine phosphoribosyltransferase, confirming formation via degradation to guanine with subsequent salvage by hypoxanthine-guanine phosphoribosyltransferase. Thymocytes also formed a small amount of deoxyguanosine triphosphate (dGTP), presumably through direct phosphorylation by deoxycytidine kinase. Incorporation of dGuo into GTP was effectively inhibited in all instances under PNP deficiency conditions and dGTP levels increased up to 10-fold in thymocytes, but tonsil-derived B or T lymphocytes and unfractionated PBL still accumulated no detectable dGTP. E and platelets formed low amounts of dGTP under these conditions. Preincubation with adenine (50 microM) to reverse any Guo-induced toxicity reduced the incorporation of dGuo into GTP without inhibitor in all cell types with intact adenine phosphoribosyltransferase, but had no effect on dGTP accumulation in thymocytes, with or without inhibitor, thus excluding any indirect formation of dGTP via the de novo route. The rapid metabolism of dGuo to GTP, in the absence of PNP inhibition and subsequent effects of the altered GTP concentrations on cellular metabolism, may account for the differing responses reported by investigators with the use of low dGuo concentrations (enhancing), compared with high (inhibitory), concentrations in mitogen-stimulated lymphocyte studies. The exclusive ability of thymocytes to accumulate significant amounts of dGTP, and inability of B cells to do so, provides a logical explanation for the selective T cell immunodeficiency in PNP deficiency.
Anti-proliferative effect of IFN-gamma in immune regulation. IV. Murine CTL clones produce IL-3 and GM-CSF, the activity of which is masked by the inhibitory action of secreted IFN-gamma.
Authors: Gajewski T F , Fitch F W
Published: December 2022

We have demonstrated recently that rIFN-gamma inhibits the proliferation of murine bone marrow cells stimulated with rIL-3 or recombinant granulocyte-macrophage (GM)-CSF. In light of this finding, three murine CD8+ CTL clones whose supernatants had been shown previously not to contain detectable levels of CSF activity but which contained a relatively high level of IFN-gamma were reassessed for their ability to secrete IL-3 and GM-CSF. Supernatants from CTL clones activated with anti-CD3 mAb failed to stimulate the IL-3-dependent cell line FDCP1. However, these supernatants were indeed able to stimulate the proliferation of FDCP1 cells if anti-IFN-gamma mAb was present. This stimulatory activity was specifically neutralized by anti-IL-3 mAb. Supernatants from two of the three clones stimulated the proliferation of a GM-CSF-responsive HT-2 cell line, and this activity was neutralized by anti-GM-CSF antibody. The otherwise modest ability of CTL supernatants to stimulate the proliferation of fresh bone marrow cells was augmented considerably in the presence of anti-IFN-gamma mAb, and this activity was appropriately blocked by anti-IL-3 and anti-GM-CSF antibodies. mRNA for IL-3 and GM-CSF, as well as for IFN-gamma and TNF-alpha, was detected in cells that secreted those lymphokines, and the time course of appearance of each mRNA correlated with secretion of the appropriate lymphokine activity. However, the time course of mRNA accumulation for each lymphokine was distinct, the order of expression of these genes apparently being TNF-alpha, then IFN-gamma and GM-CSF, and finally IL-3. Our results emphasize that potential interactions among lymphokines must be considered when interpreting data obtained from lymphokine bioassays and suggest an immunoregulatory role for CTL through the secretion of several of the same lymphokines produced by HTL.
A molecular and structural analysis of the VH and VK regions of monoclonal antibodies bearing the A48 regulatory idiotype.
Authors: Victor-Kobrin C , Barak Z T , Bonilla F A , Kobrin B , Sanz I , French D , Rothe J , Bona C
Published: December 2022

The results presented in this paper explore the molecular basis for expression of the A48 regulatory Id (RI). A48 RI+ mAb derived from idiotypically manipulated mice molecularly resembled the A48 and UPC 10 prototypes of this system by utilizing a VHX24-Vk10 combination. Id expression by these antibodies was not restricted by a particular D region sequence, JH, or JK segment, but quantitative differences in Id expression were associated with utilization of different members of the VK10 germ-line gene families. The VL sequences of these A48 RI+ mAb has identified amino acid residues lying in four different idiotope-determining regions which may contribute to the structural correlate of this Id. A comparative sequence analysis of the VH regions of these VHX24 utilizing A48 RI+ mAb with several A48 RI+ mAb utilizing VHJ558 or VH7183 VH genes as well as a hybrid transfectoma antibody derived from two A48 RI-, VHJ558 utilizing hybridomas, all suggested that four nonconsecutive positions which lie outside the idiotope-determining regions may contribute structural elements toward expression of this Id. The VH and VL regions of the A48RI+, VHX24-Vk 10+ mAb showed low to moderate levels of somatic mutation which showed different patterns of distribution between the complementary determining region (CDR) and framework regions in the H and L chains. Although the VK sequences contained 50% of the replacement mutations in the CDR, with a replacement/silent mutation ratio of 10, the CDR of the VH sequences contained only 31% of the replacement mutations with a replacement/silent mutation ratio of 0.69.
Extracellular adenosine nucleotides stimulate protein kinase C activity and human neutrophil activation.
Authors: Balazovich K J , Boxer L A
Published: December 2022

We studied the effect of adenosine nucleotides on several aspects of the functional activation of human peripheral blood polymorphonuclear leukocytes (PMN). Radiolabeled ATP bound to PMN in a manner suggesting the existence of specific binding sites because: 1) binding was reversed (92 +/- 6%) by 100-fold excess concentrations of unlabeled ATP but minimally by either ADP (43 +/- 12%) or GTP (37 +/- 8%); and 2) binding saturation was achieved (i.e., specific binding did not increase) above 250 microM ATP. Binding studies revealed that significant ATP hydrolysis occurred, even at low temperatures and in the presence of phosphatase inhibitors. Adenosine nucleotides activated signal transduction mechanisms in PMN because: 1) 1 to 100 microM ATP and 5'-adenylylimidodiphosphate (AMP-PNP) stimulated increased production of 1,2-diacylglycerols; 2) ATP (0.5 to 500 microM) and ADP (0.1 to 10 mM) induced increased insoluble protein kinase (PKC) activity in a dose-dependent manner when used at concentrations greater than 50 microM; 3) ATP (greater than or equal to 50 microM) induced a shift in the solubility of phorbol receptors from mostly soluble (89% in untreated cells) to mostly insoluble (68%), whereas ADP, GTP, and GDP were effective at higher concentrations; and 4) greater than or equal to 50 microM ATP stimulated increased phosphorylation of endogenous PMN proteins. AMP-PNP induced PKC activity and phosphoprotein changes that were qualitatively similar to those observed when PMN were treated with ATP, suggesting that extracellular ATP hydrolysis is not required for signal transduction to activate PKC. Functionally, ATP stimulated the secretion of specific (but not azurophil) granules because vitamin B12-binding protein and low levels of lysozyme, but not beta-glucuronidase, were released; qualitatively similar results were obtained by using AMP-PNP. These results suggest that certain adenosine nucleotides employed at physiologically relevant concentrations stimulate increased 1,2-diacylglycerol production, PKC activity, granule secretion, and endogenous phosphoprotein formation in a manner that is independent of extracellular ATP hydrolysis.
Anti-idiotype-induced, lipopolysaccharide-specific antibody response to Pseudomonas aeruginosa.
Authors: Schreiber J R , Patawaran M , Tosi M , Lennon J , Pier G B
Published: December 2022

Antibody directed to the O-specific polysaccharide (Ps) side chain of Pseudomonas aeruginosa LPS provides immunotype-specific protection against infection by virtue of enhancing opsonophagocytosis. We have developed a syngeneic anti-idiotypic antibody (mAb2) directed to a functionally active monoclonal immunotype 1 Ps-antibody (mAb1). The mAb2 performed as a molecular mimic of Ps as evidenced by 1) blocking of mAb1/mAb2 interaction by Ps, 2) blocking of mAb1/Ps binding by mAb2, 3) cross-species binding of mAb2 to human Ps antibodies from individuals immunized with the same immunotype 1 Ps, and 4) induction of anti-LPS antibody by immunization with mAb2 in syngeneic mice. Our studies thus show that an anti-idiotypic antibody may functionally mimic the O-polysaccharide of P. aeruginosa LPS, and bind to cross-reactive Id present in human Ps antibodies. We have further shown that this anti-idiotypic antibody induces anti-LPS antibody when used as an Ag in syngeneic mice, suggesting that this approach may eventually be used to successfully immunize humans.
Adoptive transfer of CD8+ T cells from immune animals does not transfer immunity to blood stage Plasmodium yoelii malaria.
Authors: Vinetz J M , Kumar S , Good M F , Fowlkes B J , Berzofsky J A , Miller L H
Published: December 2022

The malaria parasite, Plasmodium yoelii 17X, causes a self-limited, nonlethal infection characterized, in the blood stage, by preferential invasion of reticulocytes. Previous studies have suggested that immunity to the blood stage infection may be related to enhanced levels of class I MHC Ag on the parasitized reticulocyte surface and can be adoptively transferred to immunodeficient mice by immune CD8+ T cells in the absence of CD4+ T cells. To further examine the mechanisms of CD8+ T cell involvement in immunity to blood stage P. yoelii infection, we performed in vivo CD8 depletion and adoptive transfer experiments. Depletion of CD8+ T cells during primary blood stage infection in BALB/c mice did not diminish the ability of the mice to resolve their infections. Spleen cells from immune BALB/c and C57BL/10 mice were transferred to BALB/c-nu/nu and C57BL/10-nu/nu mice, respectively. The recipient mice were CD4 depleted in vivo to kill any transferred CD4+ T cells. The mice failed to control the infection. Populations of CD4-, CD8+ T cells were transferred from immune CBA/CaJ donors to in vivo CD4-depleted CBA/CaJ recipients. The mice were unable to control the infection. Although immune unfractionated spleen cells transferred rapid protection in all three mouse strains and immune CD4+ T cells transferred immunity in the two mouse strains studied, CD8+ T cells by themselves were neither protective nor did they enhance immunity.