bluuebunny/crossref_metadata_2025 huggingface dataset:
A systematic molecular analysis of the T cell-stimulating antigens from Mycobacterium leprae with T cell clones of leprosy patients. Identification of a novel M. leprae HSP 70 fragment by M. leprae-specific T cells.
Authors: Janson A A , Klatser P R , van der Zee R , Cornelisse Y E , de Vries R R , Thole J E , Ottenhoff T H
Published: December 2022

Both protective immunity and immunopathology induced by mycobacteria are dependent on Ag-specific, CD4+ MHC class II-restricted T lymphocytes. The identification of Ag recognized by T cells is fundamental to the understanding of protective and pathologic immunity as well as to the design of effective immunoprophylaxis and immunotherapy strategies. Although some T cell clones are known to respond to recombinant mycobacterial heat shock proteins (hsp) like hsp3 65, the specificity of most T cells has remained unknown. We therefore have undertaken a specificity analysis of 48 well defined Mycobacterium leprae- and/or Mycobacterium tuberculosis-reactive (Th-1-like) T cell clones. Most clones (n = 44) were derived from different leprosy patients, and the remainder from one healthy control. Their HLA restriction molecules were DR2, DR3, DR4, DR5, DR7, DQ, or DP. T cell clones were stimulated with large numbers (n = 20 to 40) of mycobacterial SDS-PAGE-separated fractions bound to nitrocellulose. Each clone recognized a single fraction or peak with a particular Mr range. Some of the clones (n = 7) recognized the fraction that contained the hsp 65 as confirmed with the recombinant Ag. Most clones (n = 41), however, responded to Ag other than the hsp 65. Nine clones responded to a 67- to 80-kDa fraction. Five of them responded also to an ATP-purified, 70-kDa M. leprae protein, but only one of these five (that was HLA-DR2 restricted and cross-reactive with M. tuberculosis) recognized the recombinant C-terminal half (amino acids 278-621) of the M. leprae hsp 70 molecule and also recognized the recombinant M. tuberculosis hsp 70. We therefore have used the 5' part of the M. leprae hsp 70 gene that we have cloned recently. This fragment (that encodes amino acids 6-279) was indeed recognized by the other four M. leprae-specific T cells that were all HLA-DR3 restricted and did not cross-react with the highly homologous (95%) M. tuberculosis hsp 70. These results suggest that this novel fragment is a relevant T cell-stimulating Ag for leprosy patients. A panel of other recombinant Ag, including hsp 18 was tested. The majority of T cell clones appeared to recognize antigenic fractions distinct from hsp. In conclusion, T cells of leprosy patients see a large variety of different Ag including non-hsp, and one newly recognized moiety is the N-terminal M. leprae hsp 70 fragment.(ABSTRACT TRUNCATED AT 400 WORDS)
In vitro stimulation of immune functions by lipids derived from macrophages exposed to bacterial peptidoglycan.
Authors: Wehner N G , Gray G R
Published: December 2022

It has previously been established that the processing of Bacillus subtilis cell walls by the macrophage-like cell line RAW264 leads to the formation of peptidoglycolipids containing glycopeptides of bacterial origin. In view of the immunologic activities associated with lipophilic muramyl peptide derivatives, lipid extracts derived from macrophages exposed to bacterial cell walls were assayed for mitogenicity. The crude lipid extract derived from RAW264 cells exposed to bacterial cell walls gave a strong mitogenic response when delivered as liposomes to murine splenocytes, whereas lipids derived from macrophages not exposed to bacterial cell walls did not give a mitogenic response. Fractionation of the RAW264 lipids into neutral lipids, glycolipids, and phospholipids demonstrated that mitogenicity was associated primarily with the phospholipid fraction and thus correlated with the presence of the aforementioned peptidoglycolipids. Mitogenicity was specific to the lipids derived from macrophages exposed to bacterial cell walls and was not observed in lipids derived from macrophages that had either been nonspecifically activated by exposure to Con A or had been allowed to phagocytize latex beads. The mitogenic response was found to be dependent on the presence of macrophages in the splenocyte population and to, at least in part, involve macrophage activation.
Transcriptional regulation of cytokine genes in nontransformed T cells. Apparent constitutive signals in run-on assays can be caused by repeat sequences.
Authors: Brorson K A , Beverly B , Kang S M , Lenardo M , Schwartz R H
Published: December 2022

Cytokines are immunoregulatory proteins that are secreted by T lymphocytes and other cells upon activation. A controversy exists as to whether the induction of cytokine production is mediated at the transcriptional level by the initiation of RNA synthesis or at the post-transcriptional level by the enhancement of mRNA stability. We show that in a nontransformed T cell clone the induction of all of the cytokines that are analyzed in this report is mediated transcriptionally. We also found that a constitutive signal was seen in resting cells when the probes used in the nuclear run-on assay contained some potential cross-hybridizing sequences, for example a poly (A) tract in a cDNA probe. This observation could explain the constitutive signals seen in other studies that claim cytokine production is regulated by differential mRNA stability.
The CD19 complex of B lymphocytes. Activation of phospholipase C by a protein tyrosine kinase-dependent pathway that can be enhanced by the membrane IgM complex.
Authors: Carter R H , Tuveson D A , Park D J , Rhee S G , Fearon D T
Published: December 2022

We have investigated the mechanism by which the membrane protein complex of the B lymphocyte that contains CD19 and CR2 activates phospholipase C (PLC) to induce a rise in [CA2+]i. The CD19 complex resembled the membrane IgM complex in that three protein tyrosine kinase inhibitors suppressed increases in [Ca2+]i and inositol bisphosphate and inositol triphosphate generation. However, the activation of PLC by the CD19 complex could be distinguished from that by the membrane IgM complex by slower kinetics of generation of inositol phosphates, resistance to inhibition by activators of protein kinase C, and different pattern of tyrosine-phosphorylated cellular substrates. Western blot analysis of lysates from cells stimulated by the CD19 complex demonstrated a single new phosphotyrosine-containing protein of 85 kDa, whereas multiple other phosphotyrosine-containing proteins were present in cells activated by the mIgM complex. In particular, PLC-gamma 1, which is a substrate for the protein tyrosine kinase activated by the mIgM complex, was not tyrosine-phosphorylated in cells stimulated by the CD19 complex. Cross-linking the two complexes together caused a synergistic increase in [CA2+]i which was neither suppressed by activation of protein kinase C nor associated with increased tyrosine-phosphorylation of PLC, characteristic of the CD19 pathway. Therefore, the B cell has two signal transduction complexes, associated with membrane IgM and CD19, that activate PLC by different mechanisms and that can synergistically interact to enhance this function by the CD19 pathway.
Membranes from both Th1 and Th2 T cell clones stimulate B cell proliferation and prepare B cells for lymphokine-induced differentiation to secrete Ig.
Authors: Hodgkin P D , Yamashita L C , Seymour B , Coffman R L , Kehry M R
Published: December 2022

Plasma membranes from the mitogen-activated mouse Th2 cell clone D10.G4.1 have recently been shown to provide the cell contact-dependent signals necessary for the induction of small B cell proliferation. Together with the Th2-derived lymphokines IL-4 and IL-5, these membranes stimulate production of Ig isotypes identical to those produced when B cells were stimulated by intact Th2 cells. In contrast, Th1 clones are poor inducers of Ig production in vitro. This could be solely due to differences in the lymphokines released by Th1 and Th2 cells or to differences in the cell-cell contact signals delivered by activated Th1 and Th2 cells. We report that membranes from three different activated Th1 clones induced strong Ag-independent proliferation of small dense B cells. The level of B cell proliferation was enhanced approximately fourfold by the addition of lymphokine-containing supernatant from Con A-activated Th2 cells and was unaffected by any of the lymphokine-containing supernatants from Con A-activated Th1 clones. As with D10.G4.1 membranes, Th1 membranes alone induced B cell proliferation but not secretion of Ig. However, addition of supernatant from Con A-activated D10.G41 cells, but not any supernatants from Con A-activated Th1 cells, induced Ig secretion of all isotypes. These effects were shown to not simply result from increased B cell numbers after stimulation with Th2 lymphokines. Thus, Th1 cell clones seem to poorly induce antibody responses entirely because of their lymphokine repertoire and not because of differences or deficiencies in the ability of these cells to deliver cell contact-dependent signals to B cells.
High levels of CD45 are coordinately expressed with CD4 and CD8 on avian thymocytes.
Authors: Paramithiotis E , Tkalec L , Ratcliffe M J
Published: December 2022

In this paper we describe the avian homolog of mammalian CD45. We show that this Ag is expressed on all leukocytes but not on erythroid cells or their immediate precursors. Immunoprecipitations demonstrated that B lineage cells from the bursa of Fabricius expressed a higher molecular mass variant (215 kDa) than did T lineage cells from the thymus (190 kDa), and crucially, these high molecular mass molecules had intrinsic phosphotyrosine phosphatase activity characteristic of mammalian CD45. We show that levels of CD45 expression as detected by mAb LT40 in the avian thymus are heterogeneous and further that mAb LT40 can deplete all phosphotyrosine phosphatase activity from thymocyte membrane preparations. Therefore total levels of CD45 are heterogeneous among avian thymocytes. Specifically, 87 to 89% of thymocytes expressed fourfold higher levels of surface CD45 (CD45hi) than the remaining 11 to 13% (CD45lo). The CD45lo population contained exclusively thymocytes with the phenotype CD3-4-8lo, characteristic of the immediate precursors to the CD3-4+8+ thymic population which are CD45hi. The shift from low to high levels of surface CD45 expression therefore occurred at the same stage as the transition from CD4-8lo to CD4+8+ and before the expression of CD3. The protein tyrosine kinase activity associated with CD4 and CD8 (p56lck) and the phosphatase activity of CD45 have been implicated elsewhere in jointly regulating peripheral T cell signal transduction and subsequent cellular responses. The coordinated expression of high levels of CD45 with both CD4 and CD8 in the avian thymus supports the possibility that these molecules may function together in regulating thymocyte growth and/or differentiation.
IL-4 promotes anti-Ig-mediated protein kinase C translocation and reverses phorbol ester-mediated protein kinase C down-regulation in murine B cells.
Authors: Harnett M M , Holman M J , Klaus G G
Published: December 2022

Co-stimulation of B lymphocytes with IL-4 plus nonmitogenic concentrations of anti-Ig antibodies, or protein kinase C (PKC) activators, drives resting B cells into DNA synthesis. Although cross-linking of the sIg receptors provokes the generation of the intracellular second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, the molecular mechanism utilized by IL-4R in murine B cells has not, as yet, been defined. In human B cells IL-4 has been shown to induce a transient rise in IP3 followed by a sustained elevation of cAMP. However, in murine B cells, IL-4 does not induce the release of IP3, Ca2+ mobilization, PKC translocation, or indeed modify signaling via the phosphoinositide pathway induced by ligation of sIg receptors. We now present evidence that, in murine B cells, IL-4 synergizes with nonmitogenic concentrations of anti-Ig to provoke translocation of PKC from the cytosol to membranes. In addition, the lymphokine up-regulates PKC levels and activity and prevents phorbol ester-induced PKC down-regulation in B cells. We therefore propose that (unknown) signals generated via IL-4R potentiate and/or prolong sIg-induced PKC activation. These observations may therefore provide a biochemical basis for explaining how IL-4 and anti-Ig synergize to induce B cell activation.
The cell-mediated response to schistosomal antigens at the clonal level. In vivo functions of cloned murine egg antigen-specific CD4+ T helper type 1 lymphocytes.
Authors: Chikunguwo S M , Kanazawa T , Dayal Y , Stadecker M J
Published: December 2022

It is now well established that the granulomatous inflammation surrounding the eggs of Schistosoma mansoni is mediated by Th lymphocytes. Our laboratory has recently cloned murine CD4+ Th cells specific for schistosomal egg Ag (SEA). In the current study, SEA-specific IL-2-producing Th1 clones were tested for their ability to mediate local delayed-type hypersensitivity (DTH) reactions, as well as granuloma formation in vivo. Marked delayed-onset erythema and induration developed in footpads of normal syngeneic hosts injected with SEA together with SEA-specific Th1 clones. Histologic examination of these lesions revealed typical, predominantly mononuclear, cell infiltrates characteristic of DTH reactions. Conversely, no reactions were observed in allogeneic hosts, in the absence of SEA, or with the use of a control Th1 clone. Moreover, adoptive transfer of cloned SEA-specific Th1 cells to normal syngeneic mice mediated, in 4 days, the formation of vigorous granulomas around schistosomal eggs embolized in the lungs. Such granulomas, which were quantitated by computer-assisted morphometric analysis, were comparable in size to those elicited by lung-embolized eggs in SEA/CFA-immunized mice. In contrast, significantly smaller granulomas were observed in normal recipients of eggs plus a control Th1 clone or of eggs alone. Our data indicate that Ag-specific, MHC-restricted, local DTH reactions, as well as egg granuloma formation in vivo, can be mediated by monoclonal SEA-specific Th1 cells. They suggest that T cell sensitization to only small numbers of SEA determinants may be sufficient to elicit the hepatointestinal granulomatous inflammation associated with schistosomiasis.
Expression of protein kinase C genes in hemopoietic cells is cell-type- and B cell-differentiation stage specific.
Authors: Mischak H , Kolch W , Goodnight J , Davidson W F , Rapp U , Rose-John S , Mushinski J F
Published: December 2022

We have studied the expression of mRNA encoding all known protein kinase C (PKC) isozymes (alpha, beta, gamma, delta, epsilon, zeta, and eta) in murine tumor cell lines that exemplify hemopoietic cells arrested at different stages of development as well as in normal hemopoietic cells. We demonstrate that some of the isozymes, PKC-alpha, -beta, and -eta, are differentially expressed in different lineages. PKC-alpha and -beta generally are not detectable in myeloid cell lines, where PKC-delta is the predominant isoform. Both PKC-alpha and -beta are abundant in most T and B lymphocytic lines, but steady state levels of PKC-beta mRNA are lowest in plasma cell tumors, which exemplify the terminally differentiated B lymphocyte. In contrast, the levels of PKC-alpha mRNA remain high in plasma cell tumors, and a novel, 2.5-kb PKC-alpha mRNA gains prominence. PKC-eta mRNA is the major PKC isoform expressed in T lymphocytes, but it also is highly abundant in some myeloid lines. PKC-delta is expressed at high levels in all the lines we studied, whereas PKC-epsilon and -zeta are found in most cells but only at rather low levels. Analysis of myeloid clones derived from bipotential B lineage progenitor cell lines suggests that the B cell phenotype is associated with the expression of PKC-alpha. The close correlation of protein levels with mRNA levels indicates that PKC expression in hemopoietic cells is mainly regulated at the level of mRNA. The lineage- and differentiation stage-specific patterns of PKC-isozyme expression presented here suggest the involvement of specific PKC isozymes in differentiation as well as lineage determination of hemopoietic cells.
An immune response defect due to low levels of class II cell surface expression. Analysis of antigen presentation and positive selection.
Authors: Gilfillan S , Aiso S , Smilek D , Woodland D L , Palmer E , McDevitt H O
Published: December 2022

The effects of quantitative differences in class II cell surface expression have been difficult to address in intact animals. This study uses several lines of H-2s/s mice carrying an A beta k transgene that differ significantly in terms of class II cell surface expression. Due to inefficient chain pairing, mice carrying 60 to 65 copies of this transgene express only low levels of A alpha s/A beta k on the cell surface, and cell surface expression of the endogenous A alpha s/A beta s complex (and total Ia) is severely reduced (to 7-15% control levels). The significant decrease in class II cell surface expression in the thymic cortex of these mice did not affect the frequency of peripheral T cells expressing at least 10 distinct TCR V beta chains. However, T cell proliferative responses to the A alpha s/A beta s-restricted peptide MBP 89-101 were abrogated in high copy number A beta k mice. Experiments using bone marrow chimeras demonstrated that both inefficient Ag presentation and failure to positively select appropriate T cells contributed to this lack of response. Inefficient Ag presentation was clearly the dominant defect, and the density of class II cell surface expression required for positive selection appeared to be quite low.
Transmembrane signals generated through MHC class II, CD19, CD20, CD39, and CD40 antigens induce LFA-1-dependent and independent adhesion in human B cells through a tyrosine kinase-dependent pathway.
Authors: Kansas G S , Tedder T F
Published: December 2022

Transmembrane signals generated following mAb binding to CD19, CD20, CD39, CD40, CD43, Leu-13 Ag, and HLA-D region gene products induced rapid and strong homotypic adhesion in a panel of human B cell lines. Lower levels of adhesion were also observed after engagement of CD21, CD22, and CD23. Adhesion induced by mAb binding to these Ag was identical with respect to the kinetics of adhesion and the morphology of the resulting cellular aggregates, and was distinct from PMA-induced adhesion in both of these properties. Adhesion was not observed in response to mAb binding to MHC class I, CD24, CD38, CD44, CD45RA, or CD72. In contrast to B cell lines, homotypic adhesion was not induced in two pre-B cell lines, in spite of their high level expression of CD19 and HLA-D. Adhesion induced by suboptimal stimulation through these surface Ag or by PMA was mediated primarily through LFA-1 and ICAM-1. However, optimal stimulation through CD19, CD20, CD39, CD40, and HLA-D induced strong homotypic adhesion that was not blocked by anti-LFA-1 mAb. This alternate pathway of adhesion was also observed in LFA-1-deficient cell lines and in the presence of EDTA, suggesting that adhesion was not mediated by integrins. Adhesion in response to engagement of cell-surface Ag was unaffected by H7 or genestein, but was significantly inhibited by staurosporine, and was completely ablated by sphingosine and herbimycin. These studies indicate that engagement of multiple B cell-surface molecules initiates a signal transduction cascade that involves tyrosine kinases but not protein kinase C, and which leads to homotypic adhesion. Furthermore, adhesion was mediated by at least two distinct cell-surface adhesion receptors: LFA-1/ICAM-1 and a heretofore unknown adhesion receptor.
Rapid signaling to B cells by antigen-specific T cells requires CD18/CD54 interaction.
Authors: Lane P J , McConnell F M , Clark E A , Mellins E
Published: December 2022

This study reports early B and T cell signaling events during cognate interactions between a human B cell line pulsed with peptide and an Ag-specific T cell clone. As has been previously reported, peptide in the context of the appropriate class II molecule stimulated a rise in intracellular calcium [Ca2+]i in the Ag-specific T cell clone. The activation of the T cell clone was associated with a reciprocal rise in [Ca2+]i in the B cells. Engagement of receptors on the B cell surface by the T cell also was associated with inositol phospholipid turnover comparable to that elicited by stimulation through sIg. Early signaling events in B cells can therefore be stimulated in cognate interactions with Ag-specific T cells, without the direct engagement of Ig receptors. A class II deficient B lymphoblastoid mutant, 6.1.6, which was incapable of presenting peptide to the T cell clone, could be stimulated to produce a rise in [Ca2+]i if the T cell clone was activated by monoclonal antibodies to CD3. Therefore, the interaction of class II molecules on the B cell with the TCR and/or the CD4 accessory molecule was not essential for T-dependent B cell activation. However, T-dependent signalling of B cells was profoundly inhibited by mAb to CD18 (beta-chain of LFA-1) on the T cell or CD54 (ICAM-1) on the B cell, demonstrating the importance of this pair of adhesion molecules in early T-B cell interactions.
Differential effects of neutrophil-activating peptide 1/IL-8 and its homologues on leukocyte adhesion and phagocytosis.
Authors: Detmers P A , Powell D E , Walz A , Clark-Lewis I , Baggiolini M , Cohn Z A
Published: December 2022

Several structural homologues of the chemotactic peptide neutrophil-activating peptide 1/IL-8 (NAP-1/IL-8) were tested for their ability to influence the expression and function of adhesion-promoting receptors on human polymorphonuclear leukocytes (PMN). NAP-2, melanoma growth stimulatory activity, and two forms of NAP-1/IL-8 (ser-NAP-1/IL-8 and ala-NAP-1/IL-8, consisting of 72 and 77 amino acids, respectively), each caused an increase in the expression of CD11b/CD18 (CR3) and CR1, which was accompanied by a decrease in the expression of leukocyte adhesion molecule-1 (LAM-1, LECAM-1). The binding activity of CD11b/CD18 was also enhanced 3- to 10-fold by these peptides, but enhanced function was transient: binding of erythrocytes coated with C3bi reached a maximum by 30 min and declined thereafter. Ser-NAP-1/IL-8, ala-NAP-1/IL-8, NAP-2, and melanoma growth stimulatory activity also caused a two- to threefold enhancement of the phagocytosis of IgG-coated erythrocytes (EIgG) by PMN without causing a large increase in the expression of Fc gamma receptors. Enhanced phagocytosis of EIgG appeared to be mediated through CD11b/CD18, because F(ab')2 fragments of an antibody directed against CD18 inhibited NAP-1/IL-8-stimulated ingestion of EIgG. The four active peptides caused a rapid, transient increase in the amount of F-actin within PMN, indicating that they are capable of influencing the structure of the microfilamentous cytoskeleton, which participates in phagocytosis. Two other NAP-1/IL-8-related peptides, platelet factor 4 and connective tissue-activating peptide III, were without effect on expression of CD11b/CD18, CR1, and LAM-1, binding activity of CD11b/CD18, or Fc-mediated phagocytosis, and increased actin polymerization only slightly. Our observations indicate that several members of the NAP-1/IL-8 family of peptides were capable of promoting integrin-mediated adhesion and Fc-mediated phagocytosis, processes important in the recruitment of PMN to sites of inflammation and antimicrobial responses of PMN.
Production of IL-1 receptor antagonist by human in vitro-derived macrophages. Effects of lipopolysaccharide and granulocyte-macrophage colony-stimulating factor.
Authors: Janson R W , Hance K R , Arend W P
Published: December 2022

The objective of these experiments was to evaluate the production of IL-1ra, a specific receptor antagonist of IL-1, by human in vitro-derived macrophages, a model for differentiated macrophages. IL-1ra protein levels in supernatants and lysates of cultured cells were determined by a specific ELISA. Relative steady-state IL-1ra mRNA levels were measured using a specific cDNA probe. Human monocytes were differentiated by 6 days culture in either medium or granulocyte-macrophage colony-stimulating factor (GM-CSF), after which the effects of subsequent LPS and/or GM-CSF on the production of IL-1ra were evaluated. In vitro-derived macrophages cultured in medium for 6 days constitutively produced IL-1ra protein during the 24-h period of the 7th day in culture. The constitutive production of IL-1ra by medium-aged cells correlated with low steady-state IL-1ra mRNA levels determined over this same time period. In contrast, cells cultured for 6 days in GM-CSF synthesized significantly increased levels of IL-1ra protein during the 7th day in culture but the secreted levels remained unchanged. Cells differentiated in GM-CSF displayed enhanced steady-state levels of IL-1ra mRNA in comparison with cells aged in medium. Stimulation of in vitro-derived macrophages aged for 6 days in medium or in GM-CSF, with LPS or adherent IgG, did not result in increased levels of IL-1ra protein production in comparison with non-LPS stimulated cells. The IL-1ra protein detected in the supernatants of cells differentiated in GM-CSF was biologically active in the IL-1-augmented murine thymocyte proliferation assay. By Western blot analysis, the IL-1ra protein in the in vitro-derived macrophage supernatants was predominantly the 22- to 24-kDa glycosylated species, whereas the lysates contained additional lower molecular weight forms. These results suggest that as monocytes differentiate in vitro into macrophages, they constitutively produce IL-1ra protein and that this production is enhanced by the continuous presence of GM-CSF.
The relationship between polymerization of complement component C9 and membrane channel formation.
Authors: DiScipio R G
Published: December 2022

C9 was studied with the objective to clarify the relationship between the process of C9 polymerization and membrane channel formation. Conditions that favor C9 polymerization include low ionic strength and calcium ion in the buffer. Moreover, polymerization is dependent on the concentration of C9. Calcium ion evokes about a threefold increase in the affinity constant for C9 self-association, and at 0 degrees C it imparts reversible amphiphilic properties in the molecule. These were discerned by measuring increases in the degree of reversible nonspecific binding of C9 to hydrophobic (tyramine-zymosan) and hydrophilic (arginyl-glutamyl-zymosan) supports as well as to erythrocytes. At 0 degrees C the hydrophilic-to-amphiphilic alteration of C9 is reversible, but upon incubation at 37 degrees C this transition is rendered permanent with the formation of poly(C9). A functional relationship between C9 polymerization and cytolysis was demonstrated by showing that polymerizing C9 can lyse reduced and alkylated erythrocytes. By studying comparative radiolabeling of tyrosine side chains within thrombin-nicked C9 and its polymerized form, it was demonstrated that upon polymerization the membrane-binding site of C9 becomes exposed. It is concluded that the process of circular polymerization of C9 causes a hydrophilic-to-amphiphilic transition that is required for membrane perforation and channel formation.
IFN-alpha induces autocrine production of IL-6 in myeloma cell lines.
Authors: Jourdan M , Zhang X G , Portier M , Boiron J M , Bataille R , Klein B
Published: December 2022

IL-6 is a major tumor growth factor in human multiple myeloma. Myeloma cell lines, which have the same phenotypic characteristics and Ig gene rearrangements as the original fresh myeloma cells and whose growth is strictly dependent on exogenous IL-6 similar to fresh myeloma cells, have been reproducibly established. We show here that IFN-alpha stimulated the growth of five of six of these human myeloma cell lines by inducing an autocrine production of IL-6 in myeloma cells. Indeed, IFN-alpha induced IL-6 mRNA accumulation and IL-6 production in myeloma cells and the IFN-alpha-induced growth of these cells was inhibited by anti-IL-6 mAb. Moreover, IFN-alpha made possible the rapid emergence of autonomously growing myeloma cell sublines, which produced IL-6 as an autocrine growth factor. As IFN-alpha has a potential therapeutical interest for multiple myeloma, the present study opens up new directions for studying its effects on the myeloma clone in vivo.
Physiologic activation of T cells via the T cell receptor induces NF-kappa B.
Authors: Jamieson C , McCaffrey P G , Rao A , Sen R
Published: December 2022

The transcription factor NF-kappa B has been implicated in the mitogen-induced expression of several genes that are critical for the immunologic function of T cells such as those encoding IL-2 and the IL-2R alpha chain (IL-2R alpha). We show here that NF-kappa B is induced in T cells activated by Ag, anti-CD3 antibody, or allogeneic stimulation. The induction of NF-kappa B via the TCR was dependent on protein kinase C. IL-2, which also activates IL-2R alpha expression and proliferation in T cells, was not able to induce NF-kappa B. TCR-mediated induction of NF-kappa B suggests a central role for this factor in activated T cells and also provides a mechanism for activation of latent HIV provirus during the normal immune response.
Cerebral vascular endothelial cells are effective targets for in vitro lysis by encephalitogenic T lymphocytes.
Authors: McCarron R M , Racke M , Spatz M , McFarlin D E
Published: December 2022

Lysis of cerebral vascular endothelial cells (EC) by CD4-positive, myelin basic protein-specific encephalitogenic T cell lines was investigated. Unstimulated EC were not lysed, but culture in the presence of murine rIFN-gamma resulted in the expression of class II MHC (Ia) molecules and the concomitant ability to function as effective target cells for lysis. The possible requirement for Ia molecules was further demonstrated by antibody-blocking experiments. Lysis of EC targets also required the presence of specific Ag (myelin basic protein); PPD-specific T cell lines also lysed the PPD-pulsed EC. In all cases, lysis was directly proportional to E:T ratios. In addition, continuous passage of T cell lines resulted in the concomitant loss of encephalitogenicity and ability to affect EC lysis, indicating a possible relationship between these two factors. These results demonstrate that CD4+ T cells interact with cerebral vascular EC. It is suggested that such interactions may be important in the pathogenesis of diseases involving migrations of these cells across the blood-brain barrier.
Induction of intracellular Ca2+ mobilization and cytotoxicity by hybrid mouse monoclonal antibodies. Fc gamma RII regulation of Fc gamma RI-triggered functions or signaling?
Authors: Koolwijk P , Van de Winkel J G , Pfefferkorn L C , Jacobs C W , Otten I , Spierenburg G T , Bast B J
Published: December 2022

We studied the interaction of bispecific mouse mAb with human IgG Fc receptors, and assessed their ability to activate the monocytic cell line U937. Binding of monomeric hybrid anti-HuIgA1/HRP mAb to the high-affinity IgG receptor, Fc gamma RI, on U937 cells was only observed when mAb with one or more mIgG2a H chains (hybrid mIgG1-2a, mIgG2a-2b, and mIgG2a-2a) were used. These Fc gamma RI-bound hybrid mAb were capable of enhancing the internal free cytosolic Ca2+ concentration ([Ca2+]i) in U937 cells only when bound mIgG were cross-linked using F(ab')2 fragments of goat anti-mIg antibody. A hybrid mIgG1-2a mAb were cross-linked using goat anti-mIgG1 antibody, showing that the hybrid mAb themselves mediate the induction of Ca2+ increase. Remarkably, anti-Fc gamma RII mAb IV.3 was able to inhibit the Ca2+ increase induced via mIgG2a-1 or mIgG1-2a hybrid mAb completely, despite the fact that we could not detect any effect of IV.3 on binding of monomeric hybrid mIgG1-2a or mIgG2a-1 mAb to U937. The hybrid mAb were also able to induce lysis of HuIgA1-coated E using U937 effector cells. This lysis was completely inhibited by preincubation of U937 cells with mIgG2a mAb TB-3, which blocks Fc gamma RI via its Fc-part ("Kurlander phenomenon"). In contrast, Fc gamma RII-blocking mAb IV.3 and CIKM5 caused a significant enhancement of the antibody-dependent cellular cytotoxicity (ADCC) activity mediated by hybrid mIgG1-2a and mIgG2a-2b mAb. This enhancement did not occur when the parental anti-HuIgA1/2a or the hybrid anti-HuIgA1/HRP/2a-2a mAb were evaluated for ADCC activity. These findings suggest that hybrid mAb not only can bind to Fc gamma RI, but can mediate functional activation of myeloid cells. Given the effect of mAb IV.3 on [Ca2+]i changes and ADCC triggered through IgG1-2a mAb, we suggest that Fc gamma RII may have a role in the regulation of Fc gamma RI-triggered functions or signaling.
Eosinophil activation on biologic surfaces. Production of O2- in response to physiologic soluble stimuli is differentially modulated by extracellular matrix components and endothelial cells.
Authors: Dri P , Cramer R , Spessotto P , Romano M , Patriarca P
Published: December 2022

Production of O2- in response to FMLP, TNF, IFN-gamma, platelet activating factor, LPS, substance P, and PMA by human eosinophils in suspension and in contact with polystyrene ELISA plastic (PL) or biologic surfaces was studied. Monolayers of human endothelial cells (HEC) or PL coated with FCS, fibronectin, laminin, collagen types I and IV, fibrinogen, or fibrin were used as biologic surfaces. Only PMA and FMLP stimulated O2- generation by eosinophils in suspension. Eosinophils residing on HEC monolayers, either untreated or treated with LPS, were unresponsive to all stimuli except PMA. PMA induced O2- generation by eosinophils on all surfaces; FMLP on all surfaces but HEC monolayers; TNF and platelet-activating factor only on PL, fibrinogen, and fibrin; LPS and substance P only on PL. PMA was equally effective on eosinophils on surfaces and in suspension, whereas the effect of FMLP was greater on eosinophils on surfaces than on eosinophils in suspension. IFN-gamma was ineffective on any of the surfaces tested. These results indicate that biologic surfaces may profoundly affect the ability of eosinophils to respond with a respiratory burst to physiologically relevant soluble stimuli, the effect varying according to the nature of both the stimulus and the surface. Since the respiratory burst generates products of oxygen reduction that are toxic to several tissue components, it follows that biologic surfaces may modulate eosinophil-induced tissue injury.
Differential synthesis of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine and platelet-activating factor by human inflammatory cells.
Authors: Triggiani M , Schleimer R P , Warner J A , Chilton F H
Published: December 2022

The synthesis of platelet-activating factor (PAF) and of the 1-acyl analogue of PAF, 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-2-acetyl-GPC) was examined in seven human cell preparations (lung mast cell, basophil, endothelial cell, neutrophil, eosinophil, lung macrophage, platelet) and in human lung fragments. Cells were activated by either an appropriate receptor-mediated stimulus or by ionophore A23187 in the presence of [3H]acetate. All cell types, with the exception of the platelet, responded to stimulation with at least a twofold increase in the formation of labeled 1-radyl-2-acetyl-GPC as compared with control values. A23187 was the more potent stimulus in all cell types examined except the lung mast cell, in which anti-IgE consistently induced the synthesis of more 1-radyl-2-acetyl-GPC. Human lung fragments stimulated by anti-IgE, Ag Amb a I (after passive sensitization), or A23187 also incorporated [3H]acetate into 1-radyl-2-acetyl-GPC. Subclass analysis of 1-radyl-2-acetyl-GPC produced by each cell indicated that the cell types examined can be divided into two groups according to the predominant type of 1-radyl-2-acetyl-GPC produced. Some cell types (mast cell, basophil, endothelial cell) produced predominantly 1-acyl-2-acetyl-GPC, whereas others (neutrophil, eosinophil, lung macrophage) produced almost exclusively PAF. In some cell types, such as the lung mast cell and the basophil, A23187 stimulation increased the synthesis of PAF relative to 1-acyl-2-acetyl-GPC as compared with anti-IgE stimulation. In the lung fragments, [3H]acetate was predominantly incorporated into 1-acyl-2-acetyl-GPC upon IgE-mediated stimulation (anti-IgE, Amb a I) and into PAF upon A23187 stimulation. The differential production of these two phospholipids was confirmed by determining their sensitivity to lipase A1 and phospholipase A2 hydrolysis and by HPLC. These data demonstrate that 1-acyl-2-acetyl-GPC can be synthesized by a variety of human cells involved in the inflammatory reaction. This finding raises fundamental questions about the biologic role of this molecule and the factors regulating its synthesis within inflammatory cells.
A single peptide from the major outer membrane protein of Chlamydia trachomatis elicits T cell help for the production of antibodies to protective determinants.
Authors: Allen J E , Locksley R M , Stephens R S
Published: December 2022

The protective immune response to infection with Chlamydia trachomatis is associated with antibody reactivity to serovar-specific determinants on the major outer membrane protein (MOMP). Because this immunity is T cell dependent, it is essential to define those Th cell determinants that promote natural boosting of the protective antibody response. The gene for MOMP of serovar B was separated into nine overlapping fragments that represent the five C and four V regions. These fragments were expressed as fusion peptides with GST and used to identify the regions of the MOMP that contain T cell determinants recognized in BALB/c mice. We identified peptides that elicit a T cell response to Chlamydia by immunizing mice with the fusion peptides and testing the proliferative response of T cells in vitro to intact organism. For analysis of determinants seen after infection, animals were inoculated with live organism and the T cell proliferative response to each fusion peptide was measured in vitro. In contrast to proliferative analysis in which several regions of the MOMP elicited T cell responses, functional analysis demonstrated that a single fusion peptide, containing V segment three, elicited T cell help in vivo for the production of high titered antisera, specific for protective determinants on the MOMP.
Specific adoptive immunotherapy mediated by tumor-draining lymph node cells sequentially activated with anti-CD3 and IL-2.
Authors: Yoshizawa H , Chang A E , Shu S
Published: December 2022

Lymph nodes (LN) draining progressively growing tumors contain tumor-sensitized but not fully functional preeffector lymphocytes. These cells could acquire therapeutic efficacy and be expanded upon sequential culture with anti-CD3 mAb for 2 days followed by incubation in IL-2 for 3 days. Using the weakly immunogenic MCA 106 and MCA 205 murine sarcomas, we have further defined conditions of this anti-CD3/IL-2 activation with which preeffector cells differentiated into immune effector cells. In vitro activation and expansion of effector cells required sequential but independent stimulation with anti-CD3 and IL-2 because the simultaneous presence of both anti-CD3 and IL-2 at either stage did not enhance the efficacy of activation. Generation of effector cells by this two-stage activation was critically dependent on the optimal concentrations of anti-CD3 (1.0 microgram/ml) and IL-2 (2-10 U/ml). However, these conditions were not optimal for inducing the greatest cellular proliferation. In adoptive immunotherapy experiments, although the transfer of anti-CD3/IL-2-activated cells alone could mediate the regression of established metastases, the concomitant administration of IL-2 enhanced the in vivo activity of these cells. More importantly, tumor regression mediated by the anti-CD3/IL-2-activated cells was found to be immunologically specific. The specificity was determined by the tumor that stimulated the preeffector cell response. In spite of their in vivo antitumor effects, the anti-CD3/IL-2-activated tumor-draining LN cells did not exhibit detectable in vitro cytotoxicity against the tumor target in the 4-h 51Cr-release assay. In mice bearing progressive tumor, draining LN contained most preeffector cells. Some preeffector cells were also detected in the spleen whereas mesenteric LN did not demonstrate any reactivity. In kinetics studies, sensitization of preeffector cells in the draining LN occurred between 4 to 6 days after tumor inoculation. As the tumor progressed, the presence of preeffector cells declined gradually suggesting a tumor-induced suppression. These results define the conditions whereby tumor-draining LN cells could be stimulated, in the absence of tumor Ag, to develop into specific therapeutic effector cells. Our findings also raise the possibility of using similar approaches for isolating immune effector cells from cancer patients for adoptive immunotherapy.
Genomic organization and chromosomal localization of the TAPA-1 gene.
Authors: Andria M L , Hsieh C L , Oren R , Francke U , Levy S
Published: December 2022

TAPA-1 is a 26-kDa integral membrane protein expressed on many human cell types. Antibodies against TAPA-1 induce homotypic aggregation of cells and can inhibit their growth. The murine homologue of TAPA-1 was cloned from both cDNA and genomic DNA libraries. A very high level of homology was found between human and mouse TAPA-1. The 5' untranslated region of the TAPA-1 gene resembles housekeeping gene promoters with respect to G + C content and the presence of potential Sp1 binding sites. The chromosomal localization of human and murine TAPA-1 genes was determined by Southern blot experiments using DNA from somatic cell hybrids. The genes were found to be part of a conserved syntenic group in mouse chromosome 7 and the short arm of human chromosome 11. The organization of the TAPA-1 gene and the projection of the exon boundaries on the proposed protein structure are presented.
Autoantibody-encoding kappa L chain genes frequently rearranged in lambda L chain-expressing chronic lymphocytic leukemia.
Authors: Rassenti L Z , Pratt L F , Chen P P , Carson D A , Kipps T J
Published: December 2022

Patients with kappa L chain expressing chronic lymphocytic leukemia (CLL) frequently have leukemia cells reactive with a murine mAb, designated 17.109. Raised against a monoclonal IgM rheumatoid factor autoantibody, this mAb recognizes a major kappa-L chain-associated cross reactive Id, designated 17.109-CRI. Molecular studies reveal that the 17.109-CRI in CLL is a serologic marker for expression of a conserved kappa L chain V region gene (V Kappa gene) of the V Kappa 3 subgroup, designated Humkv325. We isolated an upstream gene fragment of Humkv325 to examine for Ig gene rearrangements of this and other closely related V Kappa 3 genes by Southern analyses. Consistent with Humkv325 encoding the 17.109-CRI, we find that the genomic DNA from all 17.109-reactive leukemia cell populations have gene rearrangements that are detected using this probe. In addition, we observe V Kappa 3 gene rearrangements frequently in the genomic DNA of lambda L chain-expressing leukemia cells. Of the genomic DNA from 33 lambda-L chain-expressing CLL samples, 8 (24%) had additional nongerm-line bands detected with the Humkv325 probe. Consistent with these bands representing Ig gene rearrangements, the additional band in each but one sample also hybridized with probes specific for the J Kappa region and/or the kappa-deleting element. Using the polymerase chain reaction (PCR), we examined the genomic DNA from all lambda L chain-expressing CLL for V Kappa 3 gene rearrangements to J Kappa and/or Kde. PCR on each DNA sample with V Kappa 3 gene rearrangements detected by Southern analysis generated gene fragments that hybridized specifically with oligonucleotides corresponding to framework or CDR of the Humkv325 gene. Nucleic acid sequence analyses of representative samples confirmed that these DNA contained abortive Humkv325 gene rearrangements. PCR for rearranged V Kappa 3 genes in the DNA of other lambda-L chain-expressing CLL either did not generate any PCR product or produced fragments that failed to hybridize with all Humkv325 oligonucleotide probes. Nucleic acid sequence analyses of the latter demonstrated that these represent abortive V Kappa gene rearrangements involving another conserved V Kappa 3 gene, designated Vg. These studies indicate that Humkv325 and Vg frequently may undergo Ig gene rearrangement independent of their expression. As such, the frequent use of Humkv325 in CLL may be secondary, in part, to an enhanced propensity of this V Kappa 3 gene to undergo genetic rearrangement during B cell ontogeny.
Natural killer cell clones can efficiently process and present protein antigens.
Authors: Roncarolo M G , Bigler M , Haanen J B , Yssel H , Bacchetta R , de Vries J E , Spits H
Published: December 2022

NK cell clones obtained from three different donors were tested for their ability to present soluble proteins to Ag-specific T cell clones. All NK clones were CD2+CD3-CD56+, whereas the expression of CD16 varied from clone to clone. The NK cell clones were able to process and present tetanus toxoid (TT) to TT-specific T cell clones in a class II HLA restricted manner. The capacity of NK cell clones to function as APC was also observed using the house dust mite allergen Der p I and the Der p I-derived peptide Val89-Cys117. As with EBV-transformed B cell line, NK cell clones could present the peptide 3-13 derived from the 65-kDa heat shock protein of Mycobacterium leprae, but they were unable to present the whole M. leprae Ag. Freshly isolated NK cells, IL-2-activated NK cells, and NK cell lines expanded in vitro could also process and present TT. The ability of the different NK populations to act as accessory cells correlated with their levels of class II HLA expression. These data demonstrate that NK cell clones can efficiently function as APC, however they may be restricted in the types of Ag that they can process.
Subpopulations of B cells in germinal centers. III. HJ6, a monoclonal antibody, binds globoside and a subpopulation of germinal center B cells.
Authors: Madassery J V , Gillard B , Marcus D M , Nahm M H
Published: December 2022

To identify surface Ag uniquely expressed on human germinal center B cells, we produced a mouse mAb, HJ6. When tonsillar lymphocytes were examined, HJ6 did not label T cells and labeled only about half of PNA+ B cells that were HK23-. HJ6 did not label mononuclear cells from peripheral blood, splenocytes, and any of 29 cell lines including 23 B cell lines. This binding pattern of HJ6 was very similar to that of a mAb named 5B5. It was shown previously that 5B5 bound a glycolipid named CTH (CD77) and its Ag was expressed on HK23- PNA+ tonsillar lymphocytes and Burkitt's lymphoma cell lines. Despite the similarity, HJ6 differed from 5B5: HJ6 did not stain Burkitt's lymphoma cell lines and stained PNA+ tonsillar lymphocytes in the presence of a large concentration of galactose. When its binding to isolated glycolipids was studied, HJ6 was found to bind globoside and Forssman Ag and not to other glycolipids including CTH. When its binding to neutral glycolipids extracted from tonsillar lymphocytes was studied, HJ6 bound only globoside; Forssman Ag was not detected in tonsillar lymphocytes. Taken together, we conclude that globoside is a B cell Ag expressed on a subpopulation of germinal center B cells.