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G J Nossal

Publications and source records attributed to G J Nossal.

At least 91 records · Page 5Linked to original sources
PubMed1987

Recombinant T cell replacing factor (interleukin 5) acts with antigen to promote the growth and differentiation of single hapten-specific B lymphocytes.

The role of murine recombinant T cell replacing factor (rTRF) (interleukin 5) in the early activation, proliferation, and antibody-forming cell (AFC) clone formation of single fluorescein (FLU)-specific B cells was examined in vitro. FLU-specific B cells were selected by their adherence to FLU-gelatin and then cultured in 10-microliters wells with or without rTRF in the presence or absence of the T-independent antigen FLU-polymerized flagellin (FLU-POL). rTRF acting alone was unable to induce early B cell activation as assessed by significant cell enlargement after 24 hr in culture. When acting in the presence of FLU-POL, however, a greater number of B cells were induced to enlarge than with FLU-POL alone. When FLU-specific B cells were cultured in the presence of FLU-POL, the addition of rTRF markedly increased the frequencies of both proliferating clones and AFC clones above that induced by FLU-POL alone. Furthermore, in the presence of FLU-POL, the activity of rTRF was comparable to that seen with the mixture of B cell growth and differentiation factors contained within the supernatant from concanavalin A-stimulated EL4 cells. However, rTRF exerted little activity when acting alone in contrast to the medium conditioned by concanavalin A-stimulated EL4 cells which showed some activity in the absence of FLU-POL. rTRF acting with FLU-POL also promoted AFC clone development among single B cells stimulated in the presence of 3T3 fibroblast filler cells. Thus rTRF can be added to the list of B cell active factors (including recombinant murine interleukin 1 and recombinant human interleukin 2) that act in the concomitant presence of antigen to induce both growth and differentiation among single hapten-specific murine splenic B cells. This stands in contrast to the activity seen with interleukin 4 (formerly termed B cell stimulatory factor 1) which acts to promote early activation and proliferation but not IgM secretion.

Animals↗
PubMed1987

Effects of antigens and lymphokines on early activation of single hapten-specific B lymphocytes.

An assay was developed to monitor early activation of single fluorescein-specific B cells obtained from the spleens of nonimmunized adult mice by prefractionation on hapten gelatin. Early activation was assessed as a significant increase in the diameter of individual B cells after 24 hr in vitro. Significant enlargement of the single B cells was induced within 24 hr by either T-independent antigens acting alone or a crude source of B cell growth and differentiation factors (EL-BGDF-pik) acting alone. In contrast, T-dependent antigens acting alone were ineffective. When selected T-independent antigens and EL-BGDF-pik acted together, a greater number of B cells were induced to enlarge. B cell stimulatory factor 1 (BSF 1) behaved in a similar manner as EL-BGDF-pik, inducing early B cell enlargement both in the absence and more so in the presence of antigen. Both EL-BGDF-pik and BSF 1 enhanced the survival of single hapten-specific B cells during the 24-hr period. Interleukin 1 was unable to cause B cell enlargement when acting alone, although it was able to augment B cell enlargement induced by antigen. Interleukin 2 did not induce cell enlargement in either the presence or absence of antigen. Activation was demonstrated among cells of all sizes, regardless of the stimulus, although a greater response was demonstrated amongst the larger cell population. The addition of 3T3 filler cells enhanced early B cell activation and cell survival during the 24-hr period. The 24-hr B cell enlargement assay as applied to isolated single cells provides an unequivocal approach to the analysis of early B cell activation, adding a further parameter for the dissection of the precise roles of antigen and the various factors in the B cell differentiation pathway.

Animals↗
PubMed1987

How is tolerance generated?

There are three major, overlapping theories that account for immunological tolerance. The first is that the repertoires of T and B lymphocytes are somehow purged, actually or functionally, of potentially self-reactive immunocytes. There are three subvariants of this theory, namely clonal deletion, clonal abortion and clonal anergy. Clonal anergy, an antigen-driven down-regulation of immunocyte responsiveness, is preferred on experimental grounds. The second theory is that self-reactive lymphocytes exist but are constantly held in check by suppressor cells. The suppressor cells have not been shown to possess the capacity to discriminate between self and not self. The third theory is that self antigens, through reasons of accessibility and processing, never enter the afferent limb of immune induction effectively. The contributions which each postulated mechanism make are different and all three may enter the picture in some tolerance models. Repertoire purging, if it exists, must be incomplete, because self-reactive B and T cells can readily be detected and stimulated in vitro. For repertoire purging to survive as a concept it must be redefined in terms of the type of antigen to which it is supposed to apply (e.g. ubiquitous cell-surface antigens) and the affinity cut-off point below which it is not reasonable to expect purging. Some of the technical issues impeding speedy experimental solutions centre on the permissive character of the antibody-mediated or cell-mediated lysis assays in current use.

B-Lymphocytes↗
PubMed1987

T-cell-replacing factor (interleukin 5) induces expression of interleukin 2 receptors on murine splenic B cells.

Small, resting B lymphocytes express few, if any, interleukin 2 (IL-2) receptors, but activated B cells may express such receptors. This paper examines the requirements for receptor expression. Normal murine splenocyte populations were enriched for B cells and cultured at relatively low density. IL-2 receptor expression was studied by measuring the binding of the anti-IL-2 receptor monoclonal antibody PC61. Lymphoblasts arising through stimulation by Escherichia coli lipopolysaccharide failed to express IL-2 receptors. B cells cultured with conditioned medium from concanavalin A-stimulated EL4 thymoma cells, with or without LPS, displayed IL-2 receptors. This bioactivity of EL4 conditioned medium could not be replaced by any concentration of B-cell-stimulatory factor 1 (IL-4), IL-1, IL-2, or IL-3 tested. However, the recently cloned lymphokine T-cell-replacing factor (IL-5) was a potent inducer of IL-2 receptor expression, as was the probably identical material known as eosinophil differentiation factor. The receptors so induced appeared to be functional, as receptor-expressing (but not control) lymphoblasts, responded to IL-2 by proliferation, indicative of high-affinity-receptor expression.

Animals↗
PubMed1987

Single cell studies on the role of B-cell stimulatory factor 1 in B-cell activation.

The role of the T-cell-derived lymphokine B-cell stimulatory factor 1 (BSF-1) in the early activation, proliferation, and antibody-forming cell (AFC) clone formation of single fluorescein-specific B lymphocytes isolated from normal mouse spleens by hapten-gelatin adherence has been studied in vitro. BSF-1 acting alone induced early B-cell activation, as assessed by a significant increase in cell diameter of single B cells cultured for 24 hr. A small but significant number of these B cells formed proliferating clones, some of which secreted antibody. When acting with the specific antigen fluorescein-polymerized flagellin, BSF-1 augmented early cell enlargement and markedly enhanced proliferation, but it did not increase the frequency of AFC clones stimulated by fluorescein-polymerized flagellin alone. The further addition of recombinant murine interleukin 1 (IL-1) marginally enhanced proliferation caused by antigen plus BSF-1. No synergy was observed between BSF-1 and IL-1 for antibody formation. In the presence of fibroblast filler cells, BSF-1 substantially inhibited AFC clone development achieved by antigen plus IL-1. BSF-1 was also found to be inhibitory to AFC clone development stimulated by specific antigen acting with either recombinant human interleukin 2 (IL-2) or with IL-2 plus IL-1, both in the presence or absence of filler cells. The results suggest that BSF-1 plays a complex role in the regulation of the B-cell activation pathway by enhancing early activation and antigen-specific proliferation as well as inhibiting the effects of other B-cell factors on antibody formation. BSF-1 is the only cytokine so far tested in the single B-cell system that acts with antigen to promote proliferation without concomitant antibody production.

Animals↗
PubMed1987

Clonal analysis of the anti-DNA repertoire of murine B lymphocytes.

The present studies characterize at the clonal level the repertoire of lipopolysaccharide-responsive murine B lymphocytes committed to the production of antibodies reactive with denatured DNA. This repertoire is vast in normal mice as 1-5% of total mitogen-induced antibody-forming cell clones secreted denatured DNA-reactive antibodies when the splenocyte donors were CBA (Ighj), BALB/c (Igha), C57BL/6 (Ighb), CBA nu/nu, and C57BL/6 nu/nu athymic mice. The autoimmune NZB (Ighe) strain did not display elevated proportions of anti-denatured DNA antibody-forming cell precursors. Cross-reactions shown by CBA anti-denatured DNA antibodies suggest that many antibodies might derive significant binding energy from interaction with the bases or similar hydrophobic moieties. Cross-reactions with other tested polynucleotides were frequent, but cross-reactions with phospholipids and phosphocholine were undetectable. Most anti-DNA antibodies bound preferentially or exclusively to single-stranded denatured DNA as compared to double-stranded native DNA. The frequency of anti-denatured DNA antibody-forming cell precursors among CBA peritoneal cells was not elevated. Fluorescence-activated cell sorter-selected Ly-1-positive NZB splenic B cells were not enriched, and Ly-1 negative B cells were not depleted of anti-DNA antibody-forming cell precursors. These results show that antibody-forming cell precursors specific for denatured DNA are not restricted to the Ly-1 positive B-cell subset.

Animals↗
PubMed1987

T-independent activation of single B cells: an orderly analysis of overlapping stages in the activation pathway.

This review has three chief purposes. It describes a microculture system in which single, hapten-specific B lymphocytes can be microscopically observed, cultured and assayed for antibody production in isolation and thus are the unequivocal target of ligands present in the culture fluid. It defines the respective roles of antigens and cytokines acting singly or in combination in the four discernible phases of the immunoproliferative cascade, namely activation, clonal expansion, IgM antibody secretion and isotype switching. It then argues that this precise stepwise analysis can yield useful information concerning important immunological situations, such as experimentally induced immunological tolerance or the effects of constitutive expression of the c-myc oncogene. Evidence is presented that initial activation of the resting B cell in "T-independent" triggering can be achieved either by attachment of a molecule that has B-cell stimulatory properties, such as FLU-LPS or FLU-polymerized flagellin (FLU-POL) or by the lymphokine interleukin 4 (IL-4). IL-4 + FLU-POL is somewhat more effective than either agent alone. IL-4 alone or, better, FLU-POL + IL-4 can stimulate clonal proliferation of the B cell, but FLU-POL alone does not achieve this. Moreover, IL-4 or FLU-POL + IL-4 lead to very little antibody formation. None of IL-1, IL-2 or IL-5 acting alone causes either activation or proliferation. IgM antibody formation is stimulated most strongly by FLU-POL + IL-5, somewhat less strongly by FLU-POL + IL-1 + IL-2 and rather weakly with antigen plus only one of the latter cytokines. The cloning efficiency in the single cell system, and the median clone size can be markedly enhanced by the addition of small numbers of fibroblast or other filler cells to the cultures. While filler cell-free clones do not progress to the stage of isotype switching, filler cell-supported ones can do so in up to 30% of cases. The only cloned lymphokine which has so far been found to promote such switching is IL-4, and the fact that it is at least as powerful as a T-cell supernatant may mean that it is the only agent active in this particular system. However, the detailed pattern of secreted isotypes is different from that seen when MHC-restricted, carrier-specific T cells act on hapten-specific B cells. Hapten-specific B cells from animals rendered neonatally tolerant to FLU-HGG exhibit anergy in the single cell system.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibody-Producing Cells↗
PubMed1987

Bone marrow pre-B cells and the clonal anergy theory of immunologic tolerance.

This review begins with a summary of a decade's research from the author's own laboratory which documents the fact that B lymphocytes can receive and store negative, down-regulatory signals from an encounter with antigen, and that the sensitivity to such negative signalling depends critically on maturational status, the most immature B cells being the most susceptible. The review then examines the relationship between these experimentally-induced models of immunologic tolerance, with the pre-B to B cell transition as the critical stage for examination, and the real-life phenomenon of self-tolerance. It makes the point that no repertoire-purging mechanism to ensure self-tolerance can afford to be too effective, for fear of purging too many useful cells, given the number and variability of self-antigens. The review then examines certain dilemmas posed by recent findings in cellular and molecular immunology. These include: 1) the preferential use of particular VH gene families by B cells at different stages of the differentiation process; 2) the apparent frequency of B lymphocytes with the potential for antiself-reactivity in the B cell repertoire; and 3) the existence of a new type of B cell, the Ly-1-positive B cell, with peculiar characteristics. These findings are considered within the particular contexts of pre-B-to-B cell transition and tolerance induction through clonal anergy mechanisms.

Animals↗
PubMed1987

Immunoregulation: the key to transplantation and autoimmunity.

Immune responses depend on a unique set of recognition structures, namely, antibody molecules embedded in the B cell membrane for antibody formation and alpha-beta chain heterodimers of the T cell receptor for cellular immune receptors. These structures are coded for by genes, which undergo rearrangements during the differentiation of B- and T-lymphocytes known as somatic translocations. The end result of this process is the creation of separate repertoires of B and T cells, each single cell displaying a unique receptor. Antigen acts by selecting preexisting antigen-reactive cells for division and further differentiation. Accessory cells such as macrophages are essential for the initiation of most immune responses. Different subsets of T-lymphocytes play a vital regulatory role, not only in controlling cell-mediated immunity as such, but also in guiding B cell function. Understanding the roles of accessory cells, regulatory T cells, and the molecules on their surface is essential for understanding immunoregulation. At present, immunosuppression in organ transplantation and therapy of autoimmune diseases are largely nonspecific and empirical. As the rules of the interactive immunoproliferative cascade are progressively unraveled, more targeted immune manipulation will become possible, and some future avenues of this sort are described.

Animals↗
PubMed1986

The Florey lecture, 1986. The regulatory biology of antibody formation.

The regulatory biology of antibody formation entered a new phase of study with the development of selective theories of immunity. The discovery of the 'one cell - one antibody' dogma and the demonstration that only a small minority of B cells possessed receptors specific for a given antigen were consistent with Burnet's clonal selection hypothesis, which was later formally proven by preparing antigen-specific lymphocytes and inducing clonal activation in vitro. Clonal analysis has aided precise study of immunoregulation for both B and T lymphocytes. Clonal activation of B cells in the absence of T cells is now possible with high cloning efficiency. It requires the combined action of certain antigens and growth factors, collectively termed B-cell stimulatory factors (BSFS). Single cell analysis has shown that most BSFS so far tested, in contrast to most claims in the literature, possess the capacity (in synergy with antigen) to: stimulate B cells out of the G0 phase into active cell cycle; promote sequential mitotic divisions; and induce differentiation to active secretory status. This is clearly true for IL-1, IL-2, and BSF-p2. These multiple actions resemble those of the colony-stimulating factors in haemopoiesis. Regulation of antibody production by T lymphocytes can also be profitably analysed in clonal systems. The immunoregulatory problem of tolerance can also be analysed by means of clonal techniques. Studies are summarized which indicate that T-cell-mediated suppression and functional silencing of toleragen-specific lymphocytes are both cooperatively involved in many tolerance models. For the B lymphocyte, tolerance can be induced without an actual deletion of the cell involved; rather, the tolerant cell appears to have received and stored a negative signal, rendering it unresponsive to normally immunogenic stimuli. Thus, a state termed 'clonal anergy' has been induced within the cell. Functional clonal deletion has also been noted in several models to T-lymphocyte tolerance, but here it is not known whether clonal anergy or actual death of the relevant cell is at work. Self-tolerance sufficient to be consistent with good health need not mean a total absence of cells with any degree of self-reactivity. Indeed, it is clear that some B cells capable of forming antibody with some degree of affinity for self-constituents exist in the body, and can be activated, for example by lipopolysaccharide. The requirement is to limit the amount, affinity and duration of autoantibody production. A model suggesting how this may be achieved is presented.

Animals↗