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G J Nossal

Publications and source records attributed to G J Nossal.

At least 73 records · Page 4Linked to original sources
PubMed1990

A regulatory role for the soluble IL-2 receptor via competition with the p75 cell-surface form of the receptor for IL-2.

Murine T and B lymphocytes can be induced to release soluble interleukin 2 receptors (sIL2R). This receptor is believed to be a truncated form of the p55 chain of the cell membrane-associated receptor. It has been speculated that this receptor may play an immunoregulatory role via competition for IL-2 with the high-affinity (p55/75 heterodimer) IL-2 receptor. Of crucial importance to this hypothesis are both the concentration of the receptor and its affinity of binding for interleukin 2. We report the measurement of the affinity of sIL2R derived from stimulated normal murine splenocytes for IL-2. We also report the quantification of an enzyme linked immunosorbent assay (ELISA) for sIL2R via measurement of the sIL2R concentration in normal murine splenocyte conditioned medium using a radioimmunometric assay and Scatchard analysis. This method of sIL2R quantification is preferable to sIL2R purification and subsequent concentration estimation as used by previous investigators as any purification process risks destruction of some epitopes. Using the above conditioned medium as a standard we have tested supernatants from several cell lines and sera from several different mouse strains for sIL2R. As would be expected this method of quantification yielded a markedly different value for serum sIL2R levels in normal mice than that obtained by previous investigators. Our results indicate that it is very unlikely that sIL2R competes with the high-affinity form of the IL-2 receptor for IL-2. However, it is possible that it competes for IL-2 with the medium-affinity p75 form of the IL-2 receptor and as such is important in restricting unwanted non-specific (bystander) activation of p75 expressing cells. Evidence from both our previous work as well as from the literature is presented to support this hypothesis.

Animals↗
PubMed1989

Immunologic tolerance: collaboration between antigen and lymphokines.

Immunologic tolerance is the process whereby limits are placed on the degree to which lymphocytes respond to an animal's inherent antigens. It is a quantitative rather than an absolute term, as some autoantibody formation is common. Contrary to early hopes, it is not due to some single, simple causative mechanism confined to early developmental stages of the fetal immune system. Rather, self-tolerance results from a variety of complementary mechanisms and feedback loops in the immune system and is thus best seen as part of the general process of immunoregulation.

Animals↗
PubMed1989

Interleukins 4 and 5 control expression of IL-2 receptor on murine B cells through independent induction of its two chains.

Resting B cells express few, if any, receptors for interleukin-2 (IL-2), whereas activated B cells can express receptors for and respond to IL-2. IL-2 receptors can exist on the cell surface in three different forms; the complete high-affinity receptor, a heterodimer consisting of a chain of relative molecular mass (Mr) 70-75,000 (70-75K) and a chain of Mr 55K; the 70-75K chain alone, with intermediate affinity for IL-2; or the 55K chain alone, with low affinity for IL-2. We have previously reported that IL-5-stimulated B cells are induced to express the 55K chain. We report here evidence for the differential regulation of the expression of the two chains, namely that IL-4 and IL-5 can independently induce expression of the 70-75K and 55K chains respectively on murine B cells. As expected, cells stimulated to express the 55K chain alone are unresponsive to IL-2, whereas cells stimulated to express either the 70-75K chain or the 70-75/55K heterodimer respond to IL-2, at a high and low ligand concentration respectively, with a marked increase in proliferation. This orchestration of receptor expression and factor responsiveness may represent a novel activation pathway for B cells, where the two chains of a compound receptor are shown to be independently regulated.

Animals↗
PubMed1989

Inhibition of antibody production at high cell density following mitogen stimulation and isotype switching in vitro.

We have investigated antibody production in 3T3 filler cell-containing murine B lymphocyte cultures, stimulated with LPS and an IL-4-containing lymphokine mixture. At low cell density cultures produced 1.8 +/- 0.6 ng of IgM and 4.2 +/- 1.7 ng of IgG1 per input B cell. It was found that 21.7 +/- 3.5% of spleen cells, or approximately 43% of B cells, produce IgM under these conditions, and 11.9 +/- 5.5% spleen cells, approximately 24% of B cells, produced IgG1. Therefore, the mean IgM production per IgM-positive clone was 4.2 ng, and the mean IgG1 production per IgG1-positive clone was 17.6 ng. A cell density of about 10,000 B cells/ml was found to produce maximal antibody per input cell. A 32-fold increase above the maximum cell density resulted in a 600-fold decrease in IgG1 production per B cell. IgM production was also found to be inhibited above this concentration of cells, but to a six-fold lesser extent. Cell proliferation in dense cultures was also found to be diminished in a cell concentration-dependent manner, partially accounting for the observed inhibition phenomenon. The replenishment of media, LPS and growth factors was able to lessen the inhibition of dense cultures, but not to maximal levels. Overall, this work identified the upper limit of cell density for in vitro cloning of B lymphocytes for isotype switch and repertoire analysis. The most important conclusion is that antibody production is grossly suboptimal at the cell densities frequently used in the literature.

Animals↗
PubMed1989

Sudden appearance of anti-protein IgG1-forming cell precursors early during primary immunization.

The anti-keyhole limpet hemocyanin (KLH) B-cell repertoire of unimmunized adult mice was examined by culture of splenocytes (generally 100-3000) at limiting dilution. Cells were polyclonally stimulated with Escherichia coli lipopolysaccharide (LPS) and an interleukin-4-containing lymphokine mixture in the presence of 3T3 fibroblast filler cells. After 7 days of culture, supernatants were examined for their content of anti-KLH IgM and IgG1 antibody by an enzyme-linked immunosorbent assay (ELISA). Parallel cultures of smaller numbers (generally 1-15) of splenocytes were examined to determine the cloning efficiency of B cells in terms of total IgM and IgG1 production. Whereas one spleen cell in 370 produced clones secreting anti-KLH IgM, only 1% of these produced IgG1 that could bind to KLH, despite the fact that about half of the clones switched to IgG1 production with these stimuli. In mice immunized with KLH, this situation did not change until day 5, when there was a sudden, explosive emergence of B cells that could form clones secreting anti-KLH IgG1. The absolute number of such cells in the spleen was found to rise by a factor of 350 between days 3 and 7 of immunization. Moreover, the median amount of IgG1 antibody formed per clone and binding to KLH also rose markedly. In contrast, neither the numbers nor the median KLH-binding antibody content of anti-KLH IgM clones changed significantly after immunization. The results show that the repertoire of anti-protein B cells detected through IgM formation in ELISA consists chiefly of cells producing antibody of low avidity and of doubtful in vivo significance. Assuming that the small proportion of these cells making antibody that is of sufficient avidity to bind as the IgG1 isotype are the ancestors of the many such cells found on day 7 of the primary immune response, one would have to postulate a very high recruitment and/or division rate to account for the increase in numbers and avidity that occurs. It is possible that the anti-KLH IgG1 precursors that suddenly emerge are the results of early variable region gene (V) mutations in B cells. Moreover, it is not excluded that they represent products of a subset of B cells different from those that give rise to the primary in vitro anti-KLH IgM response. The findings have implications for theories of B-cell tolerance.

Animals↗
PubMed1989

Immunological tolerance then and now: was the Medawar school right?

As perhaps the staunchest advocates of repertoire purging as the central mechanism of immunological tolerance, we note with satisfaction a spate of recent, elegant papers which suggest an intrathymic clonal abortion model as the explanation for at least some examples of T-cell tolerance. This view agrees with the classical formulation of the Billingham-Brent-Medawar school of tolerance as a specific, central failure of immune responsiveness. Repertoire purging within the B-lymphocyte compartment remains much more controversial. There is no doubt that experimental models exist where the B cell is the reversible target of tolerance induction. The question is, in view of the ease of inducing autoantibody formation both in vivo and in vitro, just how relevant are such clonal anergy mechanisms to authentic self-tolerance? Arguments are presented that there must be two windows of tolerance susceptibility in the ontogeny of the B cell; one while it is maturing in the bone marrow, to prevent autoreactivity of high affinity to important accessible self-antigens; and a second soon after activation of pre-memory cells by exogenous antigen, to prevent fortuitous mutations towards high-affinity anti-self-reactivity establishing a forbidden clone.

Animals↗
PubMed1988

Clonal analysis of autoantibody-producing cell precursors in the preimmune B cell repertoire.

Evidence for anti-self-reactivity in the preimmune B cell repertoire has been well documented. The present study aimed to determine the frequency of antibody-forming cell precursors in this repertoire whose Ig V regions impart reactivity to "self" or autologous Ag. Clones were activated in vitro with LPS and their secreted IgM antibody was assayed for reactivity by direct binding to cell surface or intracellular Ag. An IL-4-containing lymphokine mixture was added to the clonal cultures to induce the secretion of IgG1. The reactivity of secreted IgG1 with Ag would more closely resemble the binding required to activate B cells through their monomeric surface IgM and/or IgD. The results indicate a high frequency of precursors secreting IgM with reactivity to intracellular Ag, namely 1 in 37 +/- 6 B cells, with a marked paucity of response to cell surface molecules. The repertoire was markedly deficient in precursors secreting IgG1 able to bind to intracellular Ag, with only one clone detected by the screening of 3.0 x 10(6) spleen cells. No positives were detected for cell surface Ag. This suggested that the frequency of clones in the preimmune repertoire that express IgR with sufficient affinity to bind "self" molecules must be very low.

Animals↗
PubMed1988

Analysis of the B lymphocyte repertoire by polyclonal activation. Hindrance by clones yielding antibodies which bind promiscuously to plastic.

We here describe a form of 'noise' in the ELISA as commonly performed on antigen-coated microtiter trays that represents a major hindrance to the accurate enumeration of infrequent antibody-forming cell (AFC) precursors (AFCp) specific for epitopes on monomeric proteins. Supernatants from cultures of lipopolysaccharide-stimulated murine splenocytes, when split into aliquots and separately assayed, scored as positive in parallel on ELISA trays coated with unrelated proteins and on uncoated trays. Some properties of such coincident false positives (CFP) noted were: (1) optical density (OD) ranges for CFP and non-CFP overlapped; (2) different members of CFP triplets on differently coated assay trays usually had similar OD values; (3) CFP-generating culture supernatants did not contain unusually high immunoglobulin concentrations; and (4) numbers of CFP-forming supernatants increased with increasing input cells/culture consistent with causation by single AFCp present at an approximate mean frequency of 1 in 6600 CBA splenocytes. It is proposed that CFP are due to AFC clones that secrete antibody reactive with some epitope(s) present in the assay tray itself. Repertoire elements with such 'anti-plastic' characteristics are rarer than anti-keyhole limpet hemocyanin (KLH) AFCp, but at least as frequent as anti-bovine serum albumin (BSA) or anti-transferrin (TFN) AFCp.

Animals↗
PubMed1988

A significant proportion of normal resting B cells are induced to secrete immunoglobulin through contact with anti-receptor antibody-activated helper T cells in clonal cultures.

This report describes single-cell techniques to address the nature of a cellular interaction in which activated T lymphocytes stimulate small resting B cells to develop into antibody-forming cell clones in the absence of any surface immunoglobulin ligand or an antigen bridge. The cloned T helper cell line E9.D4 was stimulated with the anti-V beta 8 antibody F23.1 bound to the plastic of Terasaki 10-ul culture wells. When an excess of T helper lymphocytes was used (1,000 X-irradiated or 600 unirradiated, stimulated E9.D4 cells), 10-25% of B cells responded by antibody formation as judged by an enzyme-linked immunosorbent assay performed after 5 days of culture. When one of a very small number of B cells were present, the rate-limiting step to antibody-forming cell formation was the number of T cells present. Far fewer T cells sufficed for stimulation when culture trays were tilted to force T and B cells into proximity at the sulcus formed at the bottom edge of the culture wells. When T cell numbers were limiting, unirradiated T cells out-performed irradiated T cells. Some cell clones held for 7 days switched to IgG antibody production. E9.D4 supernatants were virtually ineffective in causing B cell stimulation, even when 3T3 filler cells were added to support cultures. The results suggest that cell contact, and perhaps conjugate formation, with a strongly activated T cell can cause changes in the adjacent resting B cells akin to those of Ig receptor cross-linking, following which a lymphokine flux (even one not involving IL 4 and 5) promotes antibody-forming cell development.

Animals↗
PubMed1988

Somatic mutations in B lymphocytes: new perspectives in tolerance research?

This paper extends the concept of clonal anergy developed in the author's laboratory. It has been shown that the primary population of B lymphocytes induced into clonal expansion and IgM antibody formation by mitogens contains many cells capable of autoantibody synthesis, but the affinity of binding to the self constituents, or indeed to foreign antigens, is low. The creation of high affinity antibody, which will still register strongly in an ELISA as an IgG molecule, demands not only the addition of lymphokines to cause isotype switching, but also intentional immunization of the donor mice to permit mutations in V region genes and selection of higher affinity B memory cells. This process appears to begin about 6 days after in vivo immunization. It is postulated that these mutational events occur primarily in germinal centres, and that there must be mechanisms to prevent escape of cells which, by chance, mutate not to higher affinity against an immunogen, but to higher affinity against a self constituent. If such mutants were allowed to enter the long-lived, recirculating pool of B lymphocytes, they might pose a graver threat of autoimmune disease than the low-affinity anti-self cells of the primary repertoire. Therefore, it is suggested that recently mutated germinal centre B cells represent a pool of 'pre-memory' cells, which are immature in the sense of displaying the same kind of sensitivity to negative signalling by antigen that immature B cells from newborn spleen or adult bone marrow display. If so, then the earliest phases of memory generation represent a second window of opportunity for tolerance induction within the B lymphocyte compartment.

Animals↗
PubMed1988

Soluble interleukin 2 receptors are released from the cell surface of normal murine B lymphocytes stimulated with interleukin 5.

Murine T and B lymphocytes can be induced to release soluble interleukin 2 receptors (IL2Rs). This receptor is believed to be a truncated form of the 55-kDa chain of the cell-membrane-associated receptor. It has been speculated that this receptor may play an important immunoregulatory role by binding to interleukin 2 (IL-2). We report here that interleukin 5 can induce normal murine B cells to release soluble IL2Rs. This extends our finding that interleukin 5 similarly can induce murine B cells to express functional cell-surface-associated IL2Rs. Two possible mechanisms of release of soluble IL2Rs have been suggested. Soluble IL2R could be synthesized as a secretory form of the receptor lacking the transmembrane domain or by cleavage of the extracellular domain of the cell-surface-associated IL2R at the cell surface. To investigate which mechanism was operative, we radioiodinated the cell surface of normal murine splenocytes that had been cultured for 1 day with Con A to stimulate the expression of cell-surface-associated IL2Rs and the release of soluble IL2Rs. Under the conditions used, radiolabeling of internal proteins was not apparent. Labeled cells were then recultured with Con A, conditioned medium was taken from replicate cultures at various times after radioiodination, and the specific radioactivity of released soluble IL2Rs was determined by ELISA and RIA. We demonstrate that the specific radioactivity and the kinetics of change of the specific radioactivity are consistent with the hypothesis that the soluble IL2Rs are derived from the cell-surface-associated IL2Rs rather than being released in a secretory form.

Animals↗
PubMed1988

Immunology--directions for the future.

This brief introduction highlights how far immunology has moved as an independent discipline. From its birth as a branch of medical microbiology, it now pervades virtually the whole of medicine and many of its subspecialties. The steps along this pathway are summarised, and the main challenges for the future outlined.

Allergy and Immunology↗