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G J Nossal

Publications and source records attributed to G J Nossal.

At least 19 recordsLinked to original sources
PubMed1992

Ligation-anchored PCR: a simple amplification technique with single-sided specificity.

A simple, efficient, and sensitive technique has been developed for amplification of cDNAs encoding molecules with 5' regions of unknown sequence. In this ligation-anchored PCR, T4 RNA ligase is used to covalently link an "anchor" oligonucleotide to first-strand cDNAs. These anchored cDNAs are then amplified by using one PCR primer specific for the anchor and another specific for a sequence within the molecule of interest. The anchor oligonucleotide has been especially designed to facilitate subsequent analysis and cloning of the resultant PCR products. This three-stage procedure does not require purification of product between steps and avoids many of the technical difficulties associated with established anchored PCR protocols. The efficacy of ligation-anchored PCR was demonstrated by amplification of a specific IgG1 cDNA; total RNA equivalent to as few as 100 cells yielded the expected PCR product.

Base Sequence↗
PubMed1992

Memory cell generation ablated by soluble protein antigen by means of effects on T- and B-lymphocyte compartments.

Adult C57BL/6 mice were injected with 100 micrograms of soluble, freshly deaggregated human serum albumin (HSA) to produce partial immunologic tolerance. Uninjected normal control (N) mice contain only approximately 100 B cells in their spleens with the capacity to (i) be activated in vitro into clonal proliferation by Escherichia coli lipopolysaccharide plus interleukins 2, 4, and 5, (ii) form IgG1 as well as IgM antibody, and (iii) display specificity for HSA when only IgG1 is allowed to score in an enzyme-linked immunosorbent assay (ELISA). Such N mice generate approximately 50,000 clonable anti-HSA IgG1 antibody-forming cell precursors in their spleens after T-dependent immunization with HSA absorbed onto alum and given with Bordetella pertussis adjuvant. Mice preinjected with soluble HSA (TOL) generate far fewer anti-HSA IgG1 antibody-forming cell precursors, termed anti-HSA memory cells. Splenocytes were transferred from N or TOL mice into lethally irradiated syngeneic recipients together with syngeneic bone marrow. Whereas N splenocytes generated plentiful memory cells within 2 weeks in antigenically challenged recipients, TOL splenocytes did not. Work with Ly-5 congenic mice ruled out memory cell generation from either the host or the bone marrow inoculum within this limited time. N T cells plus TOL B cells showed consistently lowered memory cell generation. TOL T cells plus N B cells showed an even greater lowering of adoptive memory cell generation. Thus the lowered response capacity of TOL mice resided in the T- and B-cell compartments. Attempts to show a suppressor component within the T-cell population were inconclusive, but a profound defect in capacity to respond to HSA in vitro was exhibited by the CD4+ T cells of TOL mice. B lymphocytes were harvested from T-dependently immunized mice 5 days after challenge, incubated with soluble HSA for 18 hr, and then adoptively transferred together with N T cells. The recently activated B cells were not rendered tolerant by this manipulation. The results argue for a major T-cell component in the process whereby soluble protein antigens ablate affinity maturation and memory cell generation.

Animals↗
PubMed1992

Functional and molecular characterization of single, (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific, IgG1+ B cells from antibody-secreting and memory B cell pathways in the C57BL/6 immune response to NP.

We have used multiparameter flow cytometry to identify a population of IgG1+ IgM- antigen-specific B cells which emerges in spleens of C57BL/6 mice following immunization with the hapten, (4-hydroxy-3-nitrophenyl)acetyl (NP). Characterization of the specificities of IgG1 antibodies produced by single, sorted IgG1+ NP+ cells in both Elispot assays and in microcultures containing lipopolysaccharide, interleukin (IL)-2, IL-4 and IL-5 indicates that the splenic IgG1+ NP+ B cell population includes both IgG1 anti-NP antibody-secreting cells and non-secreting, IgG1+ memory B cells. Each functionally discrete population of IgG1+ B cells expresses a distinctive surface phenotype defined by a wide range of B cell markers. In particular, antibody-secreting, IgG1+ cells were uniquely identified by co-expression of the matrix receptor, syndecan. The NP-specific B cell population emerging in the day 7 primary response was assessed for clonotypic diversity by amplification and direct sequencing of the rearranged V186.2 heavy chain variable region gene expressed by single, ex vivo IgG1+ NP+ lambda+ B cells. Memory B cell clones, distinguished by junctional diversity, carried either no mutation or a single mutation within rearranged V186.2, suggesting isolation of these cells at the onset of the hypermutation mechanism. This novel approach, therefore, allows the direct and unambiguous identification and characterization of individual B cell clonotypes during their initial selection and activation in antibody responses in vivo.

Animals↗
PubMed1992

Autoimmune tolerance and type 1 (insulin-dependent) diabetes mellitus.

The autoimmune process that results in Type 1 (insulin-dependent) diabetes mellitus may be viewed as a failure to develop or maintain tolerance to self-antigens expressed in the islets of Langerhans. During T-cell development in the thymus, cells that are reactive with self antigens encountered there may undergo clonal deletion or, as more recently described, clonal anergy which effectively removes these cells from the pool of mature antigen reactive T cells. For antigens not found in the thymus, tolerance to self antigens is more complex and may depend on site of antigen expression, ambient concentrations of lymphokines, and availability of antigen-presenting cells that can deliver co-stimulatory signals. Transgenic mice in which the majority of T cells express T-cell receptors against "self" antigens or in which expression of antigens is targeted to peripheral tissues have proven useful for studies of tolerance in both T- and B-cell compartments. In general, T-cell reactivity against foreign antigen expressed on Beta cells does not occur because of the failure to activate T cells reactive with the antigen, termed clonal ignorance. This may be broken with, for example, viral infection or cytokines. In one transgenic model, dendritic cells that surround the islets of Langerhans have been shown to be responsible for presentation of islet antigens to the immune system. B-cell tolerance can also involve mechanisms of clonal deletion or clonal anergy similar to that occurring with T cells. In addition, a mechanism for changing the affinity of the B-cell antigen receptor termed "receptor editing" has been described, which may play an important role in diversifying the B-cell repertoire while removing self-reactive cells. Tolerance to antigens may also be inducible. For example, monoclonal antibodies against T-cell epitopes may induce antigen-specific tolerance that is transferable to other animals, and MHC blocking peptides which can inhibit T-cell responses that are restricted by disease associated MHC molecules. In conclusion, although several possible triggers and mechanisms of autoimmune diabetes can be envisioned, none can be excluded by existing data. However, advances in understanding mechanisms of tolerance to islet and other self antigens suggest potentially useful therapeutic approaches to arresting the autoimmune response.

Animals↗
PubMed1992

Cellular and molecular mechanisms of B lymphocyte tolerance.

A paradox of immunology is that the immune system is distributed so widely in the body, as a large number of cells that discharge most of their effector functions as single cells; but, at the same time, the elements of the system are so very interdependent, not only via specialized cell clusters and microenvironments, but also by mobile feedback loops, cellular and molecular. The end result is that one cannot really understand one element of the system without understanding every other, at least to a degree. Certainly, tolerance cannot be isolated from immune activation, nor B cell from T cell tolerance, rendering the task of the reviewer somewhat thankless. This being said, the last few years have seen wonderful progress in our grasp of B cell tolerance, to which the transgenic revolution has contributed a great deal. The fact that B cell tolerance exists as an important component of self-tolerance has been firmly established, as have the limits of the process in terms of both the survival of low-affinity antiself clonotypes and the question of location and concentration of antigen required for tolerance induction. Two processes have been identified as key alternatives: clonal abortion/maturation arrest/deletion and induction of clonal anergy. The latter requires a less strong Ig receptor crosslinking signal, may be partial, and is reversible. Recognition of these facts has prompted both experimentation and speculation on possible functions of the anergic cell. One unsatisfactory area, which we have not addressed because nothing like a consensus has been reached, is T cell-mediated suppression and its possible effects on tolerant states, including anergy induction in B cells. The phenomenology of suppression is too striking to sweep under the carpet, and suppressor T cell memory in particular (Adelstein et al., 1990) requires much more investigation; however, suppression has not been shown to play a major role in any of the best-studied transgenic models. These can readily be explained on the basis of direct interactions between the B cell target for abortion or anergy and the self antigen in question. The biochemical basis of discrimination between immunity and tolerance has also progressed, but not as fast. This is understandable, as so many signaling pathways have to come together for full immune induction, and as immaturity of the signal transduction pathway plays a profound role that must be studied in normal cells, with all the attendant difficulties of cell separation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

The interleukin network and lymphoid development.

Lymphoid development differs sharply between the primary and secondary lymphoid organs. In the former, lymphocytes arise from precursors by antigen-independent processes under thymic or bone marrow microenvironmental influences and undergo extensive selective processes before being allowed to leave. In the latter, lymphocytes with receptors relevant to particular antigens undergo a second wave of proliferation and differentiation leading to the emergence of immunocytes with effector functions. Each of the two sets of events are profoundly dependent on cellular interactions. In the primary lymphoid organs, the "action" centres on stromal cell-lymphoid precursor interactions, and artificial systems permitting B cell formation are much more advanced than those for T cell development. For B cells, IL-7 and c-kit ligand (KL) are clearly important but so are as yet undefined stromal cell-derived activities. For thymic development, only fragments of the complex 3-week process of T cell formation can be mimicked in vitro and no IL has unequivocally been shown to be critical. Within the secondary lymphoid organs, where lymphocytes react to the antigenic universe, the key to regulation lies in interactions between accessory cells (dendritic cells, macrophages and their various relatives) T cells and B cells. Efforts to squeeze the relevant cytokines into sharp compartments such as activation factors, growth factors and differentiation factors have been largely unsuccessful.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

A lifetime's love affair with antibody formation.

Professor Hans Gerhard Schwick has devoted the whole of his working life to Behringwerke, the world-renowned firm that owes its origins to Emil von Behring, the discoverer of antibodies. Schwick, like myself, has witnessed the incredible flowering of immunology during his career. He, like myself, has continued to see antibodies and immunity at the very centre of the extraordinary revolution in biology that will be seen by historians as perhaps the signal accomplishment of the last quarter of the 20th century. To give honour to such a man is easy; to know best how to do it is difficult. Therefore, I have chosen to attempt a personal odyssey--to describe my lifetime's love affair with the problem of antibody formation. This paper is in no sense any kind of history of the solution of the antibody puzzle. Rather, it is a description of one person's meandering pathway through the maze, a person who, like Professor Schwick, has been an eyewitness to history. Its intention is not self-indulgence, but a tribute to a man who has been an adornment to the scientific profession.

Animals↗
PubMed1991

Molecular and cellular aspects of immunologic tolerance.

This review seeks to explain the most exciting recent data concerning the nature of self/non-self discrimination by the immune system in a manner accessible to a biochemical readership. The nature of recognition in the two great lymphocyte families, B cells and T cells, is described with special emphasis on the nature of the ligands recognized by each. The history of the field of immunologic tolerance is surveyed, as are the key experiments on conventional mice which provided a conceptual framework. This suggested that tolerance was essentially due to 'holes' in the recognition repertoires of both the T and B cell populations so that lymphocytes competent to react to self antigens were not part of the immunologic dictionary. There were essentially two ways to achieve this situation. On the one hand, self antigens might 'catch' developing lymphocytes early in their ontogeny and delete the cell, a process of clonal abortion. On the other hand, self antigens might signal lymphocytes (particularly immature cells) in a negative manner, reducing or abolishing their capacity for later responses, without causing death. This process is referred to as clonal anergy. Evidence for both processes exists. Special emphasis is placed on a wave of experimentation beginning in 1988 which imaginatively uses transgenic mouse technology to study tolerance. Transgenic manipulations can produce mice which synthesize foreign antigens in a constitutive and/or inducible manner, sometimes only in specific locations; mice which possess T or B lymphocytes almost all expressing a given receptor of known specificity; and mice which are an immunologic time bomb in that the antigen is present and so too are lymphocytes all endowed with receptors for that antigen. These experiments have vindicated the possibility of both clonal abortion and clonal anergy in both T and B cell populations, the choice of which phenomenon occurs depending on a number of operational circumstances. For T cell tolerance, clonal abortion occurs if the self antigenic determinant concerned is present within the thymus; if not, clonal anergy is more likely. For B cell tolerance, the strength of the negative signal and therefore the choice between abortion and anergy depends on the molar concentration of the self antigen, the capacity for multivalent presentation to a B cell, and the affinity of the B cell's receptor for the antigen in question. Some B cells with low affinity for self antigens certainly escape censorship and remain capable of secreting low affinity anti-self antibodies, which however do no harm.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Molecular characterization of single memory B cells.

Primary antigenic exposure results in an initial antibody response and the T cell-dependent induction of B-cell memory. Memory B-cell differentiation is characterized by somatic hypermutation in antibody variable region genes (V) and selection of B cells expressing high-affinity variants of this antigen receptor. Despite our current understanding of B-cell memory, the origin of memory B cells and the regulation of their differentiation remain elusive. This is largely due to the difficulties in observing and purifying this minor component of the immunized spleen. Further, molecular characterization of memory B cells requires hybridoma formation which restricts analyses to only those clones capable of fusion and does not allow isolation of cells in a normal physiological state. We have therefore developed a unique system which allows isolation and unambiguous enumeration of IgG1+ memory B cells, based on six-parameter flow cytometry, secretion of antibody in clonal cultures and analysis of clonally expressed V genes using the polymerase chain reaction. Here we report that single IgG1+ antigen-binding B cells from an early secondary immune response proliferate in lipopolysaccharide-driven microcultures and produce antigen-specific IgG1 antibodies. Individual B-cell clones in these cultures express somatically mutated heavy chain V genes, confirming their designation as memory B cells. Although isolated memory B cells undergo extensive proliferation in vitro, V gene sequence analysis of their individual progeny shows that further hypermutation does not occur.

Amino Acid Sequence↗
PubMed1991

B-cell selection and tolerance.

The B-lymphocyte repertoire can be divided into a primary repertoire of virgin B cells and a secondary repertoire of antigenically experienced cells. Self-tolerance requires that neither of these repertoires contains potentially active cells capable of high-affinity, anti-self antibody production. Recent work has proven that both the primary and the secondary repertoire can be functionally purged, and this mechanism may involve failure of appearance of anti-self clonotypes (clonal abortion) or defects in activation (clonal anergy).

Animals↗
PubMed1990

Soluble antigen abrogates the appearance of anti-protein IgG1-forming cell precursors during primary immunization.

The anti-human serum albumin (HSA) B-cell repertoire of C57BL/6 mice was examined by culturing splenocytes at limiting dilution following polyclonal stimulation with Escherichia coli lipopolysaccharide and a lymphokine mixture. The frequency of anti-HSA precursors was determined before and after immunization with alum-precipitated HSA and 10(9) killed Bordetella pertussis organisms, by submitting clonal supernatants to an ELISA. Anti-HSA IgG1-forming precursors were rare in unimmunized spleens, representing approximately equal to 1 in 500,000 splenocytes or only approximately equal to 100 cells per spleen. Between day 5 and day 7 after immunization, this figure increased to approximately equal to 20,000 cells per spleen. Over the following 3 weeks, there was a progressive increase in the mean optical density generated in the clonal ELISA, presumably due to affinity maturation of the B-cell population. When freshly deaggregated HSA was injected before or even up to 4 days after challenge immunization, the appearance of anti-HSA IgG1-forming cell precursors was largely prevented. The effect was most marked with 5 mg or 1 mg of soluble HSA, but impressive partial effects could be seen with as little as 10 micrograms of HSA if administered before challenge immunization. Most of the few clones seen after the higher doses of the toleragen appeared to make antibody of low affinity. The capacity to influence the B-cell pool by soluble antigen administered just 1-2 days before the sudden appearance of IgG1 precursors argues against the totality of the effect being due to T-cell-mediated suppression and in favor of a direct effect on B cells.

Animals↗
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↗