Search PubMed⌕ Search

Biomedical subjects

G J Nossal

Publications and source records attributed to G J Nossal.

At least 37 records · Page 2Linked to original sources
PubMed1995

FAS is highly expressed in the germinal center but is not required for regulation of the B-cell response to antigen.

In establishing the memory B-cell population and maintaining self-tolerance during an immune response, apoptosis mediates the removal of early, low-affinity antibody-forming cells, unselected germinal center (GC) cells, and, potentially, self-reactive B cells. To address the role of the apoptosis-signaling cell surface molecule FAS in the B-cell response to antigen, we have examined the T-cell-dependent B-cell response to the carrier-conjugated hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) in lpr mice in which the fas gene is mutated. High levels of FAS were expressed on normal GC B cells but the absence of FAS did not perturb the progressive decline in numbers of either GC B cells or extrafollicular antibody-forming cells. Furthermore, the rate of formation and eventual size of the NP-specific memory B-cell population in lpr mice were normal. The accumulation of cells with affinity-enhancing mutations and the appearance of high-affinity anti-NP IgG1 antibody in the serum were also normal in lpr mice. Thus, although high levels of FAS are expressed on GC B cells, FAS is not required for GC selection or for regulation of the major antigen-specific B-cell compartments. The results suggest that the size and composition of B-cell compartments in the humoral immune response are regulated by mechanisms that do not require FAS.

Amino Acid Sequence↗
PubMed1995

Soluble antigen can impede affinity maturation and the germinal center reaction but enhance extrafollicular immunoglobulin production.

The primary immune response to T cell-dependent Ags develops in two pathways. These comprise the extrafollicular pathway, in which foci of Ab-secreting cells develop, and the intrafollicular pathway that gives rise to germinal centers and affinity maturation. We have previously shown that de-aggregated (4-hydroxy-3-nitrophenyl) acetyl (NP) coupled to the protein carrier human serum albumin (HSA), (NP-HSA), injected 6 days after challenge immunization with aggregated NP-HSA, resulted in impaired development of NP-specific, higher-affinity cells. Studies presented here describe the cellular basis underlying this impairment of affinity maturation. Using multiparameter flow cytometry, we show that mice injected with soluble NP-HSA ("tolerant" mice) develop significantly fewer NP-binding IgG1+ B220+ cells of germinal center origin than do the control ("immune") mice. In addition, using immunohistology, we noted that the spleens of tolerant mice had a marked reduction in the number of germinal centers that contained lambda-bearing cells, these being characteristic of the NP response in C57BL/6 mice. Curiously, germinal centers in the spleens of tolerant mice had more than twice the volumes of those in the immune spleens. In contrast to its effect on the germinal center pathway, soluble Ag enhanced the extrafollicular pathway, reflected by the increased numbers of B cells secreting IgM and IgG1 Abs specific for NP, HSA, and undefined Ags. Thus, soluble NP-HSA given after challenge immunization can impede affinity maturation of NP-specific cells, but enhance the extrafollicular pathway. These results are discussed in the context of the known capacity of some persisting Ags, e.g., malaria parasites, to frustrate affinity maturation and memory cell generation.

Animals↗
PubMed1995

Soluble antigen can cause enhanced apoptosis of germinal-centre B cells.

Germinal centres are dynamic microenvironments of B-lymphocyte differentiation, which develop in secondary lymphoid tissues during immune responses. Within germinal centres, activated B lymphocytes proliferate and point mutations are rapidly introduced into the genes encoding their immunoglobulin receptors. As a result, new specificities of B cells are created, including those with a heightened capacity to bind the immunizing antigen. Immunoglobulin gene mutation can also lead to reactivity to self antigens. It has been suggested that any newly formed self-reactive B cells are eliminated within the germinal centre in order to avoid autoimmunity. Here we present evidence that antigen-specific, high-affinity, germinal-centre B cells are rapidly killed by apoptosis in situ when they encounter soluble antigen. The effect seems to act directly on the B cells, rather than through helper T cells. Furthermore, the apoptosis is unique to germinal-centre cells, and is only incompletely impeded by constitutive expression of the proto-oncogene bcl-2. This phenomenon may reflect clonal deletion of self-reactive B cells within germinal centres.

Animals↗
PubMed1995

B1 and B2 cells differ in their potential to switch immunoglobulin isotype.

The ability of purified B1a (Ly-1 B) and B2 cells to switch immunoglobulin isotype was assessed by limiting dilution analysis in two in vitro culture systems. When stimulated in the presence of interleukins-4 and -5 by either lipopolysaccharide or CD40 ligand, the frequency of IgG1 precursors in the B1a population was at most one third that of IgM precursors. In B2 cells, however, the frequency of IgG1 precursors was up to seven times that of IgM precursors. B1a cells were shown to respond to interleukin-4 by virtue of up-regulating major histocompatibility complex class II expression when exposed to the cytokine, precluding non-responsiveness as a reason for not switching to IgG1. Indeed, interleukin-4 was found to specifically induce transcription of the germ-line IgG1 constant region locus in B1a cells as it did in B2 cells. Collectively these results suggest that the ability of B1 cells to respond to isotype switch commitment factors such as interleukin-4 may be secondary to the production of IgM by these cells.

Animals↗
PubMed1995

Choices following antigen entry: antibody formation or immunologic tolerance?

The research briefly summarized here covers 38 years of work in cellular immunology, chiefly devoted to understanding how antigen subserves the antithetical functions of stimulating antibody formation and inducing tolerance. The three interlocking themes running through the work are the development of methods to beat the problem of heterogeneity among lymphocytes by studying single cells and single clones; critical pursuit of antigen-trapping patterns within lymphoid tissues and analysis of consequent cellular events; and construction of models of tolerance that permit the detection of events subtler than direct purging of the B cell repertoire. One of the best features of this adventure has been its international character, the interaction with colleagues in many countries, and the influence that this work, despite its entirely fundamental character, has exerted in the practical world of preventive and clinical medicine.

Allergy and Immunology↗
PubMed1994

A form of immunologic tolerance through impairment of germinal center development.

Primary immunization with the T-cell-dependent antigen (4-hydroxy-3-nitrophenyl)acetyl (NP) coupled to human serum albumin results in the development of two pathways of B-cell development, the extrafollicular pathway and the germinal center pathway. Soluble, deaggregated NP-human serum albumin injected before immunization results in a marked diminution of clonable higher-affinity antibody-forming cell precursors--i.e., a form of immunologic tolerance within the secondary B-cell repertoire. We describe here the cellular changes in the spleen that underlie this tolerance. Using multiparameter flow cytometry, we show that tolerant mice develop far fewer NP-binding, peanut agglutinin-positive, or germinal center cells than the control immunized mice; 14 days after challenge control spleens have approximately 2 x 10(5) such cell per spleen, whereas the tolerant mice have approximately 1 x 10(4) cells. Furthermore, we demonstrate by immunohistology a reduction in the number of germinal centers containing lambda-bearing cells, characteristic of the response of C57BL/6 mice to NP. Taken together, these data suggest an impairment of germinal center development in the tolerant mice.

Animals↗
PubMed1994

Negative selection of lymphocytes.

Almost by definition, negative selection of T and B lymphocytes cannot be absolute. Given that both sets of receptors are derived by stochastic processes, recognition of epitopes by lymphocyte receptors will not be an all or none affair but a relative one. Too effective a mechanism of negative selection would have resulted in deletion of the whole repertoire, as all specificities would have cross-reacted with some self-epitope at least to some degree. This review has documented some of the influences impacting on emerging T and B cell repertoires that result in a removal of the most dangerous self-reactive cells and the progressive quantitative and qualitative increase, through positive clonal selection, of other cells specific for the actual foreign antigens encountered by each individual. T and B lymphocytes pass through a stage where their natural reaction to antigen is one of negative selection and on to a later stage where the cell is more likely to become activated. Geography plays a role in this; the primary lymphoid organs are designed largely to exclude foreign antigens and to present self-antigens, whereas the secondary lymphoid organs are designed to filter out and concentrate foreign material and to promote costimulatory intercellular immune interactions. Ontogeny of individual cells also plays its role, probably through the progressive assembly of the full receptor signaling machinery, incomplete arrays promoting a negative rather than a positive signal. However, the differing susceptibilities of immature and mature cells to silencing by deletion or anergy are relative rather than absolute. Negative signaling may involve immediate or somewhat delayed death of the anti-self-cell, and in some cases the bad cell may have the chance of editing its receptor to create one lacking anti-self-reactivity. Alternatively, the cell may receive a nonlethal down-regulatory signal and may be induced into a state of anergy. Such anergic cells may have a reduced life span, showing that anergy and deletion may shade into each other. Upon the removal of antigen (an unlikely event for cells anergic to an authentic self-antigen), the state of anergy is reversible. The strength of the receptor cross-linking signal may chiefly determine whether deletion (strong cross-linking) or anergy (weaker cross-linking) supervenes. Some lymphocytes with self-reactivity ignore the self-antigen in question because of low affinity, poor accessibility, lack of suitable presentation, or absence of appropriate help.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Bcl-2 increases memory B cell recruitment but does not perturb selection in germinal centers.

To address the role of apoptosis in the humoral immune response, we have examined a well-characterized T cell-dependent B cell response in mice expressing transgenic Bcl-2 in their B lymphocytes. The selection of somatic mutants and the appearance of high affinity antibodies was not affected by constitutive Bcl-2 expression. Such expression did, however, disproportionately increase the antigen-specific memory B cell pool, suggesting that the final size of the memory compartment may be regulated by an apoptotic process, which, in turn, can be influenced by Bcl-2. In addition, transgenic mice showed prolonged survival of foci of early antibody-producing cells, suggesting their removal is mediated by apoptosis that can be blocked by Bcl-2.

Animals↗
PubMed1994

Twenty-five years of germinal centre physiology: implications for tolerance in the secondary B cell repertoire.

Twenty-five years ago, when I delivered the inaugural lecture of the Scandinavian Society for Immunology, my mind was filled with germinal centres because of the extraordinary antigen-capturing mechanism there, which depended on follicular dendritic cells trapping the antigen and holding it in an extracellular location for long periods of time, following which the germinal centre reaction developed around the antigen depot. It was apparent that germinal centres had much to do with immunological memory, but few functional details of their role were known. In the intervening period, a great deal of knowledge has accumulated about germinal centres, which have become known as the sites of extensive B cell proliferation, immunoglobulin variable region gene hypermutation, and selection of better antigen-binding variants leading to affinity maturation in the antibody response. Our laboratory's interest was reawakened when we came to examine the hypothesis that B lymphocytes destined to develop into memory B cells might pass through a 'second window' of tolerance susceptibility if they encountered antigen in the absence of T-cell help. We have constructed a model of tolerance dependent on the injection of soluble deaggregated antigen before or even up to 6 days after T cell-dependent challenge immunization. The model was intended to mimic what might happen if a B cell, developing correctly in a germinal centre, fortuitously mutates to acquire cross-reactivity to a soluble self antigen. We have shown that the surrogate self-antigen can profoundly impair the germinal centre process and virtually stop the emergence of high affinity memory B cells.

Animals↗
PubMed1993

Tolerance and ways to break it.

The overall picture as regards cellular mechanisms in immunologic tolerance is thus clear. Thymic negative selection is an important and dominant mechanism for both CD4+ and CD8+ T cells for those antigens (and they may be very many indeed), the peptides of which get expressed within the thymus. The induction of anergy among peripheral T lymphocytes may represent an ancillary mechanism in some cases, but this is not as clear as it appeared 2 or 3 years ago. Evidently, in many cases, T cells simply ignore antigens present only within specialized organs, and these T cells, even if only of low affinity for the antigen in question, could be provoked into autoimmunity if sufficient help is provided, for example, through localized production of IL-2 or through provision of cross-reactive help. B-cell tolerance is also proven and involves deletional mechanisms (most likely maturation arrest) or functional inactivation (clonal anergy). The former phenomenon dominates for self-antigens that strongly cross-link the B cell's Ig receptors and the latter for weaker negative signals. Despite these two mechanisms, clonal ignorance prevails for many self-antigens. The secondary B-cell repertoire is also largely free of anti-self B cells, lack of T-cell help being a major factor in preventing the development of anti-self memory B cells. Therefore, to have the best chance of creating an immunogenic antitumor vaccine, a few simple and rather obvious rules must be followed. The antigen in question must be presented in such a way as to be palatable to "professional" antigen-presenting cells, particularly dendritic cells and macrophages. Immunotherapy protocols should avoid at all costs the widespread distribution of the antigen in question through the extracellular fluids. Indeed, it is possible that widespread circulation of a tumor-associated antigen through the serum and lymph because of large-scale shedding from the tumor cell may already have created a substantial degree of tolerance to the antigen in question in both T- and B-cell populations. If that has happened, it becomes particularly important to create a cancer vaccine capable of inducing help. This may well involve coupling of the most important epitopes to some highly immunogenic and foreign carrier. A wide choice of adjuvants is available for the designer of tumor vaccines, and in this regard the cancer immunotherapy community has much to learn from the rapidly developing field of infectious disease vaccinology using recombinant and other vaccines.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Antigen-driven B cell differentiation in vivo.

The secretion of specific antibodies and the development of somatically mutated memory B cells in germinal centers are consequences of T cell-dependent challenge with the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP). Using six-parameter flow cytometry and single cell molecular analysis we can directly monitor the extent of somatic hypermutation in individual responsive (isotype switched) antigen-specific B cells. The current study provides a direct quantitative assessment of recruitment into the antibody-secreting compartment on the one hand, and the germinal center pathway to memory on the other. Cellular expansion in both compartments is exponential and independent during the first week after challenge. The first evidence of somatic mutation, towards the end of the first week, was restricted to the germinal center pathway. Furthermore, germinal center cells express a significantly shorter third hypervariable region (CDR3), even when unmutated, than their antibody-secreting counterparts, suggesting a secondary selection event may occur at the bifurcation of these two pathways in vivo. By the end of the second week, the majority of mutated clones express a shorter CDR3 and affinity-increasing mutations as evidence of further selection after somatic mutation. These data provide evidence for substantial proliferation within germinal centers before the initiation of somatic mutation and the subsequent selection of a significant frequency of mutated clonotypes into the memory compartment.

Amino Acid Sequence↗
PubMed1993

Soluble antigen profoundly reduces memory B-cell numbers even when given after challenge immunization.

The splenic B-cell repertoire of unimmunized C57BL/6 mice can be examined for anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) B cells of relatively high affinity by using a dual strategy. First, limiting numbers of splenocytes are polyclonally activated by Escherichia coli lipopolysaccharide and a mixture of interleukins 2, 4, and 5 in the presence of 3T3 filler cells, thus ensuring that many B-cell clones switch to IgG1 antibody production. Second, an enzyme-linked immunosorbent assay is geared to register only higher-affinity antibody by (i) detecting only bivalent IgG1 antibody and ignoring IgM and (ii) using a lowly substituted NP-conjugated protein as the capture layer. Naive spleens contain very few higher-affinity anti-NP B cells thus defined, but thymus (T)-dependent immunization causes the appearance of approximately 10(5) per spleen within 2 weeks. The development of these clonable anti-NP antibody-forming cell precursors can be virtually eliminated by a single injection of 1 mg of soluble, freshly deaggregated NP2-human serum albumin (HSA). This toleragen works not only if injected prior to challenge immunization, but even if given up to 6 days later. Soluble HSA works partially but not nearly as well as NP2-HSA, suggesting the possibility that the toleragen must act on T and B cells. NP conjugated to irrelevant carriers achieved partial tolerance in only one of four experiments. The studies demonstrate the need for continuing T-cell help throughout the process of memory B-cell generation. They also show that those recently activated T cells involved in this process can be silenced in vivo by soluble toleragen.

Animals↗