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G J Nossal

Publications and source records attributed to G J Nossal.

At least 109 records · Page 6Linked to original sources
PubMed1985

An ELISA assay efficiently detects clonal antibody formation by single, hapten-specific B lymphocytes.

Spleen cells from non-immunized adult mice were fractionated on thin layers of fluorescein (FLU)-gelatin to yield FLU-specific B cells. These B cells were cultured either singly or in very small numbers in 10 microliter microcultures with 0.1 microgram/ml of the T cell-independent (TI) antigen FLU-E. coli lipopolysaccharide (FLU-LPS). Cultures were either filler cell-free, or supported by the addition of 10(5) CBA/N thymus cells per well. At 4-6 days, culture supernatants were assayed for the presence of anti-FLU antibody either by an enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay (RIA). With the filler cell-free cultures, B cell proliferation was scored microscopically before removal of culture supernatant. The cultured cells from each well were assayed for their capacity to form directly hemolytic FLU-specific plaques. In the filler cell-free system, the ELISA was much more sensitive than the plaque assay in identifying antibody-forming cell (AFC) clones, with over 10% of fractionated B cells yielding clones secreting detectable antibody, though with a low mean optical density (OD). This value represented over 80% of the proliferating clones. In the more efficient, filler cell-supported system, the difference between the 2 read-out methods was smaller. Here, one half of the hapten-specific B cells formed AFC clones, the highest cloning efficiency yet reported for an antigen-driven system. Comparative studies showed the RIA to be only marginally more sensitive than the ELISA, and not nearly as convenient for routine use.

Animals↗
PubMed1985

Perspectives on the regulatory biology of the B lymphocyte.

B lymphocytes with receptors specific for a particular hapten have been prepared through an antigen-affinity procedure. Methods have been developed for the clonal stimulation of these cells in vitro, with a single, hapten-specific B cell as the unequivocal target. Many stimulatory combinations involve multivalent antigen and one or more antigen non-specific, non-MHC restricted T lymphocyte-derived growth and differentiation factors. These factors, of which there are at least 4 or 5, are progressively being defined and should soon become available through recombinant DNA technology. Judicious use of factor combinations and selected antigens should soon answer whether "T-independent" and "T-dependent" B cells are truly separate subsets. A contact between multivalent antigen and immature B cell in the absence of these co-stimulatory factors can lead to the receipt and storage of a negative signal by the B cell. The B cell is not killed, but rather rendered anergic. Whether clonal anergy among B cells is an important mechanism in physiologic self-tolerance remains to be determined.

Animals↗
PubMed1985

A high-efficiency cloning system for single hapten-specific B lymphocytes that is suitable for assay of putative growth and differentiation factors.

Fluorescein (FLU)-specific murine splenic B lymphocytes from nonimmunized adult mice were prepared by the hapten-gelatin fractionation technique and cultured singly or in very small numbers in 10-microliters culture wells. Growth and differentiation to antibody-secreting status were promoted by polymeric FLU-conjugated antigens with or without added T-lymphocyte-derived conditioned media or purified cytokines. In some cultures, 3T3 fibroblasts or CBA/N thymocytes provided a source of filler cells. Anti-FLU antibody formation was detected by a sensitive enzyme-linked immunosorbent assay (ELISA). With an optimal number (around 300) of 3T3 cells per well, up to 77% of the B cells could be induced to produce detectable antibody. The ELISA permitted detection of antibody formation in essentially all wells where B-cell proliferation occurred, and it was more efficient in detecting antibody-forming clones than the hemolytic plaque assay, whether filler cells were present or not. When 10 B cells rather than 1 were included per well, the ELISA, detecting absorbance in standard fashion, provided a useful method for assessment of B-cell growth- and differentiation-promoting factors (BGDF). It was found that 3T3 cells gave less background stimulation than thymus cells, permitting the detection of as little as 1/100th as much BGDF as with thymocytes, thus offering a dynamic range of up to 30 between control absorbance in the absence of factors and the optimal factor level. Use of 3T3 cells also avoids a potential lymphokine cascade. The system has confirmed that interleukin-2 acts as a BGDF, but it has failed to establish an effect of interferon-gamma on B cells. It has also shown the inactivity of a variety of hemopoietic growth factors on B lymphocytes. This system thus promises to be a useful tool in the further analysis of B-lymphocyte activation.

Animals↗
PubMed1985

Interleukin 1 can act as a B-cell growth and differentiation factor.

Splenic B lymphocytes specifically reactive to the hapten fluorescein (FLU) were prepared from nonimmune adult mice by affinity fractionation on hapten-gelatin. These FLU-specific B cells were cultured as single cells or in small numbers in 10-microliter wells either in the absence of any feeder, filler, or accessory cell or in the presence of 3T3 fibroblasts acting as filler cells. A selected batch of a "T-cell-independent" antigen, FLU-Ficoll, which induces growth and differentiation only in the presence of lymphokines or cytokines acting as B-cell growth and differentiation factors (BGDF), was used as the antigenic stimulus. It was found that murine interleukin 1 prepared by recombinant DNA technology was an effective, although weak, BGDF when acting with antigen on B cells cultured either under filler cell-free conditions or in the presence of 3T3 cells. When the murine interleukin 1 was used in combination with recombinant human interleukin 2, itself a weak but effective BGDF in the system, an additive effect was observed. The results challenge the notion that interleukin 1 is exclusively or even primarily an activating cytokine. This system, in which pure factors are able to act with specific antigen on single hapten-specific B cells, will prove helpful for the further dissection of the respective roles of the various factors that can act on B cells.

Animals↗
PubMed1985

The induction of immunologic tolerance in newborn mice by spleen cells differing in H-2K or H-2D, but not "I-J," genotype.

Newborn mice of various strains belonging to the B10A series of recombinants received injections of spleen cells from adult donors to induce immunologic tolerance to antigens of the major histocompatibility complex (MHC). Donor-host combinations were chosen so as to provide differences at H-2K or H-2D, together with various portions of the Ia region. The experiments were predicated on the hypothesis that differences at "I-J" might be required for activation of suppressor cells--thus for the induction of the tolerant state. Tolerance was assessed both by skin grafting and by enumeration of antiallogeneic cytotoxic T lymphocyte precursors (CTL-P) through in vitro limiting-dilution cloning analysis. Host mice that differed from the donor strain only at H-2D, or at H-2K and H-2I-A, were rendered tolerant just as readily as those that differed at "I-J" plus H-2D or "I-J" plus H-2K and H-2I-A. The hypothesis that "I-J" differences are essential for tolerance induction was thus clearly negated.

Animals↗
PubMed1985

The biotechnology revolution and new vaccines.

The author reviews the principles underlying immunization and explains the basis of currently available vaccines. He then describes 2 new biotechnologies, namely peptide synthesis and genetic engineering, which have the capacity to produce both molecular vaccines and live, attenuated vaccines, of an extraordinary range. He mentions the limitations in the strength of some of the experimental vaccines and research being performed to overcome these. Examples are given of some of the vaccines in the research pipeline -- such as those against malaria, hepatitis B and a variety of diarrheal diseases including typhoid, cholera, and bacillary dysentery. He makes reference to new ways of using old vaccines, such as an inhaled aerosolized version of the measles vaccine.

Delivery of Health Care↗
PubMed1984

The double cascade of lymphoid proliferation: current challenges and problems areas.

Two largely but not entirely separate cascades of proliferation and differentiation are involved in immunity. First, processes that are mainly independent of the entry of antigen into the body generate the two types of lymphocytes, T cells and B cells. This lymphoneogenesis proceeds in the thymus and the bone marrow, respectively. It creates families and subfamilies of nondividing cells that do nothing but patrol the body until antigen enters. Each lymphocyte set represents a repertoire of recognition units preequipped with receptors for antigen, and each individual lymphocyte carries its own particular specificity as a result of a somatic rearrangement of genes. When an antigenic stimulus is given, the second and antigen-dependent wave of proliferation and differentiation occurs. This involves clonal selection of particular lymphocytes within the repertoire with specificity for the antigen, and the generation of both immunologic effector cells and memory cells. This second cellular cascade takes place chiefly in the secondary lymphoid tissues, e.g., lymph nodes or spleen. The developing immune system learns to discriminate self from not-self. The paper outlines the chief mechanisms operative in immunologic tolerance and also briefly addresses the fact that T lymphocytes must learn to "see" antigens in association with a "self" major-histocompatibility-complex antigen. Both proliferative cascades depend on intercellular interactions, but much more is known about those operative in antigen-driven lymphocyte activation. Here there is a cyclical process in which antigen-trapping cells stimulate helper/inducer T cells, which in turn stimulate cytotoxic/suppressor T cells or B cells. Some T-cell products can augment the antigen-trapping cells' inductive role.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Human interleukin 2 can promote the growth and differentiation of single hapten-specific B cells in the presence of specific antigen.

B lymphocytes specifically reactive to the hapten fluorescein (FLU) were prepared from normal adult murine spleen by the hapten-gelatin affinity procedure. They were placed in 10 microliter of microcultures singly or in small numbers in the absence of any feeder, filler, or accessory cell. The "T cell-independent" antigen FLU-conjugated polymerized flagellin (FLU-POL) or a selected batch of FLU-conjugated Ficoll were used, and these stimulated division and differentiation only in the concomitant presence of lymphokines acting as B-cell growth and differentiation factors (BGDF). It was found that human interleukin 2 (IL-2), prepared by recombinant DNA technology (r-IL-2), was an effective, albeit rather weak, BGDF in this system. When an IL-2-free source of BGDF was used with the antigenic stimulus, addition of r-IL-2 did not augment the response, nor did removal of IL-2 from the crude lymphokine mixture diminish the BGDF activity.

Animals↗
PubMed1984

Single-cell studies on hapten-specific B cells: response to T-cell-dependent antigens.

The effectiveness of the hapten-gelatin antigen-affinity fractionation technique for selection of hapten-specific B cells activatable by "T-cell-dependent" (TD) stimuli was assessed. Normal adult murine spleen cells were fractionated on fluorescein (Flu)-gelatin layers and the adherent cells were cultured singly or in small numbers with various sources of syngeneic keyhole limpet hemocyanin (KLH)-primed T lymphocytes. Conditions were defined under which the addition of Flu-KLH caused optimal clonal proliferation and differentiation of B cells into anti-Flu directly hemolytic plaque-forming cells (pfc). It was found that 3-5% of the Flu-specific B cells could be activated, versus 1 in 5000 unfractionated spleen cells. The mean enrichment factor for fractionation was 179, almost identical to that seen when the stimulus is "T-cell-independent" (TI), showing that the method is capable of isolating B cells responsive to antigenic stimuli requiring specific T-cell help. Efforts were made to determine whether TD B cells constituted a separate population from TI B cells by determining clone frequencies using Flu-KLH, the TI antigen Flu-polymerized flagellin (Flu-POL), or a mixture of both for stimulation. With Flu-POL alone and with the mixed stimulus 2-3 times more pfc clones were produced than with Flu-KLH, yet evidence for separate B-cell subsets was not obtained because of strong "bystander" stimulation due to the presence of the carrier-primed T cells in a confined volume of 10 microliters.

Animals↗
PubMed1984

A reappraisal of "T-independent" antigens. I. Effect of lymphokines on the response of single adult hapten-specific B lymphocytes.

Four supposedly T-independent antigens, fluorescein (FLU)-E. coli lipopolysaccharide (LPS), FLU-Brucella abortus (FLU-BA), FLU-Ficoll, and FLU-polymerized flagellin (FLU-POL), were tested for their capacity to stimulate B cell proliferation and antibody formation. Single, isolated FLU-specific adult murine splenic B lymphocytes were used as the unequivocal target cell in 10-microliter cultures unsupported by accessory, feeder or filler cells. The stimulatory capacity of four supposed mitogens, LPS, dextran sulfate (DXS), BA, and Ficoll, and of one T-dependent antigen, FLU-keyhole limpet hemocyanin (FLU-KLH), was also studied. Stimuli were used over a wide range of concentrations with or without added T cell-derived, antigen-nonspecific factors promoting B cell growth and differentiation (BGDA). The results allowed triggering stimuli to be divided into four groups: 1) FLU-KLH failed to stimulate single B cells even in the presence of BGDA as did unconjugated Ficoll; 2) FLU-POL was stimulatory only when BGDA was also present, as was unconjugated BA; 3) FLU-BA was slightly stimulatory in the absence of BGDA, but much more so in the presence of BGDA, as was a low concentration of LPS; 4) FLU-LPS and FLU-Ficoll were powerfully stimulatory over a 100,000-fold range of concentrations, and at no concentration did BGDA affect their capacity. LPS or LPS + DXS at high concentrations behaved similarly. The differing behavior of the various conjugates did not correlate either with their supposed "TI-1" or "TI-2" status or with the mitogenic properties of the carrier portion of the antigen. For example, FLU-Ficoll at 0.01 microgram/ml caused 28% of FLU-specific B cells to form proliferating clones, 70% of which secreted immunoglobulin, but a 1000-fold higher concentration of Ficoll failed to stimulate single cells. It is tempting to regard stimuli in group 4 as truly T-independent. However, the intentional addition of thymus filler cells to single B cells triggered by these agents markedly raised antibody formation. Until the nature of this further "helper" effect is understood, it would be unwise to ignore the possibility that T cells or accessory cells are involved.

Animals↗
PubMed1984

A reappraisal of "T-independent" antigens. II. Studies on single, hapten-specific B cells from neonatal CBA/H or CBA/N mice fail to support classification into TI-1 and TI-2 categories.

Spleen cells from adult CBA/H or CBA/N mice, or from neonatal CBA/H mice, were fractionated on thin layers of fluorescein (FLU)-gelatin to yield FLU-specific B lymphocytes. A single cell, or small numbers ranging from 1 to 10, were cultured in 10-microliter microcultures together with various antigens and mitogens. The results were compared with those of bulk culture or limiting dilution cultures supported by thymus filler cells. B cell growth and differentiation-promoting conditioned media (BGDA) were added to some cultures. The CBA/N results gave no support to the commonly used classification of T cell-independent (TI) antigens into TI-1 and TI-2 categories. A typical supposed TI-1 antigen, FLU-LPS, strongly stimulated normal adult single FLU-specific B cells to proliferate and form antibody, but virtually failed to trigger CBA/N B cells of comparable antigen-binding avidity. The same was true of LPS or LPS plus dextran sulfate acting as mitogens. The allegedly TI-2 antigen FLU-Ficoll, although still triggering comparatively poor responses, was actually marginally more active than FLU-LPS. FLU-Brucella abortus (FLU-BA) + BGDA gave the best results with single CBA/N B cells, but still induced only 1.27% of cells to develop into antibody-forming clones vs 12.2% with CBA/H cells. The results obtained with single neonatal B cells also lent no support to the distinction between TI-1 and TI-2. Both "TI-1" and "TI-2" stimuli caused adequate proliferation, one "TI-2" antigen stimulating 23.2% of the cells. None of the antigens caused good antibody formation, however, probably because multivalent antigens can deliver signals impeding the differentiation of immature B cells. It is therefore suggested that the classification of TI-1 antigens into two subcategories be abandoned, at least for the time being.

Aging↗
PubMed1983

Clonal anergy: inhibition of antigen-driven proliferation among single B lymphocytes from tolerant animals, and partial breakage of anergy by mitogens.

Mice were injected with fluoresceinated human gamma globulin (FLU-HGG) either at 2-3 days of age or as pregnant females. At 2 weeks of age, the spleen cells of the injected suckling mice or offspring were fractionated on FLU-gelatin dishes to yield FLU-binding B cells. These B cells were then cloned in microcultures using one of two recently described systems in which single B cells grow in the absence of feeder or filler cells, namely following stimulation with FLU-polymerized flagellin (FLU-POL) and conditioned media containing B cell growth and differentiation factor(s); or mitogenic activation by a mixture of E. coli lipopolysaccharide (LPS) and dextran sulfate (DxS). As such cultures permit visualization of clonal proliferation as well as ultimate harvesting of cultures for assay of hemolytic plaque-forming cells, it was possible to ask whether the lesion in the tolerant state affected the B cell's capacity to divide, to differentiate to antibody secretion, or both. The results indicated that, when stimulated with antigen, the anergic cells could neither divide nor differentiate. However, when the strong mitogen mixture was used, clonal anergy was partially broken. The cells proliferated, and a small proportion of them differentiated into anti-FLU antibody-forming cells. A marked variation in antigen-binding avidity of the FLU-binding cells made it difficult to quantitate the degree of uncoupling of proliferation and differentiation among tolerant, LPS plus DxS-stimulated cells. Nevertheless, a partial reversibility of clonal anergy must affect views on mechanisms of self-tolerance.

Animals↗
PubMed1983

Functional clonal deletion of cytotoxic T-lymphocyte precursors in chimeric thymus produced in vitro from embryonic Anlagen.

Chimeric thymus, formed by fusing the prelymphoid third pharyngeal pouches of fetal mice with fetal liver, have been allowed to develop entirely in vitro. Syngeneic and allogeneic chimeras were prepared and both types of thymus were shown to contain substantial numbers of functional cytotoxic T lymphocyte precursors reactive against "third party" alloantigens. However, alloreactivity specific for H-2 antigens present on either the third pharyngeal pouch or the fetal liver was minimal. In three different allogeneic chimeric thymuses, the frequencies of cytotoxic T lymphocyte precursors reactive to H-2 antigens present on the third pharyngeal pouches were reduced to 1%, 4%, and 0% of control values, whereas, in the one allogeneic chimera tested for alloreactivity to H-2 antigens present on the fetal liver, the cytotoxic T lymphocyte precursor frequency was reduced to less than 1% of control values. The phenotype of the H-2 tolerance is shown to be one of functional clonal deletion of the cytotoxic T lymphocyte precursor.

Animals↗
PubMed1983

Clones of cytotoxic T lymphocytes reactive to haptenated allogeneic cells: precursor frequency and characteristics as determined by a split-culture approach.

CBA (H-2k) responder spleen cells have been cultured at limit dilution with trinitrophenyl (TNP)-modified BALB/c (H-2d) stimulator cells and a source of T-cell growth factor in order to generate cytotoxic effector clones. After culture, such clones were split into two to four replicates and each was assayed against a different target. This allowed identification of clones capable of lysing TNP-modified P815 (H-2d) targets but not unmodified P815 targets. Thus, clones specific for TNP and allogeneic restriction elements were detected without the need to use techniques that deplete the responder population of alloreactive cells. Cytotoxic T lymphocyte precursors (CTL-P) specific for TNP-modified P815 (major histocompatibility complex-nonidentical) targets were identified, at a low frequency (28.2 x 10(-6)) compared to CTL-P for TNP-modified C1.18 (H-2k) (identical) targets (224 x 10(-6)). The hapten specificity, H-2 restriction specificity, and Thy-1 status of these clones have been examined. Fourteen percent of CBA CTL-P reactive to TNP-modified P815 targets also showed reactivity to NIP-modified P815 targets, and 86% of CBA clones reactive to TNP-modified P815 targets ("allo-TNP-reactive" clones) failed to show reactivity to TNP-modified C1.18 targets--i.e., showed a restriction preference for allo rather than self. All such H-2d-restricted, TNP-specific clones were uniformly sensitive to anti-Thy-1 antibody and complement. Among the H-2k responders studied, we have not demonstrated CTL-P reactive to TNP-modified syngeneic cells which also react with H-2d cells or NIP-modified H-2d cells among 168 clones analyzed. This suggests that such clones, if present, are relatively rare.

Animals↗