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G J Nossal

Publications and source records attributed to G J Nossal.

At least 127 records · Page 7Linked to original sources
PubMed1983

Analysis of true anti-hapten cytotoxic clones in limit dilution microcultures after correction for "anti-self" activity: precursor frequencies, Ly-2 and Thy-1 phenotype, specificity, and statistical methods.

By the use of split culture techniques we have been able to demonstrate conclusively that the "anti-self" activity and the spontaneous anti-hapten activity within an apparent anti-hapten cytotoxic T cell response is a clonal phenomenon and is not caused by cross-reactivity between anti-self and true anti-hapten clones. With the knowledge of this clonality we have been able formally to prove that the true hapten-generated, hapten-specific response can be obtained by subtracting the response generated by "self" but directed against modified targets from the response generated by modified self and directed against modified targets. Unbiased statistical estimators, which do not make assumptions about the origin (0 responder cells, 100% negative cultures), have been developed to plot accurately limit dilution data (maximal likelihood estimator and minimal chi-square estimator), and the analysis demonstrates that all the components of an apparent anti-hapten response obey zero order (single hit) kinetics. By specifically identifying true anti-hapten and true anti-self clones, we have been able to study the phenotype of their precursors as well as the effectors themselves at the clonal level. Precursors of true anti-hapten and anti-self clones are Thy-1+, Ly-2+. However, anti-hapten and anti-self effector cells show marked clonal variation with respect to Ly-2, as some clones are almost completely insensitive to anti-Ly-2 and complement whereas others show minimal to complete sensitivity. All anti-hapten clones are completely sensitive to anti-Thy-1 and complement, whereas about one-third of anti-self clones show only partial sensitivity, with the most lytic clones showing the most sensitivity. Hapten-specificity and anti-self-specificity have been examined clonally. Ten percent of anti-TNP clones recognize NIP, 10% of anti-NIP clones recognize TNP, and 20% of anti-self clones recognize an allo-target. These figures are in accordance with the overall specificity of effectors generated in bulk culture in the presence of CAS (concanavalin A-stimulated spleen cell conditioned medium). However, hapten specificity and H-2 restriction of bulk generated effectors are improved if CAS is omitted from cultures.

Animals↗
PubMed1983

Single cell studies on hapten-specific B lymphocytes: differential cloning efficiency of cells of various sizes.

Three separate forms of in vitro stimulation were assessed for their capacity to activate hapten-gelatin fractionated, fluorescein- (Flu) specific murine splenic B lymphocytes. They were: a) Flu-polymerized flagellin (Flu-POL) acting on a single Flu-specific B cell in microculture in the absence of "filler" or feeder cells, but in the presence of T cell-derived B cell growth and differentiation factor(s) (BGDF); b) a mixture of mitogens, E. coli lipopolysaccharide (LPS) and dextran sulfate, acting on a single Flu-specific B cell in the absence of added BGDF; and c) Flu-POL plus BGDF acting on single Flu-specific B cells as in a but with thymus filler cells also present. System c was markedly superior in causing antibody formation, 15 to 22% of cells forming a clone of Flu-specific antibody-forming cells (AFC), in contrast to 6% for system b and 3 to 6% for system a. Each stimulus was applied to single cells that had been size fractionated into samples of increasing size by using the forward light-scattering parameter of the fluorescence-activated cell sorter. Surprisingly, the smaller sized fractions proliferated poorly in system a and contributed less than 10% of the antibody-forming potential of the total population. The smaller cells proliferated better in system b, but only 10 to 15% of proliferating clones generated Flu-specific AFC, whereas the larger cells contributed 86% of the total AFC response. Even in system c, only 6% of the small cells formed AFC clones compared with 41% of the larger cells. It thus appears that the smaller half of murine B lymphocytes is relatively resistant to activation into proliferation and differentiation by "T-independent" antigens; when activated by mitogens, they clone less efficiently than larger cells. Despite these limitations, system c could generate a total of up to four hapten-specific AFC for every B cell placed into culture, making it the most efficient system of specific antibody formation yet described.

Animals↗
PubMed1983

Functional clonal deletion and suppression as complementary mechanisms operative in adult hapten-induced cytotoxic T cell tolerance.

Immunologic tolerance to the hapten TNP was induced in adult mice through the i.v. injection of reactive TNBS. To probe the cellular basis of the tolerant state, splenic cytotoxic T lymphocyte precursors (CTL-P) were stimulated in vitro with haptenated, x-irradiated syngeneic spleen cells in the presence or absence of exogenously added growth factors derived from Concanavalin A-stimulated spleen cell conditioned medium (CAS). The cultures were either conventional bulk cultures or limit dilution cloning cultures. For the latter, cytotoxicity was assessed through a semi-automated, radioautographic 111In-release assay. Suppressive potential was assessed by mixing spleen cells from tolerant mice with normal spleen cells before culture. In the absence of CAS, bulk cultures showed profound tolerance, and suppressive capacity was clearly evident. Suppression was dependent on the presence of TNP-self during culture and affected the generation of CTL from CTL-P and not the effector function of CTL. Cyclophosphamide treatment did not prevent tolerance induction. In the presence of CAS, bulk cultures still showed marked tolerance, but mixing experiments now yielded no evidence of suppression. As documented previously, limit dilution cultures of tolerant spleen cells in the presence of CAS showed a functional clonal deletion of hapten-specific CTL-P. In the absence of CAS, limit dilution cultures became dependent on helper T cells as the limiting element. Tolerant populations showed a diminution of activatable helper T lymphocyte precursors (HTL-P), which may have been due to a functional clonal deletion of HTL-P and/or a concomitant activation of suppressor T cells. Adoptive transfer studies showed that cells from tolerant mice did not detectably influence the number of hapten-specific CTL-P in the spleens of host animals. Taken together, the results suggest that both functional clonal deletion of CTL-P and suppression of HTL-P contribute to the tolerant state induced.

Animals↗
PubMed1983

Characteristics of tolerance induction among adult hapten-specific T lymphocyte precursors revealed by clonal analysis.

Thaptens 2,4,6-trinitrobenzene sulfonic acid (TNBS) and 3-iodo-4-hydroxy-5-nitrophenyl-acetic acid succinamide ester (NIP) on murine splenic and thymic cytotoxic T lymphocyte precursors (CTLp) was studied. Both BALB/c (H-2d) and CBA (H-2k) CTLp can be rendered unresponsive to subsequent in vitro stimulation with either hapten, provided the mice were pretreated with the correct dose of reactive hapten. The unresponsiveness was shown not to be due to toxicity, because treated animals can mount an alloreactive response equivalent to untreated animals. A degree of cross-reactivity in tolerance induction, however, was demonstrated between the two different haptens. Limit dilution analysis of tolerant lymphoid populations reveals that such cells obey single hit (zero order) kinetics and that there is effective deletion of the CTLp. After correction for "anti-self" responses, the anti-TNP CTLp frequency in TNBS-treated BALB/c mice was 10.7 X 10(-6) in contrast to 101.1 X 10(-6) in control animals (89.4% tolerance). Similarly, after correction for "anti-self" responses, the anti-NIP CTLp frequency in NIP-treated animals was 4.7 X 10(-6), in comparison to 42.8 X 10(-6) in control animals (89.0% tolerance). Kinetic studies show that tolerance occurs within 24 hr. This, together with the limit dilution data, argues against the role of suppressor cells, over the cellular range studied.

Animals↗
PubMed1982

A method for the accurate determination of anti-hapten cytotoxic T lymphocyte precursors: correction for apparent 'anti-self' reactivity.

A method is described for accurately determining the frequency of precursors of hapten specific cytotoxic T cells. The method is based on a standard Poisson analysis of limit dilution cultures, but makes a correction of 'anti-self' reacting clones and for spontaneously arising clones that recognise modified self. These corrections are shown to be especially important when low hapten densities are used, where there may be more than a 10-fold difference between the corrected and uncorrected frequency estimates. Determined levels of antigen specificity and of H-2 restriction are significantly enhanced by application of this method.

Animals↗
PubMed1982

Proliferation and differentiation of single hapten-specific B lymphocytes is promoted by T-cell factor(s) distinct from T-cell growth factor.

Hapten-specific B lymphocytes reactive to fluorescein were prepared from mouse spleen, placed singly in 10-microliters culture wells, and stimulated with fluorescein-polymerized flagellin in the presence of conditioned media (CM) from various concanavalin A-stimulated cloned T-cell tumors or hybridomas. Antigen plus appropriate CM triggered 5-9% of the B cells into both clonal proliferation and differentiation into antibody-forming cells. Antigen alone stimulated 0.5-0.8% of B cells and CM alone stimulated less than 0.1%. This bioactivity was termed B-cell growth and differentiation factor(s) (BGDF). Four CM rich in T-cell growth factor (TCGF)--namely, CM from spleen and the lines EL4, T6, and 123--contained BGDF. The lines T19.1 and WEHI-3 lacked BGDF and TCGF. Four lines of evidence suggested that BGDF and TCGF were distinct molecules. First, the BGDF/TCGF ratios in the various CM varied. Second, on gel filtration, TCGF eluted as a sharp peak corresponding to a Mr of about 35,000, whereas BGDF eluted over a range corresponding to a Mr of 25,000-60,000. Third, the activity of TCGF in EL4-CM was markedly reduced by treatment with guanidine HCl while BGDF activity was not. Fourth, BGDF showed more heterogeneity than TCGF on hydrophobic chromatography. All CM or fractions active in promoting B-cell division also promoted differentiation to antibody-forming cells. These results provide unequivocal evidence that antigen and a T-cell product can synergize to directly activate a single B lymphocyte.

Animals↗
PubMed1982

Clonal anergy: the universally anergic B lymphocyte.

The clonal anergy theory of induction of immunological tolerance states that differentiating B lymphocytes that encounter multivalent antigen at the pre-B to B cell transition stage can receive and store a negative signal, which renders them anergic to later triggering stimuli. The theory was tested by using an anti-mu chain monoclonal antibody, E4, as a model tolerogen. The fluorescence-activated cell sorter was used to select B cell-free cell populations from adult murine bone marrow or newborn spleen, and later, to analyze B cell neogenesis in vitro. The presence of E4 at greater than or equal to 1 microgram/ml was required to impede the development of normal numbers of B cells with full receptor status. The subsequent capacity of these B cells to respond in vitro to mitogens was assessed in a filter-cell free microculture system that allows single B cells to proliferate and differentiate. Concentrations of E4 far below those required to affect B cell neogenesis had profound inhibitory effects on the subsequent functional capacity of the B cells. In fact, 10(-3) micrograms/ml of E4 markedly impaired both proliferation and antibody formation, and 10(-1) micrograms/ml, which had no effect on Ig receptor development, abrogated functional capacity. Thus B cells formed in the presence of E4 at 10(-1) micrograms/ml, though possessing the receptor status typical of B cells, were functionally entirely anergic. Exposure to E4 appeared to accelerate the spontaneous death rate of newly formed B cells in vitro. Whether the anergic cell would also have a shortened life span in vivo is not known.

Animals↗
PubMed1981

Antibody production by single, hapten-specific B lymphocytes: an antigen-driven cloning system free of filler or accessory cells.

CBA mouse spleen cells were subjected to hapten affinity fractionation on thin layers of fluorescein (FLU)-gelatin. This procedure yields 97% B cells with varying FLU-binding avidities. One to 30 cells were placed in 10-microliter microcultures without any filler or accessory cells. the T-independent antigen polymerized flagellin coupled to FLU (FLU-POL) was ineffective in stimulating these cells to clonal proliferation or antibody production when used alone. Unpurified preparations rich in interleukins also failed to stimulate the cells. When specific antigen, but not in irrelevant hapten-POL, was combined with the interleukins, clonal proliferation was stimulated and most clones produced anti-FLU antibody-forming cells. The frequency of antibody-forming clones was only slightly lower than that in a system using antigen plus filler cells. In the absence of added interleukins, the mitogens Escherichia coli lipopolysaccharide plus dextran sulfate induced equivalent antibody production. However, a higher frequency of clonal proliferation was noted. Added interleukins did not aid these mitogen-driven responses. Such an antigen-dependent cloning system, free of filler and accessory cells, should permit more precise analysis of the respective roles of antigens and interleukins in the physiology of antibody-forming clone formation.

Animals↗
PubMed1981

Functional clonal deletion in immunological tolerance to major histocompatibility complex antigens.

CBA (H-2k) mice were rendered tolerant to H-2d antigens by injection of (CBA X BALB/c)F1 spleen cells at birth. At intervals of 2 days to 12 weeks, the frequencies of anti-H-2d cytotoxic T lymphocyte precursor cells (CTL-P) in thymus and spleen were determined by using a limiting-dilution microculture assay system for CTL-P. This assay, utilizing irradiated H-2d stimulator cells and concanavalin A-induced spleen cell conditioned medium, was shown to be linear over the range 30 to 100,000 responder cells and uninfluenced by IJ-positive cells. A profound and long-lasting deficit in activatable CTL-P, first demonstrable by day 5 of life in the thymus and day 8-10 in the spleen, developed in mice rendered tolerant, reaching a greater than 95% reduction by 6 weeks. Functional clonal deletion thus seems to be at least as important in the tolerant state as suppressor T cells. Repeated in vivo administration of anti-IJk serum partially inhibited clonal deletion, suggesting either that suppressor T cells are actively involved in producing clonal deletion or that IJk-bearing cells in the donor inoculum or the host represent an important factor.

Age Factors↗
PubMed1980

Relative sensitivity of fetal and newborn mice to induction of hapten-specific B cell tolerance.

Mice were rendered tolerant to the hapten fluorescein (FLU) by a single injection of FLU-human gamma globulin (FLU5HGG) 2-3 d after birth or via the maternal circulation at 14.5 d of fetal life. After 7-9 d, the degree of functional nonresponsiveness induced in vivo among splenic FLU-specific B cells of tolerized mice was assessed by limiting-dilution analysis in vitro, and the serum levels of trace-labeled tolerogen were determined. When tolerogen was introduced before the appearance of any B cells, and was thus present during the pre-B to B cell transition stage, a concentration of 5.4 x 10(-13) M effectively silenced 50% of the clonable anti-FLU PFC precursors; but a similar reduction on newborns required a minimal tolerogen concentration of 1.3 x 10(-10) M, > 300-fold less than has previously been shown to equally affect adult B cells, but at least 240-fold more than in the in utero situation. Neonatally induced tolerance using a relatively high tolerogen dose lasted approximately 12 wk.

Animals↗
PubMed1980

Clonal anergy: persistence in tolerant mice of antigen-binding B lymphocytes incapable of responding to antigen or mitogen.

The purpose of these experiments was to determine the degree of reduction in the number of antigen-binding B lymphocytes in the spleens of mice that had been rendered tolerant in the perinatal period. Newborn or pregnant mice were injected with fluorescein (Flu) coupled onto human gamma globulin, and the spleen cells of the neonatally injected mice, or of the offspring of the pregnant mice, were analyzed 1-6 weeks later. Tolerogen doses were chosen so as to achieve either a two-thirds reduction (low dose) in the number of anti-Flu B cells capable of yielding anti-hapten plaque-forming cell clones after in vitro stimulation, or as representing a supra-optimal tolerogenic stimulus (high dose). Antigen-binding B cells were studied by a two-cycle procedure, namely an initial cycle of binding to Flu-gelatin thin layers, followed by analysis of the binding cells in the fluorescence-activated cell sorter (FACS) after suitable staining with Flu-protein conjugates. With the high dose of tolerogen, a modest diminution in Flu-binding cell numbers down to 56-71% of control values could be induced. When these residual Flu-specific B cells were analyzed in the FACS to quantitate their spectrum of Flu-binding avidities, profiles identical to those of controls were obtained. The reduction proved transient in nature, binding cell numbers having returned to 80% of normal by 2 weeks and to normal by 6 weeks. Nevertheless, the Flu-specific B cells were incapable of responding to antigen or mitogen by antibody formation. With the low dose of tolerogen, despite the desired degree of functional silencing of Flu-specific B cells, the numbers and avidity spectra of antigen-binding cells were entirely normal in both the neonatally injected and in utero-injected groups. The results indicate that tolerance induced amongst immature B lymphocytes is not due to a physical elimination of the relevant B cell clones or to a modulation or blockade of their surface Ig receptors. Rather, it is due to the recognition and storage of negative signals amongst cells that continue to display a normal complement of receptors. We therefore propose that the term "clonal anergy" is a more accurate description than either "clonal deletion" or "clonal abortion."

Animals↗
PubMed1979

In vitro model for natural tolerance to self-antigens. Inhibition of the development of surface-immunoglobulin-negative lymphocytes into T-dependent responsive B cells by antigen.

Neonatal and adult splenic cell suspensions were labeled with fluorescein isothiocynate-anti-Ig and fractionated into surface-immunoglobulin- (s-Ig) positive and s-Ig-negative subpopulations by the fluorescence-activated cell sorter. The subpopulations were then tested by splenic focus assay for both frequency and tolerance susceptibility of clonable 2,4,-dinitrophenol (DNP) precursors. It was shown that both adult, and neonatal, s-Ig-negative subsets contained clonable DNP-specific B-cell precursors. However, because these precursors result in fewer clones secreting IgG, they appeared to be less mature than the s-Ig-positive precursors. In the absence of helper T cells, it was found that exposure of s-Ig-negative lymphocytes to tolerogen during the process in which they were acquiring surface receptors resulted in nearly total abrogation of potential DNP clones. This finding provides compelling evidence for clonal abortion.

Animals↗