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This paper uses B-lymphocyte cloning methods to quantify the effects of anti-μ chain antibody on immature and mature B cells. Nude mouse spleen lymphocytes were incubated with various concentrations of sheep anti-mouse μ chain antibody for times varying from 10 min to 24 h. They were then washed and plated in the agar B-cell colony formation assay. Five to six days later, control B cells had developed into colonies with a plating efficiency of about 5%. B cells from newborn mice pretreated with anti-μ yielded fewer colonies. Remarkably low concentrations sufficed to inhibit subsequent mitogenesis. For example, 3 μg/ml acting for 1 h or 0.1 μg/ml acting for 24 h gave > 50% inhibition. Adult B cells were about thirty-fold more resistant to negative signalling. Immature cells became more profoundly inhibited as anti-μ treatment was prolonged. Anti-Ia or anti-H2 antibodies, in the absence of complement, did not deliver a negative signal. Anti-μ pretreatment also reduced the capacity of immature B cells to form clones of anti-hapten antibody-forming cells in a liquid microculture system where the triggering stimulus was a T-cell independent antigen. Mature 'T-independent' B cells were not inhibited. Populations of hapten-specific B cells prepared by the hapten-gelatin method were investigated in the agar cloning system. Pretreatment of immature cells with anti-μ reduced their capacity to form colonies, this subpopulation of cells behaving like unfractionated B cells. Furthermore, hapten--HGG delivered a negative signal also. Mature hapten-specific cells or unfractionated immature spleen cells formed normal numbers of colonies following hapten HGG treatment. Overall, the studies support the view that anti-μ antibody and hapten--HGG deliver strong negative signals to immature but not mature cells with appropriate receptors. The value of anti-μ as a model, universal tolerogen was supported. Fluorescence-activated cell sorter (FACS) analysis was performed to study the relationships between functional inhibition and Ig receptor modulation. We confirmed that the IgM receptors of immature B cells are more readily modulated by anti-μ antibody than those of mature cells. Furthermore, the receptor regeneration could be partially inhibited amongst immature but not mature B cells. There was not a close quantitative relationship between the degree of modulation and the degree of functional inhibition. The results did not support the view that irreversible receptor modulation as such was the cause of functional inhibition. [ABSTRACT FROM AUTHOR]

This study addresses itself to the problem of antibody formation in vitro by mouse splenic B lymphocytes enriched for reactivity to the hapten NIP by the hapten-gelatine binding and melting technique of Haas and Layton (1975). Small numbers of NIP-gelatine-bound B cells were placed in micro- cultures either by bulk dispensing of dilute cell suspensions, or by micromanipulation under direct microscopic visualization. Antibody formation was induced by the T cell-independent hapten-protein conjugate NIP-polymerized flagellin, using 104 thymus cells per microlitre as 'filler' cells. The frequency of precursors of NIP-specific antibody- forming cells among bound cells was about 2.2 × 10-2 (one cell in forty-five) by both statistical and direct evaluation, after adjustment for a background frequency of 6-10 × 10-8 precursors in the thymus filler cells. Single clones commenced antibody secretion asynchronously, as shown by the fact that the incidence of positive cultures continued to rise over the whole three days of culture, and that very small clones of one to four plaque-forming cells (PFC) were still found on day 3. The mean PFC number per positive culture rose from 1.2 at day 1 to 4.7 at day 2 and about 20 at day 3. Negative feedback, presumably mediated by antigen-antibody complexes, was shown to be a powerful effect in microcultures containing bound cells. It was evident with 250 cells per well (five clones) and the effect was progressive, such that with 10⊃ bound cells per well, plaque numbers were 40-50 per cent of their potential optimum; with 104, 10 per cent, and 105, 1 per cent. With routine techniques of harvesting of cultured cells, a failure to disperse PFC uniformly into single cells was noted. Groups of two or more large cells of very similar morphology were frequently observed at the centre of haemolytic plaques. Micromanipulation studies revealed that essentially all large cells at the centre of plaques were PFC. It was found that a routine plaque count underestimated the true PFC number per culture by a factor of 2. In the absence of negative feedback, the system is capable of generating one PFC per two input B lymphocytes. This is by far the most efficient plaque- generating system yet described, and contrasts with a yield of one PFC per 104 input cells in most typical tissue culture systems. Reasons for believing that both the culture and the enrichment systems can be further improved are discussed, and the potential of the method for understanding the cell and molecular biology of lymphocyte triggering is described. [ABSTRACT FROM AUTHOR]

The capacity of CBA mouse B lymphocytes to bind polyvalent rabbit anti-mouse immunoglobulin antibody, even when this reagent is present in low concentration, was used as an index of a lymphocyte's B cell status. Various mouse tissues were held for 30 minutes at 0° with 0·2 μg/ml of 125I-labelled anti-Ig, and smear preparations of washed cells were examined, following radioautography, for their content of B cells. The survey covered two areas of B cell differentiation, namely the spontaneous emergence of B cells in the mouse foetus, and the development of B cells in lethally irradiated mice that had received early foetal liver as a source of haematogenous stem cells. The foetal survey suggested a multifocal origin of B cells, commencing 3 days before birth, in all major sites of erythromyelopoiesis, namely liver, spleen and bone marrow. Lymph nodes showed B cells later than did these organs, and thymus contained virtually no B cells at any stage. A rapid influx of B cells into lymph nodes took place shortly after birth. The CBA mouse is born with somewhat over 105 B lymphocytes in toto, of which the majority are in liver, spleen and blood. The irradiation-recovery study showed: (1) that B cells are, statistically, more sensitive to high dose irradiation than T cells; (2) that a B cell-free stem cell source can repopulate the B cell pool; (3) that repopulation is surprisingly slow; and (4) that numerical and functional recovery of B cells parallel each other to a reasonable degree. The significance of the results is discussed from the viewpoints of lymphocyte differentiation and immunological tolerance. [ABSTRACT FROM AUTHOR]