A modification of the hemolytic plaque assay using protein A-coated red cells is described which makes use of the fact that the Fc portion of IgG binds to protein A. A number of murine plasmacytomas secreting different classes of Ig have been tested for plaque formation with these indicator red cells. In the presence of complement-binding antibodies specific for the corresponding class of secreted Ig, between 10 and 70% of all plated myeloma cells formed plaques. The assay shows a prozone effect in excess of antibody, suggesting that complexes of antibody and secreted Ig effect lysis of the target cells. This assay can be used to enumerate cells secreting any molecules for which complement-binding antibodies are available.

771should permit an analysis of the mechanism of this form of suppression. The nature of the suppressive factor in this system will be of interest, as already there are reports of two antigen-specific T cell suppressor factors; a non-immunoglobulin factor, of mol. wt. 40-50 000 [30] active in vivo, and the hypothetic IgT in supraoptimal concentrations [3 11, suppressive in vitro.

We have suggested before that thymus-independent immune responses to certain antigens are caused by special properties of the antigens concerned, namely that they are B-cell mitogens (1). Actually all thymus-independent antigens (TI) 1 we have tested so far were found to be competent to activate DNA and polyclonal antibody synthesis in nonprimed B cells cultured in vitro (2). Since the TI antigens can directly induce division and differentiation in B cells, it is evident that T-B cells cooperation would not be required for the induction of specific immune responses. However, there is an apparent paradox between the ability of a mitogen to induce an antigen-specific immune response to its own antigenic determinants, and its ability to activate polyclonal antibody synthesis in all B cells, irrespective of their specificities. The paradox can be easily explained by assuming two fundamentally different types of antigen binding to the cell surfaces. At low (immunogenic) concentrations, TI antigens would be preferentially bound by lg receptors on specific antigen-binding B cells, whereas at high (mitogenic) concentrations also, other B cells (lacking specific receptors for the antigenic determinants of these molecules) would bind a sufficient number of molecules to become activated. Once triggering concentrations were reached on the cell surface, the cell would be activated and secrete the immunoglobulin for which it is genetically programmed. In terms of this argument, T-cell independence could be considered the most sensitive test for B-cell mitogenicity, since it is the "physiological" expression of direct B-cell activation.The present experiments were designed to test this hypothesis critically, by use of the hapten 4-hydroxy-3,5-dinitrophenyl (NNP) coupled to the B-cell mitogen lipopolysaccharide (LPS) (3).

Two parameters of the unresponsiveness to lipopolysaccharides from Escherichia coli (LPS) displayed by C3H/HeJ mice, namely endotoxemia and the intraperitoneal extravascular leukocyte responses to small doses of LPS, have been studied by Sultzer and found to be under polygenic control (1, 2) . Recently, however, Watson and Riblet (3) presented evidence interpreted as indicating that a single gene was responsible for influencing both mitogenic (polyclonal) and immunogenic (specific) responses to LPS, by analysing crosses between LPS low-responder mice and high-responder strains. Since the B-cell unresponsiveness of C3H/HeJ mice has been shown to depend upon a pure defect in the subpopulation of B cells which interacts with and responds to LPS in the conventional strains,' the finding of Watson and Riblet is of great importance . Thus, it seems possible that the defective gene in C3H/HeJ mice would code for the B-cell surface structure involved in cell triggering, both in polyclonal (4) and in specific (3, 5, 6) antibody responses. According to our current view in B-cell activation, such "mitogen receptor" is the only structure on the B-cell surface which is competent to deliver activating signals to the resting cell (7). If this were the case, C3H/HeJ mice would be of great importance for elucidating the molecular mechanisms governing the generation and delivery of triggering signals. We report in this paper evidence on the inheritance of the genetical defect displayed by C3H/HeJ mice with regard to the direct B-cell responses to LPS as evidenced by polyclonal and specific responses as well . Materials and MethodsC3H/HeJ (H-2') mice were obtained from Jackson Laboratories, Bar Harbor, Maine, and bred in our colony for the last 3 yr. Other mice obtained from that source were also directly used in some experiments . The high-responder strains used in these experiments were C3H/Tif (H-2'°) obtained from Bomholtgaard, Rye, Denmark, and B10.5M (H-2°) from our own colony .LPS from Escherichia coli 055:135 obtained by phenol-water extraction (8) was used throughout these experiments . The hapten (4-hydroxy-3,5-dinitrophenyl)acetyl (NNP) was conjugated to LPS as previously described (9) and the biological characterization of the conjugate used in the present

No abstract

The responses to the polyclonal B‐cell activators dextran sulphate (DxS), lipo polysaccharide (LPS), and purified protein derivative from tuberculin (PPD) were followed along the differentiation process of fetal liver cells to mature B cells in irradiated hosts It was shown that these cells sequentially gained responsiveness to DxS. LPS, and PPD, in that order. It was also shown that the result of activation depends only on the differentiation degree of the target cells at the turn they are activated. Thus, more primitive cells can only divide, and most of the DxS‐. LPS‐, or PPD‐induced blasts are lg‐negative soon after the cells become responsive to each ligand. The response of more differentiated cells is characterized also by high‐rate antibody synthesis. These results provide a unique possibility of using polyclonal B‐cell activators as well‐defined functional markers for sub‐populations of B cells and shed a new light on the problem of immune B‐cell triggering

No abstract

Immunological tolerance was induced in adult mice by the injection of 5 mg of deaggregated hapten-protein conjugate. The tolerant state was confirmed 4-19 days later by the failure of such animals to mount an immune response against an aggregated form of the same thymus-dependent hapten-protein conjugate as well as by the inability of spleen cells from tolerant animals to respond to a thymus-independent hapten-carrier conjugate. Even though the animals were fully tolerant, their spleen cells were activated by lipopolysaccharide (LPS) in vitro to produce normal numbers of plaque-forming cells against the hapten. The finding that spleen cells from tolerant animals could be activated by LPS into synthesis of antibodies against the tolerogen indicates that tolerance to thymus-dependent antigens does not affect B cells, but presumably only T cells. It is suggested that the only stringent test for the existence of B-cell tolerance is the inability of polyclonal B-cell activators to activate antibody synthesis against the tolerogen. The findings make it unlikely that B-cell tolerance to autologous thymus-dependent antigens exists and further indicate that such antigens cannot deliver activating or tolerogenic signals to B cells, although they are competent to combine with and block the Ig receptors.