Inhibition of mitogenic stimulation of mouse I y m phocytes by ant i-mouse i m munogl obu I in antibodies 1. Mode of actionAntibodies with specificities for mouse p-chains and mouse K-and X-chains inhibit the stimulation of the major fraction of splenic lymphocytes from nude mice by lipopolysaccharide (LPS), by the purified protein derivative of tuberculin (PPD) and by fetal calf serum (FCS) t o differentiate into immunoglobulin (1g)-secreting, plaque-forming cells (PFC). Antibodies with specificities for mouse y-chains or with Ig-unrelated specificities (anti-ovalbumin, anti-E. coli 0-galactosidase) d o not elicit this inhibition. Bivalent pepsin (Fab')* fragments mediate the same inhibition at the same concentrations, while monovalent papain Fab fragments of such anti-Ig antibodies have a 100-fold lower capacity t o inhibit differentiation into PFC. Rabbit as well as chicken anti-Ig antibodies are inhibitory; this indicates that the inhibition is not complementmediated. Cells stimulated for 6 h by LPS can no longer be inhibited by anti-Ig antibodies from differentiating to PFC, a finding which indicates that changes take place in the mitogen-stimulated cells which may involve surface-bound Ig molecules. Inhibition can be abrogated, but only prior t o the addition of mitogen to the cells, by proteolytic treatment of the cells or by dissociation of the anti-Ig antibodies in excess medium with time from the cells.Inhibition of DNA synthesis by anti-Ig antibodies in splenic lymphocytes is more complex and will be described in a subsequent publication,

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).

holmNormal mouse spleen cells initiate synthesis of immunoglobulins in vitro in the presence of fetal calf serum and in the absence of specific antigen following the removal of regulatory suppressor(s) present in normal syngeneic mouse serum.

Two major events occur in the response of the immune system to antigenic stimulation, proliferation of cells and secretion of soluble products. The relative importance of these two events is not well known. Mitogens have been found to provide valuable tools for analysis of these problems (for review, see reference 1). The substances most well studied are phytohemagglutinin (PHA) 1 and concanavalin A, which have been found to selectively activate thymus-derived (T) cells (2-6), and lipopolysaccharides of bacterial origin (LPS), which have been found to selectively activate bursal equivalent or bone marrow-derived (B) cells (2, 7). Pokeweed mitogen seems to activate both cell types (4).Recently we reported (8) that purified protein derivative (PPD) tuberculin, which has previously been associated only with a number of supposed T cell activities in immune situations, induces D N A synthesis in nonimmune mouse B cells, i.e., acts as a B cell mitogen. The present work reports our analysis of a second major activity of PPD, namely induction of immunoglobulin synthesis.