The in vitro secondary stimulation of the production of anti-hapten antibody has been analyzed with a view to elucidating the role of the carrier molecule and cell-to-cell interactions in this response. Stimulation was carried out on fragment cultures of the spleens of irradiated BALB/c mice which had been reconstituted with 3–4 x 107 spleen cells from isologous mice previously immunized with DNP-Hy. The results indicated that the response was maximized by stimulation with DNP-Hy, the homologous complex, however anti-DNP antibody production could be obtained by stimulation with DNP on several nonhomologous carriers including poly-D-lysine, a poor immunogen. The results also indicated that while DNP-Hy and DNP-nonhomologous-carrier complexes were stimulatory at equally low DNP concentrations, at DNP concentrations over 10–6 M DNP-Hy was stimulatory, while DNP on nonhomologous carriers was inhibitory. The results are interpreted as indicating that: (a) the affinity of the antigen-cell interaction is more likely determined by multivalent binding than by carrier recognition, (b) that a stimulatory interaction of a polyvalent antigen with a B-lymphocyte cannot be excluded, (c) that if cell-to-cell interaction is necessary for stimulation, then both cells may recognize the same determinant, and (d) that the marked enhancement of antigenic stimulation attributable to carrier recognition may result from stimulatory interactions of cells recognizing different antigenic determinants. A mechanism is postulated whereby stimulation is dependent on the formation of stable complexes resulting from two cells sharing in the binding of numerous antigen molecules. Cells recognizing carrier determinants would increase the probability of such interactions particularly at high antigen concentrations.

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