Spleen cells of normal mice which were stimulated with either T-dependent or T-independent antigens and dextran sulfate (DxS) in microcultures at limiting dilution show at low cell densities an increased frequency of responding units (B cells) with both types of antigens. At higher cell densities, DxS induces a suppression of the immune response as assessed by a declining frequency of expressed B cells. When T cells were decreased in number, the stimulating effect of DxS on T-independent responses remained unaltered while the suppressive effect (dependent on cell density) was eliminated. It is suggested that DxS lowers the threshold of activation for regulatory T cells, i.e. of cells which under standard in vitro conditions remain unactivated, and which in the presence of DxS become activated. We conclude that the regulatory cells are rare and that they are not present in most low cell density cultures. The net effect is a potent activation in dilute cultures and lack of response in dense cultures.
The requirement of B cells activated by mitogen (dextran sulfate plus lipopolysaccharide) for accessory cells was studied by partition analysis. Small numbers of splenic B cells were activated to clonal growth, as determined by visual inspection, and to immunoglobulin (Ig) synthesis, as determined by release of Ig into the culture fluid. By placing irradiated adherent cells in the periphery of the microculture wells and forcing responding cells to different areas of the well (slant experiments), it was observed that no cell contact was necessary for B cell activation, and that "promoted" contact ("Rock and Roll" experiments) does not increase the efficiency of activation. Sequential microcultures suggest that only some irradiated adherent cells act as accessory cells, but they can perform this function to more than one B cell. Attempts to perform limiting dilution analysis by varying irradiated adherent cell input showed non-single-hit behavior. When the data were rearranged, taking into account the distribution of irradiated adherent cells, then single-hit behavior with about 1 to 5% of irradiated adherent cells acting as an accessory cells for B cell clonal activation was observed. The evidence suggests that an uncommon irradiated adherent cell releases a soluble factor necessary for B cell activation and/or clonal proliferation.
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