When normal mouse spleen, cells in suspension are cultured in vitro in the presence of polymer from S. adelaide flagellin, an immune response can be obtained as measured at the level of single antibody-forming cells. Cultures were stimulated with different doses of antigen, ranging from 0.2 ng to 3 µg/ml of tissue culture fluid and it was found that the peak number of approximately 500 antibody-forming cells per 106 harvested cells by day 4 was antigen dose dependent, 2–20 ng/ml being the optimal concentration. When more than 1 µg/ml of polymer from S. adelaide together with either 20 ng/ml of polymer from S. waycross or with 4 x 106 sheep erythrocytes were placed in the system, unresponsiveness to S. adelaide, but immunity to the other antigens occurred simultaneously. Cells made immunologically tolerant in vitro to S. adelaide H antigens were transferred into syngeneic lethally irradiated recipients and challenged with the same antigen. The adoptive immune capacity in these mice, as measured at the level of the immunologically competent cell was reduced by 80–90% as compared with relevant controls. Attempts to induce low zone tolerance in vitro were without success. To study the kinetics of tolerance induction in vitro, cells were cultured with tolerogenic doses of antigen for various periods of time, washed, and subsequently cultured with immunogenic doses of antigen for 4 days. It was found, that immunological tolerance may be induced to a significant degree in vitro within a period of 15 min. Similar results were obtained when spleen cells were exposed for various lengths of time to tolerogenic doses of antigen but at a temperature of 4°C instead of 37°C. The results are taken as suggestive evidence that the initial step in tolerance induction is related to the direct interaction between the surface of immune competent cells and antigen molecules.

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