Antibody-mediated suppression of the immune response in vitro. II. A new approach to the phenomenon of immunological tolerance.

Immunological tolerance to H antigens of Salmonella adelaide may be induced in vitro by the exposure of mouse spleen cells for 6 hr to an immunogenic dose of polymerized flagellin in the presence of low concentrations of specific antibody. Such antibody-mediated tolerance requires an optimal antigen: antibody ratio for its induction. A shift in this ratio in favor of the antibody concentration results in failure of tolerance induction and leads to immune suppression commonly known as antibody-mediated feedback inhibition which is not analogous to immunological tolerance. Fragment A of flagellin fails to induce immunological tolerance in vitro. Tolerance to polymerized flagellin may however be induced in vitro, provided the spleen cells are exposed to fragment A in the presence of specific antibody for 6 hr. The results are discussed in the light of current theories of the mechanism of tolerance induction.

Immunological tolerance in vitro: kinetic studies at the cellular level.

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 microg/ml of tissue culture fluid and it was found that the peak number of approximately 500 antibody-forming cells per 10(6) harvested cells by day 4 was antigen dose dependent, 2-20 ng/ml being the optimal concentration. When more than 1 microg/ml of polymer from S. adelaide together with either 20 ng/ml of polymer from S. waycross or with 4 x 10(6) 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 degrees C instead of 37 degrees 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.

Effect of lipopolysaccharide on the induction of tolerance to polymerized flagellin.

The effect of bacterial lipopolysaccharide (LPS) on the induction of tolerance to the T-independent polymerized flagellar protein (POL) of Salmonella adelaide strain SW1338 was studied in vitro. In contrast to published results with T-dependent antigens, LPS failed to prevent or reverse tolerance induction to POL under conditions in which tolerance was reversible by other means. Moreover, the addition of high doses of LPS to slightly supra-immunogenic concentrations of POL augmented-tolerance induction. These results suggest that the mitogenic properties of LPS are insufficient to convert a tolerogenic dose of POL into an immunogenic one and, therefore, that LPS may act only on T-dependent B cell responses. Since both POL (1338) and LPS are T-independent, mitogenic and so-called "polyclonal B cell activators," these data have implications for signal discrimination in the immune response.