Induction of immunity and tolerance in vitro by hapten protein conjugates. I. The relationship between the degree of hapten conjugation and the immunogenicity of dinitrophenylated polymerized flagellin.
Of many dinitrophenylated (DNP) protein conjugates tested, only DNP conjugated to polymerized flagellin (DNP-POL) (or the structurally related bacterial flagella) elicited a primary anti-DNP response in vitro. Other DNP proteins, such as DNP-monomeric flagellin (DNP-MON), were capable of inducing secondary responses in vitro. The capacity of DNP-POL to immunize spleen cell suspensions devoid of thymus-derived cells was the reason for the greater immunogenicity of DNP-POL, since even large numbers of flagellin-reactive activated thymus cells did not increase the anti-DNP response of normal spleen cells immunized with DNP-POL, whereas the thymus-dependent response to DNP-MON was markedly increased. The capacity of various batches of DNP-POL to immunize normal spleen cells in vitro varied markedly, depending on the number of DNP groups conjugated. DNP-POL with few DNP groups conjugated was immunogenic, but even at very high concentrations did not induce tolerance. In contrast, highly conjugated DNP-POL did not immunize, but readily induced tolerance. DNP-POL with intermediate degrees of conjugation were, like unconjugated polymeric flagellin, capable of inducing both immunity and tolerance. Since DNP-POL immunizes bone marrow-derived lymphocytes (B cells) directly the reduced response was not due to a masking of carrier determinants, necessary for cell collaboration. By using mixed DNP-5-(dimethylamino)-1-naphthalyl (dansyl)-POL conjugates, it was found that the inhibitory effect of a high degree of hapten conjugated was hapten specific. Depolymerization of DNP-POL to DNP-MON, which does not induce primary anti-DNP responses, was excluded by centrifugation analysis and electron microscopy. The relationship of the degree of hapten conjugation on DNP-POL to the capacity to induce tolerance and immunity in B cells has clarified the mechanism of immunological triggering of these cells. A model of the mechanism of "signal" discrimination between immunity and tolerance in B cells, based on these findings, is proposed.