Studies were made on the in vitro initiation of primary and secondary antibody responses to DNP-protein or DNP-poly-l-amino acid conjugates using the Millipore filter well technique for spleen organ cultures. Antibodies to DNP were assayed by inactivation of DNP-conjugated T4 bacteriophages. The production of a primary response to DNP after immunization with α-DNP-poly-l-lysine was inhibited by either free DNP-lysine or free poly-l-lysine. Hence, specific cell receptors recognizing both the hapten and the carrier interact with the immunogen in the primary response. The inhibition of the response by the affinity labeling reagent to DNP, BADE, suggests that the cell receptors are antibody-like molecules which thus exist prior to the experimental induction of antibody response. Spleens from mice immunized against RSA, then stimulated in vitro with DNP-RSA manifested a higher response to DNP than spleen cultures from animals which had not been primed with the carrier. Free carrier molecules prevented this anti-DNP response. The effect of preimmunization against the carrier is specific: Spleen cultures from donors primed against RSA did not manifest increased response to DNP after in vitro stimulation with DNP-Hcy, DNP-HSA, or DNP-GPA. To study the cellular basis of the carrier effect, the effect of suppressing cell replication was tested. Vinblastine given simultaneously with RSA inhibited the response to the subsequent in vitro stimulation with DNP-RSA. When given just 24 hr after the carrier, however, it did not inhibit this response. This is interpreted as an indication that the crucial event in the carrier effect is the stimulation of just one cycle of DNA replication, which might be necessary for more carrier receptors to be produced per cell. The results are discussed in relation to the bicellular interaction in antibody production.
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