The requirement for the expression of previously unexpressed genes in the generation of T and B antigen-binding cells and the changes in sIg isotype following in vitro immunization

Abstract

The generation of antigen-binding cells (ABC) and plaque-forming cells (PFC) specific for sheep erythrocytes (SRBC) in a primary in vitro response was inhibited by 5-bromo-2′-deoxyuridine (BUdR) present in cultures during the 24 h period immediately preceding harvest. ABC and PFC numbers were greatly reduced by BUdR at a concentration that did not significantly affect cell viability, cell recovery, or the synthesis of total DNA, RNA, or protein, but was sufficient to inhibit the expression of previously unexpressed genes.

The effect of BUdR on ABC was seen no earlier than day 3. It lasted through day 6, with maximum inhibition (80–90%) occurring in the day 3–4 interval, coincident with the peak of the ABC response. PFC were inhibited from day 3 to day 5 (the peak of the PFC response), after which time their sensitivity to BUdR was abruptly lost. ABC were inhibited on days 4, 5, and 6, by as little as 0.5 μg/ml, while on any day, PFC were insensitive to concentrations of BUdR below 2.5 μg/ml. A ten-fold molar excess of thymidine blocked the inhibitory effect of the drug.

Though maximum inhibition of both T and B ABC numbers by BUdR occurred on day 4, its effect on B cells was greater. BUdR had no effect on the numbers of T and B non-antigen-binding cells.

BUdR did not preferentially inhibit surface IgM (sIgM)-bearing ABC though it did prevent the appearance of sIgG receptors on ABC, which normally occurred by day 3, and the loss of sIgD receptors on ABC which was normally complete by day 4. Thus, BUdR decreased the expression of receptors on T ABC as well as B ABC, and prevented the shift in the surface Ig isotypes on B ABC that normally accompanied antigen-driven differentiation.