Effect of hapten valency and carrier composition on the tolerogenic potential of hapten-protein conjugates
Pike, B.E.; Battye, F.L.; Nossal, G.J.
Journal of Immunology 126(1): 89-94
1981
ISSN/ISBN: 0022-1767 PMID: 6161171 Document Number: 168880
The effects of hapten valency and carrier composition on the induction of hapten-specific B lymphocyte tolerance by hapten-protein conjugates were explored. Spleen cells from newborn or adult mice were incubated for 24 h with fluorescein (FLU) coupled to human .gamma.-globulin (HGG), the F(ab')2 fragment of HGG (FAB) or to bovine serum albumin (BSA). Conjugates possessing a mean of 2, 5, 8 or 12 FLU residues per molecule of protein were prepared. After in vitro tolerance induction, the proportion of B cells capable of generating FLU-specific plaque-forming cell (PFC) clones was determined by a limit dilution micro-culture assay. With all conjugates, the newborn cells were more sensitive to negative signaling than the adult cells. The relative ratios of concentration required to tolerize 50% of the B cells varied from conjugate to conjugate. For all 3 carriers, the higher the hapten density, the lower was the molarity required for tolerance induction. The Fc piece appeared to play some role in making HGG the most effective of the carriers, since FLU-FAB conjugates were less tolerogenic than FLU-HGG conjugates; this difference could be partially overcome by increasing the hapten density. FLU5BSA behaved much like FLU5FAB, but for reasons that are unclear, FLU12BSA was much less tolerogenic than FLU12FAB. The amounts of FLU actually bound to FLU-specific cells after reaction with the various conjugates was studied. FLU-specific cells were prepared by the hapten-gelatin method, reacted with the putative tolerogens and analyzed in the fluorescence-activated cell sorter (FACS). The FLU-FAB and FLU-BSA conjugates bound cells to a degree reflecting the FLU molarity in the reaction mixture. The FLU-HGG conjugates bound 3- to 5-fold better, again suggesting a role for the Fc piece. There was a significant variance in the tolerogenic potential of the conjugates for equivalent amounts of FLU bound, suggesting that high hapten density and possession of an Fc piece can exert effects beyond the increased total amount of hapten binding to membrane Ig receptors that they engender.