IgM-bearing B cell affinity subpopulations possess differential antigen sensitivity in rainbow trout

The need for accurate assessments of in vitro generated antibody prompted examination of the effect of antigen on secreted antibody concentrations and affinities. It was found that the antigen concentrations commonly employed for in vitro stimulation were able to significantly compromise IgM titer and affinity estimates in rainbow trout. Specifically, IgM titers were underestimated with the high affinity antibodies being specifically blocked. To remedy this, pulsed antigen cultures were employed, and it was found to reveal more accurate IgM antibody titers and affinity estimates. Additionally, pulsed dose responses provided evidence that high antigen concentrations specifically suppressed high affinity B cell induction. Optimal expression of high affinity antibodies required exposure to lower concentrations of antigen. Each affinity subpopulation appeared to possess a graded sensitivity to each dose of antigen, revealing the complex dynamic for differential IgM-bearing B cell induction that is possible during a response. These results reveal not only the need for antigen removal prior to in vitro antibody secretion, but also the possible role of high zone immunological tolerance on IgM affinity maturation in rainbow trout.