Analysis of immunoglobulin (Ig) isotype diversity and IgM/D memory in the response to phenyl-oxazolone

The distribution of immunoglobulin (Ig) isotypes within specific B cell clones in vivo after immunization is not well defined. Using an IgV(H)/CDR3- and isotype-specific reverse transcription polymerase chain reaction method, we have carried out a survey of the diversification of the isotype in a splenic response to phenyl-oxazolone (phOx) on a chicken serum albumin carrier. The phOx-specific V(H) (V(H)Ox-1 with specific CDR3 motif) is associated with all of the heavy chains (mu, delta, alpha, gamma, and epsilon) after simple immunization with antigen in alum. The kinetics of expression of each isotype are distinct and reproducible. Focusing mainly on the expression of secretory Ig transcripts, IgM, IgG1, and IgE are found after priming, whereas IgD and IgA appear after boosting. Secretory IgD transcripts are found reproducibly at moderate levels and may, therefore, contribute significantly to the secreted Ig response in mice. Most crucially, we find enhanced levels of secretory IgM/V(H)Ox-1 transcripts (with 'phOx-specific' CDR3) after boosting, strongly indicating the existence of IgM memory cells that give rise to an enhanced specific IgM secretion in the secondary response.