While successive isotype switching can occur from IgM to IgE via IgGl, little is known about the pattern of germline transcript expression in normal B cells committed to switching to multiple isotypes. In this study we define the relationship between germline transcript expression and immunoglobulin isotype expression and secretion in murine splenic B cells. Following 7 days stimulation with LPS and IL-4, 10% of single cells secrete IgE and of these only 1 in l0 secrete IgE alone. In contrast, when cells were stimulated with LPS and IL-4 for 48 hr prior to sorting into clonal cultures, 71% secreted IgE alone. In an attempt to isolate switch intermediates, IgGl+ IgM- cells were sorted and upon re-culture secreted predominantly IgGl alone or IgGl and IgE. The frequency of cells expressing germline ε transcripts was 34% for surface IgGl+ IgM- expressing cells and 14% for surface IgGl-IgM+ negative cells. Furthermore, analysis of single surface IgGl+IgM- cells demonstrated that these cells can co-express germline γl and ε transcripts. Sorting of IgGl-IgM+ cells according to surface expression of the integral membrane proteoglycan, Syndecan, demonstrated that IgM+Syndecan+ cells had a lower frequency of germline transcript expression and a lower frequency of isotype switching (32%) compared to IgM+Syndecan- cells (83%). Collectively, these finding show that murine B cells can switch successively from IgM to IgE via IgGl and that IgGl expressing intermediates express germline transcripts. Furthermore, Syndecan expression appears to be linked to germline C(H) transcription and isotype switch commitment.
- Germline transcription
- Isotype switching