S region transcription per se promotes basal IgE class switch recombination but additional factors regulate the efficiency of the process. (1)
During an immune response, B lymphocytes can change the class of immunoglobulins they produce from IgM to another class (IgG, IgE or IgA) without altering their variable region (antigen) specificity. Class switch recombination (CSR) results in the deletion of the Cu gene and all intervening sequences accompanied by the placement of the target CH gene in a position that allows its expression in conjunction with the assembled variable region gene. IgM+ B cells can be induced in vitro to switch to specific immunoglobulin isotypes by stimulation with mitogens and addition of cytokines. For example, murine splenic B cells can be specifically stimulated to produce IgG3 and IgG2b by culture in media containing bacterial lipopolysaccharide (LPS), IgE and IgG1 by culturing with LPS plus interleukin-4 (IL-4), or IgA by culturing with LPS and transforming growth factor-b1.
The EPKO construct allows replacement of this fragment with a neor gene that is transcriptionally oriented in the reverse direction relative to Ik and a cassette containing the EA intronic enhancer elements (also in opposite orientation) plus a VH gene promoter. The EPKO 203 clone was selected for growth in increased concentrations of G418 to yield three independent clones (EPDKO 1, 2 and 5) that were homozygous for the mutant allele.
In all chimeras, EPKO and EPDKO ES cells gave rise to both mature B and T cells, as evidenced by the presence of fractions of spleen IgM+/B220+ and CD4+ or CD8+ cells comparable to normal controls.
Mature splenic B cells from the chimeras were cultured for 5 days in the presence of 25 ug/ml of LPS with or without 50 ng/ml of recombinant mouse IL-4. A significant fraction of normal, LPS-stimulated B cells expressed surface IgG2b (sIgG2b) and IgG3. Following culture in the presence of LPS plus IL-4, EPKO cultures, like those of normal B cells, had significant numbers of sIgG1+ and sIgE+ cells and few sIgG2b+ or sIgG3+ cells. Very few cells in the EPDKO populations treated with LPS plus IL-4 were sIgE+ while the level of sIgGl+ cells remained unchanged.
No cytoplasmic IgE+ cells were detected in LPS cultures of normal B cells, whereas cytoplasmic IgE+ cells were detectable in LPS +IL-4-treated cultures of normal B cells at a frequency of 4%. In EPKO cultures treated with LPS and IL-4, IgE producing cells were present at substantial levels, but generally at a lower frequency (2%).
In the supernatants from normal B cells stimulated with LPS+IL-4 we detected 300-900 ng/ml of IgE at day 5, with levels increasing 2- to 4-fold by day 7. Analyses of the total IgM and IgG content in the various culture supernatants described above did not reveal any difference between homozygous mutants, heterozygous mutants and normal controls.
On day 3 of stimulation the germline e transcripts (1.7 kb species) and the mature VDJ-Ce transcripts (2.3 kb species) were at approximately the same abundance, while at day 5 the mature VDJ-Ce transcript was largely predominant. Numerous species of Ce-hybridizing transcripts in the total RNA of EPKO and EPDKO cells grown with either LPS alone or LPS +IL-4. RNA from LPS- or LPS+IL-4-stimulated single or double mutants contained two major Ce- and Se- hybridizing species of -5 and 7 kb.
Of 16 EPKO hybridomas, 15 showed novel bands that corresponded to Su-Se rearrangements. However, all these hybridomas retained sequences upstream of the EPKO Se region, indicating that the detected rearrangements had occurred on the normal allele while the EPKO allele remained in germline configuration.
The EPDKO hybridomas express normal levels of e mRNA transcripts plus detectable levels of germline transcripts from the mutant (unrearranged) allele.
