Increased Targeting of Donor Switch Region and IgE in S g1-Deficient B Cells.(1)
In mice, S regions are organized in the following order: 59-Sm-Sg3-Sg1-Sg2b-Sg2a-Sε-Sa-39 and consist of a tandem array of short repeats ranging from 1 to 10 kb in length. Each S region is preceded by intronic (I) promoters. Im (59 of Sm) is constitutively transcribed, whereas transcription and accessibility of downstream S regions depend on the particular stimuli that B cells receive. S regions are essential for CSR and contain tandem re- petitive hot spots, such as TGGGG, GGGGT, GGGCT, GAGCT, and AGCT. The numbers of tandem repetitive sequences within S regions determine how efficiently a particular S region can recruit AID to carry out CSR. In the mouse, Sm has the highest number of tandem repeats, whereas Sg1 is the largest (10 kb) and Sε is the shortest S region with the fewest tandem repeats. Shortening the size of a particular S region or replacing it with a less efficient one reduces the rate of switching to the modified S region.
The Sg1 region (10 kb) was replaced with a 0.5-kb inert I sequence containing two ISceI sites. A 160-fold decrease in levels of IgG1+ B cells in WT compared with DSg1 splenocytes was shown. DSg1 B cells could not secrete any detectable levels of IgG1. Levels of CSR to IgG3, and IgG2b are not significantly different between WT and DSg1 B cells.
56 and 31 internal Sm deletions, respectively, out of 72 DSg1 and 50 heterozygous hybridomas, whereas only 17 internal Sm deletions were observed among 72 WT hybridomas analyzed.
A 10-fold increase for mutations in DSg1 hybridomas (7.44 x 10^-4), compared with WT (0.71 x 10^-4) hybridomas was observed. A modest increase (almost 2-fold) in SHM in DSg1 mice (3.2 mutations/10,000 bp) compared with WT littermates (1.8 mutations/10,000 bp).
Approximately 50% of the analyzed IgG1+ heterozygous colonies showed Sm-Sε CSR in their nonproductive allele. ∼30% of DSg1 B cells undergo Sm-Sε CSR in their nonproductive alleles, a marked increase.
A 2.5-fold increase in the levels of IgE Ab inDSg1 samples compared with WT. DSg1 samples did not express any significant amounts of IgG1 Ab, whereas the levels of IgM Ab were comparable between WT and DSg1 splenocytes stimulated with anti-CD40/IL4. TNP-OVA–specific IgE Ab, compared with WT mice, on day 42. Again, DSg1 mice failed to produce any significant levels of TNP-OVA–specific IgG1 Ab ∼50-fold lower than that of the WT at day 42.
Consistent with our general TNP-OVA immunization model, higher levels of IgE were seen in the DSg1 mice than in the WT group, suggesting that lack of Sg1 affects the level of IgE isotype switching.
Sequential switching in Su-Sg1-Sε does not play an important role in IgE isotype switching because in the absence of Sg1, IgE levels, if anything, are elevated.