In vivo and in vitro IgE isotype switching in human B lymphocytes: Evidence for a predominantly direct IgM to IgE class switch program (1)
Isotype switching is accompanied by deletion of the intervening DNA as a circular excision product and leaves a molecular footprint in the genome in form of a composite S region. Molecular analysis of these products in the mouse has indicated that Ig isotype switching to a number of CH genes may progress directly or successively. For example, in vivo and in vitro IgM to IgE (or IgA) switching in murine B cells may proceed directly, mediated via Su – Se recombination, or sequentially mediated via Su-Sr1 and Sr1 – Se intermediates.
Molecular analysis of in vitro IgE switched B cells and in vivo switched B cells from patients with hyper-IgE has unveiled both direct IgM -> IgE as well as sequential IgM -> IgG -> IgE switch programs. The data from in vitro switched B cells suggest that the direct IgM -> IgE program predominates.
For induction of in vitro IgE isotype switching we employed two different culture systems. In one system, B cell-enriched fractions were cultured in the presence of 2.5 x 10^5/ml irradiated (2400 rad) EL4 thymoma cells,and PMA(5ng/ml). In the second system, B cell-enriched fractions were further depleted of natural killer cells and residual monocytes and cultured in the presence of autologous CD4+ T cells, and an anti-CD3 mAb (100 ng/ml).
A two-step PCR amplification of the composite Su/Se region present in the genomic DNA of the U266 cell line consistently generated a single diagnostic fragment of 600bp. In first round of PCR amplifications using genomic DNA from the IgM-secreting cell lines we occasionally detected faint ethidium bromide staining fragments that were undetecta- ble upon the second round of PCR, even after probing of Southern blots with a Se-specific probe.
The IgE response in these patients is dominated by a restricted set of clonally expanded B cells. Although composite human Su/Ss regions may theoretically be up to 5 kb in length, the observation that virtually all of the S recombination donor sites clus- tered within the 5′ end of Sp (including Su/Se fragments containing small 3′ portions of Se), fragments of up to 2700 bp may be expected. None of the sequences contained identifiable stretches of S regions from other CH genes or of unknown origin, compatible with a direct human IgM to IgE switch program. This observation is in striking contrast with findings in the mouse, where switching t o Cu-distal CH genes often proceeds via successive S-S recombination involving various combinations of S regions. 30-100% of Ig isotype switching to IgE induced in vitro by stimulation with LPS and IL-4.
Virtually all Sp recombination sites were located at the 5′ end of the Su region, between positions240 and 756. The single exception, B5-4A2, contained a large deletion at the 5′ end of Su and its recombination site maps around position 3280. In striking contrast, the Se switch acceptor sites are dispersed over the entire 3-kb Se region.
Of note, murine B cells switched from IgM to IgA upon stimulation with LPS and TGFb contain Su donor sites clustered in a 3′ subregion of Su, whereas a switch from IgM to IgG1 after stimulation with LPS and IL-4 results in a dispersed pattern of Su breakpoints.
