The murine IgG1/IgE class switch program (1)
In murine B cells immunoglobulin class switching to IgGl and IgE is controlled by the same cytokine, interleukin-4 (IL-4). In hybridomas and LPS blasts expressing IgG2b, IgG2a, IgE, or IgA, for chromosomal DNA as well as for the circular excision products of class switch recombination (switch circles).
For LPS cultures, spleen cells were depleted of Thy-1+ cells by magnetic cell sorting using the MACS. The “Thy-1-” cell population contained less than 1% Thy-1+ cells. These cells were cultured for 5 days with LPS and various concentrations of affinity-purified recombinant IL-4. By day 5 , 95 % of the viable cells were B cell plasma cells, i.e. cytoplasmically stainable with anti-x or anti-h immunoglobulin light chain antibodies. Class switching to IgG3 is severely inhibited at low doses of IL-4 (from 8.3% IgG3+ without IL-4 to 0.08% with 25 units of IL-4).The frequency of IgM+ cells is only moderately reduced in the presence of IL-4, corresponding to the proportional increase in IgG1-producing cells.
The maximum frequencies of cells expressing IgG1 or IgE differ by an order of magnitude. Up to 35 % of the cells are IgG1+ (23 % in the experiment), while the frequency of IgE+ cells was always below 3% (1.4%). For both IgGl and IgE the frequency reaches a plateau at about 200 units of IL-4, and is not much different at 750 units. However, at low doses of IL-4, only switching to IgG1 is induced. For IgE, approximately fivefold higher concentrations of IL-4 are required compared to IgG1 to reach half maximal switch frequency (11 % IgG1 at 25 units, – 0.6 % IgE at 125 units).
“L.I.” series: hybridomas gener- ated after 5days of culture; D16 series: hybridomas generated after 16days of culture. Of 12 IgE+ hybridomas, 1 showed switch recombination of S, to S, on both alleles.The majority of IgE+hybridomas, 9 out of 12,had performed switch recombination of S, to S,1 on the silent allele. Of the remaining two IgE+hybridomas L.I.23 showed an Su, to Sr3, and L.I.24 most probably an Su to Sa switch recombination on the inactive allele.
For switch inhibition with anti-IgG1, spleen cells were cultivated in the presence of LPS or LPS/IL-4 and 100 pg/ml affinity-purified goat anti-mouse IgG1. The antisera had been tested for cross-reactivity with the other isotypes. In general, cross-reactions were below 0.1%, except 3 % cross-reactivity of the anti-IgG1 serum with IgG3. These cross-reactions could account for a slight suppression of IgG3-expressing cells from 6.4% t o 4.7% in LPS and from 0.5% to 0.3% in LPS/IL-4 cultures. In the presence of these antisera, IgM+ cells in both LPS an LPS/IL-4 cultures were not suppressed at all. IgG2b cells were not suppressed in LPS cultures and were slightly suppressed, from 3.1 +/- 0.15% to 2.25 +/- 0.33%, in LPS/IL-4 cultures, perhaps reflecting sequential switching via IgG1 in some IgG2b+ cells. The appearance of IgG: cells was drastically reduced in LPS/IL-4 cultures by anti-IgG1, from 34.8 +/- 2% to 6.52 +/- 0.23%, i.e. an inhibition of more than 80%. Switching to IgE can also be inhibited by antibodies to IgG1. The frequency is reduced from 1.77 +/- 0.05% to 0.52 +/- 0.02% in LPS/IL-4 cultures, i.e. more than 70 % of the cells induced to switch to IgE by IL-4 in vitro had switched to IgG1 before and expressed it on the surface.
