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. 1998 Apr 6;187(7):1081-91.
doi: 10.1084/jem.187.7.1081.

Transgene expression of bcl-xL permits anti-immunoglobulin (Ig)-induced proliferation in xid B cells

N Solvason  1 , W W Wu, N Kabra, F Lund-Johansen, M G Roncarolo, T W Behrens, D A Grillot, G Nunez, E Lees, M Howard
Affiliations

Transgene expression of bcl-xL permits anti-immunoglobulin (Ig)-induced proliferation in xid B cells

N Solvason et al. J Exp Med. .

Abstract

Mutations in the tyrosine kinase, Btk, result in a mild immunodeficiency in mice (xid). While B lymphocytes from xid mice do not proliferate to anti-immunoglobulin (Ig), we show here induction of the complete complement of cell cycle regulatory molecules, though the level of induction is about half that detected in normal B cells. Cell cycle analysis reveals that anti-Ig stimulated xid B cells enter S phase, but fail to complete the cell cycle, exhibiting a high rate of apoptosis. This correlated with a decreased ability to induce the anti-apoptosis regulatory protein, Bcl-xL. Ectopic expression of Bcl-xL in xid B cells permitted anti-Ig induced cell cycle progression demonstrating dual requirements for induction of anti-apoptotic proteins plus cell cycle regulatory proteins during antigen receptor mediated proliferation. Furthermore, our results link one of the immunodeficient traits caused by mutant Btk with the failure to properly regulate Bcl-xL.

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Figures

Figure 1
Figure 1
Defective anti-IgM–triggered proliferation in xid B cells. B cells from xid (black bar) and CBA/Ca (gray bar) were stimulated with mAbs against CD40 (A) or F(ab′)2 anti-IgM (B) and pulsed with [3H]thymidine for 1 h at the time indicated. Standard errors are shown and results are representative of three independent experiments.
Figure 4
Figure 4
In vitro kinase assays from anti-IgM–stimulated B cells. Stimulated B cells were collected at the time points indicated and used to measure (A) cdk2- and (B) cdc2-associated specific kinase activity using Histone H1 as the substrate in the presence of [32P]gATP. Kinase activity was quantitated by comparing the amount of 32P incorporated into the substrate by phosphorimager analysis. Kinase activity detected in xid (black bars) and CBA/Ca (gray bars) are shown as arbitrary units. Similar results were obtained in three independent experiments.
Figure 5
Figure 5
BrdU incorporation and PI analysis of anti-IgM–stimulated B cells from CBA/Ca and xid B mice. The diagram at the top indicates the cell cycle position of cells at the time the cells were harvested. This analysis identifies cells in G0 (G0), S phase (S), and those which have completed S phase and are now in the second cell cycle G0 phase (2nd G0), and apoptotic cells (Apo). B cells from CBA/Ca (top row) and xid (bottom row) mice were stimulated with 25 μg/ml of anti-IgM in the presence of BrdU. Cells were harvested at time points indicated and fixed to permit intranuclear staining of DNA with PI. The initial entry of B cells from xid and CBA/Ca is indicated with an arrow. Analysis was performed on FACSCalibur® using CellQuest software.
Figure 6
Figure 6
Analysis of Bcl-2 and related proteins. B cells were stimulated as described in the legend to Fig. 2 and were collected at the time points indicated. Western blots were prepared and screened with antibodies specific for Bcl-2 (A), Bax (B) or Bcl-xL (C). Horseradish peroxidase conjugates were used as secondary reagents followed by visualization with enhanced chemiluminescence.
Figure 7
Figure 7
BrdU and PI analysis of stimulated B cells from xid and F1 (xid.bcl-x-87) offspring. B cells from xid, female F1 (xid.bcl-x-87), and male F1 (xid.bcl-x-87) mice were stimulated with 25 μg/ml anti-IgM in the presence of BrdU and collected at the indicated time points. Cells were prepared and analyzed as described in the legend to Fig. 5. Arrows indicate the initial entry of cells into S phase.
Figure 8
Figure 8
Comparison of in vitro viability. Splenic B cells from CBA/Ca, xid, male F1 (xid.bcl-x-87), and female F1 (xid.bcl-x-87) mice were cultured in 96-well flat-bottomed wells at 106 cells/ml in RPMI with 10% FCS for times indicated. From days 1 to 6, viability of spleen B cells was assessed by Trypan blue exclusion. Results are representative of three independent experiments.
Figure 9
Figure 9
Serum immunoglobulin levels of IgM and IgG3. Sera was collected from 8-wk-old xid (n = 5), CBA/Ca (n = 4), F1 female (n = 5), and F1 male (n = 6) mice. Biotin-conjugated anti-IgG3 and anti-IgM reagents were used in combination with horseradish peroxidase–conjugated streptavidin in ELISAs to quantitate serum Ig levels. Serial dilutions of each sample were made and compared with standard curves generated by IgM or IgG3 preparations of known concentrations. Asterisks indicate mice carrying the bcl-x transgene.
Figure
Figure
Figures 2 and 3. Induction of cell cycle regulatory proteins in anti-IgM–stimulated B cells from xid and CBA/Ca mice. B cells were stimulated with 25 μg/ml anti-IgM and pellets were collected at the indicated time points after stimulation. Figure 2: Western blots were prepared and sequentially screened with antibodies against cyclin D2 (A), cyclin D3 (B), cdk4 (C), cdk6 (D), and Rb (E). Figure 3: The same Western blot was sequentially screened with antibodies against cyclin E (A), cyclin A (B), cyclin B (C), cdk2 (D), and cdc2 (E). Horseradish peroxidase–conjugated secondary reagents were used in combination with enhanced chemiluminescence to visualize results. The cell line, P3X, was used as positive control.
Figure
Figure
Figures 2 and 3. Induction of cell cycle regulatory proteins in anti-IgM–stimulated B cells from xid and CBA/Ca mice. B cells were stimulated with 25 μg/ml anti-IgM and pellets were collected at the indicated time points after stimulation. Figure 2: Western blots were prepared and sequentially screened with antibodies against cyclin D2 (A), cyclin D3 (B), cdk4 (C), cdk6 (D), and Rb (E). Figure 3: The same Western blot was sequentially screened with antibodies against cyclin E (A), cyclin A (B), cyclin B (C), cdk2 (D), and cdc2 (E). Horseradish peroxidase–conjugated secondary reagents were used in combination with enhanced chemiluminescence to visualize results. The cell line, P3X, was used as positive control.
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