Effects of Culture on B Cell Surface Marker Phenotype

To determine if the cultured hbcl-2 transgenic B cells maintained their surface phenotype during culture, we measured the expression of several surface markers on these cells. Human bcl-2 transgenic B cells cultured for up to six days essentially retained their surface phenotype, as assessed by expression of IgM, B220, and CD23 (Fig. 9). The percentage of cells determined positive for these markers averaged 85%, 99%, and 92% for fresh cells and 80%, 100%, and 76% for day 6 cultured cells for IgM, B220, and CD23, respectively, suggesting an apparent decline in CD23 expression in culture. Absolute fluorescence intensity values for cells stained on different days cannot be directly compared; however, the ratios of median fluorescence intensities for stains and isotype-matched controls gave an indication of relative levels of specific marker expression. The histograms shown (Fig. 9), and data from other experiments, suggested an overall reduction in CD23 expression after six days in culture compared with fresh cells (Fig. 4). The day 6 cultured cells showed a broader range of CD23 expression, with increased numbers of lower expressing cells; however, we did not investigate this observation further. Day 6 cultured hbcl-2 transgenic B cells had similar levels of chemokine receptor expression compared to freshly isolated wild-type B lymphocytes (data not shown), and hbcl-2 transgenic B cells sustained CXCR4 and CXCR5 expression for up to six days in culture (Fig.10). In accordance with this, migration responses to the chemokines BLC and SDF-1a were also sustained in cultured hbcl-2 transgenic B cells (day 1, 3, and 6) and were similar to those seen with fresh B cells (see below; Functional Responses in Cultured Human Bcl-2 Transgenic B Cells). CXCR5 was normally expressed at a higher level than CXCR4 (median fluorescence intensities usually 3- to 6-fold and 1.5- to 2-fold isotype control intensities, respectively). The majority of freshly isolated wild-type and hbcl-2 transgenic splenic B cells lacked expression of the activation markers CD69 and CD86 (Fig. 7). Culture of hbcl-2 transgenic B cells for up to six days in the absence of stimulation did not cause any increase in expression of these activation markers (see below; Functional Responses in Cultured Human Bcl-2 Transgenic B Cells).

Fig. 9. Surface phenotype of cultured hbcl-2 transgenic B cells. Purified splenic hbcl-2 transgenic B cells were cultured for 6 days then harvested and stained with anti-IgM-FITC, anti-CD23-PE, anti-B220-PcP, anti-CD11b-APC, and anti-CD3e-APC fluorophore-coupled antibodies. Samples were acquired and analyzed for four-color staining by FACS cytometry, using Cell Quest software. The histograms shown were gated on live cells (using forward and side scatter profiles), and plots of fluorescence intensities for specific stains (listed on x-axes, in purple fill) and isotype control stains (green lines) are shown.

Fig. 10. Chemokine receptor expression of cultured hbcl-2 transgenic B cells. Expression of the chemokine receptors CXCR5 and CXCR4 on fresh versus cultured hbcl-2 transgenic B cells was examined using FACS cytometry analysis. (A) Anti-CXCR5-PE fluorescence intensity, shown on the x-axis, showed the expression of the chemokine receptor CXCR5 (purple-filled histograms) was stable in hbcl-2 transgenic B cells, cultured for up to six days in 10% FBS. The green-line histograms show fluorescence intensity using isotype-matched fluorophore-coupled negative control antibodies. Antibodies were used at 1 mg/106 cells, and 0.25 x 106 cells were stained in 100 ml. (B) CXCR4 staining on fresh and day 3 cultured hbcl-2 transgenic B cells was also stable. Anti-CXCR4-FITC fluorescence intensity is shown in purple fills, with isotype-matched controls shown as green lines. Antibodies were used at 1 mg/106 cells, and 0.25 x 106 cells were stained in 50 ml of buffer (using the buffers described in AfCS protocol PP00000018). Representative staining for three hbcl-2 splenic B cell preparations (A) and (B) is shown.

Human bcl-2 was readily detected by fluorescence cytometry analysis of the hbcl-2 transgenic B cells, as expected, and its expression was maintained in culture, measured at up to six days (data not shown). Mature resting B cells also expressed endogenous mouse bcl-2 protein (8), and we observed that mouse bcl-2 was expressed at similar levels in hbcl-2 transgenic and wild-type B cells (data not shown). Only B cells from hbcl-2 transgenic mice expressed human bcl-2. As a consequence, other contaminating cell types (<5%) failed to survive in culture. Hence, the purity of hbcl-2 transgenic B cells increased from greater than 96% (Table 1) at isolation to 100% after one to six days in culture (data not shown). In conclusion, hbcl-2 transgenic expression is maintained in the hbcl-2 B cells in culture, imparting a selective survival advantage on these cells versus any contaminating immune cells. This allows the establishment of pure cultures of resting mature splenic B cells.