Surface Phenotype of hBcl-2 Transgenic B Cells

We compared the expression of surface antigens on hbcl-2 transgenic B cells to expression on wild-type mature splenic B cells. In accordance with earlier reports by Strasser and colleagues (5, 7), we observed that the expression levels of IgM, IgD, CD23 and B220 on splenic hbcl-2 transgenic B cells were comparable to levels observed on wild-type splenic B cells (Fig. 3). Therefore, the majority of splenic hbcl-2 transgenic B cells were mature B2 B cells (IgM⁺, IgD⁺⁺⁺, CD23⁺⁺ and B220⁺⁺⁺; Fig. 3A and 3C, Hardy and Hayakawa,16), similar to wild-type splenic B cells (Fig. 3B and 3D). Marginal zone (MZ) B cells (CD21⁺⁺⁺, CD23^-; region R1, Fig. 4) were reduced in hbcl-2 transgenic mice. Marginal zone B cells comprised approximately 5% of the wild-type splenic B cell population but were barely detected in hbcl-2 transgenic splenic B cells (R1 = <1%). Follicular, mature B2 cells (CD23⁺⁺, CD21⁺⁺; region R2, Fig. 4) were 89% and 88%, respectively, in hbcl-2 transgenic and wild-type splenic B cell populations. Although CD23 expression levels appeared normal, CD21 levels on hbcl-2 transgenic B cells were reduced compared to wild-type B cells (median fluorescence intensity more than twofold less). The sum of the transitional (T2 and T1) and B1 B cell subpopulations was 7% for hbcl-2 transgenic and 5% for wild-type splenic B cell populations (Fig 4, top panels, regions R3 and R4; plots gated for live B220 positive cells).

Fig. 3. FACS cytometry analysis of hbcl-2 transgenic versus wild-type splenic B cell subsets. Purified splenic B cells were stained with anti-IgM-FITC, anti-CD23-PE, anti-B220-PcP, anti-CD11b-APC, and anti-CD3e-APC fluorophore-coupled antibodies. Samples were then analyzed by FACS cytometry, using Cell Quest software. The top left histogram of each panel shows dot plots of live cells stained with anti-CD23-PE versus anti-B220-PcP (A and B) and anti-IgD-PE versus anti-IgM-FITC (C and D) for purified splenic B cell populations from both hbcl-2 transgenic and wild-type mice. Isotype control dot plots, stained with anti-keyhole limpet hemocyanin (KLH) antibodies coupled to PE and/or FITC fluorophores are shown in the top right histogram of each panel. The cell classification criteria, according to expression of surface receptors, were taken from Hardy and Hayakawa (16). The expression or lack of expression of a specific protein is indicated by + or -, respectively; cell subsets, which either can or cannot express a specific protein, are designated +/- [noted in Fig. 4]. Positive expression boundaries are marked by either horizontal lines (M1) on the histogram plots, or right-hand, and upper quadrants for x and y axes respectively, marked on the dot plots. The relative strength of expression for a given protein is indicated by the number of + signs from one to four, ++++ being the highest level of expression observed in the expression range for the population. The majority of splenic B cells were mature B2 B cells (IgM+, IgD+++, CD23++ and B220+++ for both strains. The histogram plots show the intensity of staining with the specific antibodies designated on the x-axis (purple fills) with isotype control antibodies (green lines) on the same plots. Representative experiments are shown for staining on 17 B cell preparations from hbcl-2 transgenic and 7 wild-type litter-matched mice.

Fig. 4. Marginal zone B cells were reduced in hbcl-2 transgenic splenic B cells. Purified splenic B cells were stained with anti-CD21-FITC, anti-CD23-PE, anti-B220-PcP, anti-CD11b-APC, and anti-CD3e-APC fluorophore-coupled antibodies. Samples were then acquired and analyzed for four-color staining using FACS cytometry, with Cell Quest software. The top two panels were gated on live B cells (using forward- and side-scatter profiles and positive B220 staining) and show dot plots of anti-CD21-FITC versus anti-CD23-PE staining for both hbcl-2 transgenic and wild-type B cells. The regions R1 to R4 designate recognized subsets of splenic B cells. R1 = Marginal zone (MZ) B cells (CD21+++, CD23-); R2 = Follicular B cells (CD23++, CD21++), R3 and R4 = Transitional, T2 or T1, and B1 subsets of B cells (CD21+CD23+, CD21+/-CD23-, CD21+/-CD23++/-, respectively). The lower four panels, also gated on live B cells, show the intensity of staining with specific antibodies (purple-fill histograms) and isotype control antibodies (green lines). Representative experiments are shown for staining on four separate spleen preparations.

In summary, hbcl-2 transgenic splenic B cells are comparable to wild-type splenic B cells in that the majority of cells express the surface receptor characteristics of mature B2 B cells. The main difference detected in the hbcl-2 B cell population compared with wild-type B cells was the absence/reduction of MZ B cells, as was also recently reported by Brunner and colleagues in a different strain of bcl-2 transgenic mouse (17). Importantly, the transgenic expression of bcl-2 does not lead to an accumulation of B cells that are IgM^-/-, consistent with evidence that bcl-2 expression alone is not sufficient for the persistence of mature B cells, which also requires signaling through the pre-BCR or BCR during development (18).