Functional Responses in Freshly Isolated hBcl-2 Transgenic and Wild-Type B Cells

To determine if hbcl-2 transgenic B cells were functionally similar to wild-type B cells, we compared outputs following BCR ligation, including intracellular calcium ([Ca²⁺]_i) flux, protein phosphorylation, upregulation of activation markers (CD86 and CD69), and gene expression changes (see below; Gene Expression Changes in Response to In Vitro Culture and Anti-IgM Stimulation). In the two populations, activation of the BCR with anti-IgM caused comparable [Ca²⁺]_i flux (Fig. 5), protein phosphorylation (Fig. 6), and upregulated expression of the activation markers CD69 and CD86 (Fig. 7). We also measured proliferation responses to LPS, anti-CD40, and IL-4, in addition to anti-IgM (Fig. 8) and examined migration responses to the chemokines SDF-1a and BLC, comparing hbcl-2 transgenic B cells to wild-type B cells (see below; Functional Responses in Cultured Human Bcl-2 Transgenic B Cells). Both ³[H]-thymidine incorporation and cell count indicators of proliferative responses by hbcl-2 transgenic B cells in response to LPS, anti-IgM, anti-CD40, and IL-4 (singly and in pair combinations) were generally less than those in wild-type splenic B cells (Fig. 8). Importantly, LPS produced the greatest increase in ³[H]-thymidine incorporation for both hbcl-2 transgenic and wild-type B cells over multiple experiments and was the only stimulus able to consistently increase hbcl-2 B cell numbers within two days of stimulation. In contrast, for wild-type B cells, anti-CD40 alone or in combination with anti-IgM or IL-4 produced an increase in cell numbers above those obtained for unstimulated cells, which died within two days. The reduced proliferation response observed here with hbcl-2 transgenic B cells is consistent with previously described effects of overexpression of bcl-2, resulting in delayed transition of B cells from the quiescent state into the cell cycle (19).

Fig. 5. Calcium flux responses in fresh hbcl-2 transgenic and wild-type B cells. Fluo-3-loaded B cells were plated in 96-well black-walled plates and allowed to settle on the bottom of the wells (106 cells/well). A FLEXstation fluorometer (Molecular Devices) was used to excite and take emission readings for individual wells from the bottom of black-walled clear-bottom 96-well assay plates. The numbers 1 and 2 denote replicate wells read in a single experiment. Baselines were recorded prior to the robotic addition of anti-IgM at 20 sec, followed by 5mM ionomycin/10 mM EGTA/1mM thapsigargin at 300 sec (to give a minimum fluorescence value for loaded cells), and 25 mM CaCl2 at 600 sec (to give a maximum fluorescence value for loaded cells). Relative fluorescence unit traces (readings taken every 1.26 sec) were converted to calculated [Ca2+]i values using the min and max values measured for each well with the formula given in Methods and Protocols. The converted [Ca2+]i traces shown are for two wells each of wild-type versus hbcl-2 transgenic B cells. Negative control wells (HBSS buffer added at 20 sec) were run for both populations. Average peak height [Ca2+]i values for wild-type B cells were HBSS controls 126 nM and anti-IgM 588 nM; for bcl-2 transgenic B cells, values were HBSS controls 106 nM and anti-IgM 446 nM. A representative experiment of three experiments performed on different B cell preparations is shown.

Fig. 6. Stimulation of protein phosphorylation following anti-IgM stimulation of fresh wild-type and hbcl-2 transgenic B cells. B cells were plated in 12-well tissue-culture plates and stimulated according to AfCS protocol PP00000010. Briefly, 1.8 X 107 fresh wild-type or hbcl-2 transgenic B cells were stimulated with anti-IgM at 50 mg/ml for 0, 2.5, and 5 min or 0, 2.5, 5, 15, and 30 min, respectively, then lysed in SDS-PAGE sample buffer containing phosphatase inhibitors and processed for SDS-PAGE and Western blotting. hBcl-2 transgenic splenic B cells are slightly smaller than wild-type B cells (detected by FACS analysis of cell forward scatter), and protein yields from these cells averaged 6.9 mg and 9.9 mg per million cells, respectively. Equivalent amounts of protein, 20 mg per lane, were run on SDS-PAGE gels. Antibody mixes for probing Western blots were developed by the AfCS. The top blot was probed with phosphospecific antibody mix 1, which contained phosphospecific antibodies for stat-6, p90RSK, Akt, and ERK-1 and -2 (PS00000312; positive (+) control A, PS00000083). The lower blot was probed with phosphospecific antibody mix 2, which contained phosphospecific antibodies for PKCm, Stat-3, NFkB, JNK, and p38, listed in order of decreasing protein size as observed on blots (PS00000334; positive (+) control B, PS00000085).

Fig. 7. Upregulation of activation markers CD86 and CD69 in hbcl-2 transgenic versus wild-type B cells. B cells were isolated and plated in 12-well culture dishes for 1 hr (PP00000010). B cells were then either nonstimulated or stimulated with anti-IgM (50 mg/ml). At 24 hr poststimulation, hbcl-2 transgenic (panel A) and wild-type (panel B) B cells were harvested and processed for antibody staining to detect expression of the activation markers CD69 and CD86. B cells were stained with anti-CD86-FITC and anti-CD69-PE antibodies and analyzed using FACS cytometry. Histogram plot analyses were gated for live cells and show specific fluorescent signals observed for either anti-CD69 or anti-CD86 staining, as listed on the x-axes (shown with purple-fill traces), and isotype controls (shown with green-line traces). Representative data from three separate experiments is shown.

Fig. 8. Proliferation responses of fresh wild-type and hbcl-2 transgenic B cells. Freshly isolated wild-type (blue) or hbcl-2 transgenic (yellow) B cells were plated at 6 x 106/ml, 100 ml/well, in 96-well plates. Duplicate plates for 3[H]-thymidine incorporation assays and hand counts were harvested separately on day 1 (graphs A and B) and day 2 poststimulation (graphs C and D), respectively. Stimuli used were LPS (20 mg/ml), goat polyclonal anti-mouse IgM (50 mg/ml), hamster IgM anti-CD40 (at 20 mg/ml), IL-4 (50 U/ml), and hamster IgM isotype control antibody, which had no significant effect (data not shown). For combinations of anti-IgM and anti-CD40 or anti-CD40 and IL-4, the same doses that were used for single ligand stimulation were used in the combination. A single experiment is shown, including average values for quadruplicate wells for 3[H]-thymidine incorporation ( +SD error bars) and average count values from duplicate wells (2 counts each) for hand counts in each experiment. Similar results were obtained in three separate experiments.