AfCS Research Reports

To determine if hbcl-2 transgenic B cells retained their functional responses in culture, we first measured signaling responses to BCR stimulation in cells cultured for up to six days. These included intracellular calcium flux and the phosphorylation of signaling proteins, including PKCm, Akt, ERK1/2, p38MAPK, JNK, p90RSK, NFkB p65, STAT3, and STAT6. Functional responses by hbcl-2 transgenic B cells were sustained for up to six days in culture (Fig. 11 and 12). The activation markers CD69 and CD86 were also upregulated in response to anti-IgM in cultured hbcl-2 transgenic B cells (Fig. 13). B cell proliferative responses to anti-IgM, IL-4, and/or anti-CD40, or LPS were determined by measurement of ³[H]-thymidine incorporation and by viable cell counts assessed by trypan blue exclusion (Fig. 14). Day 6 cultured hbcl-2 transgenic B cells showed the same relative ³[H]-thymidine incorporation responses to these specific stimuli that were observed with fresh hbcl-2 transgenic or wild-type B cells. As with fresh hbcl-2 transgenic B cells, day 6 cultured cells showed no increase in cell numbers at two days poststimulation for any stimulus (anti-IgM, anti-CD40, and IL-4) other than LPS (Fig. 8 and 14). Finally, migration was assessed in response to the chemokines SDF-1a and BLC (the ligands for CXCR4 and CXCR5, respectively) and was also not diminished by culture of these cells (Fig. 15).

Fig. 11. Calcium flux in response to anti-IgM stimulation in cultured hbcl-2 transgenic B cells. Cultured day 6 hbcl-2 transgenic B cells were harvested and counted. Viable cell number was assessed by trypan blue exclusion. Cells were loaded with fluo-3 indicator dye for 30 min in tubes then plated into 96-well plates and allowed to equilibrate for 30-min at 37^oC prior to assay. Anti-IgM or vehicle control (HBSS buffer) was robotically added to quadruplicate wells (individual wells denoted by numbers 1 to 4) in the assay plate simultaneously. Quadruplicate samples for a single experiment are shown; similar [Ca²⁺]_i flux responses were observed in three separate experiments.

Fig. 12. Protein phosphorylation responses to anti-IgM stimulation in cultured hbcl-2 transgenic B cells. Cultured day 6 hbcl-2 transgenic B cells were harvested and counted. Viable cell number was assessed by trypan blue exclusion. Viable cells were replated in 12-well tissue-culture plates and rested for 1 hr prior to stimulation with anti-IgM at 50 mg/ml. Cells were stimulated according to AfCS protocol PP00000010. Briefly, 18 x 10⁶ viable cultured hbcl-2 transgenic B cells were stimulated for 0, 5, or 15 min and then lysed in SDS-PAGE sample buffer, containing protease and phosphatase inhibitors, and processed for SDS-PAGE and Western blotting. Equivalent amounts of protein, 20 mg per lane, were run on SDS-PAGE gels. Antibody mixes developed by the AfCS (see Fig. 5 legend) were used to probe blots. Mix 1 contained phosphospecific antibodies for stat-6, p90RSK, Akt, and ERK-1 and -2. Mix 2 contained phosphospecific antibodies for PKCm, Stat-3, NFkB, JNK, and p38 (listed in order of decreasing protein size as observed on blots).

Fig. 13. Upregulation of activation markers CD86 and CD69 in response to anti-IgM stimulation in cultured hbcl-2 transgenic B cells. hBcl-2 transgenic B cells were cultured for six days in medium containing 10% FBS then harvested and counted to determine viability (using trypan blue exclusion). Cells were replated in 24-well plates (1 x 10⁶/well in 1 ml) and stimulated with anti-IgM (50 mg/ml) overnight. The next day, cells were harvested and stained with anti-CD86-FITC and anti-CD69-PE (shown in purple-filled histograms) or appropriate fluorophore-coupled isotype negative control antibodies (shown with green-line histograms). Stained cells were analyzed by FACS cytometry; the histograms shown were gated for all live cells. The results shown are from a single experiment, replicate experiments (2) produced similar results.

Fig. 14. Proliferation responses in cultured hbcl-2 transgenic B cells. Cultured day 6 hbcl-2 transgenic B cells were harvested, viability counts were taken, and cells were replated at 6 x 10⁶ viable cells/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 (A) and day 2 poststimulation (B), 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; average values for quadruplicate wells for ³[H]-thymidine incorporation (+SD error bars) and average count values from duplicate wells for hand counts in each experiment are given. Similar results were obtained in three separate experiments.

Fig. 15. Cultured hbcl-2 transgenic B cells migrated in response to BLC (CXCL13), the ligand for CXCR5, and SDF-1a (CXCL12), the ligand for CXCR4. (A and B) Transwell migration experiments were performed over 4-hr periods with 1 x 10⁶ cells per sample. Average migrations from multiple experiments are shown. SEM error bars are for three or more repeat experiments; within each experiment, transwell samples were run in duplicate. The percentage of cells migrating was calculated using the positive control maximum (i.e., cells pipetted directly into the lower chamber of the transwell and harvested alongside test samples). (A) Graphs show dose response data for fresh (wild-type or hbcl-2 transgenic) and cultured (hbcl-2 transgenic) B cells to the chemokine BLC. (B) The histogram shows the responses of day 6 cultured bcl-2 transgenic B cells migrating to near optimal doses of BLC (102 nM = 1 mg/ml) or SDF (62.5 nM = 500 ng/ml). Graphs show the averages of three experiments ( SEM) on different B cell preparations (duplicate transwells per experiment), except (A) hbcl-2 day 3, which shows the average of two experiments (hence, no error bars given).