Table 3.
B cell survival and proliferation during different B cell differentiation conditions.
| Isolated CD19+ B cells (TD) | ||||||
|---|---|---|---|---|---|---|
| Condition I | Condition II | Condition III | ||||
| Starting B cell number | 25.000 | 2500 | 250 | |||
| 3T3-CD40L cell | 10.000 | 10.000 | 10.000 | |||
| Ratio B cell : 3T3-CD40L cell | 1 : 0.4 | 1 : 4 | 1 : 40 | |||
| Cytokines | IL-21 | IL-21 | IL-21 | |||
| Mean CD19+ live cells | ||||||
| Day 0 | 25.000 | 2500 | 250 | |||
| Day 6 | 12438 | ± 3120; n=4 | 9736 | ± 1566; n=4 | 1656 | ± 224; n=4 |
| Day 9 | 8012 | ± 2643; n=4 | 8899 | ± 3196; n=4 | 6769 | ± 1305; n=4 |
| Mean amplification (compared to starting B cell number) | ||||||
| Day 6 | 0.5 | ± 0.1; n=4 | 3.9 | ± 0.6; n=4 | 6.6 | ± 0.9; n=4 |
| Day 9 | 0.3 | ± 0.1; n=4 | 3.6 | ± 1.3; n=4 | 27.1 | ± 5.2; n=4 |
| PBMCs (TD) | ||||||
| Condition I.2 | Condition II.2 | Condition III.2 | ||||
| Starting B cell number | 25.000 | 2500 | 250 | |||
| 3T3-CD40L cell | 10.000 | 10.000 | 10.000 | |||
| Ratio B cell : 3T3-CD40L cell | 1 : 0.4 | 1 : 4 | 1 : 40 | |||
| Cytokines | IL-21 | IL-21 | IL-21 | |||
| Mean CD19+ live cells | ||||||
| Day 0 | 25.000 | 2500 | 250 | |||
| Day 6 | 8601 | ± 1397; n=3 | 14487 | ± 4320; n=3 | 4139 | ± 311; n=3 |
| Day 9 | 6019 | ± 695; n=3 | 7819 | ± 2713; n=3 | 12826 | ± 3750; n=3 |
| Mean amplification (compared to starting B cell number) | ||||||
| Day 6 | 0.3 | ± 0.1; n=3 | 5.8 | ± 1.7; n=3 | 16.6 | - |
| Day 9 | 0.2 | ± 0.1; n=3 | 4.2 | ± 1.1; n=3 | 51.3 | - |
| Isolated CD19+ B cells (TI) | ||||||
| Condition IV | Condition V | Condition VI | ||||
| Starting B cell number | 25.000 | 2500 | 250 | |||
| TLR ligand | CpG | CpG | CpG | |||
| Cytokines | IL-2 | IL-2 | IL-2 | |||
| Mean CD19+ live cells | ||||||
| Day 0 | 25.000 | 2500 | 250 | |||
| Day 6 | 4751 | ± 1397; n=4 | 85 | ± 22.7; n=4 | 17 | ± 2.3; n=4 |
| Day 9 | 1571 | ± 695; n=4 | 29 | ± 13.8; n=4 | 2 | ± 0.8; n=4 |
| Mean amplification (compared to starting B cell number) | ||||||
| Day 6 | 0.2 | ± 0.1; n=4 | - | - | - | - |
| Day 9 | 0.1 | ± 0.0; n=4 | - | - | - | - |
| PBMCs (TI) | ||||||
| Condition IV.2 | Condition V.2 | Condition VI.2 | ||||
| Starting B cell number | 25.000 | 2500 | 250 | |||
| TLR ligand | CpG | CpG | CpG | |||
| Cytokines | IL-2 | IL-2 | IL-2 | |||
| Mean CD19+ live cells | ||||||
| Day 0 | 25.000 | 2500 | 250 | |||
| Day 6 | 8978 | ± 1985; n=3 | 3823 | ± 1983; n=3 | 58 | ± 18; n=3 |
| Day 9 | 7841 | ± 2127; n=3 | 2463 | ± 1055; n=3 | 47 | ± 26; n=3 |
| Mean amplification (compared to starting B cell number) | ||||||
| Day 6 | 0.4 | ± 0.1; n=3 | 1.5 | ± 0.8; n=3 | - | - |
| Day 9 | 0.3 | ± 0.1; n=3 | 1.0 | ± 0.4; n=3 | - | - |
Purified B cells or non-purified B cells (PBMC cultures) were cultured using a 1-step culture system for 6 and 9 days with either TD (CD40L + IL-21) or TI (CpG + IL-2) stimuli. On day 6 and day 9 cell counts and viability were determined using flow cytometry with fluorochrome-conjugated Live/Dead and anti-CD19. Results are shown as the mean ± SEM of n = 4 (B cell cultures) or n = 3 (PBMC cultures) donors. – indicates no further analysis due to <1000 CD19+ live events.