Generation of plasma cells from memory B cells
| . | B-cell amplification and differentiation (days 0-4; step 1) . | Plasmablastic differentiation (days 4-7; step 2) . | Plasma cell differentiation (days 7-10; step 3) . |
|---|---|---|---|
| Activation | sCD40L + ODN | — | — |
| Cytokines | IL-2 + IL-10 + IL-15 | IL-2 + IL-6 + IL-10 + IL-15 | IL6 + IL-15 + IFN-α |
| Mean cell amplification | 6.1 ± 1.8; n = 19 | 3.7 ± 1.3; n = 18 | 0.51 ± 0.09; n = 17 |
| Cell viability, percentage | 94 | 81 | 38 |
| CD20+CD38− activated B cells, % | 42.3 ± 12.2; n = 13 | 11.7 ± 5.1; n = 13 | 2.2 ± 1.2; n = 13 |
| CD20+CD38+ intermediate cells, % | 16.4 ± 12.5; n = 13 | 20.1 ± 8.8; n = 13 | 13.6 ± 8.5; n = 13 |
| CD20−CD38++ plasmablasts/plasma cells, % | 19.5 ± 5.5; n = 13 | 56.6 ± 7.7; n = 13 | 79.0 ± 8.8; n = 13 |
| CD20−CD38+CD138+ plasma cells, % | 2.1 ± 0.9; n = 13 | 15.9 ± 6.2; n = 13 | 54.8 ± 8.7; n = 13 |
| Yield of plasmablast/plasma cell generation for one starting memory B cells | NA | Plasmablasts /plasma cells: 12.3 ± 6.1; n = 13 | Plasma cells: 6.3 ± 3.3; n = 13 |
| . | B-cell amplification and differentiation (days 0-4; step 1) . | Plasmablastic differentiation (days 4-7; step 2) . | Plasma cell differentiation (days 7-10; step 3) . |
|---|---|---|---|
| Activation | sCD40L + ODN | — | — |
| Cytokines | IL-2 + IL-10 + IL-15 | IL-2 + IL-6 + IL-10 + IL-15 | IL6 + IL-15 + IFN-α |
| Mean cell amplification | 6.1 ± 1.8; n = 19 | 3.7 ± 1.3; n = 18 | 0.51 ± 0.09; n = 17 |
| Cell viability, percentage | 94 | 81 | 38 |
| CD20+CD38− activated B cells, % | 42.3 ± 12.2; n = 13 | 11.7 ± 5.1; n = 13 | 2.2 ± 1.2; n = 13 |
| CD20+CD38+ intermediate cells, % | 16.4 ± 12.5; n = 13 | 20.1 ± 8.8; n = 13 | 13.6 ± 8.5; n = 13 |
| CD20−CD38++ plasmablasts/plasma cells, % | 19.5 ± 5.5; n = 13 | 56.6 ± 7.7; n = 13 | 79.0 ± 8.8; n = 13 |
| CD20−CD38+CD138+ plasma cells, % | 2.1 ± 0.9; n = 13 | 15.9 ± 6.2; n = 13 | 54.8 ± 8.7; n = 13 |
| Yield of plasmablast/plasma cell generation for one starting memory B cells | NA | Plasmablasts /plasma cells: 12.3 ± 6.1; n = 13 | Plasma cells: 6.3 ± 3.3; n = 13 |
Purified memory B cells were cultured for 10 days using a 3-step culture system. In step 1, B cells were activated for 4 days with sCD40L and ODN and IL-2 + IL-10 + IL-15. In step 2, plasmablast differentiation was further promoted removing sCD40L and ODN and adding IL-6 together with IL-2 + IL-10 + IL-15. In step 3, plasma cell differentiation was induced for 3 days, removing IL-2 and IL-10 and adding IFN-α together with IL-6 + IL-15. At the end of every step, cell counts and viability were determined and cell phenotype was assayed with fluorochrome-conjugated anti-CD20, CD38, or CD138 mAbs, or isotype-matched control mAbs. Flow cytometry was performed with a FACScan device. Results are shown as the mean ± SD of n experiments.
— indicates no sCD40L or ODN; and NA, not applicable.