The effect of cytokines (A) and different stimuli (B) on the cloning frequency and the cytokine profile of human CD8 T-cell clones
| Donor Number . | Method . | Cytokine Present . | Cloning Frequency . | Average Cytokine Production (pg/mL) . | Percentage Clones . | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| IFN-γ . | IL-4 . | IL-5 . | IL-10 . | Tc1 . | Tc2 . | Tc0 . | ||||
| (A)* 1 | Direct | IL-2 | 0.23 | 13 018 | 420 | 2974 | 5227 | 85 | 0 | 15 |
| Limiting | IL-2 + IL-4 | 0.24 | 12 064 | 203 | 792 | 0 | 31 | 0 | 69 | |
| Dilution | IL-4 | 0.38 | 13 160 | 837 | 7336 | 703 | 39 | 0 | 61 | |
| (A) 2 | Direct | IL-2 | 0.53 | 19 562 | 1005 | 18 337 | 1012 | 75 | 0 | 25 |
| Limiting | IL-2 + IL-12 | 0.61 | 28 000 | 204 | 726 | 151 | 88 | 0 | 12 | |
| Dilution | — | — | — | — | — | — | — | — | — | |
| (B)† 1 | Bulk culture with | |||||||||
| Anti-CD3/CD28 | IL-2 + IL-4 | 0.33 | 9388 | 505 | 6182 | 2854 | 80 | 10 | 10 | |
| Anti-CD28 | IL-2 + IL-4 | 0.37 | 13 520 | 1663 | 7549 | 9002 | 74 | 13 | 13 | |
| Donor Number . | Method . | Cytokine Present . | Cloning Frequency . | Average Cytokine Production (pg/mL) . | Percentage Clones . | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| IFN-γ . | IL-4 . | IL-5 . | IL-10 . | Tc1 . | Tc2 . | Tc0 . | ||||
| (A)* 1 | Direct | IL-2 | 0.23 | 13 018 | 420 | 2974 | 5227 | 85 | 0 | 15 |
| Limiting | IL-2 + IL-4 | 0.24 | 12 064 | 203 | 792 | 0 | 31 | 0 | 69 | |
| Dilution | IL-4 | 0.38 | 13 160 | 837 | 7336 | 703 | 39 | 0 | 61 | |
| (A) 2 | Direct | IL-2 | 0.53 | 19 562 | 1005 | 18 337 | 1012 | 75 | 0 | 25 |
| Limiting | IL-2 + IL-12 | 0.61 | 28 000 | 204 | 726 | 151 | 88 | 0 | 12 | |
| Dilution | — | — | — | — | — | — | — | — | — | |
| (B)† 1 | Bulk culture with | |||||||||
| Anti-CD3/CD28 | IL-2 + IL-4 | 0.33 | 9388 | 505 | 6182 | 2854 | 80 | 10 | 10 | |
| Anti-CD28 | IL-2 + IL-4 | 0.37 | 13 520 | 1663 | 7549 | 9002 | 74 | 13 | 13 | |
CD8 clones were generated from peripheral blood CD8 cells by limiting dilution using PHA and autologous feeders for stimulation.
To study the effects of polarizing cytokines on the cloning outcome, clones were generated in the presence of IL-2, IL-4, IL-4 + IL-2, and IL-12 + IL-2.
CD8 cells were stimulated in bulk culture with immobilized anti-CD3 and anti-CD28 or anti-CD28 alone in the presence of IL-4 and IL-2 before limiting dilution. After 3 stimulation cycles, generated CD8 clones were restimulated for 18 hours with PMA and ionomycin (as described in Materials and Methods), and secreted cytokines in the supernatants were measured by ELISA. At least 30 clones were analyzed under each condition. Clones were classified based on the production of IL-4 and IFN-γ. Mean cytokine production was calculated for clones producing a particular cytokine.