IL-5, IL-13, and IFN-γ accumulation in CD56+and CD56− natural killer cells from secondary cultures
| Donor3-150 . | Cells . | Day 0 . | IL-2 + IL-12 + Daudi, day 8 . | ||||
|---|---|---|---|---|---|---|---|
| IFN-γ+ . | IL-13+ . | IL-5+3-151 . | IFN-γ+(IL-13+, IL-5+) . | IL-13+ (IL-5+, IFN-γ+) . | IL-5+(IFN-γ+) . | ||
| 1. CD3− | Total | 23-152 | 42 | 16 | 40 (37, 8)3-153 | 54 (33, 28) | 18 (17) |
| CD56+3-155 | 4 | 9 | 2 | 39 (38, 5) | 56 (29, 27) | 16 (12) | |
| CD56− | 0 | 85 | 33 | 42 (38, 10) | 52 (40, 31) | 21 (19) | |
| 2. CD3− | Total | 18 | 7 | 2 | 42 (31, 2) | 27 (15, 48) | 4 (25) |
| CD56+ | 20 | 2 | 1 | 46 (30, 2) | 27 (15, 52) | 4 (25) | |
| CD56− | 1 | 50 | 12 | 12 (42, 8) | 22 (32, 23) | 7 (14) | |
| Donor3-150 . | Cells . | Day 0 . | IL-2 + IL-12 + Daudi, day 8 . | ||||
|---|---|---|---|---|---|---|---|
| IFN-γ+ . | IL-13+ . | IL-5+3-151 . | IFN-γ+(IL-13+, IL-5+) . | IL-13+ (IL-5+, IFN-γ+) . | IL-5+(IFN-γ+) . | ||
| 1. CD3− | Total | 23-152 | 42 | 16 | 40 (37, 8)3-153 | 54 (33, 28) | 18 (17) |
| CD56+3-155 | 4 | 9 | 2 | 39 (38, 5) | 56 (29, 27) | 16 (12) | |
| CD56− | 0 | 85 | 33 | 42 (38, 10) | 52 (40, 31) | 21 (19) | |
| 2. CD3− | Total | 18 | 7 | 2 | 42 (31, 2) | 27 (15, 48) | 4 (25) |
| CD56+ | 20 | 2 | 1 | 46 (30, 2) | 27 (15, 52) | 4 (25) | |
| CD56− | 1 | 50 | 12 | 12 (42, 8) | 22 (32, 23) | 7 (14) | |
| . | Cells . | Day 0 . | IL-2 + IL-12 + Daudi, day 9 . | IL-2 + IL-12, day 9 . | ||||
|---|---|---|---|---|---|---|---|---|
| IFN-γ+ . | IL-13+ . | IL-5+ . | IFN-γ+(IL-13+) . | IL-13+(IFN-γ+) . | IFN-γ+(IL-13+) . | IL-13+ (IFN-γ+) . | ||
| 3. CD3−/CD56− | Total | 0.1 | 51.7 | nd3-154 | 3.8 (79) | 55.4 (5.4) | 0.6 (50.0) | 57.7 (0.5) |
| CD56+ | —3-159 | — | — | 1.7 (53) | 37.2 (2.4) | 0.4 (0.0) | 3.1 (0.0) | |
| CD56− | 0.1 | 51.7 | nd | 5.8 (86) | 72.8 (6.9) | 0.6 (50.0) | 63.7 (0.5) | |
| . | Cells . | Day 0 . | IL-2 + IL-12 + Daudi, day 9 . | IL-2 + IL-12, day 9 . | ||||
|---|---|---|---|---|---|---|---|---|
| IFN-γ+ . | IL-13+ . | IL-5+ . | IFN-γ+(IL-13+) . | IL-13+(IFN-γ+) . | IFN-γ+(IL-13+) . | IL-13+ (IFN-γ+) . | ||
| 3. CD3−/CD56− | Total | 0.1 | 51.7 | nd3-154 | 3.8 (79) | 55.4 (5.4) | 0.6 (50.0) | 57.7 (0.5) |
| CD56+ | —3-159 | — | — | 1.7 (53) | 37.2 (2.4) | 0.4 (0.0) | 3.1 (0.0) | |
| CD56− | 0.1 | 51.7 | nd | 5.8 (86) | 72.8 (6.9) | 0.6 (50.0) | 63.7 (0.5) | |
For abbreviations, see Table 1.
Cells from primary cultures of CD34+ cells with Flt3-L and IL-2 from 2 independent donors (1 and 2) were cultured for 8 days in secondary culture with IL-12 (2 ng/mL), IL-2 (50 U/mL) and 50-Gy-irradiated Daudi feeder cells. CD161+/CD56− cells from similar cultures from a third donor were prepared by sorting, after sensitization with anti-CD3, anti-CD5, and anti-CD56 mAbs. Upon reanalysis, the proportion of CD161+ cells in this donor was more than 99%, with no detectable CD56+ cells. Intracellular cytokines and surface phenotype (CD56 or CD56 and CD161) were analyzed simultaneously before (day 0, 3-color immunofluorescence with anti-CD3, anti-CD56 alone, or with anti-CD161, and anti-IFN-γ, anti-IL-5, or IL-13 mAbs) and at the end of the culture (day 8 or 9, 4-color immunofluorescence with anti-CD3, anti-CD56 alone, or with anti-CD161, anti-IFN-γ, and anti-IL-5 or IL-13 mAbs) on cells stimulated as in Figure 4. Analysis was performed on gated CD3− cells.
All IL-5+ cells were always included within the IL-13+ population.
Figures are percent cells positive for the cytokine indicated above them.
Numbers in parentheses are the percentages of cells within the indicated cytokine+ population producing also the cytokines indicated in the parentheses above them.
The percentages of CD3−/CD56+cells within total CD161+ NK cells in donor 1 were 56 on day 0 and 53 on day 8; in donor 2 they were 90 on day 0 and 87 on day 8. On day 0, before secondary culture, more than 99% of the cells from donor 3 were CD3−/CD161+/CD56−; these contained no detectable CD56+ or IFN-γ+/IL-13+ cells. After a 9-day culture, the proportion of CD161+/CD56+ cells increased to 49% with IL-2 and IL-12 and Daudi and to 10% with IL-2 and IL-12 only.
Not done.
Not applicable (0% CD56+ cells).