Effect of FasLs Transfection on Apoptosis Induced by Anti-Fas or Anti-CD3 MoAb
| . | Control . | Anti-Fas . | Control . | Anti-Fas . | Control . | Anti-CD3 . | Control . | Anti-CD3 . |
|---|---|---|---|---|---|---|---|---|
| 1 | 11.7 | 9.3 | 6.5 | 9.0 | 0.1 | 1.3 | 2.6 | 40 |
| 2 | 5.0 | 4.5 | 6.0 | 8.1 | 0.1 | −1.1 | 2.2 | 38 |
| 3 | 6.5 | 6.0 | 8.3 | 10.0 | 0.2 | −1.2 | 3.7 | 51 |
| 4 | 4.7 | 5.2 | 9.0 | 10.0 | 0.2 | 0.6 | 1.7 | 50.1 |
| 5 | 9.4 | 5.5 | 10.7 | 12.0 | 0.1 | −1.1 | 3.1 | 52.4 |
| 6 | 3.3 | 4.1 | 7.8 | 10.1 | 0.2 | 0.7 | 3.5 | 49.1 |
| 7 | 10.7 | 39.5* | 5.8 | 30.5* | 0.1 | 14.6* | 4.2 | 39.5 |
| 8 | 10.5 | 36.8* | 8.9 | 35.3* | 0.1 | 16.6*,† | 1.4 | 46 |
| 9 | 7.4 | 31.6* | 5.0 | 27.2* | 0.3 | 18.5*,† | 2.8 | 51 |
| 10 | 8.3 | 38.9* | 6.9 | 31.0* | 0.1 | 14.9* | 4.0 | 43 |
| . | Control . | Anti-Fas . | Control . | Anti-Fas . | Control . | Anti-CD3 . | Control . | Anti-CD3 . |
|---|---|---|---|---|---|---|---|---|
| 1 | 11.7 | 9.3 | 6.5 | 9.0 | 0.1 | 1.3 | 2.6 | 40 |
| 2 | 5.0 | 4.5 | 6.0 | 8.1 | 0.1 | −1.1 | 2.2 | 38 |
| 3 | 6.5 | 6.0 | 8.3 | 10.0 | 0.2 | −1.2 | 3.7 | 51 |
| 4 | 4.7 | 5.2 | 9.0 | 10.0 | 0.2 | 0.6 | 1.7 | 50.1 |
| 5 | 9.4 | 5.5 | 10.7 | 12.0 | 0.1 | −1.1 | 3.1 | 52.4 |
| 6 | 3.3 | 4.1 | 7.8 | 10.1 | 0.2 | 0.7 | 3.5 | 49.1 |
| 7 | 10.7 | 39.5* | 5.8 | 30.5* | 0.1 | 14.6* | 4.2 | 39.5 |
| 8 | 10.5 | 36.8* | 8.9 | 35.3* | 0.1 | 16.6*,† | 1.4 | 46 |
| 9 | 7.4 | 31.6* | 5.0 | 27.2* | 0.3 | 18.5*,† | 2.8 | 51 |
| 10 | 8.3 | 38.9* | 6.9 | 31.0* | 0.1 | 14.9* | 4.0 | 43 |
FasLs expression protects T cells from anti-Fas–activated apoptosis. FasLs-transfected (clones 1 through 6) or empty vector-transfected (clones 7 through 10) clones were triggered by anti-Fas MoAb (1 μg/mL). The percentage of apoptosis (column 2, untreated control; column 3, anti-Fas–treated cells) was evaluated as described in Materials and Methods. Supernatant of FasLs-transfected clones inhibits anti-Fas–activated apoptosis. Anti-Fas–induced cell death (column 8, untreated control; column 9, anti-CD3–treated cells) was evaluated against normal untransfected 3DO cells in the presence of supernatants (100 μL supernatant added in the assay, see also Materials and Methods) from cultures of FasLs-transfected (clones 1 through 6) or empty vector-transfected (clones 7 through 10) clones. FasLs expression inhibits the cytotoxic activity of activated T cells against Fas+ P815 target cells. Cytotoxic activity of unstimulated (control, column 6) or anti-CD3–activated (column 7) clones was evaluated in a cytotoxicity assay against the Fas+ P815 cell line. FasLs-transfected (clones 1 through 6) or empty vector-transfected (clones 7 through 10) clones were triggered with anti-CD3 antibody and used as effectors in the cytotoxicity assay. The percentage of cytotoxicity, at the E:T ratio of 25:1, was calculated as described in Materials and Methods. Results are the average of 3 experiments; the standard errors, which are less than 10% of the values, are omitted for clarity. Expression of FasL in FasLs- or empty vector-transfected clones. Cells were cultured in 96-well plates coated with anti-CD3 MoAb (1 μg/mL), and FasL expression was evaluated by flow cytometry analysis after 15 hours of culture. Numbers represent the percentage of FasL+ cells of a representative experiment.
P < .01 as compared with control (respective control values are in the left column).
Blocking antimouse FasL MoAb (MFL4, 5 μg/mL) was used in 2 clones to control that the observed cytotoxic effect of anti-CD3 triggered cells was FasL-dependent. The addition of the MoAb inhibited the cytotoxicity down to 5.6% (clone 8) and 4.1% (clone 9), respectively, compared with controls (without anti-FasL blocking MoAb) giving 17.9% (clone 8) and 21.5% (clone 9).