Results indicating that the veto effect is not mediated by contaminating CD34− cells and requires cell contact
| Cells* . | MLR against donor A . | MLR against donor B . | ||
|---|---|---|---|---|
| CTL activity (% responding cultures)† . | Veto activity (% inhibition)‡ . | CTL activity (% responding cultures)† . | Veto activity (% inhibition)‡ . | |
| None | 87.5 | — | 93.75 | — |
| CD34+ | 18.75 | 79 | 62.5 | 33 |
| CD34− | 100 | 0 | Not done | — |
| CD34+ in transwell | 87.5 | 0 | 100 | 0 |
| Cells* . | MLR against donor A . | MLR against donor B . | ||
|---|---|---|---|---|
| CTL activity (% responding cultures)† . | Veto activity (% inhibition)‡ . | CTL activity (% responding cultures)† . | Veto activity (% inhibition)‡ . | |
| None | 87.5 | — | 93.75 | — |
| CD34+ | 18.75 | 79 | 62.5 | 33 |
| CD34− | 100 | 0 | Not done | — |
| CD34+ in transwell | 87.5 | 0 | 100 | 0 |
The role of alloreactivity was evaluated by testing the veto effect of the CD34+ cells in comparison to the CD34− cells, which included a significant number of alloreactive T cells compared with the CD34+cells. The role of cell contact was evaluated by using the transwell assay. Briefly, the responder cells to be tested for their cytotoxic T-lymphocyte (CTL) response were cultured with irradiated stimulator cells in the lower chamber of the transwell plate. The CD34+ cells were cultured with responder cells in the upper chamber.
Cells from donor A were added to a 5-day mixed lymphocyte reaction (MLR) against stimulator cells from donor A or B.
The CTL activity was tested at the end of a 7-day limiting-dilution culture in microtiter plates. Wells were scored positive for CTL activity when chromium release exceeded the mean spontaneous release value by at least 3 SDs from the mean. Data are the percentages of responding cultures at a cell concentration of 1 × 104 responder cells/well.
Veto activity of tested cells was evaluated by assessment of their capacity to inhibit alloreactive CTL precursors in the MLR to which they were added at a 0.5 ratio of veto to responder cells.