T cells cultured in the presence of CD34+ cells do not exhibit suppressive activity in a secondary MLR. A 6-day MLR was established in which responder cells (1 × 106/mL) were cultured with irradiated allogeneic stimulator cells (1 × 106/mL) of the CD34+ cell donor or a third-party donor in the absence (▨) or presence (▪) of CD34+ cells (0.5 × 106/mL). The cultures were then harvested. (A) A fraction of the recovered cells were recultured for 7 days in limiting dilution cultures, and the CTL activity was determined by 51Cr-release assay. The CTL-p frequency (f) was calculated as described in “Materials and methods.” In the second fraction of the recovered cells, T cells were isolated using E rosetting with sheep red blood cells. (B) A second 6-day MLR was carried out in which naive responder cells (1 × 106/mL) from a new donor were stimulated against stimulator cells (1 × 106/mL) of the CD34+ cell donor or the primary third-party donor. The isolated T cells from the primary MLR were added (▨, ▪) or not (□) to the secondary MLR at a T–responder cell ratio of 1:1. The responder cells were then recultured for 8 days under limiting dilution, and the CTL-p frequency was determined. The statistical significance of the difference between the slopes (f) of 2 regression lines was evaluated as described in “Materials and methods.” Asterisks indicate statistical significance. **P < .001 when compared by t test with control cultures without CD34+ cells; *P < .005 when compared by t test with control cultures without addition of isolated T cells. The total CTL-p number (n) per primary bulk culture was calculated according to the yield of effector cells by the end of the primary culture period. (A) MLR against donor stimulators. In the absence of CD34+ cells (▨)n = 1162, in the presence of CD34+ cells (▪)n = 48. MLR against third-party stimulators. In the absence of CD34+ cells (▨)n = 621, in the presence of CD34+ cells (▪) n = 1749. (B) MLR against donor stimulators. In the absence of T cells (□) n = 1540, in the presence of T cells from culture without stem cells (▨) n = 11 560, in the presence of T cells from stem cell culture (▪) n = 1518. MLR against third-party stimulators. In the absence of T cells (□)n = 966, in the presence of T cells from culture without stem cells (▨)n = 1588, in the presence of T cells from stem cell culture (▪)n = 1765. The fWM was calculated as described in “Materials and methods”: (A) MLR against donor stimulators. In the absence of CD34+ cells (▨) fWM = 1.9 × 10-4, in the presence of CD34+ cells (▪) fWM = 1.1 × 10-5. MLR against third-party stimulators. In the absence of CD34+ cells (▨) fWM = 2.8 × 10-4, in the presence of CD34+ cells (▪) fWM = 2.4 × 10-4. V(fWM) values were below 4.5 × 10-9. (B) MLR against donor stimulators. In the absence of T cells (□) fWM = 5.5 × 10-4, in the presence of T cells from culture without stem cells (▨) fWM = 1.4 × 10-3, in the presence of T cells from stem cell culture (▪) fWM = 2.6 × 10-4. MLR against third-party stimulators. In the absence of T cells (□) fWM = 8.6 × 10-5, in the presence of T cells from culture without stem cells (▨) fWM = 3.2 × 10-4, in the presence of T cells from stem cell culture (▪) fWM = 3.7 × 10-4. V(fWM) values were below 1.2 × 10-7. Results represent 1 experiment.
