Clinical grade CD25/71 depletion of alloreactive T cells under GMP conditions using the CliniMACS system results in a 2-log reduction of alloreactivity (n = 5). PBMCs were cocultured with HLA-mismatched matured DCs (R:S ratio of 10:1) for 4 days in T-175 flasks. Cells were stained using biotinylated CD25 and CD71 antibodies followed by depletion using antibiotin immunomagnetic beads. Depletion was performed using standard CliniMACS tubing set and the CliniMACS system. (A) Flow cytometric analysis showing up-regulation of CD3⁺/25⁺ cells from a median of 2.55% to 8.17% and CD3⁺/71⁺ cells from 0.24% (0.07%-0.47%) to 1.77% (1.18%-3.9%) after 4 days of coculture and effective depletion of activated cells after allodepletion (median CD3⁺/25⁺, 0.05%; range, 0.02%-0.69%; median CD3⁺/71⁺, 0.05%; range, 0%-0.14%). (B) Residual alloreactivity of allodepleted cells in delayed 2° MLR. Individual data on all 5 depletions showing residual proliferative responses to host (□) and third-party (■) PBMCs are shown. (C) Cumulative data on residual alloreactivity in 2° MLR. Allodepleted T cells showed a median proliferative response to host PBMCs of 0.39% ± 0.063% compared with unmanipulated PBMCs with preservation of reactivity to third-party PBMCs (62% ± 0.478%).