Third-party alloreactivity is preserved after depletion of alloreactive cells stimulated with single-donor apheresis products. PBMCs from a healthy donor were stimulated with irradiated PBMCs obtained from clinically harvested apheresis products from 2 donors (designated A and B). Following 7 days of stimulation, the CD4^hiCD38⁺ T-cell population was depleted using the gating strategy shown in the panels on the left. After depletion of alloantigen-specific CD4^hiCD38⁺ T cells, repeat stimulation with the original donor resulted in activation of CD4⁺ T cells similar to that seen following autologous restimulation. However, residual cells remained capable of responding to either a third-party donor (eg, 3.7% of CD4⁺ T cells still responded functionally to donor B, and 1.21% of CD4⁺ T cells responded to CMV stimulation following depletion of cells originally stimulated with donor A, top). Results are representative of 2 similar experiments from healthy donors.