Specific depletion of alloreactive and/or CMV-specific CD4⁺ T cells. (A) The baseline frequencies of responder CD4⁺ T cells from a healthy CMV-seropositive donor responding to 7 days of stimulation with autologous PBMCs, pooled allogeneic PBMCs, and CMV antigens are shown. In each case, the TNFα+ frequency within the CD4⁺ T-cell population is shown (upper right quadrants). (B) Specific depletion of CD4^hiCD38⁺ T cells following allogeneic or CMV stimulation. Following primary stimulation with either pooled allogeneic PBMCs (top) or CMV lysates (bottom), high-speed sorting was used to deplete the cells contained within the CD4^hiCD38⁺ population (black squares). Residual cells, including CD4^intCD38^– cells and all CD8⁺ T cells, were then restimulated with autologous PBMCs, pooled allogeneic PBMCs, and CMV antigens. Following primary allogeneic stimulation and depletion of CD4^hiCD38⁺ cells, secondary stimulation induced a CMV-specific CD4⁺ T-cell response slightly higher than that at baseline (2.99% versus 1.44%), while secondary stimulation with the allogeneic PBMC pool was reduced to a level similar to that following control autologous stimulation (0.22% versus 0.23%). Conversely, depletion of CD4^hiCD38⁺ cells following primary CMV stimulation resulted in preserved secondary CD4⁺ T-cell responses to allogeneic stimulation (5.49%) but not CMV stimulation (0.17% versus 0.16% following autologous stimulation). As shown in the panels on the right, depletion of the CD4^hiCD38⁺ T-cell subset following either allogeneic or CMV stimulation also resulted in the loss of a detectable CD8⁺ T-cell response following secondary restimulation with the same antigen (0.23% for allogeneic and 0.19% after CMV stimulation), similar to that in control samples (data not shown). Data shown are representative of 5 depletion experiments following allogeneic stimulation and 4 experiments depleting CMV-specific T cells from unique donors. (C) Aggregate results from 9 separate experiments are shown (5 from unique donors depleting allogeneic CD4^hiCD38⁺ responders, 4 from depleting CMV-specific CD4^hiCD38⁺ responders). Displayed are the medians and standard deviations of TNFα-producing cells within CD4⁺ and CD8⁺ T-cell subsets prior to either allogeneic depletion (top left panels) or CMV depletion (bottom left panels). Following depletion of either allogeneic or CMV-specific CD4^hiCD38⁺ T cells, responses to the primary stimulus in both CD4⁺ and CD8⁺ T-cell compartments during secondary stimulation were reduced to a level indistinguishable from that seen following control autologous stimulation (right panels).