Exposure of GVHD patient PBMCs to photodepletion cells increases Treg numbers. PBMCs from GVHD patients were cultured with untreated (untx) or photodepletion (PD)–exposed autologous PBMCs at a 1:1 effector:target ratio. At days 1, 3, and 6 of coculture, (A) percentages and (B) absolute numbers of FOXP3⁺ cells were determined by flow cytometry (mean ± SEM of 5 individual experiments). Black lines correspond to the addition of untreated PBMCs, and grey lines correspond to the addition of photodepletion-exposed PBMCs. (C) Treg proliferation was evaluated by measuring Ki-67 expression (mean ± SEM) and (D) EdU incorporation (representative example) in CD4⁺FOXP3⁺ cells after the coculture of GVHD patient cells with untreated (clear) or photodepletion-exposed (filled bars) GVHD PBMCs (3 independent experiments). (E) CD4⁺CD25⁻ cells were isolated and exposed (or not) to photodepletion. Three days after photodepletion, surviving cells were characterized for the presence of FOXP3. (F) CD4⁺CD25⁻ cells were isolated and stained with MitoTracker Deep Red. These cells were then cultured alone or with photodepleted CD4⁺CD25⁺ cells, in the presence of IL-2, or irradiated histoincompatible PBMCs and IL-2. (G) PBMCs from GVHD patients were untreated or exposed to photodepletion in the presence or absence of 50μM verapamil. Survival of CD4⁺ cells according to CD25 expression levels was assessed by 7-AAD staining. (H) GVHD PBMCs either untreated (clear), exposed to photodepletion alone (filled), or exposed to photodepletion in the presence of verapamil (striped bars) were then incubated with untreated autologous PBMCs, and proliferation was assessed by measuring ³H-thymidine incorporation. Bars represent mean ± SEM of 3 independent experiments. **P < .01 and ***P < .001 vs untreated targets alone (0:1) cells.