Phenotype of CD4⁺CD25^high T cells from patients with cGVHD. (A) Representative histogram plots are shown of flow sorted CD4⁺CD25^high T cells (thick solid line) and CD4⁺CD25^- T cells (thin solid line), which were fixed, permeabilized, and stained with PE-conjugated anti-CTLA-4 or isotype control (thin dotted line) in a patient with (left) or without (right) cGVHD. Mean cell fluorescence is shown as the figure to the top right of each histogram (n = 6 cGVHD; n = 5 no GVHD). (B) CD40L mean cellular fluorescence (± SEM) shown for both CD4⁺CD25^high cells (▪) and CD4⁺CD25^- cells (□) in patients with or without cGVHD and healthy volunteers. (C) CD62L expression of CD4⁺CD25^high T cells expressed as mean cellular fluorescence (± SEM). Significant differences indicated below the histograms (cGVHD n = 10; no GVHD n = 8; healthy volunteers n = 5). (D) Results of add-back experiments in a representative patient with cGVHD (top, n = 8) and a healthy volunteer (bottom, n = 3); 1 × 10⁴ flow-sorted CD4⁺CD25^high (25⁺) and 1 × 10⁴ CD4⁺CD25^- T cells (25^-), alone or in coculture (1:1 ratio of 25⁺ or 25^- cells, total 2 × 10⁴ cells/well, denoted as 25^-/- or 25^+/-), were stimulated with PHA (2 μg/mL) in the presence of 1 × 10⁴/well irradiated autologous accessory cells (ACs). Proliferations at 72 hours by [H³]-thymidine incorporation are shown in the left panels and IFN-γ production by cytokine ELISA in the right panels. These experiments were also performed with the addition of neutralizing antibody to IL-10 (α-IL10). Similar results were observed in patients without GVHD (n = 6). (E) CD4⁺CD25^- cells were stimulated as above in coculture with different numbers of CD4⁺CD25^high T cells derived from a patient with cGVHD and a control. Percentage inhibition of proliferation at 72 hours is shown. (F) Autologous CD4⁺ T cells were stimulated with immobilized anti-CD3 monoclonal antibody (1 μg/mL) in the presence or absence of CD4⁺CD25^high T cell clones at a 1:1 ratio and at 72 hours, proliferation was measured as above. Mean proliferations (± SEM) of individual clones derived from one patient are shown. (G) IL-10 was added to PHA-stimulated CD4⁺ cells in the presence of irradiated autologous ACs and in the presence or absence of neutralizing antibody to IL-10. Percentage inhibition of proliferation at 72 hours is shown.