Frequency of CD25⁺Foxp3⁺ cells in the CD4⁺ T cell population in patients with chronic myeloproliferative neoplasms. (A) Frequencies of CD25⁺Foxp3⁺CD4⁺ T cells in patients with chronic myeloproliferative neoplasms who were untreated (4 ET, 11 PV, and 3 PMF patients), treated with hydroxyurea (Hydrea; 1 ET and 10 PV patients), or treated with IFN-α (8 ET, 10 PV, and 1 PMF patient) compared with healthy donors. In the IFN-α–treated group, 12 patients were in complete hematologic remission, 5 had a partial hematologic response, and 1 patient did not respond to therapy. No patients were in major molecular remission. The gating strategy is shown in panel B, and frequencies are reported as number of cells in gate CD25⁺⁺ and CD25⁺ as percentage of CD4⁺ T cells. (B) Flow cytometric analysis showing the frequency of CD4⁺CD25⁺Foxp3⁺ cells in a 63-year-old woman with PV before and after 12 months of IFN-α treatment. The plot shows CD4⁺ cells previously gated on CD3⁺ cells in the lymphoid compartment. Gates represent CD25^highFoxp3⁺cells and CD25^intermediateFoxp3⁺ cells, respectively. Both Foxp3⁺ cells with CD25^high and CD25^intermediate expression expanded significantly during IFN-α2a treatment. (C) The frequency of CD4⁺CD25⁺Foxp3⁺ cells before and after 12-14 months of treatment with IFN-α2a in 6 JAK2-positive CMPN patients (4 ET and 2 PV). (D-E) Gating strategy for single cell sorting. Fraction A: CD4⁺CD25^highCD127⁻; fraction B: CD4⁺CD25^intermediateCD127⁻; Fraction C: CD4⁺CD25⁻CD127⁺. The histogram shows that > 90% of cells in fractions A and B expressed Foxp3; however, Foxp3 expression was discretely higher in fraction A. (F) Diagram showing inhibition of proliferating CD8⁺ T cells in the different fractions in 3 patients undergoing IFN-α2 treatment. Results are given as percentage inhibition of CD8⁺ T cells compared with positive controls. The suppressor/responder ratio was 1:3 in patient 2 and 1:2 in patients 1 and 3. In patient 1, more inhibitory activity was observed in fraction A than in fraction B. In patient 2, the inhibitory activity was very strong in fraction B compared with fraction A. Patient 3 had no or minimal suppression, and in fact, a discrete increase in proliferation was observed in fraction B.