Treg frequency and function in patients with chronic ITP. (A) Representative dot plot of CD25 and Foxp3 reactivity of gated CD4⁺ T-cell population in PBMCs. Isotype control for anti-Foxp3 antibody was used to set the gates for calculating the percentage of the Foxp3⁺CD25^hi cells within the CD4⁺ subset. (B) The percentages of Foxp3⁺CD25^hi cells in controls and patients with chronic ITP as calculated by the gating strategy shown in panel A. (C) Representative dot plot of CD4 and CD25 in total PBMCs with the box showing the CD4⁺CD25^hi population. Below the dot plot is the histogram showing the levels of Foxp3 reactivity in the CD4⁺CD25^hi subset expressed as percentage. The gating was set using isotype control for anti-Foxp3 antibody. (D) The levels of Foxp3 reactivity in controls and patients shown as percentages based on gating strategy in panel C. Horizontal bars represent the medians in panels B and D. (E) The mean percentage inhibition of the proliferative response of stimulated CD4⁺CD25⁻ cells by autologous CD4⁺CD25^hi cells from 13 of the chronic ITP patients (■) and 10 healthy controls (□) was calculated (“Proliferation assay”). CD4⁺CD25^hi and CD4⁺CD25⁻ sorted cells were stimulated with plate-bound 0.1 μg/mL anti-CD3 antibodies and allogeneic accessory cells, alone or cocultured at various suppressor-responder ratios (1:1, 1:4, and 1:16). Addition of CD4⁺CD25^hi cells at 1:1 ratios to CD4⁺CD25⁻ cells inhibited proliferative responses and decreasing the number of CD4⁺CD25^hi cells resulted in less inhibition of proliferation. For comparison, percentage inhibition of proliferation of CD4⁺CD25^hi when stimulated alone (1:0) is also shown. Regulatory T cells from patients with chronic ITP showed significantly less suppressor activity. Error bars depict standard error of the mean. (F) CD4⁺CD25⁻ responder T cells from healthy donors (n = 5) were cocultured with CD4⁺CD25^hi cells from patients with chronic ITP (n = 5) using the same conditions as in panel E, and mean percentage inhibition was calculated for the various responder-suppressor ratios (△). For comparison, the mean percentage inhibition of autologous proliferative responses of patients (n = 13) and controls (n = 10) from panel E is shown. The P values reflect the difference between the suppression of proliferation of CD4⁺CD25⁻ responder T cells from healthy donors using CD4⁺CD25^hi from healthy controls versus CD4⁺CD25^hi from patients. (G) Responder cells from patients with chronic ITP (n = 3) were cocultured with CD4⁺CD25^hi from healthy controls (n = 3) as in panel E, and mean percentage inhibition was calculated (◇). For comparison, the mean percentage inhibition of autologous proliferative responses from panel E is shown. The P values reflect the difference between the suppression of proliferation of CD4⁺CD25⁻ responder T cells from patients with chronic ITP using CD4⁺CD25^hi from healthy controls versus autologous patient CD4⁺CD25^hi.