Circulating Treg-suppressive activity in patients before and on treatment with thrombopoietic agents. Suppression of proliferation by CD4⁺CD25^hi Treg was analyzed in patients before treatment (n = 10) and in patients on treatment with thrombopoietic agents (n = 9) as well as normal healthy controls (n = 9). Sorted populations of CD4⁺CD25⁻ T cells were stimulated with plate-bound 0.1 μg/mL anti-CD3 antibodies and allogeneic accessory cells, alone or cocultured at various ratios (solid lines indicate 1:1; and dotted lines, 1:4) with autologous sorted CD4⁺CD25^hi cells, and mean inhibition was calculated as described in “Proliferation/Treg cell suppression assay.” Although this is a cross-sectional study with different patients in the pretreatment and on treatment groups, we have joined the pretreatment and on treatment data points by a line (solid or dotted) to highlight the difference in the suppressive activity between the 2 groups. Suppression measured at a 1:1 ratio of CD4⁺CD25⁻ to CD4⁺CD25^hi in patients before treatment was 53% ± 7% but was higher in patients on treatment (63% ± 4%). Similarly, at 1:4, the pretreatment group had a lower suppressive activity (35% ± 7%) compared with on treatment group of 51% ± 6% (overall P = .045). For comparison, the healthy volunteer controls' suppressive activity at 1:1 ratio (70% ± 2%) and at 1:4 ratio (50% ± 5%) is also indicated. The raw proliferation data are shown in supplemental Figure 2.