In vitro and in vivo Treg-suppressive activity of responders versus nonresponders. (A) Representative dot plot of CD25 and Foxp3 reactivity of gated CD4⁺ T-cell population in mouse splenocytes. Isotype control for anti-Foxp3 antibody was used to set the gates for calculating the percentage of the Foxp3⁺CD25⁺ cells within the CD4⁺ subset. (B) The percentages of Foxp3⁺CD25⁺ cells in splenocytes of nontransfused (“Nontransfused controls”) as well as nonresponder and responder mice as calculated by the gating strategy shown in panel A. (C) Sorted splenic suppressor Treg (CD4⁺CD25⁺) and T effector (CD4⁺CD25⁻) cells were stimulated with accessory cells from responders and anti-CD3 antibodies alone or cocultured at various suppressor/T effector ratios (CD4⁺CD25⁺/CD4⁺CD25⁻ of 1:1, 1:2, 1:4, 1:8, and 1:16). Proliferation was measured by incorporation of [³H]thymidine. The mean percentage proliferative responses of stimulated CD4⁺CD25⁻ cells by autologous CD4⁺CD25⁺ cells from responders (●) and nonresponders (○) was calculated as (cpm incorporated in the coculture)/cpm of effector T cells alone) × 100%. Proliferative responses of T effectors were thus normalized to 100%. Addition of CD4⁺CD25⁺ cells at 1:1 ratios to CD4⁺CD25⁻ T effector cells inhibited proliferative responses, and decreasing the number of CD4⁺CD25⁺ cells resulted in more proliferation (less suppression). For comparison, percentage proliferation of CD4⁺CD25⁺ when stimulated alone (1:0) is also shown. Error bars represent SEM. The P values show the statistically significant differences in suppression of proliferation between responders and nonresponders at the indicated suppressor/effector ratios. In Figure S1 (available on the Blood website; see the Supplemental Materials link at the top of the online article), the comparative proliferation of mice treated with CpG-ODN alone for 1 week is shown. (D) CD4⁺CD25⁻ nonresponder T effector cells were cocultured with CD4⁺CD25⁺ Tregs from responders () and CD4⁺CD25⁻ responder T effector cells were cocultured with CD4⁺CD25⁺ Tregs from nonresponders (), and mean percentage proliferation was calculated for the 1:16 suppressor/effector T-cell ratio. For comparison, the mean percentage autologous proliferative responses of nonresponders (□) and responders (■) from panel C are shown. The indicated P values reflect the difference between the proliferation of nonresponder CD4⁺CD25⁻ using CD4⁺CD25⁺ from responders and nonresponders as well as differences between the proliferation of responder CD4⁺CD25⁻ using CD4⁺CD25⁺ from responders and nonresponders. Tregs from nonresponders were equally effective in suppressing T effector cells from responders and nonresponders (P = .5; not indicated in the figure). Similarly, responder Tregs suppressed T effector cells from responders and nonresponders to the same degree (P = .9; not indicated in the figure). (E) Groups of mice were adoptively transferred with CD4⁺CD25⁺ from splenocytes of nonresponder (“+ CD4⁺CD25⁺ Non-responder”) and responder (“+ CD4⁺CD25⁺ Responder”) syngeneic mice. After 24 hours, the mice were given intravenous transfusions of buffy-coated/granulocyte-depleted huGPA red cells (equivalent to 1-2 packed units) with CpG-ODN adjuvant followed by weekly transfusions of red cells alone for another 3 weeks. After that, levels of alloantibodies (IgG) in the plasma were measured using diluted plasma (1 in 4) followed by analysis using flow cytometry and are expressed in fluorescent units on the y-axis. Levels of alloanti-huGPA in untransfused (“control”) and nonadoptively transferred transfused mice (“RBC transfused”) from Figure 1A is also shown. The P values indicate the difference in alloantibody levels in nonadoptively transferred transfused mice (“RBC transfused”) and mice adoptively transferred with nonresponder Tregs (P = .005) as well as the difference in alloantibody levels in mice adoptively transferred with Tregs from nonresponders (“+ CD4⁺CD25⁺ Non-responder”) versus responders (“+ CD4⁺CD25⁺ Responder”; P = .004). In Figure S2, alloanti-huGPA levels after adoptive transfer of control, nonsuppressive CD4⁺CD25⁻ sorted cells from responders and nonresponders are shown. (F) Mice were given intravenous transfusion of buffy-coated/granulocyte-depleted red cells from human Duffy Fy^b (huFy^b) transgenic mice (equivalent to 1-2 packed units) with CpG-ODN adjuvant followed by weekly transfusions of red cells alone for another 2 weeks. The mice were then given a single transfusion of huGPA RBCs (no CpG). The presence of IgG-specific alloanti-GPA and allo-Fy^b in plasma from mice was then measured using diluted plasma (1 in 4) and huGPA or huFy^b transgenic red cells, followed by analysis using flow cytometry, and is expressed in fluorescent units on the y-axis and x-axis, respectively. The correlative relationship between the 2 sets of values is indicated by Pearson r and P values. The upper and lower 95% confidence interval bands of the regression line (—) are shown by the dotted lines.