Functional analysis of CD4⁺CD25^high T cells. Highly purified CD4⁺CD25^– T cells were stimulated by allogeneic irradiated PBMCs in either the presence or absence of highly purified CD4⁺CD25^highT cells derived from MM patients or healthy donors (both allogeneic). T_reg cells from 22 controls and 7 MM patients were assessed in these MLRs. As a function of T-cell inhibition, proliferation (A) and IFN-γ production (B) were measured. Alternatively, T_reg cell–induced reduction of proliferation by CD4⁺CD25^– T cells stimulated with beads coated with anti-CD3 and anti-CD28 mAbs was assessed by flow cytometry (E). Panel A shows representative experiments froma healthy control and an MM patient. White bars (PB) indicate background proliferation of irradiated allogeneic PBMCs; light gray bars (T_conv), background proliferation of CD4⁺CD25^– conventional T cells; gray bars (PB+T_conv), alloantigen-induced proliferation of CD4⁺CD25^– conventional T cells; dark gray bars (PB+T_reg), background proliferation of CD4⁺CD25^high T_reg cells; and black bars (PB+T_conv+T_reg), proliferation of CD4⁺CD25^– conventional T cells in the presence of CD4⁺CD25^highT_reg cells, at a 1:1 ratio, (*P < .001, Student t test). Error bars represent SD. (B) Measurement of IFN-γ by cytokine bead array in the supernatants from cultures described in panel A. (C) Inhibition of proliferation of CD4⁺CD25^– conventional T cells by CD4⁺CD25^highT_reg cells at different ratios (responders to suppressors) from a healthy donor (○) and an MM patient (▪). (D) Percentages of inhibition of proliferation of CD4⁺CD25^– conventional T cells by CD4⁺CD25^highT_reg cells at a 1:1 ratio in all healthy donors (n = 22) and MM patients (n = 7). (E) Proliferation of allogeneic CD4⁺CD25^– T cells alone or in presence of CD4⁺CD25^highT cells derived from a healthy donor, an MGUS patient, and an MM patient. One representative experiment of at least 3 for each group is shown here. Percentage of proliferating cells is indicated within the figure.