Figure 3.
Figure 3. Functional analysis of CD4+CD25hi T cells. Highly purified CD4+CD25- T cells were stimulated by allogeneic irradiated PBMCs or DCs either in the presence or absence of highly purified CD4+CD25hi T cells derived from patients with CLL or healthy donors (both allogeneic). Treg cells from 16 controls, 10 patients pretreated with fludarabine, and 8 patients never treated with fludarabine were assessed in these MLRs. As a function of T-cell inhibition, proliferation (A) and IFN-γ production (B) were measured. Panel A shows representative experiments from a control, a CLL patient pretreated with fludarabine (flud), and a patient never treated with fludarabine (w/o flud). Open bars (PB) indicate background proliferation of irradiated allogeneic PBMCs; light gray bars (PB+Tconv), alloantigen induced proliferation of CD4+CD25- conventional T cells; dark gray bars (PB+Treg), background proliferation of CD4+CD25hi Treg cells; and black bars (PB+Tconv+Treg), proliferation of CD4+CD25- conventional T cells in the presence of CD4+CD25hi Treg cells at a 1:1 ratio (error bars represent SD; *P < .01 Student t test). (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+CD25hi Treg cells at different ratios (responders to suppressors) from a healthy donor (○), a fludarabine-treated CLL patient (•), and a CLL patient never treated with fludarabine (▪). Representative experiments are shown here, error bars represent SD. (D) Percentages of inhibition of proliferation of CD4+CD25- conventional T cells by CD4+CD25hi Treg cells at a 1:1 ratio from all healthy donors (n = 16), fludarabine-treated patients (n = 10), or patients with CLL never treated with fludarabine (n = 8) (*P < .001 Student t test).

Functional analysis of CD4+CD25hi T cells. Highly purified CD4+CD25- T cells were stimulated by allogeneic irradiated PBMCs or DCs either in the presence or absence of highly purified CD4+CD25hi T cells derived from patients with CLL or healthy donors (both allogeneic). Treg cells from 16 controls, 10 patients pretreated with fludarabine, and 8 patients never treated with fludarabine were assessed in these MLRs. As a function of T-cell inhibition, proliferation (A) and IFN-γ production (B) were measured. Panel A shows representative experiments from a control, a CLL patient pretreated with fludarabine (flud), and a patient never treated with fludarabine (w/o flud). Open bars (PB) indicate background proliferation of irradiated allogeneic PBMCs; light gray bars (PB+Tconv), alloantigen induced proliferation of CD4+CD25- conventional T cells; dark gray bars (PB+Treg), background proliferation of CD4+CD25hi Treg cells; and black bars (PB+Tconv+Treg), proliferation of CD4+CD25- conventional T cells in the presence of CD4+CD25hi Treg cells at a 1:1 ratio (error bars represent SD; *P < .01 Student t test). (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+CD25hi Treg cells at different ratios (responders to suppressors) from a healthy donor (○), a fludarabine-treated CLL patient (•), and a CLL patient never treated with fludarabine (▪). Representative experiments are shown here, error bars represent SD. (D) Percentages of inhibition of proliferation of CD4+CD25- conventional T cells by CD4+CD25hi Treg cells at a 1:1 ratio from all healthy donors (n = 16), fludarabine-treated patients (n = 10), or patients with CLL never treated with fludarabine (n = 8) (*P < .001 Student t test).

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