Absence of CCR8 leads to diminished T_reg numbers in the presence of syngeneic DCs in vitro. (A) CD4⁺CD25⁺ WT B6 T_regs (5 × 10⁵) were cultured for 72 hours on fibronectin-coated dishes in IL-2–deficient media with varying dilutions of syngeneic WT B6 BM-derived DCs. Cells were subsequently stained with an amine-reactive viability dye, and the total number of viable T_regs determined by flow cytometry. All wells were done in duplicate. (B) CD4⁺CD25⁺ WT B6 T_regs (5 × 10⁵) were cultured for 72 hours on fibronectin-coated dishes in IL-2–deficient media with varying dilutions of syngeneic WT B6 BM-derived macrophages. Cells were subsequently stained with an amine-reactive viability dye, and the total number of viable T_regs determined by flow cytometry. All wells were done in duplicate. (C) CD4⁺CD25⁺ WT Thy1.1⁺ eGFP^– B6 T_regs (5 × 10⁵) were incubated for 72 hours with an equal number of CCR8^−/− Thy1.2⁺ eGFP⁺ B6 T_regs with or without varying dilutions of syngeneic WT eGFP^– B6 DCs in the presence or absence of supplemental IL-2 at 100 IU/mL. Absolute numbers of viable WT (Thy1.1⁺) and CCR8^−/− (eGFP⁺) T_regs were subsequently measured by flow cytometry using a Foxp3⁺Viability Dye^low gate, and the ratio of CCR8^−/− to WT T_regs then determined for each well. The ratio of CCR8^−/− T_regs to WT T_regs did not vary according to the presence or absence of IL-2 for any of the 3 DC:T_reg culture conditions (1:8, 1:32, no DCs). As a result, all of the CCR8^−/− T_reg to WT T_reg ratios from a given culture condition (with and without IL-2) were pooled and compared with a reference ratio of 1.0 using a 1-sample t test; n = 4 per culture condition. P = .013 for 1:8 cultures; P = .017 for 1:32 cultures; P = 1.0 for no DC cultures. (D) Freshly isolated CD4⁺CD25⁺ WT eGFP⁺ B6 T_regs (1 × 10⁶) were fixed in a 1% formaldehyde solution and then permeabilized with a commercially available buffer (Becton Dickinson). Cells were subsequently stained with a phycoerythrin-conjugated antibody against the phosphorylated form of Stat5 (left panel). As a negative control, freshly isolated WT eGFP⁺ B6 T_regs were stained for phospho-Stat5 but without the preceding nuclear membrane permeabilization step (right panel). (E) CD4⁺CD25⁺ WT eGFP⁺ B6 T_regs or CCR8^−/− eGFP⁺ B6 T_regs (5 × 10⁵) were incubated with 5 × 10⁴ WT eGFP^– B6 DCs. After 72 hours, the cells were fixed and permeabilized. Cells were subsequently stained for phospho-Stat5. The buffers needed for nuclear phosphorylated transcription factor staining are poorly compatible with cell surface and Foxp3 staining. As a result, T_regs were distinguished from DCs using a forward vs side scatter lymphocyte gate and by nature of their eGFP positivity. Representative flow plots of WT (left) and CCR8^−/− T_regs (right) are depicted. (F) CD4⁺CD25⁺ WT eGFP⁺ B6 T_regs or CCR8^−/− eGFP⁺ B6 T_regs (5 × 10⁵) were incubated with 5 × 10⁴ WT eGFP^– B6 DCs for 72 hours. Absolute numbers of WT and CCR8^−/− T_regs were then determined by flow cytometry using an eGFP⁺ lymphocyte gate; n = 5 WT T_reg, n = 6 CCR8^−/− T_reg. *P = .001 by Student t test. (G-I) CD4⁺CD25⁺ WT eGFP⁺ B6 T_regs or CCR8^−/− eGFP⁺ B6 T_regs (5 × 10⁵) were seeded in the lower wells of 24-well 3-µM transwell plates and cultured for 72 hours with either 5 × 10⁴ WT eGFP^– B6 DCs placed in the upper chambers (G), with no DCs (H), or with 5 × 10⁴ WT eGFP^– B6 DCs placed also in the lower chambers (I). Absolute numbers of WT and CCR8^−/− T_regs were then determined by flow cytometry using an eGFP⁺ lymphocyte gate; n = 4 per group. *P = .018 by Student t test.