CCR8 is overexpressed at the cell surface of CTCL peripheral blood tumor cells and is involved in Sézary cell activation and proliferation. Flow cytometric analyses of CCR8 expression by peripheral blood Sézary cells, regulatory T cells, healthy controls T cells, and T-cell lymphoma cell lines. (A) Gating strategy of peripheral blood Sézary cells (left panels) and mean fluorescence intensity of CCR8 expression in Sézary cells (vs control isotype) of 2 Sézary patients (medium panels). CCR8/CCR4 coexpression by Sézary cells is shown on the right panels. CCR8 was not significantly expressed by peripheral blood CD4⁺CD25hiCD127lo Tregs from Sézary patients (lower histograms). (B) CCR8 δ mean fluorescence intensity (CCR8 mAb, control isotype) in fresh Sézary cells from Sézary patients compared with healthy controls T cells. (C) Expression of CCR8 by SNK6, DERL-2, and HuT78 cell lines. (D) CCR8 stimulation by its ligands CCL18 (upper blots) and CCL1 (lower blots) induces Sézary cell activation. Freshly isolated Sézary cells were cultured for 15 or 30 minutes in medium alone or in the presence of IL-2 (100 IU/mL), CCL1 (10 ng/mL), CCL-18 (300 ng/mL), IL-2/CCL1, or IL-2/CCL18. The cells were lysed in NP40 (the content of the nuclei not visible), and expression of pErk1/2 was analyzed by western blot. (E) CCR8 stimulation by its CCL1 ligand induces Sézary cell proliferation. Fresh PBMC were incubated in CFSE and cultured over 96 hours in IL-2 (100 IU/mL), CCL-1 (10 ng/mL), or IL-2/CCL1 and the percentage of CFSE^lo cells calculated among live KIR3DL2⁺ Sézary cells. (F) CCR8 expression in freshly isolated healthy controls peripheral blood mononuclear cells before and after 3 days of in vitro CD3/28 activation. CFSE, carboxyfluorescein diacetate succinimidyl ester; mAb, monoclonal antibody; PBMC, peripheral blood mononuclear cell.