Inhibition of the TCR-CXCR4–mediated Rac1 signaling blocks cytokine production by the Sézary syndrome cell line, HUT-78, and patient isolates. (A) TCR-dependent IL-2 and IFN-γ secretion in HUT78 cells was assayed following 1 hour pretreatment with vehicle, 60 μM AMD3100 or 50 μM NSC22766 and stimulation with 2 μg/mL plate-bound OKT3 for 24 hours as in Figure 1D-E. Each bar denotes the mean cytokine production, ± SEM (n = 3; P < .05). (B) HUT78 cells were transfected with pmir-GLO empty vector or pmir-GLO-3′UTR via electroporation at 315 V, treated with 60 μM AMD3100 or 50 μM NSC23766 for 1 hour, stimulated with 2 μg/mL plate-bound OKT3 for 3.5 hours, harvested, lysed, and assayed for luciferase activity as in Figure 5F. Representative experiment is shown with each bar denoting mean relative light units ± SD (n = 3; P < .05) (unpaired Student t test). (C) HUT78 cells were treated with 60 μM AMD3100 or 100 μΜ NSC23766 for 1 hour, cultured ± 2 μg/mL plate-bound OKT3 for 24 hours, and assayed for IL-10 secretion as in Figure 1 (n = 3). (D-G) T cells isolated from residual diagnostic patient specimens (TCL-1, 3, 4) were pretreated where indicated with 50 μM NSC23766, stimulated with 1 μg/mL plate-bound OKT3 and soluble CD28 for 24 hours, and assayed for IL-2 production as in Figure 1D. (F) Graph summarizes results from panel E. Each bar denotes the percentage change in cytokine production normalized to the corresponding cytokine production by control-treated cells for each stimulation condition (n = 3; ± SEM; P < .05). (G) Patient information.