Functional expression of GPR-9-6 and chemotactic behavior of human thymocytes in response to TECK. (A) HEK293 cells were stably transfected with CCR1, CCR4, CCR5, CCR6, and GPR-9-6. Transfected cells were loaded with Fura-2AM and sequentially stimulated with the chemokines (100 nmol/L) indicated. Transient calcium mobilization was monitored by measuring relative fluorescence of Fura-2AM. (B) Concentration dependence of calcium flux. Fura-2AM–loaded GPR-9-6-expressing cells were stimulated with the indicated concentrations of TECK (1 to 100 nmol/L), and fluorescence was monitored. The peak amplitude of the calcium response was plotted. (C) TECK is a broad-spectrum chemoattractant for immature and mature human thymocyte subsets. (D) Comparison of chemotactic specificity of TECK and SDF-1 for human thymocyte subsets. Input thymocytes before chemotaxis and thymocytes migrated to control medium, TECK (3 μg/mL), and SDF-1 (200 ng/mL) are shown with the percent composition of each thymocyte subset shown in each panel. (E) TECK-induced chemotaxis of human thymocytes is sensitive to pertussis toxin. Thymocytes were pretreated with pertussis toxin (1 μg/mL) for 1 hour and used for chemotaxis in response to TECK (3 μg/mL). Percent inhibition was obtained from a formula [% Inhibition = 100 − (% Cell Migration With Pertussis Toxin Treatment − Background Migration)/(% Cell Migration Without Pertussis Toxin Treatment − Background Migration) ×100].