P2Y₁₁R activation increases cAMPi and mediates ATP-induced inhibition of CX₃CL1-elicited chemotaxis and cytotoxicity of NK cells. (A) Effect of NAD⁺ on CX₃CL1-induced NK-cell chemotaxis. Cells were preincubated with or without the indicated inhibitors, stimulated with 10 ng/mL CX₃CL1 alone in the presence of the indicated concentrations of NAD⁺ for 60 minutes and cell migration measured as indicated in “Migration assays.” *P < .05, MRS2159 or NF023 versus NF157. (B) Induction of cAMPi by ATP and NAD⁺. NK cells were incubated at 37°C for 15 minutes with 100μM ATP or NAD⁺ in the presence or absence of the indicated inhibitors, and concentration of cAMPi was measured as described in “Intracellular cAMP quantification.” (C) Inhibition of CX₃CL1-dependent NK-cell migration by 8Br-cAMP. Chemotaxis was stimulated by 10 ng/mL CX₃CL1 alone or in the presence of 100μM 8Br-cAMP in NK cells pretreated or not for 1 hour with 100μM NF157. (D) NAD⁺ and 8Br-cAMP inhibit CX₃CL1-elicited NK cytotoxicity against HUVECs. Cells were preincubated with (filled bars) or without (open bars) 100μM NF157 and stimulated with 10 ng/mL CX₃CL1 alone in the presence of 100μM NAD⁺ or 100μM 8Br-cAMP and used for cytotoxicity assay against K562 cells. (A-C) Data are mean ± SD of triplicate cultures from 3 independent experiments. (D) Data are mean ± SD of sextuplicate cultures from 3 independent experiments. All P values were calculated performing Student t test for paired samples.