RosA inhibits TCR-induced intracellular calcium mobilization and the tyrosine phosphorylation of Itk but not ZAP-70 in human peripheral blood lymphocytes.

Human PBLs from healthy volunteers were isolated as described in “Materials and methods.” (A) hPBLs were loaded with calcium-sensitive dye fura-2/am and then treated with varying concentrations of RosA for 15 minutes. Cells were stimulated, as indicated by arrows, with OKT3 ascites (1:1000) and cross-linking antibodies, and results are representative of 2 separate experiments. (B-C) hPBLs were pretreated with indicated concentrations of RosA for 4 hours and then either left unstimulated or stimulated for 3 minutes with OKT3. hPBLs were either subjected to immunoprecipitation with the rabbit polyclonal anti-Itk (B) or anti–ZAP-70 mAb (C), separated on SDS-PAGE, and immunoblotted with antiphosphotyrosine mAb (top panels). The membranes were stripped and reblotted with anti-Itk or ZAP-70 mAb (bottom panels).