Transmigration of Vδ1 and Vδ2 T cells to CXCR3 or CXCR4-directed chemokine dependency on PI-3K and CAMKII. Migration across HUVEC monolayers of Vδ2 and Vδ1 γδ T-cell lines, at 60 minutes, in the presence of 50 ng/mL IP-10/CXCL10 or SDF-1/CXCL12 as described in “Patients, materials, and methods.” (A-B) Effect of the PI-3K inhibitors LY294002 (20-0.2 μM; panel A) or wortmannin (1-100 nM; panel B). ○ indicates Vδ1 cells with SDF-1/CXCL12; •, Vδ2 cells with IP-10/CXCL10. (C-D) Effect of the CAMKII blocker KN62 (10-0.1 μM; •) or the inactive compound KN92 (10-0.1 μM, on Vδ2 cells with IP-10 (C) or Vδ1 cells with SDF-1 (D). Results are expressed as percentage of cell migration, calculated as described in “Patients, materials, and methods” (mean ± SD from 4 independent experiments with cell lines from 4 donors). In the absence of chemokines, at 60 minutes the transendothelial migration of Vδ2 or Vδ1 T cells was 15% ± 5% or 10% ± 3%, respectively (not shown). *Student t test; P < .05. (E-F) CXCR4, CXCR3, or TCR was engaged on Vδ1 or Vδ2 T cells using the specific mAbs (5 μg/mL) followed by GAM (100 μg/mL). Nil indicates GAM alone; sIg, murine immunoglobulin followed by GAM. (E) PI-3K activity tested by analyzing the phosphorylation of Akt1/PKB (pAkt) in cell lysates, using ELISA for phosphorylated Akt, normalized for total Akt, in the absence or presence of the PI-3K inhibitor LY294002 (20μM; ▦). Results are expressed as percentage units of pAkt compared with total Akt/10⁶ cells (mean ± SD of 3 independent experiments). (F) CAMKII activity assessed using the specific substrates and ³²P-γ ATP after lysis of Vδ2 or Vδ1 T cells, untreated or pretreated with the specific CAMKII blocker KN62 (10 μM; ▦) and immunoprecipitation with anti-CAMKII–specific mAbs. Results are expressed as cpm × 10^–3 and are the mean ± SD from 3 independent experiments with cell lines from 3 different donors. *Student t test; P < .05.