PTK ZAP-70 is involved in T-cell transendothelial migration induced by CXCL12.

(A) Transendothelial migration of the ZAP-70–deficient mutant Jurkat cell line (P116), transfected with either wild-type ZAP-70 cDNA (pEFneo-HA-ZAP-70) or with empty vector, was compared with the migration of Jurkat cells (JE6.1) or with CD3⁻ JR3.T3 T cells. Cells (0.5 × 10⁶) of each cell type were added in the upper well of a Transwell chamber. Medium alone or medium plus CXCL12 (100 ng/mL) was added in the lower chamber. Transendothelial migration of cells from the upper well to the lower well was measured after 90 minutes. The absolute number of each population was determined by flow cytometry using calibrated beads. (B) CXCL12-induced migration of phytohemagglutinin-activated CD4⁺ T cells obtained from a patient deficient in ZAP-70 expression. ZAP-70/GFP-retrovirus–mediated transduced cells (filled histograms) and untransduced cells (open histograms) from the same patient were added to the upper well. Medium alone or medium plus CXCL12 (100 ng/mL) was added in the lower chamber. Transendothelial migration and quantification of the cell number were performed as described in “Materials and methods.” (C) Same cells were analyzed for CXCR4 expression by flow cytometry. ZAP-70/GFP-retrovirus–mediated transduced cells (full black line) and untransduced cells (full gray line) were labeled using a phycoerythrin-conjugated anti-CXCR4 mAb. Phycoerythrin-conjugated, isotype-matched mAb (dotted line) was used as a negative control.