CCL19-, CXCL12-, or CXCL13-mediated TEM of CLL cells can be modulated by the EphA2-EFNA4 interaction. (A) CLL cells differing in EFNA4 expression (CLL nos. 1, 3, 4, 5, 8, 10, and 13) or normal B cells (3 samples; Table 1), were labeled with CFSE and added to the upper chamber of Transwell inserts (5 × 105/ well), which contained a HUVEC monolayer grown onto them and chemokines added to the lower chamber (CCL19, 500 ng/mL; CXCL12, 100 ng/mL; CXCL13, 1000 ng/mL). HUVECs or CLL/normal B cells were treated separately with EFNA4-Fc or EphA2-Fc homodimers (0.5 μg/106 cells, 30 minutes, 37°C), and chemotaxis was allowed to proceed for 2 hours. The number of cells migrated was determined by FACS. Mean values from triplicate experiments were compared with respect to control conditions (hFc-only–treated cells). The statistically significant differences are indicated by the P value. (B) Chemotaxis assays were performed as in panel A, in the absence of HUVECs and of the corresponding EFNA4-Fc treatment. Experiments were done in triplicate.
