CXCL12 requires CXCR4 to activate Wnt5A, and Wnt5A can modulate CXCR4 expression. Primary human T cells were pretreated with the CXCR4 antagonist, AMD3100 (1 μM, for 1 hour, with continued treatment for 12 hours, in the presence or absence of 100 ng/mL CXCL12). In the presence of AMD3100, CXCL12 is unable to increase Wnt5A expression (A). In CXCR4-null mutant mice, CXCL12 treatment can increase Wnt5A expression in wild-type, but not in CXCR4-null, mice (B). Stable transfection of CEM cells with a plasmid overexpressing WNT5A (pDEST12.2-WNT5A) results in a modest increase (*P < .05) in CXCR4 expression by Western blot analysis (C). Conversely, stable shRNA knockdown of WNT5A (PLKO.1-WNT5A-shRNA) in CEM cells results in a significant decrease in CXCR4 expression (D). This is confirmed by FACS analysis (E-F). RWnt5A treatment (50 ng/mL) of primary T cells resulted in increases in the levels of CXCR4 transcription (G). Transfection of primary T cells with siRNA against WNT5A, resulted in a significant reduction in CXCR4 transcription compared with control siRNA–transfected cells (H). FACS analysis demonstrates that the expression of CCR4 and CCR7, the only other chemokine receptors present on the surface of CEM cells, is not significantly affected by WNT5A overexpression. WNT5A knockdown slightly decreases CCR4, but does not significantly affect CCR7 (I). Migration of CEM cells to either of the ligands for CCR4 and CCR7, that is, TARC (J) and CCL19 (K), is not increased by the addition of rWnt5A, indicating that these effects are specific to the CXCL12/CXCR4 axis.