RWnt5A can influence CXCL12-mediated signaling in primary T cells. (A) Western blot analysis demonstrates that phospho-PKC (α, β, and γ isoforms) is up-regulated in primary human T cells by treatment with 0.2 μg/mL rWnt5A for 16 hours. This activation occurs in a biphasic manner. (B) T cells pretreated with rWnt5A for 16 hours demonstrate greater migration in response to CXCL12 in a dose-dependent manner; n (donors) = 3, *P < .01. (C) Western blot analysis demonstrates that in the presence of WNT5A siRNA, 100 ng/mL CXCL12 (12 hours of treatment) failed to increase Wnt5A expression compared with control siRNA-transfected cells. Primary human T cells were then transfected with siRNA against either WNT5A or FZD2, and 36 hours later were subjected to a Matrigel invasion assay. Primary T cells transfected with either WNT5A or FZD2 siRNA demonstrated a diminished migratory response to CXCL12 compared with those transfected with a scrambled siRNA (D); n (donors) = 3, *P < .01. The same is true of a stably transfected WNT5A shRNA cell line (CEM), in which Wnt5A expression is dramatically reduced (E inset) and so is migration in response to CXCL12 (E); *P < .01. These data are supported further by the fact that Rac activation, required for motility of T cells, is increased by rWnt5A (F) in primary T cells and cannot be activated by CXCL12 in WNT5A shRNA-transfected CEM cells (G).