Assessing function of skin homing T cell selectin-ligands via selective knockdown with siRNA (B62)

The recruitment of T cells to skin is a key feature of inflammatory diseases such as psoriasis and eczema. Skin homing T cells express cutaneous lymphocyte-associated antigen (CLA), a carbohydrate epitope that binds to E-selectin on postcapillary venules and mediates lymphocyte tethering and rolling in shear flow. E-selectin ligand (ESL) activity and CLA epitope expression have been described on P-selectin glycoprotein ligand-1 (PSGL-1) and leukosialin (CD43). CLA+ PSGL-1 serves as both a P-selectin ligand (PSL) and an ESL, whereas CLA+ CD43 is only an ESL. Although their ESL functions appear redundant, the true relative contribution of PSGL-1 and CD43 to ESL activity in human CLA+ T cells has not yet been evaluated. Using siRNA, we inhibited expression of PSGL-1, CD43, or both on CLA+ T cells by 70–80%, as measured by flow cytometry. PSGL-1 knockdown resulted in a 70% reduction of PSL but relatively unchanged ESL activity, as assessed by shear flow binding assays. CD43 knockdown did not signficantly impact either PSL or ESL activity. Surprisingly, knockdown of both PSGL-1 and CD43 did not noticeably reduce ESL activity nor CLA expression. As predicted from animal models, PSGL-1 is the major PSL on human CLA+ T cells but serves a redundant role as an ESL. The high residual amounts of CLA expression and ESL activity remaining after knockdown of both PSGL-1 and CD43 suggest that an uncharacterized ESL distinct from PSGL-1 and CD43 exists and may represent a signficant pool of ESL function on skin homing T cells.