(620f) Characterization Of H C E L L-Specific Contributions To E- And L-Selectin Ligand Activities Of Human Hematopoietic Cells

E-, L-, and P-selectin comprise a family of cell-surface, calcium-dependent lectins specialized to mediate adhesive interactions under hydrodynamic shear flow conditions, and are molecules that play critical roles in cell trafficking in normal and pathobiological conditions. We have previously reported that cells expressing the sialofucosylated CD44 glycoform termed HCELL (Hematopoietic Cell E-/L-selectin Ligand), including native CD34+ hematopoietic progenitor cells, de novo acute myeloid leukemia (AML) cells, the human AML cell line KG1a, and G-CSF-mobilized peripheral blood leukocytes, possess markedly stronger E- and L-selectin ligand activities than those lacking this molecule. However, the discrete contribution(s) of HCELL to this biology, relative to the other selectin glycoprotein ligands expressed by hematopoietic cells, PSGL-1 and CD43, has not been elucidated. To directly assess HCELL potency for E- and L-selectin binding, expression was silenced using lentiviral siRNA targeting CD44 on KG1a cells, and selectin ligand activities were subsequently compared between untreated and transduced cells. CD44 siRNA transduction led to >90% reduction in CD44 (i.e., HCELL) expression, without affecting expression of PSGL-1, CD43, or integrin subunits. Compared to untreated and vector-transduced cells, CD44-silenced cells showed striking decreases (>50%) in E-selectin-binding over physiologic shear stress levels, with parallel decreases observed for L-selectin-dependent rolling interactions. No inhibition of P-selectin ligand activity was observed, consistent with the lack of effect of CD44 silencing on the expression of PSGL-1, the only P-selectin ligand expressed by KG1a cells. These data highlight the complexity of selectin ligands displayed on human hematopoietic cells, and provide direct evidence that HCELL is the primary mediator of adhesion to E- and L-selectin on cells expressing this molecule. Continued characterization of the biological properties of HCELL should provide additional insight into its role in physiologic hematopoiesis and pathobiologic conditions, such as leukemia and vascular diseases.