ZAP-70 Expression Associated with Activation in Normal Human B Cells and B Cell Chronic Lymphocytic Leukemia.

The protein tyrosine kinase ZAP-70, known to be critical for T cell development and T cell receptor signaling, has become a potential prognostic tool for B cell chronic lymphocytic leukemia (B-CLL). Recent studies have suggested that those patients with ZAP-70 positive leukemic cells have a poorer disease prognosis than do patients whose leukemic cells lack ZAP-70 expression. In addition, ZAP-70 expression occurs more frequently in CLL B cells that express unmutated immunoglobulin (Ig) VH region genes. However, several questions remain regarding ZAP-70 expression in B-CLL. First, is ZAP-70 expression in some patients with B-CLL the consequence of malignant transformation or do normal human B cells express ZAP-70 under certain conditions of stimulation? Secondly, does the expression of ZAP-70 reflect a more activated phenotype, thus resulting in the more aggressive phenotype of ZAP-70+ B-CLL? To begin to address these questions, we focused our studies on ZAP-70 expression in normal, human B cells. CD19+ B cells were isolated by magnetic bead separation from normal human peripheral blood (PB) or tonsil and spleen surgical waste tissue. We first used three-color flow cytometry to evaluate ZAP-70 expression in CD19+ versus CD3+ lymphocytes. While PB B cells were negative for ZAP-70 expression, we were able to detect a subset of CD19+/ZAP-70+ cells in both spleen and tonsil and this subset expressed ZAP-70 at levels comparable to those of CD3+ T cells. To verify the flow cytometry results, we evaluated total ZAP-70 expression by immunoblotting and observed readily detectable ZAP-70 in both splenic and tonsillar CD19+ cells. Based on the observation that ZAP-70 was found in B cells of tonsil and spleen, but not PB, we hypothesized that ZAP-70 may be associated with activation status since tonsillar and splenic B cells will include a population of activated B cells. To examine this issue, we sorted CD19+/CD38+ from CD19+/CD38− tonsillar cells and again evaluated ZAP-70 expression. Whereas both populations expressed ZAP-70, CD38+ B cells expressed ZAP-70 at a higher level suggesting that ZAP-70 may be associated with a more activated phenotype. To address the issue of activation, we activated CD19+ PB B cells with a cocktail of CD40L, IL-10, IL-4 and IL-6 for 3 days and analyzed ZAP-70 expression. Indeed, we were able to induce ZAP-70 expression in a subpopulation of blood B cells. We also assessed ZAP-70 phosphorylation following tonsillar B cell receptor (BCR) ligation and we observed that phosphorylation of ZAP-70 (tyrosine 493) occurred within 10 min. of BCR stimulation. Finally, we assessed whether ZAP-70 expression in B-CLL could be associated with an activated cell phenotype. By stimulating Ig VH non-mutated and mutated B-CLL patient samples with the CD40L cocktail, we observed an increase in ZAP-70 expression in non-mutated CLL by immunoblotting. These results demonstrate expression of ZAP-70 in a subset of normal B cells that express an activated phenotype. Moreover, our results also suggest that ZAP-70 levels in CLL B cells may also be increased following cellular activation. Given that non-mutated type B-CLL expresses ZAP-70 more frequently than mutated type B-CLL, ZAP-70 expression may simply reflect a more activated population of leukemic cells that may correlate with progressive disease. Continuing investigation into the role of ZAP-70 in normal B cell development and B-CLL is clearly warranted.