Abstract
CD79b is B-cell surface molecule that non-covalently associates with CD79a and surface immunoglobulin (sIg), which together serve as the B-cell receptor complex (BCR). Both CD79a and CD79b have cytosolic immunoreceptor tyrosine-based activation motifs (ITAMs) that can become phosphorylated following sIg ligation, thereby allowing for recruitment to the BCR complex of cytosolic kinases, such as p72Syk , which then can initiate downstream intracellular signaling events. Compared to normal B cells, chronic lymphocytic leukemia (CLL) B cells typically expresses low levels of CD79b, which is speculated to contribute to the relatively poor capacity of CLL cells to initiate intracellular signaling following BCR ligation despite having apparently adequate levels of p72Syk. BCR signaling in CLL cells can be enhanced by expression of the zeta-associated protein of 70 kD (ZAP-70), a tyrosine kinase that initially was identified in T cells, where it plays a critical role in the phosphorylation of ITAMs of the accessory molecules of the T-cell receptor (TCR) complex for antigen following TCR ligation. We investigated for phosphorylation of CD79b following BCR ligation with F(ab)2 anti- μ antibody in CLL cell samples that did or did not express ZAP-70. All CLL cell samples expressed similar amounts of surface IgM and p72Syk, as assessed via flow cytometry and immunoblot analysis. Within 10 minutes after treatment with anti-μ the CLL cell samples that expressed ZAP-70 (n = 28) experienced a mean increase in phosphorylation of CD79b of 21.5% (± 14.0% S.D.), which was significantly greater than the 7.5% increase (± 7.9% S.D.) experienced by similarly treated CLL cell samples that did not express ZAP-70 (n = 19) (P< 0.01). Immune precipitation studies demonstrated association of CD79b with p72Syk in CLL B cells. CLL cell samples (n = 5) lacking expression of ZAP-70 were transfected with a control vector or an expression vector encoding ZAP-70, allowing us to examine the effect that engineered-expression of ZAP-70 has on CD79 phosphorylation following treatment with anti-μ. Anti-μ treatment induced significantly higher mean levels of CD79b phosphorylation in CLL samples made to express ZAP-70 (33% ± 16%) than in control mock-transfected CLL cells (4% ± 2%). This also was associated with enhanced anti-μ induced phosphorylation of p72Syk. We conclude that expression of ZAP-70 in CLL B cells enhances phosphorylation of the accessory molecules in the BCR complex following sIg ligation, potentially allowing for improved recruitment of cytosolic kinases and adapter proteins to these accessory molecules for enhanced BCR signaling.
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