CD33 (Siglec-3) Inhibitory Function: Role in the NKG2D/DAP10 Activating Pathway

Figure 5

CD33 ligation reduces Vav1 phosphorylation under crosslinking with NKG2D activating receptor in hc-NKL cells. (a) hc-NKL cells were treated with the indicated combination of antibodies against NKG2D and/or CD33, NKG2A, or MHC-I as a control, crosslinked with sheep anti-mouse Ab on ice and incubated at 37°C for 5 min. Then cellular extracts were analyzed by Western blotting to test the indicated phosphorylated intracellular proteins. Crosslinking of NKG2D plus CD33 resulted in a reduction of the amount of p-Vav1 whereas crosslinking of NKG2D plus NKG2A resulted in a reduction of both p-Vav1 and p-ERK2 intracellular mediators. Data are representative of four independent experiments. (b) Changes in p-Vav1 and p-ERK2 over time are represented as percentage of phosphorylated proteins after NKG2D plus CD33, MHC-I, or NKG2A stimulation in relation to NKG2D stimulation alone. Means and SDs from four experiments are shown. (c) Relative extent of Vav1 phosphorylation in hc-NKL cells when stimulated by crosslinking of CD16, NKG2D, NKp46, or 2B4 activating receptors.

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