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. 2022 Jan 31;12:747482. doi: 10.3389/fonc.2022.747482

Figure 5.

Figure 5

NF-κB Disinhibition by Lirilumab and Avelumab Co-Engagement is Vav1-Dependent. (A) IL-2-primed NK cells were mixed with autologous target cells and stimulated with lirilumab (aKIR) and/or avelumab (aPD-L1) by receptor crosslinking for 4 h. Whole cell lysates underwent co-immunoprecipitation for Vav1-SHIP1 binding using an anti-SHIP1 antibody (left) or were immunoblotted for phospho-Y174 Vav1, total Vav1, phospho-S536 p65, phospho-S276 p65, total p65, and β-actin (right). (B-E) Normalized intensities of (B) Vav1-SHIP1 interactivity, (C) phospho-Y174 Vav1, (D) phospho-S536 p65, and (E) phospho-S276 p65 relative to their total counterparts are reported. (F) IL-2-primed NK cells transfected with pCtrl, shVav1, and shVav1+CA-p65 were mixed with autologous target cells and stimulated as in (A) for 4 h. Lysates from isolated NK cells were immunoblotted for phospho-Y174 Vav1, total Vav1, phospho-S536 p65, phospho-S276 p65, total p65, and β-actin. (G, H) Transfected IL-2-primed NK-κB-GFP cells were mixed with autologous target cells and stimulated with plate-immobilized aKIR and/or aPD-L1 for 6 h. GFP expression in the isolated reporter NK cells was analyzed by flow cytometry. *P < 0.05, **P < 0.01 [(B–E) one-way ANOVA or (H) two-way ANOVA (mAb factor × transfection factor)]. n=3 biological replicates×3 technical replicates.