Differential requirements for Vav proteins in DAP10- and ITAM-mediated NK cell cytotoxicity - PubMed (original) (raw)

Differential requirements for Vav proteins in DAP10- and ITAM-mediated NK cell cytotoxicity

Marina Cella et al. J Exp Med. 2004.

Abstract

Natural killer (NK) cells express multiple activating receptors that initiate signaling cascades through DAP10- or immunoreceptor tyrosine-based activation motif-containing adapters, including DAP12 and FcRgamma. Among downstream signaling mediators, the guanine nucleotide exchange factor Vav1 carries out a key role in activation. However, whether Vav1 regulates only some or all NK cell-activating pathways is matter of debate. It is also possible that two other Vav family molecules, Vav2 and Vav3, are involved in NK cell activation. Here, we examine the relative contribution of each of these exchange factors to NK cell-mediated cytotoxicity using mice lacking one, two, or all three Vav proteins. We found that Vav1 deficiency is sufficient to disrupt DAP10-mediated cytotoxicity, whereas lack of Vav2 and Vav3 profoundly impairs FcRgamma- and DAP12-mediated cytotoxicity. Our results provide evidence that these three Vav proteins function specifically in distinct pathways that trigger NK cell cytotoxicity.

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Figures

Figure 1.

Figure 1.

ITAM-dependent cytotoxicity in Vav1ko mice. Cytotoxicity of Vav1ko NK cells was tested against the following: (A) EL-4 cells with or without an anti-Thy1.2 mAb, (B) Baf-3-m157 and mock-transfected Baf-3 with or without a blocking mAb against Ly49H, and (C) CHO cells with or without a blocking anti-Ly49D mAb. NK cells were cultured in IL-2 for 10–15 d, contained >98% NK1.1+/CD3− cells. Vav1ko and WT NK cells exhibited comparable percentages of Ly49H+ and Ly49D+ cells.

Figure 2.

Figure 2.

NKG2D-dependent cytotoxicity in Vav1ko mice. Cytolitic activity of Vav1ko NK cells against RMA-S, RMA-S/Rae1γ (A), and YAC-1 (B) in the presence of a blocking anti-NKG2D mAb or control mAb. Effector cells included freshly isolated NK cells (day 0) and NK cells cultured in IL-2 for 6, 15, and 30 d. Reduction of NKG2D-dependent cytotoxicity is particularly evident at day 0 (RMA-S-Rae1γ) and at day 30 (RMA-S-Rae1γ and YAC-1). Note that our RMA-S parental cell line is poorly killed by B6 NK cells although it expresses a low level of MHC class I. Different variants of RMA-S may be more susceptible to NK cell–mediated lysis.

Figure 3.

Figure 3.

ADCC, Ly49H-, and Ly49D-dependent cytotoxicity in Vav1,2,3ko and Vav2,3ko NK cells. Cytotoxicity of Vav1,2,3ko (A and B) and Vav2,3ko (D and E) was tested against EL-4 cells with or without an anti-Thy1.2 mAb as well as Baf-3-m157 and Baf-3. Cytotoxicity of Vav1,2,3ko was also tested against CHO cells with and without a blocking anti-Ly49D antibody. ADCC was abrogated in both Vav1,2,3ko and Vav2,3ko NK cells; Ly49H-dependent killing was severely impaired, whereas Ly49D-mediated CHO killing was considerably conserved (C). NK cells were cultured in IL-2 for 10–15 d. In the CHO killing experiment, Ly49D+ cells represented 35% of WT and 25% of Vav1,2,3ko NK cell cultures.

Figure 4.

Figure 4.

Effect of Vav deficiency on DAP10- and DAP12-dependent activation of ERK. (A) NKG2D–DAP10 and NKG2D–DAP12 pathways activate ERK1/2. WT, DAP10ko, and DAP12ko NK cells were incubated at 37°C for the indicated times with anti-NKG2D mAb (left). WT and DAP12ko NK cells were also conjugated with fixed CHO cells (right). NK cell lysates were analyzed by immunoblotting with antibody against active ERK1/2 (pERK1/2) and anti-ERK1/2. (B) Impact of Vav deficiency on activation of ERK1/2. WT, Vav1ko, and Vav1,2,3ko NK cells (left) were stimulated via NKG2D as described in A. WT and Vav1,2,3ko NK cells were also conjugated with CHO cells (right). NK cell lysates were analyzed as indicated in A. (C) MEK inhibitor PD98059 reduces NKG2D-, FcγRIIIA-, and Ly49H-mediated cytotoxicity. IL-2–activated NK cells were preincubated at 37°C for 30 min with PD98059 (50 μM) or DMSO and tested for cytolytic activity against RMA-S, RMA-S/Rae1γ, EL-4 cells coated with anti-Thy1.2 mAb, Baf-3, and Baf-3/m157.

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