Cutting edge: The adapters EAT-2A and -2B are positive regulators of CD244- and CD84-dependent NK cell functions in the C57BL/6 mouse

Abstract

EWS/FLI1-activated transcript 2 (EAT-2)A and EAT-2B are single SH2-domain proteins, which bind to phosphorylated tyrosines of signaling lymphocyte activation molecule family receptors in murine NK cells. While EAT-2 is a positive regulator in human cells, a negative regulatory role was attributed to the adapter in NK cells derived from EAT-2A-deficient 129Sv mice. To evaluate whether the genetic background or the presence of a selection marker in the mutant mice could influence the regulatory mode of these adapters, we generated EAT-2A-, EAT-2B-, and EAT-2A/B-deficient mice using C57BL/6 embryonic stem cells. We found that NK cells from EAT-2A- and EAT-2A/B-deficient mice were unable to kill tumor cells in a CD244- or CD84-dependent manner. Furthermore, EAT-2A/B positively regulate phosphorylation of Vav-1, which is known to be implicated in NK cell killing. Thus, as in humans, the EAT-2 adapters act as positive regulators of signaling lymphocyte activation molecule family receptor-specific NK cell functions in C57BL/6 mice.

FIGURE 1. SAP family adaptors are required for in vivo clearance of CD48+ or CD84+ targets

CFSE-labeled RMAS / CD48+, RMAS/CD48− cells [3×10⁶] (A, B and C) or P815/CD84+ cells [3×10⁶] (E and F) were injected in the peritoneum of wt, EAT-2A/B−/−, SAP−/−, EAT-2A/B × SAP−/− or CD244−/− B6 mice. After 18 hours, the tumor cells were recovered from the peritoneum and the number of tumor cells was calculated based on the percentage of CFSE+ cells by flow cytometry. The data are representative of three independent experiments. * p<0.05, ***p<0.001.

FIGURE 2. Ligation of CD244 or CD84 by CD48 or CD84 expressing targets enhances NK cytotoxicity in B6, but not EAT-2A/B−/− mice

NK cells isolated from the spleens of wt or EAT-2A/B−/− B6 were cultured in an IL-2 containing medium for seven days. Cytolytic activity was determined by the [⁵¹Cr]- or LDH-release assay. NK cell cytotoxicity against RMAS/CD48+ or RMAS/CD48− cells (LDH release assay) (A), P815/CD48+ or P815/CD48− cells (⁵¹Cr release assay) (B) or B16/CD84+ or P815/CD84+ cells (LDH release assay) (C). Pooled data from three independent experiments were shown. *p<0.05, **p<0.01. Error bars represent SD.

FIGURE 3. Defective αCD244 or αCD84 redirected killing and IFN-γ production by EAT-2A/B−/− NK cells

NK cells from the spleens of wt or EAT-2A/B−/− mice were were cultured with IL-2-containing medium for seven days and analyzed in a redirected killing assay against the FcγR+ P815 target cells either in the absence or presence of anti-CD244 (A) anti-CD84 (B) or anti-NKG2D (C) mAbs. The lytic activity of wt or EAT-2A/B−/− NK cells was tested against P815 target cells by measuring ⁵¹Cr (A and C) or LDH (B) released into the cell supernatants. Wt or EAT-2-A/B−/− NK cells purified from the spleens were cultured in the presence of IL-2. At day 7, the NK cells were stimulated with anti-CD244 (5μg/ml) (D), anti-NKG2D (5μg/ml) or anti-Ly49D (5μg/ml) (E) mAbs for 24 hours. Culture supernatants were harvested and IFN-γ production was quantified by ELISA. Pooled data from three independent experiments were shown. *p<0.05, **p<0.01. Error bars represent SD.

FIGURE 4. Phosphorylation of Vav-1 by αCD244 or by ligation with CD48 expressing targets is reduced in EAT-2A/B−/− NK cells

A. IL-expanded splenic NK cells from wt or EAT-2A/B−/− mice were stimulated with αCD244 mAb for 15 min. Cell lysates were used for immunoprecipitation (IP) with αVav-1 mAb and were probed with anti-p-Tyr mAb (4G10) and reprobed with αVav-1. B, NK cells were stimulated with CD48-expressing P815 cells for 30 min. and analyzed, as in Fig. 4A. C, Lysates of αCD244 activated NK cells form wt or EAT-2A/B−/− mice were analyzed by probing αCD244 immunoprecipitates with anti-p-Tyr mAb (4G10). CD244 was quantitated by reprobing the membrane with αCD244.