Regulation of natural cytotoxicity by the adaptor SAP and the Src-related kinase Fyn

Abstract

SAP is an adaptor protein that is expressed in NK and T cells. It is mutated in humans who have X-linked lymphoproliferative (XLP) disease. By interacting with SLAM family receptors, SAP enables tyrosine phosphorylation signaling of these receptors by its ability to recruit the Src-related kinase, Fyn. Here, we analyzed the role of SAP in NK cell functions using the SAP-deficient mouse model. Our results showed that SAP was required for the ability of NK cells to eliminate tumor cells in vitro and in vivo. This effect strongly correlated with expression of CD48 on tumor cells, the ligand of 2B4, a SLAM-related receptor expressed in NK cells. In keeping with earlier reports that studied human NK cells, we showed that SAP was necessary for the ability of 2B4 to trigger cytotoxicity and IFN-gamma secretion. In the absence of SAP, 2B4 function was shifted toward inhibition of NK cell-mediated cytotoxicity. By analyzing mice lacking Fyn, we showed that similarly to SAP, Fyn was strictly required for 2B4 function. Taken together, these results provide evidence that the 2B4-SAP-Fyn cascade defines a potent activating pathway of natural cytotoxicity. They also could help to explain the high propensity of patients who have XLP disease to develop lymphoproliferative disorders.

Figure 1.

In vivo and in vitro natural killing responses are impaired in SAP-deficient mice. (A) The elimination of tumor cells in vivo is impaired in SAP ⁻ mice. SAP ⁺ and SAP ⁻ mice were injected intraperitoneally with PKH-26–labeled RMA cells (10⁶). After 24 h, the numbers of residual RMA cells recovered from the peritoneal cavity of SAP⁺ and SAP ⁻ mice were determined by cytometry (see Materials and methods). The data presented are pooled from three independent experiments. Bars indicate mean values for each group of mice. (B) Natural cytotoxicity in vitro against a panel of target cells is restricted in SAP-deficient NK cells. The cytolytic activity of IL-2–expanded splenic NK cells from SAP ⁺ (○) and SAP ⁻ (•) mice was tested against the indicated target cells, at the mentioned NK/target cells ratios. For each target, data are presented as means from one experiment representative of three independent experiments.

Figure 2.

SAP is required for 2B4-mediated cell cytotoxicity and IFN-γ production by NK cells. (A) Association of SAP with 2B4 in mouse NK cells. IL-2–expanded splenic NK cells were treated (+) or not treated (−) with pervanadate (PV) for 10 min. 2B4 was immunoprecipitated (I.P.) and subjected to immunoblotting with anti-Ptyr antibody (top), anti-2B4 antibodies (middle), or anti-SAP antibodies (bottom). Total cell lysates were assessed in parallel. (B) The 2B4-mediated cytolytic activity by NK cells is impaired in the absence of SAP. The cytolytic activity of IL-2– expanded NK cells from SAP ⁺ (left) and SAP ⁻ (right) mice was measured in a redirected antibody-dependent cell-cytotoxicity assay. NK cells were incubated with the FcR⁺ P815 target cells in the presence of anti-2B4, anti-Ly49D, anti-NK1.1 antibodies or medium (no Ab) at the indicated NK/target cell ratios. Data are presented as means from one representative experiment of three independent experiments. (C and D) 2B4-mediated IFN-γ production by NK cells is impaired in the absence of SAP. IFN-γ in the cell-supernatants was detected by ELISA. (C) NK cells from SAP ⁺ and SAP ⁻ mice were incubated with increased concentrations of anti-2B4 antibodies in the presence of FcR⁺ P815 cells to trigger antibody- redirected stimulation of NK cells. Data are means from one representative experiment of two independent experiments. (D) IL-2–expanded NK cells from SAP ⁺ and SAP ⁻ mice were stimulated with medium, soluble anti-2B4 antibodies; irrelevant antibodies of the same isotype; or IL-12. Data are means ± SD from one representative experiment of three independent experiments.

Figure 3.

Cell surface expression of CD48, the 2B4 ligand, on target cell lines. Cell surface expression of CD48 was assessed by flow cytometry with anti-CD48 and FITC-conjugated anti–rat Ig (thick histogram). Controls were stained with FITC-conjugated anti–rat Ig alone (thin histogram).

Figure 4.

The lack of CD48 expression on tumor cells decreases the susceptibility of these cells to be killed by NK cells in vitro. (A) CD48 is not expressed on CL.1 and CL.52 cells which were derived from C4.4-25 cells (see Materials and methods). CD48 expression was assessed as in Fig. 3. (B) Lysis of CL.1 and CL.52 by NK cells. The cytolytic activity of IL-2–expanded splenic NK cells was tested against CL.1, CL.52, or the parental C4.4-25 target cells, at the indicated NK/target cell ratios. Data are presented as means from one experiment representative of three independent experiments.

Figure 5.

Influence of SAP expression on NK cell–mediated cytotoxicity toward CD48-expressing tumor cells. (A) Cell surface expression of CD48 in CL.1 and CL.52 cells stably transfected with a cDNA encoding CD48. Experiment same as in Fig. 4. (B and C) The expression of CD48 on tumor cells enhanced the susceptibility of these cells to be killed by SAP ⁺ NK cells, whereas it inhibited the killing of these cells by SAP ⁻ NK cells. The cytolytic activity of IL-2–expanded splenic NK cells from SAP ⁺ (B) and SAP ⁻ (C) mice was tested in parallel against CL.1, CL.52, CL.1-CD48, or CL.52-CD48 target cells at the indicated NK/target cell ratios. For each target, data are presented as means from one representative experiment of three independent experiments.

Figure 6.

Increase in the in vivo elimination of CD48-expressing tumor cells by NK cells. SAP ⁺, SAP ⁻, RAG-2 ^−/− and RAG-2 ^−/−γc^−/− mice were injected intraperitoneally with a mixture of equal numbers of CFSE-labeled CL.1 and CFSE-labeled CL.1-CD48 tumor cells. After 6 h, peritoneal cells were recovered, counted, and stained with PE-conjugated anti-CD48 antibody. An aliquot of the cell mixture was kept in culture for 6 h (noninjected). (A) The percentages of CL.1 and CL.1-CD48 tumor cells in the CFSE-positive cell population were evaluated by flow cytometry, based on CD48 fluorescence. Representative dot-plots of gated CFSE- positive cells are shown and the injected mice are indicated. (B) Numbers of residual CL.1 cells and CL.1-CD48 cells recovered from the peritoneal cavity of SAP ⁺, SAP ⁻, RAG-2 ^−/−, and RAG-2 ^−/−γc^−/− mice. The data presented are pooled from four independent experiments. One experiment in which only CL1-CD48 cells were injected is included. Bars indicate mean values for each group of mice.

Figure 7.

2B4-mediated cell-cytotoxicity and IFN-γ production by NK cells is impaired in the absence of Fyn. (A) The cytolytic activity of IL-2–expanded NK cells from wild-type (left) and Fyn ^{− / −} mice (right) mice was measured in a RADCC assay. Experiment same as in Fig. 2. (B) 2B4-mediated IFN-γ production by IL-2-expanded NK cells. Experiment same as in Fig. 2. (C and D) Natural cytotoxicity in vitro against a panel of target cells is restricted in Fyn-deficient NK cells. (C) Cytolytic activity of IL-2– expanded splenic NK cells from wild-type (○) and Fyn ^{− / −} (•) mice against C4.4-25, RMA/S, RMA, or CHO target cells. Experiment same as in Fig. 1. (D) Cytolytic activity of IL-2–expanded splenic NK cells from wild-type (left) and Fyn ^{− / −} (right) mice was tested in parallel against CL.1 and CL.1-CD48 target cells. Experiment same as in Fig. 5.

Figure 8.

Fyn and SAP are required for 2B4-mediated tyrosine phosphorylation signals. (A) Overall tyrosine phosphorylation. IL-2– expanded splenic NK cells from wild-type, SAP ⁻, and Fyn ^{− / −} mice were stimulated (+) or not (−) for 5 min with biotinylated anti-2B4 antibodies and avidin. Protein tyrosine phosphorylation was examined by immunoblotting of total cell lysates with anti-P.tyr antibody (top). The abundance of 2B4 in the lysates was verified by reprobing the membrane with anti-2B4 antibodies (bottom). (B) Tyrosine phosphorylation of SHIP and 2B4. Experiment same as (A). Tyrosine phosphorylation of 2B4 (top) and SHIP (middle) was analyzed by probing anti-2B4 and anti-SHIP antibody immunoprecipitates (I.P.) with anti-P.tyr antibody. The abundance of 2B4 and SHIP was verified by reprobing the membranes with anti-2B4 and anti-SHIP antibodies, respectively. (C) Expression of SAP and Fyn in NK cells from wild-type, SAP ⁻, and Fyn ^{− / −} mice. Experiment same as (A) and (B). The abundance of SAP and Fyn was verified in total cell lysates by immunoblotting with anti-SAP (top) and Fyn (bottom) antibodies.