Receptor-pairing specificity of DAP10, DAP12, FcεRIγ, and CD3ζ. BaF/3 cells were first transfected with either a control cDNA, human NKG2D, CD94 and NKG2C, or CD16, and stable transfectants were generated by maintaining the cells in appropriate drug selection. The Flag epitope–tagged DAP10, DAP12, FcεRIγ, or CD3ζ cDNA were then transfected into the indicated cell lines, and subsequent double or triple transfectants were selected by drug selection. Cells were analyzed by flow cytometry using the indicated Abs, and data are presented as histograms. Western blot analysis showed that the Flag-tagged adaptors were expressed comparably in all transfectants (data not shown). cIg, control Ig.

DAP10 and DAP12 form distinct receptor complexes in Ba/F3 transfectants. (A) A Ba/F3 transfectant expressing Flag-DAP12 and KIR2DS2 was transfected with Myc epitope–tagged DAP10 in an IRES-enhanced GFP–containing vector. Cells were sorted for GFP positive, maintained in appropriate drug selection, stained with the indicated mAbs, and analyzed by flow cytometry (left). cIg, control Ig. The triple transfectant was transfected with human NKG2D and sorted for NKG2D-positive cells. The KIR2DS2/NKG2D/Myc-DAP10/Flag-DAP12 transfectant was stained with the indicated mAb and analyzed by flow cytometry (right). (B) The KIR2DS2/NKG2D/Myc-DAP10/Flag-DAP12 Ba/F3 transfectant was surface labeled with ¹²⁵I, lysed in digitonin buffer, and immunoprecipitated with either a control Ig, anti-DAP10 antiserum, anti-DAP12 antiserum, anti-KIR mAb DX27, or anti-NKG2D mAb 1D11. The resulting immune complexes were resolved by SDS-PAGE and analyzed by autoradiography. The heterogeneous migration pattern of Myc-DAP10 is likely due to O-link glycosylation of its extracellular domain. (C) The KIR2DS2/NKG2D/Myc-DAP10/Flag-DAP12 Ba/F3 transfectant was stimulated with a control mAb, anti-KIR mAb DX27, or anti-NKG2D mAb 1D11 for 3 min at 37°C. Anti-Syk Ab immune complexes were resolved by SDS-PAGE, transferred to Immobilon membrane, probed with HRP-conjugated antiphosphotyrosine (αp-Y) mAb 4G10, and developed by using a chemiluminescent substrate.

Functional cooperation between DAP10 and DAP12 receptor complexes. (A) A normal polyclonal NK cell line was assayed for Ab-redirected cytotoxicity against ⁵¹Cr-labeled FcR⁺ P815 target cells in the presence of anti-CD56 mAb Leu 19 (used as a negative control) or anti-NKG2D mAb 1D11 (at a 1:1,000 dilution of dialyzed ascites; left). In parallel, the normal polyclonal NK cell line was cultured for 20 h with immobilized control Ig (cIg), anti-CD56 mAb Leu 19, or anti-NKG2D mAb 1D11 (at 1:1,000 dilution of dialyzed ascites), and culture supernatants were analyzed by ELISA for IFN-γ (right). E:T, effector/target. Comparable results were obtained in two independent experiments using normal, polyclonal NK cell lines. (B) The KIR2DS2-expressing NKL cells were stimulated with immobilized anti-KIR mAb DX27 (starting at a concentration of 1 μg/ml) or anti-NKG2D mAb 1D11 (starting at a dilution of 1:1,000 dialyzed ascites). (C and D) KIR2DS2⁺ NKL cells were stimulated with a fixed dilution (1:5,000) of either control ascites or anti-NKG2D 1D11 ascites, together with either a control mAb or anti-KIR mAb DX27 at final concentrations of 31, 16, 8, and 4 ng/ml (C), or 62, 31, 16, and 8 ng/ml (D). Cells were stimulated in 96-well plates at 37°C for 20 h, and the supernatants were analyzed by ELISA for IFN-γ (C) or GM-CSF (D). Each stimulation condition was conducted in triplicate and the data were representative of three independent experiments.

The TM domains of DAP10 and DAP12 confer receptor-pairing specificity. (A) The alignment of the TMs of DAP10 and DAP12, and an illustration of the TM mutants used in the study. The consensus TM segments of DAP10 and DAP12 were predicted based on analysis of the protein structures using the SOSUI program, the DAS membrane predictor server, and the TMPred program. A predicted α-helical wheel diagram (http://www.bmm.icnet.uk/people/turcotte/Java/HelixWheel/) of the DAP10 and DAP12 TM regions is shown. Amino acid 19 in the TM region corresponds to the same relative location as amino acid 1 shown in the helix, amino acid 20 corresponds to amino acid 2, amino acid 21 corresponds to amino acid 3, amino acid 22 corresponds to amino acid 4, amino acid 23 corresponds to amino acid 5, and amino acid 24 corresponds to amino acid 6. The charged residues are boxed and the identities are in bold. (B) The TM of DAP10 confers specificity for its association with NKG2D. The Flag-tagged wt and TM mutants of DAP10 and DAP12 were transfected into a Ba/F3 transfectant expressing human NKG2D. Stable transfectants were generated by using drug selection. Cells were stained with anti-NKG2D mAb and analyzed by flow cytometry. Similar staining profiles were observed by using anti-Flag mAb M2 (data not shown). cIg, control Ig. (C) The double transfectants were surface labeled with ¹²⁵I, lysed, and immunoprecipitated with either a control mAb or anti-Flag mAb M2. The top part of the gel was analyzed by autoradiography; the lower part was transferred to an Immobilon membrane and analyzed by Western blot using anti-Flag mAb M2. (D) The DAP12 TM confers specificity to pair with the DAP12 partner, CD94/NKG2C. A Ba/F3 transfectant expressing human CD94 and NKG2C was transfected with either a control vector, wt DAP12, wt DAP10, or EC10-TM12-CY10. Cells were stained with an mAb reactive with the heterodimers of CD94/NKG2A or CD94/NKG2C, and subsequently analyzed by flow cytometry.

DAP12 complexes with different ligand-binding receptors. (A) A Ba/F3 transfectant expressing human CD94/NKG2C was transfected with Flag-tagged DAP12 (left), and the resulting transfectant was further transfected with KIR2DS2. Cells were sorted for positive KIR expression, stained with the indicated mAbs, and analyzed by flow cytometry. cIg, control Ig. (B) The CD94/NKG2C/Flag-DAP12 and the CD94/NKG2C/KIR2DS2/Flag-DAP12 transfectants were surface biotinylated, lysed in digitonin lysis buffer, and the resulting lysates were immunoprecipitated with the indicated Abs. The immunoprecipitates were separated by SDS-PAGE, transferred to Immobilon membrane, probed with HRP-conjugated streptavidin, and visualized by using a chemiluminescent substrate. The KIR2DS2 mAb DX27 has been consistently shown to be a poor immunoprecipitating Ab (our unpublished observation).

Separate DAP10 and DAP12 receptor complexes in polyclonal, normal NK cells and NKL cells. (A) A short-term polyclonal NK cell line established from a normal, healthy blood donor was labeled with either ¹²⁵I (left) or biotin (right), lysed in 1% digitonin, and immunoprecipitated with the indicated Abs. Samples were analyzed by SDS-PAGE (reducing conditions) and visualized by autoradiography or chemiluminescence. Note that neither DAP10 nor DAP12 labels with ¹²⁵I in NK cells, whereas DAP12, but not DAP10, labels with biotin. cIg, control Ig. (B) The parental NKL cell line was positive when stained with anti-NKG2D mAb 1D11, but was negative when stained with anti-KIR mAb DX27 (left). NKL cells were transfected with KIR2DS2, sorted for positive KIR expression, and maintained in neomycin (right). (C) Both NKL and the KIR2DS2⁺ NKL transfectant were ¹²⁵I labeled, lysed in digitonin lysis buffer, immunoprecipitated with the indicated Abs, and the resulting immune complexes were analyzed by SDS-PAGE (reducing conditions) and autoradiography.