Supplementary Figure 1

Impact of SAP adaptors on mouse NK cell cytotoxicity towards nonhematopoietic cells.

(a) Expression of SLAM receptors and CD48 on splenic NK cells from wild-type (WT) and SAP-deficient (SAP KO) mice in 129S1/Sv background.

NK cells were identified as NKp46+CD3- cells. Blue line, receptor-specific antibody; red line, isotype control. (b,c) Natural cytotoxicity by IL-2-

activated NK cells (“LAK” cells) from WT, SAP KO (b) or EAT-2 ERT double KO (DKO; c) mice was determined using a 51Cr release assay at the

indicated effector-to-target (E:T) ratio. Percentage (%) of maximum 51Cr release is shown. Average values of duplicates are shown; error bars depict

standard deviations. Representative of 2 (a) and 3 (b,c) independent experiments.

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Supplementary Figure 2

Expression of SLAM receptors.

Expression of SLAM receptors and CD48 on HeLa and K562 cells was determined by flow cytometry. Blue line, receptor-specific antibody; filled

histogram, isotype control. Representative of 2 independent experiments.

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Supplementary Figure 3

Generation of mice lacking CD48, SLAMF6 or Ly-9.

The exon-intron structure of the genes encoding CD48 (Slamf2; a), SLAMF6 (Slamf6; b) and Ly-9 (Slamf3; c) in the mouse is shown at the top of

each panel. Exon 1 contains the initiating ATG (right-sided arrow), while a downstream exon bears the stop codon (left-sided arrow). The targeting

construct is shown below. For Slamf2 and Slamf3, the construct allows disruption and introduction of a stop codon (TGA) in exon 1. For Slamf6, it

enables deletion of exons 2-4. This deletion is expected to remove both immunoglobulin-like domains and the transmembrane region. The middle

fragment, which contains the neomycin resistance gene (neor) cassette, is bordered by frt sites (diamonds). The targeted allele containing the neor

cassette is depicted below. The neor-deleted allele, which was generated by transient expression of the Flpe recombinase, is shown at the bottom.

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Supplementary Figure 4

Expression of SLAM receptors and SAP during NK cell development.

Expression of SLAMF6 (a) and SAP (b) in various sub-populations of splenic NK cells was assessed in C57BL/6 mice, as detailed for Figure 5k.

NKP, NK precursors; iNK-1, stage 1 immature NK cells; iNK-2, stage 2 immature NK cells; mNK, mature NK cells. Shaded histograms represent

isotype controls. A graphic representation of mean fluorescent intensity (MFI) at the various stages is depicted on the right. Closed symbols: staining

with receptor-specific antibodies. Open symbols: staining with isotype controls. (c,d) Expression of the other SLAM family receptors, as well as

CD48, was assessed on splenic NK cell subsets from 129S1/Sv (c) or C57BL/6 (d) mice, as detailed for (a,b). Representative of 3 independent

experiments are shown in (a, b, c, d).

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Supplementary Figure 5

Ectopic expression of mouse SLAMF6 in YT-S cells.

(a) Expression of mouse SLAMF6 isoform 1 (mSLAMF6-1) and 2 (mSLAMF6-2) on YT-S cells was determined by flow cytometry. Cells

expressing empty vector were used as control (CT). (b,c) Natural cytotoxicity towards HeLa or K562 by YT-S-mSLAMF6-2 cells transfected with

control siRNA (siCT) or SAP-specific siRNA (siSAP), at different effector-to-target (E:T) ratios. A representative experiment is shown in (b),

whereas statistical analyses of data from multiple independent experiments at the 25:1 E:T ratio are shown in (c). Standard deviations are depicted by

error bars in (b). Error bars represent sem in (c). Data are representative of 3 (a) and 4 (b,c) independent experiments.

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Supplementary Figure 6

Role of SLAMF6 on NK cell education.

(a) Expression of SLAM receptors and other NK cell markers on parental and SLAMF6-deficient (KO) YT-S cells was determined by flow

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cytometry. SLAMF6 KO YT-S cells were generated using three different targeting vectors (named 1-3). (b,c) Natural cytotoxicity of parental or

SLAMF6 KO YT-S cells towards HeLa and K562, expressing or not human SLAMF6 (b) or CD48 (c). Assays were performed as detailed for Figure

6a. (d) Splenocytes described in Figure 6h were stimulated overnight with plate-bound anti-Ly49D antibodies. Production of IFN- by SAP KO NK

cells was then determined by intracellular staining and gating on CFSE+DX5+CD3- cells. A representative experiment is shown on the left. Numbers

in the box indicate percentages (%) of IFN-+ cells. Statistical analyses of the increase in IFN-+ cells, between cells stimulated with anti-Ly49D and

those stimulated with medium alone, in multiple independent experiments (n=7) are shown on the right. (e) YT-S cells expressing mouse DNAM-1

(mDNAM-1) were mixed with parental or SLAMF6 KO YT-S cells at the ratio 1:15. After 3 weeks, the percentages of mDNAM-1-positive YT-S

cells in the mixtures were determined by flow cytometry using mouse anti-DNAM-1 antibodies (upper panel). YT-S cells were then stimulated with

anti-mDNAM-1 antibodies, and calcium flux was assessed (lower panel). Data are representative of 2 (a), 3 (b,c), 7 (d), and 3 (e) independent

experiments. Unpaired t test (n=7) was used for d. NS, not significant.

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Supplementary Table 1. Primers for human SLAMF6 KO in CRISPR-Cas constructs. Primer name Sequence hSLAMF6 KO-1 F CACCGAGAATCCCGTTCACCATCAA hSLAMF6 KO-1 R AAACTTGATGGTGAACGGGATTCTC  hSLAMF6 KO-2 F CACCGAGAAGACAAACAGGAGCGAT hSLAMF6 KO-2 R AAACATCGCTCCTGTTTGTCTTCTC hSLAMF6 KO-3 F CACCG GGTTTCATGGGGTACTATGA hSLAMF6 KO-3 R AAACTCATAGTACCCCATGAAACCC

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Supplementary Table 2. Antibodies used in this study. Antibody against Clone Company

CD48 HM48-1

eBioscience

2B4 CD244.2 CD3 145-2C11

CD49b DX5 CD16 93 CD226 10E5 and 480.2 IFN-γ XMG1.2

CD107a 1D4B Human CD107a H4A3

MHC class I 28-14-8 CD155 TX56 CD112 3387 CD16 2.4G2

BD Bioscience Ly49D 4E5 2B4 CD244.1

Ly9.1 30C7 pErk E10

Cell Signaling p44/42 9102 pSrc Tyr416 pTyr 4G10 Millipore β-actin C4 Santa Cruz CD3 UCHT1

Biolegend

CD56 HCD56 SLAMF6 NT-7

2B4 C1.7 CD48 BJ40 CD84 CD84.1 SLAM A12 Ly-9 HLy9.1

NKp46 9E2

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