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A hematopoietic cell–driven mechanism involving SLAMF6 receptor, SAP adaptors and SHP-1 phosphatase regulates NK cell education

Abstract

Activation of natural killer (NK) cells by hematopoietic target cells is controlled by the SLAM family of receptors and by the associated SAP family of adaptors. Here we found that SLAM receptors also enhanced NK cell activation by nonhematopoietic target cells, which lack ligands for SLAM receptors. This function was mediated by SLAMF6, a homotypic SLAM receptor found on NK cells and other hematopoietic cells, and was regulated by SAP adaptors, which uncoupled SLAM receptors from phosphatase SHP-1 and diminished the effect of SLAMF6 on NK cell responsiveness toward nonhematopoietic cells. Thus, in addition to their role in NK cell activation by hematopoietic cells, the SLAM-SAP pathways influence responsiveness toward nonhematopoietic targets by a process akin to NK cell 'education'.

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Figure 1: Impact of SAP adaptors on mouse NK cell cytotoxicity toward nonhematopoietic cells.
Figure 2: Effect of SAP adaptors on cytotoxicity by human NK cell line YT-S toward nonhematopoietic target cells.
Figure 3: Activation responses in NK cells from SAP-deficient XLP patients.
Figure 4: Influence of SAP adaptors on NK cell–mediated cytokine production and in vivo tumor clearance in response to nonhematopoietic cells.
Figure 5: Roles of SLAM receptors in inhibitory effect of SAP on NK cell responsiveness.
Figure 6: SLAMF6 regulates responsiveness toward nonhematopoietic cells in a human NK cell line.
Figure 7: Effect of SAP adaptors on activating receptors involved in antibody-dependent cellular cytotoxicity and recognition of nonhematopoietic targets.
Figure 8: Molecular mechanism by which SAP suppresses NK cell responses.

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Acknowledgements

A.V. holds the Canada Research Chair in Signaling in the Immune System. We thank the members of A.V.'s lab for discussions. We acknowledge L. Yin for the SAP-KO mouse. Supported by grants from the Canadian Institutes of Health Research (CIHR) (to A.V.), the China-Canada Joint Health Research Initiative (to A.V. and Z.D.) and the Canadian Cancer Society Research Institute (CCSRI) (to A.V.). N.W. received a fellowship from Fonds de la Recherche du Québec – Santé (FRQS). R.R. received a studentship from CCSRI. L.-A.P.-Q. received a studentship from Institut de Recherches Cliniques de Montréal. Z.D. received a postdoctoral fellowship from CIHR.

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Authors

Contributions

N.W. planned and performed experiments, interpreted data and wrote the manuscript. M.-C.Z., R.R., L.-A.P.-Q., H.G., C.L., Z.D. and Z.Z. planned and performed experiments, generated reagents and interpreted data. S.L. planned and performed experiments, interpreted data and provided reagents. A.V. planned experiments, generated reagents, interpreted data, wrote the manuscript and obtained funding.

Corresponding author

Correspondence to André Veillette.

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The authors declare no competing financial interests.

Integrated supplementary information

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.

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.

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.

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).

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.

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 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-γ 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-γ+ cells. Statistical analyses of the increase in IFN-γ+ 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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Wu, N., Zhong, MC., Roncagalli, R. et al. A hematopoietic cell–driven mechanism involving SLAMF6 receptor, SAP adaptors and SHP-1 phosphatase regulates NK cell education. Nat Immunol 17, 387–396 (2016). https://doi.org/10.1038/ni.3369

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