Supplementary Figure 1

aBI-CRACC BI-CD84 BI-2B4 BI-Ly-9 BI-Ly108 BI-SLAM

CRACC

Eve

nts

CD84 2B4 Ly-9 Ly108 SLAM

Eve

nts

BI-CRACC

Fc fusion protein

Eve

nts

BI-SLAM

Fc-CRACCFc-SLAM

BI-Puro BI-CD84

CD84

Eve

nts

b c

Supplementary Figure 1. Expression and self-ligating capacity of mouse CRACC. a. Top,

mouse CRACC-specific mAb 4G2 was used to stain BI-141 T cell derivatives expressing the

indicated members of the SLAM family (open histograms). An isotype control antibody (filled

histograms) was also used. Bottom, expression of the various members of the SLAM family on

the BI-141 derivatives was confirmed by staining with the indicated antibodies (open

histograms). Staining with the relevant isotype controls was also performed (filled histograms).

The results shown are representative of at least two independent experiments. b. Specificity of

anti-CD84 monoclonal antibody. Mouse CD84-specific mAb 1D3 was used to stain BI-141 T

cell derivatives expressing CD84 (BI-CD84) or the puromycin resistance marker alone (BI-Puro)

(open histograms). An isotype control antibody (filled histograms) was also used. c. CRACC-

Fc or SLAM-Fc fusion proteins were used to stain BI-141 cells expressing CRACC or SLAM, as

indicated. The results shown are representative of at least two independent experiments.

Supplementary Figure 2a

Supplementary Figure 2. Impact of ectopic expression of CRACC on target cells. a. Indicated

target cells were infected with retroviruses encoding GFP alone (–) or in combination with

mouse CRACC (+). CRACC expression was examined by flow cytometry, using CRACC-

specific mAb 4G2 (open histograms). An isotype control antibody (filled histograms) was also

used. The results shown are from a single experiment. However, they were confirmed on

several occasions when individual target cells were used in functional assays. b. IL-2-activated

NK cells were stimulated in triplicates with the indicated target cells, expressing (+) or lacking (–

) CRACC, at a 1:1 ratio. IFN-γ production was assayed by intracellular staining. Representative

samples of the triplicates are shown. The average percentage values of IFN-γ-producing cells for

the triplicates (± s.d.) are indicated at the top right of each panel. US, unstimulated NK cells;

P+I, NK cells stimulated with phorbol myristate acetate (PMA) and ionomycin. SSC, side

scatter. These results are representative of at least four independent experiments. c. C57BL/6

mice were injected in the tail vein with B16 melanoma cells (2×105), expressing GFP alone

(B16) or together with CRACC (B16 CRACC). Mice were also injected or not with anti-

asialoGM1, as outlined in Methods. Left, photographs of lung surfaces from individual mice.

Right, average numbers of lung colonies in the different groups (mean ± s.d.). These results are

representative of at least three independent experiments.

Mutant (9 kb)

_

IB:aCRACC

IB:a2B4

IB:aSAP

2B4

EAT-2/ERT

SAP

+

CRACC

IB:aEAT-2/ERTW

T

CRACC-KO

SLAM

Ly108

CD84

Ly-9

WT CRACC-KO

CRACC

Eve

nts

Supplementary Figure 3. Generation of CRACC-deficient mice. a. The exon-intron structure

of the CRACC-encoding gene in the mouse (Slamf7) is shown at the top. Exon 1 contains the

initiating ATG (right-sided arrow), while exon 7 bears the stop codon (left-sided arrow). The

PstI sites (P) used for screening the embryonic stem (ES) cell clones by Southern blotting are

depicted. The targeting construct is shown below. This construct allows disruption and

introduction of a stop codon in exon 1. 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. The positions of the oligonucleotide

(oligo) primers used for polymerase chain reaction (PCR) screening of mouse DNA are

highlighted. b. DNA from representative ES cell clones was digested with PstI and probed by

Southern blotting with 5’ and 3’ probes. Two properly recombined clones (1C3 and 1G3), as

well as a negative control (NC) clone, are shown. WT, wild-type. The results shown are

representative of two independent experiments. c. DNA from representative mice was screened

by PCR using the oligonucleotides shown in panel a. Fragments of ~340 and ~440 nucleotides

are expected for the WT and mutant alleles, respectively. The results shown are representative

of several independent experiments (every time mice were genotyped prior to breeding or

experiments). d. The expression of SLAM family receptors (open histograms) on splenic NK

cells from the indicated mice was determined by multicolor flow cytometry. Plots are gated on

DX5+CD3– cells. An isotype control antibody (closed histograms) was also used.

Unfortunately, no 2B4-specific antibody is available to detect 2B4 from 129 mice using

multicolor flow analyses. These results are representative of at least two independent

experiments. e. Expression of CRACC, 2B4, EAT-2 and ERT, and SAP in IL-2-activated NK

cells from the indicated mice was analyzed by immunoblotting of equivalent amounts of total

cell lysates with the relevant antibodies. As the anti-EAT-2 antibody recognizes EAT-2 and

ERT, both proteins were simultaneously assayed in this immunoblot. The results shown are

representative of at least two independent experiments. f. Red blood cell-depleted splenocytes

from WT and CRACC-deficient mice (CRACC-KO) mice were stained with the indicated

antibodies. Plots are gated on DX5+CD3– cells. A representative pair of WT and CRACC-KO

mice is shown. The proportions of positive cells are shown in the upper right quadrant. A Table

depicting the average proportions (± s.d.) of the major subsets in three independent pairs of WT

and CRACC-KO mice is shown on the right. The results shown are representative of three

independent experiments.

IB:aEAT-2/ERT IB:aSAP SAPEAT-2/ERT

WT

WTDKO

SAP-KO

Supplementary Figure 4. Expression of SAP family adaptors in mutant mouse strains. a.

Expression of EAT-2 and ERT in IL-2-activated NK cells from wild-type (WT) and EAT-2,

ERT-deficient (DKO) mice was examined by immunoblotting of total cell lysates with a rat mAb

recognizing EAT-2 and ERT. b. Expression of SAP in IL-2-activated NK cells from WT and

SAP-KO mice was examined by immunoblotting of total cell lysates with a rat mAb recognizing

SAP. The results shown in a and b are representative of at least three independent experiments.

Supplementary Figure 5. Natural cytotoxicity of YT-S derivatives towards K562 cells. a.

Expression of mouse CRACC on derivatives of the target cell line K562 was analyzed by flow

cytometry using CRACC-specific mAb 4G2 (open histograms) or an isotype control antibody

(closed histograms). The results shown are from a single experiment. However, they were

confirmed on several occasions when individual target cells were used in functional assays. b,c.

YT-S cells expressing CRACC, with or without wild-type or Y2F EAT-2, were tested for their

capacity to kill K562 cells, expressing or lacking CRACC, using a standard 51Cr release assay.

All assays were done in triplicate. E:T, effector-to-target cell ratio. The results depicted in b

and c are representative of at least three independent experiments.

Supplementary Figure 6

Supplementary Figure 6. Model of CRACC-mediated activation and inhibition. The ability of

CRACC to promote NK cell activation is dependent on Y281, which is located in a typical

“immunoreceptor tyrosine-based switch motif” (ITSM) and binds EAT-2. The ability of

CRACC to inhibit NK cell activation relies on another tyrosine, Y261, which presumably

recruits one or more as yet unknown inhibitory effectors.

SUPPLEMENTARY METHODS

cDNAs, site-directed mutagenesis and constructs. cDNAs encoding CRACC (from C57BL/6

mice), Rae-1ε and CD48 were obtained from Invitrogen. For expression in target cells, the

CRACC-encoding cDNA was modified to leave only the first 4 amino acids in the cytoplasmic

domain of CRACC, to prevent the possibility of intracellular signaling by CRACC in target cells.

Conversely, the cDNAs coding for Rae-1ε and CD48 were altered to replace the

glycosylphosphatidylinositol linkage with the transmembrane and cytoplasmic domains of

CD84. This was done to standardize the membrane retention signals used by the various ligands

expressed in target cells. cDNAs encoding SLAM (Slamf1), EAT-2 (Sh2d1b1), SAP (Sh2d1a)

and SH2 domain-deleted FynT (ΔSH2 FynT) were reported elsewhere1,2. Constructs encoding

CRACC-Fc and SLAM-Fc fusion proteins were produced by PCR, using the cDNA for mouse

CRACC or SLAM and a human IgG1 cDNA (obtained from N. Beauchemin, McGill University,

Montréal, Québec, Canada) as templates. Point mutations in the cytoplasmic segment of

CRACC were introduced using the QuickChange Site-Directed Mutagenesis Kit (Stratagene).

All cDNAs were fully sequenced to make certain that they carried no undesired mutations. For

expression of CRACC in mouse or human targets, and EAT-2 in YT-S or BI-141, the relevant

cDNAs were cloned in the retroviral vector pFB-GFP, which encodes the green fluorescent

protein (GFP). For expression of CRACC, Rae-1ε or CD48 in YT-S, BI-141 or DCEK, cDNAs

were cloned in the expression plasmid pSRα-puro, which confers resistance to puromycin.

Antibodies, Fc fusion proteins and flow cytometry. Polyclonal antisera against CRACC were

generated in rabbits using a TrpE fusion protein encompassing the cytoplasmic domain of

CRACC. A mAb recognizing CRACC (clone 4G2) was generated in rats, using the CRACC-Fc

fusion protein containing the full extracellular domain of mouse CRACC. Rabbit antibodies

1

against 2B4, SLAM, mouse SAP, SHP-2, SHIP-1, Csk, FynT and phosphotyrosine, and mAbs

against EAT-2 (clone 8F12; which recognize EAT-2 and ERT), SAP (clone 1A9), SLAM (clone

12F12) or Ly108 (clone 3E11) were reported previously1,3-11. A rat mAb reacting with mouse

CD84 (clone 1D3) will be described elsewhere. Its specificity is shown in Supplementary

Figure 1b, online. A rabbit antiserum against human SAP was kindly provided by S. Latour

(Hôpital Necker, Paris, France). Polyclonal rabbit antibodies against SHP-1 (sc-287) were

purchased from Santa Cruz Biotechnology Inc. mAbs against 2B4 (clone 2B4), CD16 (clone

93), class I MHC (clone KH95), Ly49D (clone 4E5), NKG2D (clone A10), CD11b (clone

M1/70), CD49b (clone DX5), NKp46 (clone 29A1.4), CD43 (clone eBio R2/60), CD62L (clone

MEL-14), CD69 (clone H1.2F3), NKG2A/C/E (clone 20D5), Ly49G (clone AT-8), Ly49A/D

(clone eBio 12A8), CD94 (clone 18D3), CD122 (clone 5H4) or Ly-9 (clone 30C7) were

purchased from eBioscience or BD Biosciences. Soluble Fc fusion proteins were generated by

transient transfection of fusion protein constructs in Cos-1 cells, and subsequent purification of

soluble proteins from the culture supernatant using protein G Sepharose (GE Healthcare

Biosciences). Flow cytometry using antibodies or soluble Fc proteins was performed according

to standard procedures.

Cells, transfections and retroviral infections. Thymocytes, splenocytes, splenic T cells and

peritoneal macrophages were obtained from C57BL/6 mice or 129S1/Sv mice, according to

standard protocols. Briefly, thymocytes and splenocytes were obtained by making red blood

cell-depleted single cell suspension from thymus and spleen, respectively. Peritoneal cells were

generated by washing the peritoneal cavity with phosphate-buffered saline (PBS). Specific cell

populations were then identified by staining with antibodies directed against the indicated cell

surface markers and multicolor flow cytometry. For activation studies, splenic T cells were

2

purified by negative selection using purification kits from Stem Cell Technologies. For

generation of fresh ex vivo NK cells, mice were injected intra-peritoneally with the polyclonal

NK cell activator poly I:C (150 μg in 300 μl of PBS); Sigma-Aldrich) or PBS alone 18-24 h

before cell isolation, based on a protocol described elsewhere12. Cells were purified by positive

selection using a purification kit from Stem Cell Technologies. IL-2-activated NK cells were

generated by propagating splenic NK cells purified by negative selection (using a purification kit

from Stem Cell Technologies) in medium supplemented with IL-2 (1000 U/ml). They were

typically used after 5-6 days of culture in IL-2. YB2/0 (rat plasmacytoma), B16 (mouse

melanoma), CHO (Chinese hamster ovary cell), C1.1 (class I MHC-deficient variant of EL-4

mouse lymphoma), RMA-S (class I MHC-deficient mouse lymphoma), YAC-1 (mouse

thymoma), BI-141 (mouse T cell hybridoma), DCEK (L929 fibroblasts stably expressing I-Ek),

YT-S (human NK cell line), HeLa (human cervical carcinoma) and K562 (human chronic

myelogenous leukemia) were described elsewhere1,3,11,13. For expression of exogenous proteins

using retroviral plasmids, cells were infected using supernatants from GP2-293 packaging cells

transfected with the pFB-GFP-based vectors and the vesicular stomatitis virus G protein plasmid

pMD-G. Control cells were infected with retroviruses encoding GFP alone. After infection,

GFP-positive cells were purified by cell sorting. For expression of pSRα-puro-based constructs,

cells were transfected by electroporation and selected in puromycin-containing medium. Control

cells were generated by transfection with pSRα-puro alone. Cells expressing CRACC were

further purified by cell sorting using mAb 4G2. Cos-1 cell transfections were performed as

described1.

Antibody-mediated cell stimulation. IL-2-activated mouse NK cells (20×106 cells per ml)

were incubated for the indicated periods of time at 37oC with mAb 4G2 followed by rabbit anti-

3

rat IgG (Jackson ImmunoResearch Laboratories Inc.). After lysis, CRACC was

immunoprecipitated from cell lysates by addition of Staphylococcus aureus protein A (EMD

Biosciences Inc.). Unstimulated cells were processed in the same manner, except that mAb 4G2

was added only after cell lysis.

Immunoprecipitations and immunoblots. Immunoprecipitations and immunoblots were done

as specified in previous reports8,14. Immunoreactive products were revealed using 125I-protein A,

horseradish peroxidase (HRP)-coupled protein A, HRP-coupled sheep anti-mouse IgG or HRP-

coupled goat anti-rat IgG (GE Healthcare Biosciences).

Calcium fluxes. IL-2-activated NK cells (3-4×106 per ml) were loaded with Indo-1 (10 μM;

Invitrogen) for 20 minutes at 37oC. After washing the cells, they were stimulated at 37oC with

the indicated biotinylated antibodies (6 μg in 500 μl) followed by avidin (14 μg). Changes in

intracellular calcium over time were monitored using the Fluo-4 (FL4) and Fluo-5 (FL5)

channels of a BD LSR (BD Biosciences)15. Mean values are shown. As a control, cells were

stimulated with ionomycin (1 μg per ml).

Peptide binding assays. Biotinylated peptides encompassing segments of the cytoplasmic

domain of mouse CRACC were synthesized and phosphorylated, or not, by the W.M. Keck

Facility (Yale University, New Haven, CT). Peptides (10 μg; dissolved in dimethylsulfoxide)

were first coupled to agarose-avidin beads (Neutravidin; Pierce Biotechnology Inc.). Peptide-

coupled beads were then incubated with lysates (1 mg of total cellular proteins) from the

indicated cell line. After 1.5 h, beads were washed extensively to remove unbound proteins and

associated proteins were detected by immunoblotting.

T cell activation. CD4+ T cells were purified from spleen of T cell receptor AND mice by

negative selection. Cells (2×104 in 150 μl) were then incubated for 3 days in the presence of

4

mitomycin-treated DCEK cells (104), expressing or not CRACC, and various concentrations of

pidgeon cytochrome C peptide (amino-acids 88-104; synthesized at Advanced Protein

Technology Centre, Hospital for Sick Children, Toronto, Ontario, Canada). Thymidine

incorporation and cytokine production were monitored as outlined elsewhere16.

Statistical analyses. For statistical analysis, Student’s t-tests were performed with the Microsoft

Excel software.

5

References 1. Roncagalli,R. et al. Negative regulation of natural killer cell function by EAT-2, a

SAP-related adaptor. Nat. Immunol. 6, 1002-1010 (2005).

2. Latour,S. et al. Binding of SAP SH2 domain to FynT SH3 domain reveals a novel mechanism of receptor signalling in immune regulation. Nat. Cell Biol. 5, 149-154 (2003).

3. Chen,R. et al. Molecular Dissection of 2B4 Signaling: Implications for Signal Transduction by SLAM-Related Receptors. Mol. Cell Biol. 24, 5144-5156 (2004).

4. Latour,S. et al. Regulation of SLAM-mediated signal transduction by SAP, the X-linked lymphoproliferative gene product. Nat Immunol 2, 681-690 (2001).

5. Lemay,S., Davidson,D., Latour,S., & Veillette,A. Dok-3, a novel adapter molecule involved in the negative regulation of immunoreceptor signaling. Mol. Cell Biol. 20, 2743-2754 (2000).

6. Cao,M.Y. et al. Regulation of mouse PECAM-1 tyrosine phosphorylation by the Src and Csk families of protein-tyrosine kinases. J Biol Chem 273, 15765-15772 (1998).

7. Chow,L.M., Fournel,M., Davidson,D., & Veillette,A. Negative regulation of T-cell receptor signalling by tyrosine protein kinase p50csk. Nature 365, 156-160 (1993).

8. Davidson,D., Chow,L.M., Fournel,M., & Veillette,A. Differential regulation of T cell antigen responsiveness by isoforms of the src-related tyrosine protein kinase p59fyn. J Exp Med 175, 1483-1492 (1992).

9. Abraham,N., Miceli,M.C., Parnes,J.R., & Veillette,A. Enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck. Nature 350, 62-66 (1991).

10. Castro,A.G. et al. Molecular and functional characterization of mouse signaling lymphocytic activation molecule (SLAM): differential expression and responsiveness in Th1 and Th2 cells. J Immunol. 163, 5860-5870 (1999).

11. Zhong,M.C. & Veillette,A. Control of T lymphocyte signaling by ly108, a signaling lymphocytic activation molecule family receptor implicated in autoimmunity. J. Biol. Chem. 283, 19255-19264 (2008).

12. Fernandez,N.C. et al. A subset of natural killer cells achieve self-tolerance without expressing inhibitory receptors specific for self MHC molecules. Blood 105, 4416-4423 (2005).

13. Bloch-Queyrat,C. et al. Regulation of natural cytotoxicity by the adaptor SAP and the Src-related kinase Fyn. J. Exp. Med. 202, 181-192 (2005).

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14. Veillette,A., Bookman,M.A., Horak,E.M., & Bolen,J.B. The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck. Cell 55, 301-308 (1988).

15. Davidson,D., Bakinowski,M., Thomas,M.L., Horejsi,V., & Veillette,A. Phosphorylation-dependent regulation of T-cell activation by PAG/Cbp, a lipid raft-associated transmembrane adaptor. Mol. Cell Biol. 23, 2017-2028 (2003).

16. Davidson,D. et al. Genetic evidence linking SAP, the X-linked lymphoproliferative gene product, to Src-related kinase FynT in T(H)2 cytokine regulation. Immunity. 21, 707-717 (2004).

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