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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