Developmental and Comparative Immunology 37 (2012) 151–163
Contents lists available at SciVerse ScienceDirect
Developmental and Comparative Immunology
journal homepage: www.elsevier.com/locate/dci
Channel catfish leukocyte immune-type receptor mediated inhibition of
cellular cytotoxicity is facilitated by SHP-1-dependent and -independent
mechanisms
Benjamin C. Montgomery a, Herman D. Cortes a, Deborah N. Burshtyn b, James L. Stafford a,⇑
a
b
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada
a r t i c l e
i n f o
Article history:
Received 5 August 2011
Revised 8 September 2011
Accepted 9 September 2011
Available online 16 September 2011
Keywords:
Teleosts
Channel catfish
Immunoglobulin superfamily
Immunoregulatory receptors
Leukocyte immune-type receptors
Inhibitory signaling
Cytoplasmic tails
Immune tyrosine-based inhibition motifs
Cytotoxicity
Src-family protein tyrosine kinases
C-terminal Src kinase
SH2-domain containing phosphatases
Chimeric receptors
Lymphokine activated killer cells
Vaccinia virus
Natural killer cells
a b s t r a c t
Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are immunoregulatory
proteins belonging to the immunoglobulin superfamily that likely play an important role in the regulation
of teleost immune cell effector responses. IpLITRs are expressed by myeloid and lymphoid subsets and
based on their structural features can be classified as either putative stimulatory or inhibitory forms.
We have recently demonstrated at the biochemical and functional levels that stimulatory IpLITR-types
induced intracellular signaling cascades resulting in immune cell activation. Alternatively, we have shown
that putative inhibitory IpLITRs may abrogate immune cell responses by recruiting teleost Src homology 2
(SH2) domain-containing cytoplasmic phosphatases (SHP) to their tyrosine-containing cytoplasmic tails.
In the present study, we used vaccinia virus to express recombinant chimeric proteins encoding the extracellular and transmembrane regions of human KIR2DL3 fused with the cytoplasmic tails of two putative
inhibitory IpLITRs (i.e. IpLITR1.2a and IpLITR1.1b) in mouse spleen-derived cytotoxic lymphocytes. This
approach allowed us to study the specific effects of IpLITR-induced signaling on lymphocyte killing of B
cell targets (e.g. 721.221 cells) using a standard chromium release assay. Our results suggest that both
IpLITR1.2a and IpLITR1.1b are potent inhibitors of lymphocyte-mediated cellular cytotoxicity. Furthermore, using a catalytically inactive SHP-1 mutant in combination with site-directed mutagenesis and
co-immunoprecipitations, we also demonstrate that the IpLITR1.2a-mediated functional inhibitory
response is SHP-1-dependent. Alternatively, IpLITR1.1b-mediated inhibition of cellular cytotoxicity is
facilitated by both SHP-1-dependent and independent mechanisms, possibly involving the C-terminal
Src kinase (Csk). The involvement of this inhibitory kinase requires binding to a tyrosine residue encoded
in the unique membrane proximal cytoplasmic tail region of IpLITR1.1b. Overall, this represents the first
functional information for inhibitory IpLITR-types and reveals that catfish LITRs engage SHP-dependent
and -independent inhibitory signaling pathways to abrogate lymphocyte-mediated killing.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
In response to infection a complicated series of receptor-mediated signaling events are necessary for initiation, propagation,
and subsequent termination of potent immune cell effector responses. These cellular immune responses (e.g. phagocytosis,
degranulation, cytokine secretion, and cytotoxicity) are vital for
immune defense against pathogens and are in part controlled by
Abbreviations: IpLITRs, Ictalurus punctatus leukocyte immune-type receptors; SH2, Src homology 2; SHP, Src homology 2 domain-containing cytoplasmic phosphatase;
Csk, C-terminal Src kinase; ITAM, immune receptor tyrosine-based activation motif; SFK, Src-family protein tyrosine kinases; SYK, spleen tyrosine kinase; PI3-K,
phosphatidylinositol 3-kinases; IgSF, immunoglobulin superfamily; FcRs, Fc receptors; KIRs, killer cell immunoglobulin-like receptors; LILRs, leukocyte immunoglobulin-like
receptors; NKRP1, natural killer receptor P1; ITIMs, immune receptor tyrosine-based inhibition motifs; CYT, cytoplasmic tail; SHIP, SH2-domain containing inositol 5phosphatase; NK, natural killer; rVV, recombinant vaccinia virus; LAK, lymphokine activated killer; Y, tyrosine; IL-2, interleukin 2; MHC I, major histocompatibility class I;
HLA, human leukocyte antigen; mAb, monoclonal antibody; pAb, polyclonal antibody; HA, hemagglutinin; PE, phycoerythrin; HRP, Horseradish peroxidase; tr, truncated; F,
phenylalanine; WR, Western Reserve; DN, dominant-negative; TK-, thymidine kinase-deficient; MOI, multiplicity of infection; D-PBS, Dulbecco’s phosphate buffered saline;
Cr, chromium; E:T, effector to target; PEI, polyethylenimine; IP, immunoprecipitation; ED, extracellular domains; TM, transmembrane; kDa, kiloDalton; CHK, CSKhomologous kinase; Cbp/PAG, Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomains adaptor; LAIR, leukocyte-associated Ig-like
receptor-1; SIRP, signal-regulatory protein; SH2D1A, SH2 domain protein 1A; EAT-2, Ewing’s Sarcoma-activated transcript 2.
⇑ Corresponding author. Tel.: +1 780 492 9258; fax: +1 780 492 9234.
E-mail address: [email protected] (J.L. Stafford).
0145-305X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dci.2011.09.005
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B.C. Montgomery et al. / Developmental and Comparative Immunology 37 (2012) 151–163
subsets of co-expressed stimulatory and inhibitory immunoregulatory receptors that are coupled with distinct intracellular signaling modules (Barrow and Trowsdale, 2008; Lanier, 2008; Long,
1999; Takai, 2005). In general, stimulatory immunoregulatory
receptor-types activate immune cells by recruiting adaptor proteins encoding immune receptor tyrosine-based activation motifs
(ITAMs). Following receptor engagement with specific ligands,
ITAMs are phosphorylated by Src-family protein tyrosine kinases
(SFKs), which then interact with downstream mediators such as
spleen tyrosine kinase (SYK) and/or phosphatidylinositol 3-kinases (PI3Ks) that in turn potentiate a series of cellular activation
cascades (Bakker et al., 2000; Kuster et al., 1990; Gergely et al.,
1999; Lanier, 2008; Lanier et al., 1998; Underhill and Goodridge,
2007; Van den Herik-Oudijk et al., 1995; Wu et al., 2000). Many
of these stimulatory receptors are germline-encoded and belong
to either the immunoglobulin superfamily (IgSF) (Barclay, 2003)
or the C-type lectin superfamily (Weis et al., 1998). Examples of
immunoregulatory proteins with stimulatory functions include
Fc receptors (FcRs; Falk and Ravetch, 2006; Nimmerjahn and Ravetch, 2007, 2011; Takai, 2005), killer cell Ig-like receptors (KIRs;
Parham, 2004; Stanietsky and Mandelboim, 2010; Vilches and Parham, 2002), leukocyte Ig-like receptors (LILRs; Brown et al., 2004;
Katz, 2006; Thomas et al., 2010), triggering receptors expressed on
myeloid cells (Ford and McVicar, 2009), natural cytotoxicity
receptors (e.g. NKp30, NKp44, and NKp46; Biassoni et al., 2001;
Yokoyama and Plougastel, 2003), CD94/NKG2 (Gunturi et al.,
2004; López-Botet et al., 1997), NK receptor-P1 (NKRP1; Carlyle
et al., 2004), and Ly49 receptors (Anderson et al., 2001). In addition to these stimulatory forms, inhibitory receptor-types also
play an indispensible role in the regulation of cellular immune responses (Long 2008, Long et al. 1997 and Steevels and Meyaard
2011).
Inhibitory receptors establish the activation threshold of immune cells and attenuate stimulatory receptor-induced effector
functions. Like their stimulatory counterparts, these receptors are
also present within the FcR, KIR, LILR, CD94/NKG2, NKRP1, and
Ly49 families. When engaged by specific ligands, inhibitory receptors recruit cellular phosphatases, which play an important role in
down-regulating immune cell responses (Katz, 2006; Long, 1999;
Ravetch and Lanier, 2000; Vivier and Daeron, 1997). The inhibitory
capacity of these receptors is primarily dependent on the presence
of immune receptor tyrosine-based inhibition motifs (ITIMs) within their cytoplasmic tail (CYT) regions (Beebe et al., 2000; Burshtyn
et al., 1996, 1997, 1999). Ligand-induced phosphorylation of the
tyrosine residue embedded within ITIMs (S/I/V/LxYxxI/V/L; an x
indicates any amino acid) leads to the recruitment of SH2 domain-containing cytoplasmic phosphatases (SHP-1, SHP-2, and
SH2-domain containing inositol 5-phosphatase; SHIP), which
dephosphorylate various intracellular activation signaling intermediates (Burshtyn et al., 1997, 1999; Imhof et al., 2006). Recent
studies have indicated substrate specificity for inhibitory phosphatases suggesting that they are not random or non-specific inhibitors of SFK-induced phosphorylation events. For example, when
recruited to ITIMs, SHP-1 exhibits specificity for the guanine nucleotide exchange factor Vav1 resulting in its dephosphorylation and
inability to activate Rac1 (Stebbins et al., 2003). Since Vav1 plays
an important role in T cell activation events including synapse formation and receptor clustering (Tybulewicz, 2005), SHP-1-mediated dephosphorylation of Vav1 can likely block a range of
cellular immune responses including natural killer (NK) cell cytotoxicity (Long, 2008). There are also SHP-independent inhibitory
signaling mechanisms that occur in immune cells. For example,
Csk binds to phosphorylated tyrosines present in the CYT region
of ligand-engaged immunoregulatory receptors (Sayos et al.,
2004; Veillette et al., 1998; Verbrugge et al., 2006). Csk is a potent
inhibitor of cellular signaling by its targeted phosphorylation of
SFKs at a C-terminal tyrosine residue, which then induces conformational inactivation of these kinases (Okada et al., 1991). Overall,
complex mechanisms of receptor-induced inhibitory signaling
pathways are required to abrogate potent cellular immune effector
responses (Long, 2008; Ravetch and Lanier, 2000; Vivier et al.,
2004).
Although much less is known about the specific immune receptor-types and signaling events involved in controlling cellular
immunity in non-mammalian vertebrates, several immunoregulatory receptor families have been discovered in avian, amphibian,
and teleost species (Yoder and Litman, 2011). Often, these immune proteins share key structural features with mammalian
immunoregulatory proteins known to control and coordinate leukocyte responses (Yoder and Litman, 2011; Montgomery et al.,
2011). Detailed sequence analyses, examination of phylogenetic
relationships, and several functional studies have provided important information required for interspecies comparisons of vertebrate immunoregulatory receptor networks and their predicted
phylogenetic origins (Yoder and Litman, 2011). One example from
teleosts are the channel catfish LITR proteins, which represent a
large and polymorphic immune receptor family with signaling potential predicted to augment or abrogate catfish immune cell
responses (Montgomery et al., 2011; Stafford et al., 2006).
Although IpLITR ligands remain unknown, recent biochemical
and functional studies have revealed that associations with
ITAM-encoding adaptor molecules as well as ITIM-mediated
recruitment of cellular phosphatases are key requirements for
IpLITR-mediated stimulatory and inhibitory functions, respectively (Mewes et al., 2009; Montgomery et al., 2009). We have also
demonstrated that IpLITR-adaptor associations induce ITAMdependent intracellular signaling and functional responses such
as degranulation and phagocytosis in transfected immune cells,
confirming the stimulatory nature of certain IpLITR-types (Cortes
et al., 2012).
Expanding on our previous findings that putative inhibitory
IpLITRs recruited SHP-1 and SHP-2 in an ITIM-dependent fashion
(Montgomery et al., 2009), the focus of the present study was to
examine the inhibitory capabilities of two different ITIM-containing IpLITR-types by addressing the following questions: (i) do the
ITIM-bearing CYT regions of IpLITR1.2a (ABI16051) and
IpLITR1.1b (ABI16050) inhibit cellular immune responses; (ii)
what are the inhibitory signaling pathways used by IpLITR1.2a
and 1.1b; and (iii) is there an inhibitory function mediated by
the unique tyrosine-containing, membrane-proximal CYT region
of IpLITR1.1b? Herein we show that these receptors function as
potent inhibitors of lymphocyte-mediated cellular cytotoxicity
(i.e. target cell killing). Specifically, we used recombinant vaccinia
virus (rVV) to express the previously reported ‘inhibitory’ KIR/
IpLITRCYT constructs (Montgomery et al., 2009) in mouse lymphokine activated killer (LAK) cells. This allowed us to determine the
inhibitory effects of ITIM-encoding IpLITR CYT regions on cellular
cytotoxic responses. Co-expression of the KIR-LITRCYT with a catalytically inactive SHP-1 recombinant protein revealed that
IpLITR-mediated inhibition of the LAK cell killing response was
only in part a SHP-1-dependent mechanism. The inhibitory functions of KIR-LITRCYT 1.1b, which encodes the CYT region of IpLITR
1.1b (ABI16050), was not affected by the inactive SHP-1 mutant,
indicating an unexpected SHP-1-independent mechanism of immune cell inhibition. Subsequently, site-directed mutagenesis
and co-immunoprecipitations revealed that Csk is possibly an
additional player in IpLITR 1.1b-mediated abrogation of the LAK
cell killing response. Identification of phosphatase- and kinasedependent inhibitory pathways engaged by IpLITRs is unique
and sets the stage for exploring the relevance of SHP-1-dependent
and -independent inhibitory signaling pathways in teleost
immunity.
B.C. Montgomery et al. / Developmental and Comparative Immunology 37 (2012) 151–163
2. Materials and methods
2.1. Cells and antibodies
HEK 293T cells and TK- cells were grown at 37 °C and 5% CO2 in
DMEM/High Glucose (Invitrogen, Life Technologies) supplemented
with 2 mM L-glutamine (Invitrogen, Life Technologies), 100 Units/
mL penicillin (Invitrogen, Life Technologies), 100 mg/mL streptomycin (Invitrogen, Life Technologies), and 10% heat-inactivated fetal bovine serum (FBS; Hyclone). Prior to use, culture media was
filter sterilized using 0.22 lm cell culture filtering units (Corning).
Mouse LAK cells were derived from the splenocytes of C57BL/6
mice as previously described (Roussell et al., 1990) and cultured
in RPMI media (Invitrogen, Life Technologies) with 10% FBS,
50 lM 2-mercaptoethanol (Invitrogen Life Sciences), 2 mM L-glutamine, 100 Units/mL penicillin, 100 mg/mL streptomycin, and
1000 U/mL recombinant interleukin 2 (IL-2) (TECIN; Biological Resources Branch, Division of Cancer Treatment and Diagnosis, National
Cancer
Institute-Frederick Cancer
Research
and
Development Center). All mouse work was approved by the Health
Research Ethics Board at the University of Alberta. The major histocompatibility class I (MHC I)-deficient B cell lymphoma target
cell line 721.221 and 721.221 cells expressing human leukocyte
antigen (HLA)-Cw3 cells were maintained in Iscove’s media with
10% FBS and 2 mM L-glutamine. The 721.221-HLA-Cw3 transfectants were maintained in Iscove’s media supplemented with
0.5 mg/mL geneticin (Gibco).
DX27 (IgG2a) is an anti-KIR2DL2/L3/S2 antibody and was provided by Dr. Lewis Lanier (UCSF, San Franciso, CA). W6/32 (IgG2a)
a pan-HLA-reactive monoclonal antibody (mAb), L243 (IgG2a) an
anti-HLA-DR mAb and the control IgG2a (51.1) were all purified
by protein G-Agarose from hybridomas obtained from American
Type Culture Collection (ATCC). Anti-hemagglutinin (HA) mAb
and phycoerythrin (PE)-conjugated goat anti-mouse IgG were purchased from Cedarlane Laboratories. The anti-Csk rabbit antiserum
was kindly provided by Dr. André Veillette (IRCM, Montreal, Canada). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit
IgG was purchased from Bio-Rad.
2.2. Constructs and recombinant vaccinia viruses
The KIR/IpLITRCYT chimeras used in this study were previously
developed in our laboratory as described by Montgomery et al.
(2009) and are renamed as follows: KIR/IpLITR1.0 is now KIR/IpLITR1.2a, KIR/IpLITR3.0 is now KIR/IpLITR1.1b, KIR/IpLITR2.0 is now
KIR/LITR1.1b truncated (tr), and KIR/IpLITR 3X is now KIR/IpLITR
DCYT. For generation of rVV, each construct was subcloned into
the pSC66 vector using SalI and NotI restriction sites. Tyrosine-tophenylalanine (F) mutants within the CYT region of KIR/IpLITR1.1b
(tr) were generated by site-directed mutagenesis using the QuickChange Lightning site-directed mutagenesis kit (Stratagene) as
previously described (Mewes et al., 2009). Primers used to generate KIR/IpLITR FY were 50 -CTTCAGACCGATGAGCACATTTTTGACACTGTGGA-30 and its reverse complement. Primers used to
generate KIR-LITR 1.1b (tr) YF were 50 -GAACTCAGTGGGGCCGTTTT
TGCACAGGTCAT-30 and its reverse complement. Primers used to
generate KIR-LITR 1.1b (tr) FF were 50 - CTTCAGACCGATGAGCACATTTTTGACACTGTGGA-30 and its reverse complement followed
by 50 -GAACTCAGTGGGGCCGTTTTTGCACAGGTCAT-30 and its reverse complement.
All pSC66-KIR/IpLITRCYT constructs and an empty pSC66 vector
were recombined with the vaccinia virus strain Western Reserve
(WR) as described (Willey et al., 1988). Vaccinia viruses encoding
KIR2DL3 and dominant-negative (DN)-SHP-1 have been described
previously, named cl6, and HCP453S, respectively (Burshtyn et al.,
1996). All rVV were propagated in the human thymidine kinase-
153
deficient (TK)-human osteosarcoma cell line 143B TK- (ATCC,
Manassas, VA), released from the cells by sonication, and enriched
by spinning through a 36% sucrose cushion (Earl et al., 1994). Titers
in plaque-forming units were determined in TK-cells as described
(Willey et al., 1988).
2.3. Infection with recombinant vaccinia viruses
Mouse LAK cells were washed in Iscove’s medium supplemented with 2 mM L-glutamine, 1X non-essential amino acids,
0.2% bovine serum albumin (BSA), and 100 U/mL rIL-2. The cells
were infected at the indicated multiplicity of infection (MOI; see
figure legends) with rVV at 37 °C with 5% CO2. All experiments
involving virus infections were conducted in the presence of cytosine b-D-arabinofuranoside-HCl (Sigma–Aldrich) at a final concentration of 40 lg/mL.
2.4. Flow cytometry
Flow cytometry was used to assess the expression of rVV-induced chimera receptor expression. Briefly, 4 h post-infection,
LAK cells were washed once with Dulbecco’s phosphate buffered
saline (D-PBS) and counted; aliquots of 5 105 cells were placed
in 1.5 mL Eppendorf tubes. Cells were then stained with 1 lg
DX27 followed by 1 lg of goat anti-mouse IgG (H + L)-PE antibody
or by 1 lg goat anti-mouse IgG (H + L)-PE alone as described
(Montgomery et al., 2009). Surface expression of HLA-Cw3 on
721.221 B cells was also determined by flow cytometry using the
W6/32 mAb and the PE-conjugated anti-mouse IgG mAb or PEconjugated anti-mouse IgG mAb alone.
2.5. Cytolysis assay
After 4 h of virus infection, mouse LAK cells were washed,
counted, and then diluted in warm assay medium (Iscove’s medium,
5% FBS, 2 mM L-glutamine, 100 U/mL rIL-2, and 40 lg/mL cytosine
b-D-arabinofuranoside-HCl). Target cell cytolysis was measured
using the Chromium 51(51Cr) release (Zöller et al., 1977) as follows:
721.221 cells (targets) were labeled with 51Cr sodium chromate
(NEN) in the presence of 1 lg/mL of the anti-HLA-DR mAb, L243.
Labeled cells were washed twice D-PBS diluted to 2500 cells/well
and then plated with mouse LAK effector cells in triplicate at 1:1,
3:1, and 10:1 effector to target (E:T) ratios in a 96-well plate. Effectors and targets were then co-incubated for 4 h at 37 °C with 5% CO2
prior to measuring cytolysis. For antibody-blocking experiments,
mouse LAK cells were pre-incubated with 10 lg/mL of DX27 mAb
or 10 lg/mL IgG2a for 30 min at room temperature and then mixed
10:1 with target cells, in triplicate. To quantify Cr-51 release, 50 lL
of supernatant incorporated into 150 lL of scintillation fluid Optiphase Supermix (Perkin–Elmer) and analyzed in a 1450 Microbeta
Trilux (Wallac). 51Cr release was calculated as: percent lysis =
(mean sample release mean spontaneous release)/(mean total
release mean spontaneous release) 100 (Zöller et al., 1977).
2.6. Cellular transfections
Transfections were performed using HEK 293T cells seeded in
6-well tissue culture plates (Costar). Five hundred thousand cells
were seeded in 2 mL DMEM/10% FBS per well and incubated overnight prior to transfection with the KIR/IpLITRCYT chimeras and
pXM139-Csk (Cloutier and Veillette, 1996). For each expression construct, 1 lg of DNA was first diluted in 190 lL of serum-free DMEM
and then 4 lL of polyethylenimine (PEI) in vitro transfection reagent
(Sigma–Aldrich) was added. Samples were gently mixed and incubated for 20 min at room temperature. The DMEM-plasmid-PEI
solution was then evenly layered onto the cells, which were then
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B.C. Montgomery et al. / Developmental and Comparative Immunology 37 (2012) 151–163
incubated for 48 h at 37 °C to allow for protein production. Co-transfections with different expression constructs were performed as described for single transfections except 1 lg of each plasmid were
diluted in 190 lL of serum-free DMEM. Prior to seeding cells for
transfection experiments, cell viability was examined using Trypan
blue staining to ensure that cultures had >95% viable cells prior to
seeding.
2.7. Immunoprecipitations and Western blotting
Forty-eight hours post-transfection, HEK 293T cells were washed
with 2 mL D-PBS/EDTA, harvested with 0.25% trypsin, and then
stimulated with 1 mL D-PBS containing 10 mM H2O2 and 0.1 mM sodium pervanadate (Na3VO4) for 10 min at 37 °C. Pervanadate solution was then aspirated following centrifugation (400g, 6 min) of
the cells at 4 °C. Cell pellets were then lysed with 500 lL of ice-cold
immunoprecipitation (IP) buffer (50 mM Tris–HCl, 150 mM NaCl, 1%
Triton X-100, pH 7.4, supplemented with complete mini EDTA-free
protease inhibitor and phosphatase inhibitor cocktail tablets;
Roche). Following the removal of cellular debris by centrifugation
at 16,000g, cellular lysates were incubated with 2 lg of anti-HA
mAb clone HA.C5 for 14–16 h at 4 °C on a rotary mixer. Fifty microliters of pre-washed (with IP buffer) protein G Sepharose beads (GE
Healthcare) were then added to the samples and incubated for a further 2 h at 4 °C on a rotary mixer. Beads were washed three times
with 200 lL of IP buffer followed by the addition of 60 lL of 2
SDS–PAGE reducing buffer. Samples were then boiled for 10 min at
100 °C and stored at 20 °C prior to analysis. Twenty microliters
of the samples were electrophoresed on 8% SDS–PAGE gels, transferred to 0.2 lm nitrocellulose membranes (Bio-Rad) and then
stained with Ponceau S (Sigma–Aldrich) to ensure successful transfer and equivalent loading of samples. Blots were then blocked in
Tris-buffered saline (TBS) supplemented with 0.1% Tween 20% and
5% skim milk (TTBS-SKIM) for 30 min at room temperature. Membranes were then incubated 14–16 h at 4 °C with anti-HA-HRP
mAb or anti-CSK antisera diluted 1:1000 v/v in TTBS-SKIM for the
detection of immunoprecipitated proteins. For blots incubated with
anti-CSK antisera, the membranes were washed 3 with TTBS and
incubated for 1 h at room temperature with 1:5000 (v/v) of goat
anti-rabbit IgG (H + L)-HRP. After three washes with TBS, immunoreactive bands were detected using the SuperSignal West Pico
Chemiluminescent Substrate kit (Pierce Biotechnology).
2.8. Statistics
A Student’s T-test (two tails) was performed when assessing potential differences between experimental groups. P-values <0.05
were designated as statistically significant.
3. Results
3.1. Inhibition of LAK cell-mediated cytotoxicity by KIR/IpLITRCYT
chimeras
Mouse LAK cells were infected with rVV encoding an empty
pSC66 vector, the pSC66 vector encoding the full length human
KIR2DL3, or the pSC66 vectors encoding the various KIR/IpLITRCYT
chimeric receptor constructs (i.e. KIR/IpLITR1.2a, KIR/IpLITR1.1b,
or KIR/IpLITR DCYT). KIR-LITR1.2a contains the two extracellular
domains (ED) and transmembrane (TM) region of KIR2DL3 fused
to the putative inhibitory CYT region of IpLITR1.2a. The KIR/
IpLITR1.1b and KIR/IpLITR DCYT chimeras encode the full-length
CYT of IpLITR1.1b or a deleted version of the IpLITR1.1b CYT that
is devoid of all intracellular tyrosines, respectively. These various
constructs have previously been described in detail (Montgomery
et al., 2009), and a schematic representation of the ITIM- and
ITIM-like motifs encoded within the CYT regions of IpLITR1.2a
and IpLITR1.1b are presented in Supplementary Fig. 1A.
Following infections with rVV encoding the various constructs,
mouse LAK cells were monitored for surface receptor expression by
flow cytometry using the DX27 mAb as previously described
(Montgomery et al., 2009). All receptors were detected at comparable levels on the surface of LAK cells, whereas no expression was
observed in the empty vector control group of cells, pSC66
(Fig. 1A). KIR/IpLITR1.1b demonstrates slightly reduced expression
levels when compared to the other constructs. However, as described below, this did not influence its inhibitory capabilities.
Cytotoxicity assays were then performed by incubating the
rVV-infected LAK cells with 721.221 MHC class I deficient B cell
targets and 721.221 cells expressing HLA-Cw3 (Supplementary
Fig. 2) using a standard Cr-51 release assay (Zöller et al., 1977).
For the LAK cells infected with rVV containing the empty pSC66
vector we observed 721.221 cell killing that was most effective
at the highest E:T ratio tested (Fig. 1B; top panel). Similarly, empty
vector rVV-infected LAK cells (i.e. pSC66) killed 721.221 cells
expressing HLA-Cw3. Next we tested the cytotoxic response of
pSC66-KIR2DL3 infected LAK cells and observed increased killing
of 721.221 targets at the increasing E:T ratios. However, when
the 721.221 targets expressed the KIR2DL3 ligand HLA-Cw3 on
their surface (Supplementary Fig. 2), the % killing observed for
the pSC66-KIR2DL3 expressing cells was significantly reduced
(Fig. 1B). This inhibition of the killing response was statistically significant when the data from three independent cytolysis assays
were pooled (p < 0.05; Supplementary Fig. 3). As observed with
LAK cells expressing KIR2DL3, cells expressing the chimeric KIR/
IpLITRCYT constructs pSC66-KIR/IpLITR1.2a and pSC66-KIR/
IpLITR1.1b also demonstrated a significant inhibition of their cytotoxic responses when they engaged 721.221 targets expressing
HLA-Cw3 in comparison to their 721.221 MHC-I deficient counterparts (Fig 1B and Supplementary Fig. 3). When the CYT region of
IpLITR1.1b was deleted (DCYT), there was no difference between
the % killing of 721.221 cells and 721.221-HLA Cw3 target cells
(Fig. 1B and Supplementary Fig. 3).
3.2. The membrane proximal CYT region of IpLITR1.1b inhibits LAK
cell-mediated cellular cytotoxicity
As previously reported by Montgomery et al., 2009, the CYT region of IpLITR1.1b has a unique TM proximal region containing
three tyrosines (Y433, Y453, and Y463) surrounded in residues that
do not fit with the classical definition of an ITIM (Supplementary
Fig. 1). This CYT region also binds relatively little to no teleost
SHP-1 or SHP-2 in comparison to the SHP-recruiting ability of its
membrane distal ITIMs (Montgomery et al., 2009). However, when
we expressed this tyrosine-containing CYT region into LAK cells
(note: this construct is termed KIR/IpLITR1.1b (tr) in this manuscript vs. KIR/IpLITR 2.0 in Montgomery et al. (2009)), it mediated
significant (p < 0.05) abrogation of the LAK cell-mediated lysis of
721.221-HLA Cw3 cells at 3:1 and 10:1 E:T ratios (Fig. 2; bottom
panel). These levels of inhibition were comparable to those observed using the full-length IpLITR 1.1b CYT and the control receptor KIR2DL3 (Fig. 2 and Supplementary Fig. 3). The top panel of
Fig. 2 shows the surface expression levels for each of the receptors
used in the killing assays displayed in the bottom panels. In addition, the TM proximal CYT region of KIR/IpLITR 1.1b (tr) is also
shown in Supplementary Fig. 1.
3.3. Inhibition of LAK cell cytotoxicity is due to specific interactions
between KIR/IpLITRCYT chimeras with the ligand HLA-Cw3
The inhibitory functions of KIR2DL3, KIR/IpLITR1.2a, KIR/
IpLITR1.1b, and KIR/IpLITR1.1b (tr) when expressed in LAK cells
B.C. Montgomery et al. / Developmental and Comparative Immunology 37 (2012) 151–163
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Fig. 1. Surface expression and inhibition of LAK cell-mediated cellular cytotoxicity by KIR/IpLITRCYT chimeras. (A) LAK cells were infected with rVV-WR (MOI of 20) encoding
an empty pSC66 vector, wild-type KIR2DL3 or the chimeric receptors KIR/IpLITR1.2a, KIR/IpLITR1.1b, and KIR/IpLITRDCYT. The surface expression of each receptor was
determined by flow cytometry using the anti-KIR mAb DX27 followed by staining with a secondary goat anti-mouse IgG pAb conjugated to PE. The secondary pAb alone
control is indicated by the solid histogram and the anti-KIR2DL3 staining with DX27 is indicated by the empty histogram. (B) Mouse LAK cells were infected for 4 h with rVV
prior to mixing them with either 721.221 cells (black circles) or 721.221 cells expressing HLA-Cw3 (white circles) at E:T ratios of 1:1, 3:1, and 10:1. Effectors and targets were
then incubated for a further 4 h and target cell killing was measured from cellular supernatants using the Cr-51 release assay as described in Section 2. 51Cr release was
calculated as: % lysis = 100 (mean sample release mean spontaneous release)/(mean total release mean spontaneous release) and is represented as % killing on the yaxis of each graph. Data presented is a representative from three independent experiments that were performed, which gave similar results.
were due to specific interactions with HLA-Cw3, which we confirmed by performing the killing assays in the presence of the
blocking anti-KIR mAb, DX27 (Lanier et al., 1997). As shown in
Fig. 3, performing cytotoxicity assays at a 10:1 effector to target ratio, in the presence of DX27 reverted the % killing values of
721.221-HLA-Cw3 target cells (Fig. 3B; black bars) back to those
observed using the MHC-I deficient target cells, 721.221 (Fig. 3A;
black bars). However, no reversion of target cell killing by the
LAK cells was observed in the presence of the IgG2a isotype control
antibody (Fig. 3; grey bars). Also, as demonstrated earlier, pSC66
infected LAK cells and those expressing KIR/IpLITR DCYT did not
inhibit LAK cell-mediated cytotoxicity of 721.221-HLA-Cw3 target
cells; therefore no reversion of killing was observed for these cells
in the presence of DX27 (Fig. 3).
3.4. Catalytically inactive SHP-1 abrogates IpLITR1.2a- but not
IpLITR1.1b-mediated inhibitory signaling in mouse LAK cells
We previously reported that the CYT regions of IpLITR1.2a and
IpLITR1.1b bind SH2 domain-containing cytoplasmic phosphatases
(SHPs) (Montgomery et al., 2009). To test whether a catalytically
inactive SHP-1 could interfere with the inhibitory function of
KIR/IpLITRCYT chimeras in mouse LAK cells, we co-expressed a
DN-SHP-1 recombinant protein (Fig. 4A) with the various receptor
constructs using the rVV approach described above. Shown in
Fig. 4A is a schematic representation of the DN-SHP-1 used in these
experiments, illustrating the two N-terminal SH2 domains, the
protein tyrosine phosphatase domain (PTP), and the C453 to S453
mutation that renders this protein catalytically inactive (Burshtyn
et al., 1996). Also shown is a Western blot demonstrating that in
LAK cells, recombinant DN-SHP-1 can be identified as an 70 kilodalton (kDa) protein, which is slightly larger than the endogenous
SHP-1 observed in the control lane. This slight size discrepancy is
due to the nature of the SHP-1 (human) and the presence of an epitope tag (Burshtyn et al., 1996).
To normalize the level of infection between experimental
groups (i.e. to ensure that each cell was infected with similar
amounts of rVV), we co-infected LAK cells with the pSC66 empty
vector in combination with the pSC66-KIR2DL3, pSC66-KIR/IpLITR1.2a, pSC66-KIR/IpLITR1.1b, or pSC66-KIR/IpLITR1.1b (tr) constructs. When examined by flow cytometry using DX27 mAb, the
LAK cells co-infected with empty pSC66 or DN-SHP-1 in conjunction with the various KIR/IpLITRCYT chimeras demonstrated overlapping (i.e. similar) levels of receptor expression on the cell
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Fig. 2. The membrane proximal CYT region of IpLITR1.1b inhibits LAK cell-mediated
cellular cytotoxicity. Top panel: LAK cells were infected with rVV-WR (MOI of 20)
encoding an empty pSC66 vector, wild-type KIR2DL3 or the chimeric receptors KIR/
IpLITR1.1b, and KIR/IpLITR1.1b (tr). The surface expression of each receptor was
determined by flow cytometry using the anti-KIR mAb DX27 followed by staining
with a secondary goat anti-mouse IgG pAb conjugated to PE. The secondary pAb
alone control is indicated by the solid histogram and the anti-KIR2DL3 staining with
DX27 is indicated by the empty histogram. Bottom panel: mouse LAK cells were
infected for 4 h with rVV prior to mixing them with either 721.221 cells (black
circles) or 721.221 cells expressing HLA-Cw3 (white circles) at E:T ratios of 1:1, 3:1,
and 10:1. Effectors and targets were then incubated together for 4 h and target cell
killing was measured from cellular supernatants using the Cr release-51 assay as
described in Section 2. 51Cr release was calculated as: % lysis = 100 (mean sample
release mean spontaneous release)/(mean total release mean spontaneous
release) and is represented as % killing on the y-axis of each graph. Data presented
is a representative from three independent experiments that were performed,
which gave similar results.
surface (Fig. 4B; compare solid grey line with the dotted line; see
pSC66-KIR2DL3 as an example). Then using the Cr-51 release assay
to assess LAK cell cytotoxicity (Fig. 4C and D), we observed that the
catalytically inactive DN-SHP-1 significantly reversed the inhibition of 721.221 killing mediated by KIR2DL3- and KIR/IpLITR1.2aexpressing LAK cells when incubated with the HLA-Cw3 expressing
targets. For example, LAK cells expressing KIR2DL3 that were coinfected with empty pSC66 killed 30% of 721.221 cells and only
9.7% of targets if they expressed HLA-Cw3 (Fig. 4C and D). Conversely, when co-infected with DN-SHP-1, the KIR2DL3 expressing
LAK cells killed 43% of 721.221 targets and 36.5% of targets if
they expressed HLA-Cw3 (Fig. 4C and D). This reverted killing of
721.221-HLA-Cw3 targets by KIR2DL3-expressing LAK cells co-infected with empty pSC66 (9.7%) vs. DN-SHP-1 (36.5%) is statistically significant (p < 0.05; Fig. 4D). A DN-SHP-1-dependent
reversion of inhibitory receptor function was not exclusive to
KIR2DL3. When we tested the KIR/IpLITRCYT chimeras, we observed
that LAK cells expressing KIR/IpLITR1.2a and co-infected with
pSC66 killed 32.5% of 721.221 cells and only 8.9% of targets if
Fig. 3. Inhibition of cytotoxicity in LAK cells is due to specific interactions between
KIR/IpLITRCYT chimeras with the ligand HLA-Cw3. LAK cells were infected with rVVWR encoding an empty pSC66 vector, wild-type KIR2DL3 or the chimeric receptors
KIR/IpLITR1.2a, KIR/IpLITR1.1b, KIR/IpLITR1.1b (tr) at an MOI of 20. Infected LAK
cells were also incubated in the presence of an IgG2a isotype control mAb (grey
bars) or the anti-KIR2DL3 mAb DX27 (black bars) at a final concentration of 10 lg/
mL for 30 min prior to performing the cytotoxicity assay. The target cells used were
(A) 721.221 cells or (B) 721.221 expressing HLA-Cw3 and effectors and targets were
incubated at an E:T of 10:1 for 4 h prior to performing the Cr-51 release assay as
described in Section 2. 51Cr release was calculated as: % lysis = 100 (mean sample
release mean spontaneous release)/(mean total release mean spontaneous
release) and is represented as % killing on the y-axis of each graph. Data presented
is the pooled results from three independent experiments. Each bar represents the
mean ± SEM. (⁄p < 0.01 and ⁄⁄p < 0.05 when comparing IgG2a isotype (grey bars) to
anti-DX27 mAb (black bars) for each construct tested).
they expressed HLA-Cw3. However, when co-infected with DNSHP-1, the KIR/IpLITR1.2a-expressing LAK cells killed 47.9% of
721.221 targets and 38.1% of targets if they expressed HLACw3. Once again, this reverted killing of 721.221-HLA-Cw3 targets
by KIR/IpLITR1.2a-expressing LAK cells co-infected with pSC66
(8.9%) vs. DN-SHP-1 (38.1%) is statistically significant
(p < 0.05; Fig. 4D). Notably, neither KIR/IpLITR1.1b nor KIR/
IpLITR1.1b (tr) demonstrated a reversion of their inhibitory responses when the catalytically inactive DN-SHP-1 was present
(Fig. 4D). This was evident by the failure of DN-SHP-1-co-infections
to revert the killing of 721.221-HLA-Cw3 targets by KIR/
IpLITR1.1b- or KIR/IpLITR1.1b (tr)-expressing LAK cells co-infected
with empty pSC66 vs. DN-SHP-1 (Fig 4D). However, KIR/IpLITR1.1b
and KIR/IpLITR1.1b (tr) expressing LAK cells still effectively killed
MHC I-deficient targets (Fig. 4C).
3.5. KIR/IpLITRCYT chimeras bind the C-terminal Src kinase, Csk
As described above in Section 3.4, KIR/IpLITR1.1b and KIR/
IpLITR1.1b (tr) inhibit LAK cell-mediated cytotoxicity even in the
B.C. Montgomery et al. / Developmental and Comparative Immunology 37 (2012) 151–163
157
Fig. 4. Catalytically inactive SHP-1 abrogates IpLITR1.2a but not IpLITR1.1b-mediated inhibitory signaling in mouse LAK cells. (A) Schematic diagram of the DN-SHP-1
rendered catalytically inactive by a C453 to S453 mutation. The two N-terminal SH2 (Src homology 2) domains and the C-terminal protein tyrosine phosphatase (PTP) are
indicated. Also, a representative Western blot is shown indicating that DN-SHP-1 expression can be detected in the lysates of rVV-infected cells. The mock lane is a lysate from
LAK cells infected with rVV encoding the empty vector pSC66 and the DN-SHP-1 lane is a lysate from LAK cells infected with rVV encoding pSC66-DN-SHP-1 at a MOI of 20.
(B) LAK cells were co-infected with rVV-WR (MOI of 20) encoding an empty pSC66 vector or pSC66-DN-SHP-1 in conjunction with wild-type KIR2DL3 or the chimeric
receptors KIR/IpLITR1.2a, KIR/IpLITR1.1b, KIR/IpLITR1.1b (tr) and KIR/IpLITRDCYT. The surface expression of each receptor co-expressed with pSC66 or DN-SHP-1 was
determined by flow cytometry using the anti-KIR mAb DX27 followed by staining with a secondary goat anti-mouse IgG pAb conjugated to PE. The secondary pAb alone
control is indicated by the solid histogram and the anti-KIR2DL3 staining with DX27 for pSC66 or DN-SHP-1 co-expressed with the different receptors is indicated by the
empty histograms as indicated with arrows for pSC66-KIR2DL3. (C and D) Killing assays were performed with LAK cells expressing the indicated receptors co-infected with
either DN-SHP-1 (grey bars) or rVV-pSC66 (black bars). The target cells used were (C) 721.221 cells or (D) 721.221 expressing HLA-Cw3 and effectors and targets were
incubated at an E:T of 10:1 for 4 h prior to performing the Cr-51 release assay as described in Section 2. 51Cr release was calculated as: % lysis = 100 (mean sample
release mean spontaneous release)/(mean total release mean spontaneous release) and is represented as % killing on the y-axis of each graph. Data presented is the
pooled results from two independent experiments. Each bar represents the mean ± SEM. (⁄p < 0.05 and ⁄⁄p < 0.05 when comparing DN-SHP-1 (grey bars) to pSC66 (black bars)
for each construct tested).
presence of a catalytically inactive SHP-1. Since the SHP-recruitment potential of the different KIR/IpLITRCYT chimeras has already
been established (Montgomery et al., 2009), we examined whether
or not these proteins could interact with another candidate signaling molecule (i.e. Csk) to facilitate their inhibition of killing
responses. To test this, we co-transfected the KIR/IpLITRCYT chimeras with a plasmid encoding Csk into HEK 293T cells. Transfected
cells were then treated with pervanadate, lysed, and immunoprecipitated with an anti-HA mAb that recognizes the HA-tag located
at the N-terminus of the recombinant chimeras. As shown in Fig. 5
(top panel), immunoprecipitation with anti-HA mAb followed by
detection with an HRP-conjugated anti-HA mAb resulted in the
detection of bands at the predicted size for each KIR/IpLITRCYT chimera, which is similar to our previous results (Montgomery et al.,
2009). When the HA-immunoprecipitated cell lysates were then
probed with an Ab specific for Csk, no visible bands were detected
in non-transfected cells (mock) or those transfected with Csk alone
(Fig. 5; middle panel). However, HA-immunoprecipitated lysates
from HEK 293T cells co-transfected with KIR/IpLITR1.1b or KIR/
IpLITR 1.1b (tr) and Csk demonstrated strong Csk immunoreactive
bands (40 kDa) when probed with the anti-Csk pAb (Fig. 5; middle panel). In comparison, the Csk plus KIR/IpLITR1.2a and Csk plus
KIR/IpLITR DCYT co-transfected cells recruited only faint Csk
immunoreactive bands. The bottom panel of Fig. 5 shows the available Csk proteins in each of the cell lysates examined and demonstrates the difference between endogenous levels of Csk found in
HEK 293T cells (mock) vs. the levels of Csk after transfection/
overexpression.
3.6. Tyrosine residue 453 within the unique TM proximal CYT region of
IpLITR1.1b is responsible for Csk recruitment and inhibitory function
Examination of Csk recruitment and inhibition of killing by
KIR/IpLITR1.1b (tr) were performed by generating a series of
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Fig. 5. KIR/IpLITRCYT chimeras bind the C-terminal Src kinase, Csk. One million HEK
293T cells were transiently co-transfected with the KIR/LITRCYT chimeras (1.2a,
1.1b, 1.1b (tr), and DCYT) and pXM113-Csk. After 48 h the cells were treated for
15 min at 37 °C with pervanadate, lysed and then immunoprecipitated with antiHA mAb. Samples were then separated on an 8% SDS–PAGE gel under reducing
conditions, transferred to nitrocellulose and duplicate blots were probed with
either an HRP conjugated anti-HA mAb (top panel) or with anti-Csk antisera
followed by an anti-Ig pAb conjugated to HRP (middle panel). Whole cell lysates
were also analyzed for total Csk expression (bottom panel). Mock lanes had lysates
from non-transfected HEK 293T cells and Csk lanes are lysates from cells
transfected with pXM113-Csk only. Data presented is a representative blot from
two independent experiments that were performed, which gave similar results.
tyrosine-to-phenylalanine mutants and repeating the immunoprecipitation experiments and Cr-51 release assays described above.
Outlined in Supplementary Fig. 1 is the wild type (YY) and three
mutated KIR/IpLITR1.1b (tr) constructs (FY, YF, and FF) that we
generated for these experiments. When transfected into HEK
293T cells, each protein was visualized following immunoprecipitation with the anti-HA mAb (Fig. 6; top panel). As described in
Section 3.5, the wild type KIR/IpLITR1.1b (tr) construct recruited
Csk in co-immunoprecipitation experiments (Fig. 6; middle panel;
1.1b (tr) YY lane). Similarly, KIR/IpLITR1.1b (tr) FY co-immunoprecipitated more Csk protein than was observed in the control (Fig. 6;
middle panel; 1.1b (tr) YY lane vs. Csk alone lane). However, when
constructs 1.1b (tr) YF and 1.1b (tr) FF were examined, they appeared to only bind Csk at levels similar to that observed for Csk
alone transfected cells, which were lower than those observed
for 1.1b (tr) YY and 1.1b FY (Fig. 6; middle panel). The bottom panel of Fig. 6 shows the available Csk protein in each of the HEK
293T cell lysates that were used in the co-immunoprecipitations.
To examine if the differential Csk recruitment capacity observed
for the different KIR/IpLITR1.1b (tr) proteins (i.e. YY, FY, YF, and FF)
also correlated with altered inhibitory function, we infected LAK
cells with the various KIR/IpLITR 1.1b (tr) constructs and performed killing assays. Surface expression of the receptors is shown
in Fig. 7A. Interestingly, the diminished ability of KIR/IpLITR 1.1b
(tr) YF and KIR/IpLITR 1.1b FF to recruit Csk correlated precisely
with their inability to abrogate the killing of 721.221-HLA-Cw3 targets at the various E:T ratios (Fig. 7B). This is particularly evident
when compared with the inhibitory activities of KIR/IpLITR 1.1b
(tr) YY and KIR/IpLITR 1.1b (tr) FY that abrogated target cell killing
if they expressed HLA-Cw3. Pooled data from two independent
Fig. 6. Tyrosine residue 453 within the TM proximal CYT region of IpLITR1.1b is
responsible for Csk recruitment. One million HEK 293T cells were transiently cotransfected with the wild-type KIR/LITR1.1b (tr) chimera or the 1.1b (tr) chimera
with mutated tyrosines (i.e. 1.1b (tr) FY, YF, or FF) in conjunction with pXM113-Csk.
After 48 h the cells were treated with pervanadate for 15 min at 37 °C, lysed and
then immunoprecipitated with anti-HA mAb. Samples were then separated on an
8% SDS–PAGE gel under reducing conditions, transferred to nitrocellulose and
duplicate blots were probed with either an HRP conjugated anti-HA mAb (top
panel) or with anti-Csk antisera followed by an anti-Ig pAb conjugated to HRP
(middle panel). Whole cell lysates were also analyzed for total Csk expression
(bottom panel). Mock lanes were non-transfected HEK 293T cells and Csk lanes
were cells transfected with pXM113-Csk only. Data presented is a representative
blot from two independent experiments that were performed, which gave similar
results.
experiments are provided in Supplementary Fig. 4 reinforcing that
the Y330 to F330 mutation in constructs KIR/IpLITR 1.1b (tr) YF and
FF is essential for the inhibitory function of the TM proximal CYT
region of IpLITR1.1b. Note this residue corresponds to Y453 of the
native IpLITR 1.1b sequence (ABI16050).
4. Discussion
In the present study, we provide new functional information
defining the inhibitory roles of two ITIM-encoding IpLITR-types.
We infected mouse LAK cells with rVV carrying the pSC66 vector
encoding different KIR/IpLITRCYT chimeras and performed target
cell killing assays. When incubated with the MHC class I-negative
human B cell line 721.221, mouse LAK cells infected with rVVpSC66 (empty), rVV-pSC66-KIR2DL3, or the rVV-pSC66-KIR/IpLITRCYT chimeric receptors effectively killed these target cells as determined by the 51Cr release assay. Importantly, this indicated that
our virus infection protocols did not interfere with the ability of
LAK cells to engage and kill their targets. However, if the target
cells expressed the specific ligand for KIR2DL3 (i.e. HLA-Cw3),
the killing responses observed for LAK cells expressing KIR/IpLITR1.2a and KIR/IpLITR1.1b chimeras were significantly diminished;
KIR/IpLITR DCYT did not hamper target cell killing, indicating that
the CYT regions of IpLITR1.2a and IpLITR1.1b are required for their
inhibitory capacities. When we expressed the full-length human
KIR2DL3 in mouse LAK cells, its ability to abrogate cytotoxic responses was similar to that observed with IpLITR1.2a and
IpLITR1.1b and these inhibitory effects were abolished if the LAK
cells were pre-incubated with the blocking mAb DX27. Since
KIR2DL3-mediated inhibition of cellular responses is known to involve SHP-1 recruitment (Burshtyn et al., 1999), we next tested
whether or not inhibition of LAK cell killing by the IpLITR1.2a
and IpLITR1.1b CYT regions was also a SHP-1-dependent process.
This would lend support to our previous findings that IpLITR-mediated inhibitory signaling is dependent on SHP recruitment
B.C. Montgomery et al. / Developmental and Comparative Immunology 37 (2012) 151–163
159
Fig. 7. Tyrosine residue 453 within the TM proximal CYT region of IpLITR1.1b is responsible for inhibitory function. (A) LAK cells were infected with rVV-WR (MOI of 20)
encoding an empty pSC66 vector, or the chimeric receptors KIR/IpLITR1.1b (tr), KIR/IpLITR1.1b (tr) FY, KIR/IpLITR1.1b (tr) YF, and KIR/IpLITR1.1b FF. The surface expression of
each receptor was determined by flow cytometry using the anti-KIR mAb DX27 followed by staining with a secondary goat anti-mouse IgG pAb conjugated to PE. The
secondary pAb alone control is indicated by the solid histogram and the anti-KIR2DL3 staining with DX27 is indicated by the empty histogram. (B) Mouse LAK cells were
infected for 4 h with rVV prior to mixing them with either 721.221 cells (black circles) or 721.221 cells expressing HLA-Cw3 (white circles) at E:T ratios of 1:1, 3:1, and 10:1.
Effectors and targets were then incubated for 4 h at 37 °C and target cell killing was measured from cellular supernatants using the Cr-51 release assay as described in
Section 2. 51Cr release was calculated as: % lysis = 100 (mean sample release mean spontaneous release)/(mean total release mean spontaneous release) and is
represented as % killing on the y-axis of each graph. Data presented is a representative from two independent experiments that were performed, which gave similar results.
(Montgomery et al., 2009). To examine this, we co-expressed a catalytically inactive SHP-1 mutant (i.e. DN-SHP-1) in the KIR/IpLITRCYT expressing LAK cells. Binding of the inactive DN-SHP-1 to ITIMs
via its SH2 domains competitively inhibits wild-type SHP-1 binding thus resulting in a loss of inhibitory signaling (Burshtyn
et al., 1996). As expected, the DN-SHP-1 significantly reversed
the inhibitory function of pSC66-KIR2DL3, confirming that SHP-1
is required for KIR2DL3-mediated inhibition of LAK cell killing responses. When we examined the SHP-1 dependency of the two
inhibitory IpLITR-types, only IpLITR1.2a was sensitive to the presence of the DN-SHP-1, indicating that although IpLITR1.1b binds
SHP-1, its inhibitory activity is not completely dependent on this
phosphatase. Furthermore, the CYT region of this receptor encodes
a unique tyrosine-containing, membrane-proximal region that is
not present in IpLITR1.2a and does not appear to bind SHP-1 or
SHP-2 (Montgomery et al., 2009).
We used a truncated version of IpLITR1.1b (tr) in our functional
experiments to examine if this unique region had any inhibitory
function and whether or not this activity was SHP-1-dependent.
The membrane-proximal CYT region of KIR/IpLITR1.1b (tr) contains
three tyrosine residues (Y433, Y453, and Y463) that are not embedded
within ITIMs (Supplementary Fig. 1). In the order of Y433, Y453, and
Y463 these sequences are HIYDTV, AVYAQV, and ESYKNK. Unexpectedly, when KIR/IpLITR1.1b (tr) was expressed in LAK cells,
and killing assays performed, this CYT region displayed functional
inhibitory activity that was comparable to that of the inhibitory
receptor KIR2DL3 as well as the chimeric receptors KIR/IpLITR1.2a
and KIR/IpLITR1.1b. This inhibitory function was dependent on
receptor engagement with HLA-Cw3 and was not affected by the
presence of DN-SHP-1. Therefore, the membrane proximal CYT region of IpLITR1.1b exhibits ITIM- and SHP-1-independent inhibitory capacity when expressed in LAK cells. To further explore the
signaling mechanisms used by KIR/IpLITR1.1b (tr) we searched
for candidate inhibitory molecules that might be recruited to one
or all of these alternate tyrosine residues. From these searches,
we predicted that the inhibitory kinase, Csk could potentially bind
Y453, found within the sequence AVYAQV. This fits precisely with
the Csk-binding consensus sequence of Y[T/A/S][K/R/Q/N][M/I/V/
R] that was identified by others using a peptide library to identify
Csk-binding motifs (Songyang et al., 1994). Indeed, cellular transfection experiments confirmed that Csk binds to the CYT regions
of IpLITR1.1b and IpLITR1.1b (tr), which is diminished when Y453
but not Y433 was mutated to phenylalanine (note: Y463 was not
investigated in this study and based on our results has no major
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functional role in IpLITR 1.1b-mediated functions). More
importantly, functional killing assays demonstrated that a Y433 to
F433 mutation had no affect on KIR/IpLITR1.1b (tr) inhibitory functions whereas Y453 to F453 completely abrogated the inhibitory
capability of this receptor. These results suggest that the SHP-1independent activity of IpLITR1.1b is possibly mediated by a Cskdependent inhibitory signaling mechanism involving Y453 and suggests that inhibitory IpLITR-types may engage multiple pathways
in order to negatively regulate immune cell effector responses.
The relevance of these findings in teleost immune cells remains
to be explored but these results are the first functional studies
reported for inhibitory IpLITR-types and the first to suggest the
involvement of the Csk pathway as a novel inhibitory signaling
mechanism in fish. Inhibitory signaling mediators including SHP2 and SHIP were not examined in the present study and thus cannot be unequivocally excluded as potential mediators of IpLITRinduced inhibitory responses. These along with other potential
inhibitory signaling molecules (described below) need to be
explored further.
In mammals, Csk has been identified as a major endogenous
inhibitor of SFK activity and thus is a key regulator of multiple kinase-dependent cellular responses (Chong et al., 2005; Kim et al.,
2010; Okada et al., 1991). Src-family protein tyrosine kinases are
vital players in the control of cellular functions and aberrant regulation of SFK activity is associated with multiple diseases (Bjorge
et al., 2000; Frame, 2002; Irby and Yeatman, 2000; Thomas and
Brugge, 1997). Aberrant control of SFK activities in immune cells
also results in uncontrolled cellular proliferation (i.e. lymphoma)
and immune-related pathologies (i.e. autoimmunity) (Hibbs
et al., 2002; Malek et al., 1998; Okada et al., 1991). The inhibitory
actions of Csk are dependent on its ability to specifically phosphorylate a conserved tyrosine residue located at the C-terminus of
SFKs (i.e. Lyn, Fyn, Hck, etc.), which inhibits their catalytic activities
(Chong et al., 2005; Kim et al., 2010; Okada et al., 1991). A non-catalytic homolog of Csk also exists, called CSK-homologous kinase
(CHK), which also plays a pivotal role in the regulation of SFKs
but it does so via non-covalent, allosteric protein–protein interactions with target kinases and not phosphorylation of the C-terminal regulatory tyrosine (Chong et al., 2005, 2006).
Despite the knowledge that Csk is an important regulator of
various cellular signaling events, recruitment of this regulatory kinase by immunoregulatory receptors and its ability to negatively
influence cellular immune responses has only recently been recognized (Sayos et al., 2004; Verbrugge et al., 2006). This kinase-mediated inhibitory signaling in immune cells opposes the classical
phosphatase-driven attenuation of immune cell responses but is
likely as important in the regulation of immunity. Primarily located
in the cytoplasm, Csk is known to be recruited to lipid rafts in the
plasma membrane via an SH2 domain-mediated interaction with
the Csk-binding protein (Cbp)/phosphoprotein associated with glycosphingolipid-enriched microdomains adaptor (PAG) (Brdicka
et al., 2000; Kawabuchi et al., 2000). The recruitment of Csk to lipid
rafts by Cbp/PAG promotes phosphorylation of Csk and places this
regulatory kinase in close proximity to its targeted SFKs (Cary and
Cooper, 2000; Chong et al., 2005). Consequently, the Cbp/PAGmediated membrane recruitment and activation of Csk has been
implicated in the negative regulation of immune cell signaling
(Davidson et al., 2003; Kitaura et al., 2007; Yasuda et al., 2002). Recently, different ITIM-containing immunoregulatory receptortypes have also been demonstrated as Csk-binding proteins including LILR1, leukocyte-associated Ig-like receptor-1 (LAIR-1), and signal-regulatory protein (SIRPa) (Sayos et al., 2004; Veillette et al.,
1998; Verbrugge et al., 2006). Like the Csk-Cbp/PAG interactions
described above, recruitment of Csk to these inhibitory membrane
receptor-types places this protein at a location where it can effectively phosphorylate targeted SFKs rendering them inactive and
incapable of initiating the proximal signaling events required for
cellular activation such as the phosphorylation of ITAMs within
stimulatory adaptor proteins. Additionally, since SFK-mediated
phosphorylation of ITIMs is also necessary for phosphatase binding
(i.e. SHP-1, SHP-2, and/or SHIP; Long, 2008; Ravetch and Lanier,
2000; Vivier et al., 2004), concomitant or subsequent recruitment
of Csk represents a potential auto-regulatory signaling mechanism
by diminishing ITIM phosphorylation and abrogating phosphatasemediated deactivation of intracellular signaling molecules like
Vav1. This essentially would dictate how much phosphatase binding occurs during inhibitory receptor engagement and adding an
additional level of immunoregulatory receptor control of cellular
Table 1
Phosphatases, kinases, and other signaling molecules in mammals and fish.a
Moleculeb
Humanc
Zebrafishc
% Identityd
Catfishe
% Identityd
SHP-1
SHP-2
SHIP
SH2D1A
EAT-2
Csk
CHK
Vav1
c-Abl
Crk
Slp-76
Cbp/PAG
c-Src
Fyn
Lck
Yes
Lyn
Fgr
Blk
AAA36610
BAA02740
AAB49680
NP_001108409
AAM28522
NP_004374
NP_002369
AAH13361
NP_005148
NP_058431
EAW61478
NP_060910
CAA26485
NP_002028
AAH13200
NP_005424
NP_002341
NP_005239
NP_001706
CAZ68039
CAZ68072
XP_001923007
XP_001920648
NP_001189421
NP_001071067
XP_695792
XP_001119865
XP_001337899
NP_001003628
NP_999882
XP_003200660
CAF06181
AAX47959
NP_001035418
NP_001013288
NP_001004543
NP_997946
NP_001025391
62
90
56
66
40
86
63
54
69
70
38
36
91
92
66
86
76
71
69
T67588
61
TC72632
TC61594
TC62921
TC6373
45
64
42
87
TC74369
55
TC60454
TC63446
TC56935
TC61748
TC66318
TC64454
69
37
27
91
73
68
a
Signaling molecules described throughout the manuscript that play important roles as mediators of immune cell inhibition and those that are targeted during immune
cell inhibition are listed.
b
Abbreviations defined throughout the manuscript.
c
GenBank Accession numbers from http://www.ncbi.nlm.nih.gov/protein/.
d
Percent amino acid identity when compared with the human sequence.
e
TC (tentative consensus) Identifier as per DFCI (Dana Farber Cancer Institute, Harvard) Gene Index (http://compbio.dfci.harvard.edu/tgi/index.html) for channel catfish
(Ictalurus punctatus).
B.C. Montgomery et al. / Developmental and Comparative Immunology 37 (2012) 151–163
activation. Although Csk binding to LILR1, LAIR-1, and SIRPa appear to be ITIM-dependent events, there are several examples of
other proteins that bind Csk in an ITIM-independent manner
(Brdicková et al., 2003; Songyang et al., 1994), which now also includes IpLITR1.1b at Y453 within the non-ITIM ‘AVYAQV’ identified
in this study. However, it is difficult to predict precisely how Csk
interacts with all of its binding partners but a phosphorylated tyrosine is most certainly required (Brdicková et al., 2003).
While our functional data was dependent on the recruitment of
a mammalian Csk to a teleost receptor, a Csk homolog is present in
fish that shares >85% identity with the human protein (Table 1). In
fact, many of the known inhibitory receptor signaling mediators
(e.g. SHP-1, SHP-2, SHIP, Csk, CHK, c-Abl, and Crk) and their specific
targets (i.e. Vav, SLP-76, and several SFKs; reviewed in Vivier et al.
(2004)) are also present in teleosts (Table 1), indicating that key
mechanisms of immune cell inhibition are likely conserved between mammals and fish. Some of these signaling molecules have
only recently been recognized as inhibitory mediators, which provide new insights into the mechanisms of immunoregulatory
receptor signaling and new opportunities to explore these pathways in different vertebrates. Classically, ITIM-mediated signaling
requires recruitment of intracellular phosphatases (e.g. SHP-1;
Burshtyn et al., 1996; Long, 2008; Olcese et al., 1996; Vivier
et al., 2004) that dephosphorylate important proximal signaling
proteins and metabolites (e.g. Vav1, SLP-76, IP3, and PI(3,4,5)P3)
preventing the transmission of kinase-mediated signaling events
and subsequent immune cell effector functions (i.e. cytotoxicity
and cytokine secretion). However, it has also been reported that
the ITIM-containing immune receptors, KIR and CD94/NKG2A induce a tyrosine phosphorylation-dependent inhibitory pathway
(Peterson and Long, 2009) via the actions of the non-receptor tyrosine kinase c-Abl and the Crk adaptor protein. It is believed that
following ligand engagement of KIR or CD94/NKG2A, SFK-mediated tyrosine phosphorylation of their ITIMs leads to an interaction
between the c-Abl tyrosine kinase and the adaptor molecule Crk
that culminates in the phosphorylation of Crk (Peterson and Long,
2009). Phosphorylated Crk is then believed to contribute to cellular
inhibition (Peterson and Long, 2009). Interestingly, this mechanism
is concurrent with SHP-1-mediated dephosphorylation of the
guanine exchange factor Vav1 indicating that inhibitory immune
receptor signaling is much more complicated than originally described and provides a new look into ITIM-mediated inhibitory signaling events. Teleost homologs for both c-Abl and Crk also exist
(Table 1) indicating that this novel inhibitory pathway may function in the regulation of fish immune cell responses. Another inhibitory pathway mediated by immunoregulatory receptors is
dependent on immune tyrosine-based switch motifs (ITSMs; Sidorenko and Clark, 2003). While often encoded in CYT regions that
contain ITIMs, this motif binds SHP-1 and SHP-2 (Chemnitz et al.,
2004) but can also inactivate cellular signaling via a SHP-1, -2/
SHIP-independent mechanism due to the selective recruitment of
the adaptor proteins SH2 domain protein 1A (SH2D1A) and Ewing’s
sarcoma (EWS)-activated transcript 2 (EAT-2) (Sidorenko and
Clark, 2003; Thompson et al., 1999). Like the other inhibitory signaling mediators described above (SHP-1, SHP-2, SHIP, Csk, CHK,
c-Abl, and Crk), fish have SH2D1A and EAT-2 homologs (Table 1).
Furthermore, the membrane distal region of IpLITR1.1b encodes
an ITSM (Supplementary Fig. 1) that may contribute to the ability
of this receptor to influence leukocyte responses (Montgomery
et al., 2009). Clearly, more experiments are required to define the
precise SHP-independent mechanisms of IpLITR-induced cellular
inhibition.
Our results provide a unique look into the inhibitory signaling
capacity of the ITIM-encoding receptors IpLITR1.2a and IpLITR1.1b.
We have demonstrated that both SHP-1-dependent and -independent pathways participate in the inhibitory functions of these
161
proteins, which can potently abrogate lymphocyte-mediated cellular cytotoxicity. The ability of an IpLITR CYT region to inhibit the
effector function of mouse LAK cells implies that evolutionarily
conserved molecules facilitate inhibitory signaling in immune
cells. This is reinforced by the identification of teleost homologs
for a vast array of phosphatases, kinases, and adaptor molecules
believed to participate in down-regulating immune cell responses
(Table 1). Detailed studies are now required to: (i) confirm that Csk
is a SHP-independent mechanism of IpLITR-mediated cellular inhibition; (ii) to determine if other SHP-independent inhibitory signaling pathways are engaged by IpLITRs; (iii) to biochemically
and functionally characterize inhibitory signaling molecules in teleost immune cells, and; (iv) to examine the impact(s) of IpLITRmediated inhibitory signaling on fish immune defense against
pathogens. The results presented in this study are an important
first step in these directions.
Acknowledgments
This work was supported by Grants from the Natural Sciences
and Engineering Council of Canada (NSERC), Canadian Foundation
for Innovation (CFI) Leaders Opportunity Fund, Alberta Advanced
Education and Technology, Alberta Heritage Foundation for Medical Research (AHFMR) major equipment Grant, and a Faculty of Science Start-up Grant to J.L.S. Graduate teaching assistantships
awarded by the Department of Biological Sciences, University of
Alberta were provided for B.C.M. B.C.M is also funded by an NSERC
C-GSD award. D.N.B is supported by CIHR and AHFMR. We thank
Dr. Andre Véillette for providing reagents.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.dci.2011.09.005.
References
Anderson, S.K., Ortaldo, J.R., McVicar, D.W., 2001. The ever-expanding Ly49 gene
family: repertoire and signaling. Immunol. Rev. 181, 79–89.
Bakker, A.B., Wu, J., Phillips, J.H., Lanier, L.L., 2000. NK cell activation: distinct
stimulatory pathways counterbalancing inhibitory signals. Hum. Immunol. 61,
18–27.
Barclay, A.N., 2003. Membrane proteins with immunoglobulin-like domains-a
master superfamily of interaction molecules. Semin. Immunol. 14, 215–223.
Barrow, A.D., Trowsdale, J., 2008. The extended leukocyte receptor complex: diverse
ways of modulating immune responses. Immunol. Rev. 224, 98–123.
Beebe, K.D., Wang, P., Arabaci, G., Pei, D.H., 2000. Determination of the binding
specificity of the SH2 domains of protein tyrosine phosphatase SHP-1 through
the screening of a combinatorial phosphotyrosyl peptide library. Biochemistry
39, 13251–13260.
Biassoni, R., Cantoni, C., Pende, D., Sivori, S., Parolini, S., Vitale, M., et al., 2001.
Human natural killer cell receptors and co-receptors. Immunol. Rev. 181, 203–
214.
Bjorge, J.D., Jakymiw, A., Fujita, D.J., 2000. Selected glimpses into the activation and
function of Src kinase. Oncogene 19, 5620–5635.
Brdicka, T., Pavlistova, D., Leo, A., Bruyns, E., Korinek, V., Angelisova, P., et al., 2000.
Phosphoprotein associated with glycosphingolipid-enriched microdomains
(PAG), a novel ubiquitously expressed transmembrane adaptor protein, binds
the protein tyrosine kinase csk and is involved in regulation of T cell activation.
J. Exp. Med. 191, 1591–1604.
Brdicková, N., Brdicka, T., Angelisová, P., Horváth, O., Spicka, J., Hilgert, I., et al., 2003.
LIME: a new membrane Raft-associated adaptor protein involved in CD4 and
CD8 coreceptor signaling. J. Exp. Med. 198, 1453–1462.
Brown, D., Trowsdale, J., Allen, R., 2004. The LILR family: modulators of innate and
adaptive immune pathways in health and disease. Tissue Antigens 64, 215–225.
Burshtyn, D.N., Scharenberg, A.M., Wagtmann, N., Rajagopalan, S., Berrada, K., Yi, T.,
et al., 1996. Recruitment of tyrosine phosphatase HCP by the killer cell inhibitor
receptor. Immunity 4, 77–85.
Burshtyn, D.N., Yang, W., Yi, T., Long, E.O., 1997. A novel phosphotyrosine motif with
a critical amino acid at position -2 for the SH2 domain-mediated activation of
the tyrosine phosphatase SHP-1. J. Biol. Chem. 272, 13066–13072.
Burshtyn, D.N., Lam, A.S., Weston, M., Gupta, N., Warmerdam, P.A., Long, E.O., 1999.
Conserved residues amino-terminal of cytoplasmic tyrosines contribute to the
162
B.C. Montgomery et al. / Developmental and Comparative Immunology 37 (2012) 151–163
SHP-1-mediated inhibitory function of killer cell Ig-like receptors. J. Immunol.
162, 897–902.
Carlyle, J.R., Jamieson, A.M., Gasser, S., Clingan, C.S., Arase, H., Raulet, D.H., 2004.
Missing self-recognition of Ocil/Clr-b by inhibitory NKR-P1 natural killer cell
receptors. Proc. Natl. Acad. Sci. USA 101, 3527–3532.
Cary, L.A., Cooper, J.A., 2000. Molecular switches in lipid rafts. Nature 404, 945–947.
Chemnitz, J.M., Parry, R.V., Nichols, K.E., June, C.H., Riley, J.L., 2004. SHP-1 and SHP-2
associate with immunoreceptor tyrosine-based switch motif of programmed
death 1 upon primary human T cell stimulation, but only receptor ligation
prevents T cell activation. J. Immunol. 173, 945–954.
Chong, Y.-P., Mulhern, T.D., Cheng, H.-C., 2005. C-terminal kinase (Csk) and CSKhomologous kinase (CHK)-endogenous negative regulators of Src-family
protein kinases. Growth Factors 23, 233–244.
Chong, Y.-P., Chan, A.S., Chan, K.-C., Williamson, N.A., Lerner, E.C., Smithgall, T.E.,
et al., 2006. C-terminal Src kinase-homologous kinase (CHK), a unique inhibitor
inactivating multiple active conformations of Src family tyrosine kinases. J. Biol.
Chem. 281, 32988–32999.
Cloutier, J.-F., Veillette, A., 1996. Association of inhibitory tyrosine kinase p50csk
with protein tyrosine phosphatase PEP in T cells and other hemopoietic cells.
EMBO J. 15, 4909–4918.
Cortes, H.D., Montgomery, B.C., Verheijen, K., García-García, E., Stafford, J.L., 2012.
Examination of the stimulatory signaling potential of a channel catfish
leukocyte immune-type receptor and associated adaptor. Dev. Comp.
Immunol. 36, 62–73.
Davidson, D., Bakinowski, M., Thomas, M.L., Horejsi, V., Veillette, A., 2003.
Phosphorylation-dependent regulation of T-cell activation by PAG/Cbp, a lipid
raft-associated transmembrane adaptor. Mol. Cell. Biol. 23, 2017–2028.
Earl, P.L., Broder, C.C., Long, D., Lee, S.A., Peterson, J., Chakrabarti, S., et al., 1994.
Native oligomeric human immunodeficiency virus type 1 envelope glycoprotein
elicits diverse monoclonal antibody reactivities. J. Virol. 68, 3015–3026.
Falk, N., Ravetch, R.V., 2006. Fcc receptors: old friends and new family members.
Immunity 24, 19–28.
Ford, J.W., McVicar, D.W., 2009. TREM and TREM-like receptors in inflammation and
disease. Curr. Opin. Immunol. 21, 38–46.
Frame, M.C., 2002. Src in cancer: deregulation and consequences for cell behaviour.
Biochim. Biophys. Acta. 1602, 114–130.
Gergely, J., Pecht, I., Sarmay, G., 1999. Immunoreceptor tyrosine-based inhibition
motif bearing receptors regulate the immunotyrosine-based activation motifinduced activation of immune competent cells. Immunol. Lett. 68, 3–15.
Gunturi, A., Berg, R.E., Forman, J., 2004. The role of CD94/NKG2 in innate and
adaptive immunity. Immunol. Res. 30, 29–34.
Hibbs, M.L., Harder, K.W., Armes, J., Kountouri, N., Quilici, C., Casagranda, F., et al.,
2002. Sustained activation of Lyn tyrosine kinase in vivo leads to autoimmunity.
J. Exp. Med. 196, 1593–1604.
Imhof, D., Wavreille, A.-S., May, A., Zacharias, M., Tridandapani, S., Pei, D., 2006.
Sequence specificity of SHP-1 and SHP-2 Src homology 2 domains. Critical roles
of residues beyond the pY+3 position. J. Biol Chem. 281, 20271–20282.
Irby, R.B., Yeatman, T.J., 2000. Role of Src expression and activation in human
cancer. Oncogene 19, 5636–5642.
Katz, H.R., 2006. Inhibition of inflammatory responses by leukocyte Ig-like
receptors. Adv. Immunol. 91, 251–272.
Kawabuchi, M., Satomi, Y., Takao, T., Shimonishi, Y., Nada, S., Nagai, K., et al., 2000.
Transmembrane phosphoprotein Cbp regulates the activities of Src-family
tyrosine kinases. Nature 404, 999–1003.
Kim, K.I.A., Mills, R.D., Hossain, M.I., Chan, K.-C., Jarasrassamee, B., Jorissen, R.N.,
et al., 2010. Structural elements and allosteric mechanisms governing
regulation and catalysis of CSK-family kinases and their inhibition of Srcfamily kinases. Growth Factors 28, 329–350.
Kitaura, J., Kawakami, Y., Maeda-Yamamoto, M., Horejsi, V., Kawakami, T., 2007.
Dysregulation of Src family kinases in mast cells from epilepsy-resistant ASK
versus epilepsy-prone EL mice. J. Immunol. 178, 455–462.
Kuster, H., Thompson, H., Kinet, J.P., 1990. Characterization and expression of the
gene for the human Fc receptor gamma subunit: definition of a new gene
family. J. Biol. Chem. 265, 6448–6452.
Lanier, L.L., 2008. Up on the tightrope: natural killer cell activation and inhibition.
Nat. Immunol. 9, 495–502.
Lanier, L.L., Corliss, B.C., Phillips, J.H., 1997. Arousal and inhibition of human NK
cells. Immunol. Rev. 155, 145–154.
Lanier, L.L., Corliss, B.C., Wu, J., Leong, C., Phillips, J.H., 1998. Immunoreceptor DAP12
bearing a tyrosine-based activation motif is involved in activating NK cells.
Nature 391, 703–707.
Long, E.O., 1999. Regulation of immune responses through inhibitory receptors.
Annu. Rev. Immuno. 17, 875–904.
Long, E.O., 2008. Negative signalling by inhibitory receptors: the NK cell paradigm.
Immunol. Rev. 224, 70–84.
Long, E.O., Burshtyn, D.N., Clark, W.P., Peruzzi, M., Rajagopalan, S., Wagtmann, N.,
et al., 1997. Killer cell inhibitory receptors: diversity, specificity, and function.
Immunol. Rev. 155, 135–144.
López-Botet, M., Carretero, M., Pérez-Villar, J., Bellón, T., Llano, M., Navarro, F., 1997.
The CD94/NKG2 C-type lectin receptor complex: involvement in NK cellmediated recognition of HLA class I molecules. Immunol. Res. 16, 175–185.
Malek, S.N., Dordai, D.I., Reim, J., Dintzis, H., Desiderio, S., 1998. Malignant
transformation of early lymphoid progenitors in mice expressing an activated
Blk tyrosine kinase. Proc. Natl. Acad. Sci USA 95, 7351–7356.
Mewes, J., Verheijen, K., Montgomery, B.C., Stafford, J.L., 2009. Stimulatory catfish
leukocyte immune-type receptors (IpLITRs) demonstrate a unique ability to
associate with adaptor signaling proteins and participate in the formation of
homo- and heterodimers. Mol. Immunol. 47, 318–331.
Montgomery, B.C., Mewes, J., Davidson, C., Burshtyn, D.N., Stafford, J.L., 2009. Cell
surface expression of channel catfish leukocyte immune-type receptors
(IpLITRs) and recruitment of both Src homology 2 domain-containing
protein tyrosine phosphatases (SHP)-1 and SHP-2. Dev. Comp. Immunol. 33,
570–582.
Montgomery, B.C., Cortes, H.D., Mewes-Ares, J., Verheijen, K., Stafford, J.L., 2011.
Teleost IgSF immunoregulatory receptors. Dev. Comp. Immunol. 35,
120–134.
Nimmerjahn, F., Ravetch, J.V., 2007. Fc-receptors as regulators of immunity. Adv.
Immunol. 96, 179–204.
Nimmerjahn, F., Ravetch, J.V., 2011. FccRs in health and disease. Curr. Top.
Microbiol. Immunol. 350, 105–125.
Okada, M., Nada, S., Yamanashi, Y., Yamamoto, T., Nakagawa, H., 1991. Csk: a
protein-tyrosine kinase involved in regulation of Src family kinases. J. Biol.
Chem. 266, 24249–24252.
Olcese, L., Lang, P., Vély, F., Cambiaggi, A., Marguet, D., Bléry, M., et al., 1996. Human
and mouse killer-cell inhibitory receptors recruit PTP1C and PTP1D protein
tyrosine phosphatases. J. Immunol. 156, 4531–4534.
Parham, P., 2004. Killer cell immunoglobulin-like receptor diversity: balancing
signals in the natural killer cell response. Immunol. Lett. 92, 11–13.
Peterson, M.E., Long, E.O., 2009. Inhibitory receptor signaling via tyrosine
phosphorylation of the adaptor Crk. Immunity 29, 578–588.
Ravetch, J.V., Lanier, L.L., 2000. Immune Inhibitory Receptor. Science 90, 84–89.
Roussell, E., Gerrard, J.M., Greenberg, A.H., 1990. Long-term cultures of human
periperhal blood lymphocytes with recombinant human interleukin-2 generate
a population of virtually pure CD3+ CD16- CD56- large granular lymphocyte
LAK cells. Clin. Exp. Immunol. 82, 416–421.
Sayos, J., Martinez-Barriocanal, A., Kitzig, F., Bellon, T., Lopez-Botet, M., 2004.
Recruitment of C-terminal Src kinase by the leukocyte inhibitory receptor
CD85j. Biochem. Biophys. Res. Commun. 324, 640–647.
Sidorenko, S.P., Clark, E.A., 2003. The dual-function CD150 receptor subfamily: the
viral attraction. Nat. Immunol. 4, 19–24.
Songyang, Z., Shoelson, S.E., McGlade, J., Olivier, P., Pawson, T., Bustelo, X.R., et al.,
1994. Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB2, HCP, SHC, Syk, and Vav. Mol. Cell. Biol. 14, 2777–2785.
Stafford, J.L., Bengtén, E., Du Pasquier, L., McIntosh, R.D., Quiniou, S.M., Clem, L.W.,
et al., 2006. A novel family of diversified immunoregulatory receptors in
teleosts is homologous to both mammalian Fc receptors and molecules encoded
within the leukocyte receptor complex. Immunogenetics 58, 758–773.
Stanietsky, N., Mandelboim, O., 2010. Paired NK cell receptors controlling NK
cytotoxicity. FEBS Lett. 584, 4895–4900.
Stebbins, C.C., Watzl, C., Billadeau, D.D., Leibson, P.J., Burshtyn, D.N., Long, E.O.,
2003. Vav1 dephosphorylation by the tyrosine phosphatase SHP-1 as a
mechanism for inhibition of cellular cytotoxicity. Mol. Cell. Biol. 23, 6291–6299.
Steevels, T.A.M., Meyaard, L., 2011. Immune inhibitory receptors: essential
regulators of phagocyte function. Eur. J. Immunol. 41, 575–587.
Takai, T., 2005. Fc receptors and their role in immune regulation and autoimmunity.
J. Clin. Immunol. 25, 1–18.
Thomas, S.M., Brugge, J.S., 1997. Cellular functions regulated by Src family kinases.
Annu. Rev. Cell Dev. Biol. 13, 513–609.
Thomas, R., Matthias, T., Witte, T., 2010. Leukocyte immunoglobulin-like receptors
as new players in autoimmunity. Clin. Rev. Allergy Immunol. 38, 159–162.
Thompson, A.D., Teitell, M.A., Arvand, A., Denny, C.T., 1999. Divergent Ewing’s
sarcoma EWS/ETS fusions confer a common tumorigenic phenotype on NIH3T3
cells. Oncogene 30, 5506–5513.
Tybulewicz, V.L., 2005. Vav-family proteins in T-cell signalling. Curr. Opin.
Immunol. 17, 267–274.
Underhill, D.M., Goodridge, H.S., 2007. The many faces of ITAMs. Trends Immunol.
28, 66–73.
Van den Herik-Oudijk, I.E., Ter Bekke, M.W., Tempelman, M.J., Capel, P.J., Van de
Winkel, J.G., 1995. Functional differences between two Fc receptor ITAM
signaling motifs. Blood 86, 3302–3307.
Veillette, A., Thibaudeau, E., Latour, S., 1998. High expression of inhibitory receptor
SHPS-1 and its association with protein-tyrosine phosphatase SHP-1 in
macrophages. J. Biol. Chem. 273, 22719–22728.
Verbrugge, A., Rijkers, E.S.K., De Ruiter, T., Meyaard, L., 2006. Leukocyte associated
Ig-like receptor-1 has SH-2 domain-containing phosphatase independent
function and recruits C-terminal Src kinase. Eur. J. Immmunol. 36, 190–198.
Vilches, C., Parham, P., 2002. KIR: diverse, rapidly evolving receptors of innate and
adaptive immunity. Annu. Rev. Immunol. 20, 17–51.
Vivier, E., Daeron, M., 1997. Immunoreceptor tyrosine-based inhibition motifs.
Immunol. Today 18, 286–291.
Vivier, E., Nunès, J.A., Vély, F., 2004. Natural killer cell signaling pathways. Science
306, 1517–1519.
Weis, W.I., Taylor, M.E., Drickamer, K., 1998. The C-type lectin superfamily in the
immune system. Immunol. Rev. 163, 19–34.
Willey, R.L., Smith, D.H., Lasky, L.A., Theodore, T.S., Earl, P.L., Moss, B., 1988. In vitro
mutagenesis identifies a region within the envelope gene of the human
immunodeficiency virus that is critical for infectivity. J. Virol. 62, 139–147.
Wu, J., Cherwinski, H., Spies, T., Phillips, J.H., Lanier, L.L., 2000. DAP10 and DAP12
form distinct, but functionally cooperative, receptor complexes in natural killer
cells. J. Exp. Med. 192, 1059–1068.
Yasuda, K., Nagafuku, M., Shima, T., Okada, M., Yagi, T., Yamada, T., et al., 2002.
Cutting edge: Fyn is essential for tyrosine phosphorylation of Csk-binding
B.C. Montgomery et al. / Developmental and Comparative Immunology 37 (2012) 151–163
protein/phosphoprotein associated with glycolipid-enriched microdomains in
lipid rafts in resting T cells. J. Immunol. 169, 2813–2817.
Yoder, J.A., Litman, G.W., 2011. The phylogenetic origins of natural killer receptors
and recognition: relationships, possibilities, and realities. Immunogenetics 63,
123–141.
163
Yokoyama, W.M., Plougastel, B.F.M., 2003. Immune functions encoded by the
natural killer complex. Nat. Rev. Immunol. 3, 304–316.
Zöller, M., Price, M.R., Baldwin, R.W., 1977. Evaluation of 51Cr release for detecting
cell-mediated cytotoxic responses to solid chemically induced rat tumours. Br. J.
Cancer 25, 834–843.