KIR family, belonging to the type II KIR2D subgroup, which con-
tains one other member named KIR2DL4 (2DL4) (21, 22).
KIR2DL5 is structurally related to 2DL4, showing 79% amino
acid sequence homology. Both receptors have a distinct configu-
ration of extracellular Ig-like domains (D0 –D2) and a longer cy-
toplasmic tail than that of classical type I inhibitory KIRs.
KIR2DL5 has two tyrosines in the cytoplasmic domain, no
charged amino acids in the transmembrane domain, and is found in
50% of all humans, while 2DL4 is the only long KIR that has a
single cytoplasmic tyrosine, a positively charged transmembrane
arginine, and is encoded by a gene found in virtually all individuals
(22, 23). Interestingly, both 2DL5 and 2DL4 are highly conserved
among rhesus monkeys, chimpanzees, and humans, suggesting
their physiological importance in NK cell functions (24 –26). The
ligand of 2DL4 is thought to be the nonclassical class I MHC
molecule, HLA-G, which is normally expressed only on fetally
derived trophoblast cells that invade the maternal decidua in preg-
nant women (27). In contrast, the ligand specificity and function of
2DL5 have not been addressed to date, although the structural
similarities with 2DL4 suggest similar ligand recognition patterns.
Despite the cytoplasmic ITIM, 2DL4 was shown to function as an
activating receptor to selectively produce IFN-
␥, apparently
through association with an undefined transmembrane accessory
protein (28, 29). Interestingly, mutation of the charged residue in
the transmembrane domain of 2DL4 converted it from an activat-
ing to an inhibitory receptor, probably due to dissociation from the
putative activating accessory signaling protein (30). In addition,
the cytoplasmic domain of 2DL4 was shown to inhibit NK cell
cytotoxicity when fused with extracellular and transmembrane do-
mains of KIR3DL1 (3DL1), which appears to be due to exclusive
recruitment of SHP-2, but not SHP-1 (31). These results suggest
that 2DL4 may play both activating and inhibitory roles in NK cell
biology, although only activating function has been demonstrated
for the full-length wild-type receptor, which is not hindered by the
cytoplasmic ITIM (28 –30). The function of 2DL5, however, has
not been tested.
The two tyrosine-based sequences in the cytoplasmic domain of
2DL5 are characteristic of a classical N-terminal ITIM and another
sequence similar to the immunoreceptor tyrosine-based switch mo-
tif (ITSM; T/SxYxxV/I). The ITSM sequence has been shown to
exist in several human immunoreceptors, including 2B4, signaling
lymphocyte activation molecule (SLAM), CD84, and Ly-9 (32,
33), and has been shown to recruit both the SLAM-associated pro-
tein (SAP) (otherwise known as SH2-containing adapter protein
1A (SH2D1A)) and SHP-2 (34, 35). Although 2B4 recruits both
proteins when expressed in nonlymphoid cells, all published ex-
periments have failed to detect SHP-2 binding to 2B4 in NK cells
(35, 36), even in the absence of SAP (37). Because SHP-2 is also
involved in KIR function, it is of interest to determine whether the
ITSM-like sequence of 2DL5 affects SHP-2 recruitment and in-
hibitory function of the receptor in human NK cells.
In this study, we directly compared the inhibitory function and
PTP recruitment capacities of the cytoplasmic domains of the clas-
sical KIR, 3DL1, with 2DL5 and 2DL4 using a chimeric receptor
approach. We defined the importance of two ITIM sequences for
optimal KIR inhibition, because 2DL5 inhibition seems to be
weaker than that of 3DL1. Our results indicate that 2DL5 functions
as an inhibitory receptor, sharing similar inhibitory capacity with
that of the cytoplasmic domain of 2DL4. Using cytotoxicity and
conjugate formation assays and DN forms of phosphatases, we
demonstrate that different inhibitory KIRs have qualitatively dis-
tinct inhibitory capacities, which cannot be entirely predicted from
a strictly biochemical approach.
Materials and Methods
Cell culture and Abs
The IL-2-dependent NK-like cell line, NK-92 (a gift from C. Lutz, Uni-
versity of Iowa, Iowa City, IA), was cultured, as previously described (31).
Cells were passed with fresh IL-2 every 4 days. The murine mastocytoma,
P815, was cultured, as previously described (31). Abs were purified with
protein G from the hybridomas for the anti-3DL1 mAb, DX9, and the
anti-CD56 mAb, B159.5.2, which were kindly provided by L. Lanier (Uni-
versity of California, San Francisco, CA) and B. Perussia (Thomas Jeffer-
son University, Philadelphia, PA), respectively. The anti-FLAG mAb, M2,
was purchased from Sigma-Aldrich (St. Louis, MO). Anti-SAP polyclonal
Abs raised against the entire recombinant protein were obtained from Ex-
alpha (Boston, MA).
cDNA constructs
KIR cDNA constructs were ligated into the retroviral expression vector,
pBMN-NoGFP (green fluorescence protein), derived from pBMN-IRES-
EGFP (enhanced green fluorescence protein) (38), using BamHI, XhoI, and
NotI restriction sites, as previously described (31). The 3DL1 cDNA
(NKAT3; GenBank accession number, L41269) was obtained from M.
Colonna (Washington University, St. Louis, MO). The deletion mutant of
3DL1 (272P
⌬), which was truncated before the first ITIM and lacks most
of the cytoplasmic domain, was described previously (31). To generate the
chimeric 3DL1/L5 receptor, the cytoplasmic domain was amplified by PCR
from the human 2DL5.1 cDNA (AF204903), kindly provided by C.
Vilches (Inmunologia, Hospital Universitario Puerta de Hierro, Madrid,
Spain) and cloned into the pCR2.1-TOPO vector (Invitrogen, San Diego,
CA) using the following primers: sense containing BspHI site (underlined),
5
Ј-CT GCT GTC ATG AAC CAA GAG CCT GC-3Ј; antisense containing
XhoI site, 5
Ј-ACT GTA CTC GAG GCT AAG CAA AGG AGT GTG
TC-3
Ј. The extracellular and transmembrane domains of 3DL1 were am-
plified by PCR using the sense primer (
BamHI), 5
Ј-TCG ACT GGA TCC
ACC ATG TCG CTC ATG GTC GTC AGC ATG-3
Ј, and the antisense
primer (BspHI), 5
Ј-AGG CTC TTG GTT CAT GAC AGC AGC AT-3Ј.
The BamHI-BspHI fragment of 3DL1 and the BspHI-XhoI fragment of
2DL5 were cloned into the pBMN-NoGFP vector. The ITIM tyrosine in the
2DL5 cytoplasmic domain was mutated to phenylalanine by PCR using the
sense primer, 5
Ј-CT CAG GAG GTG ACA TTT GCA CAG TTG GAT
CAC-3
Ј, and the antisense primer, 5Ј-GTG ATC CAA CTG TGC AAA
TGT CAC CTC CTG AG-3
Ј. To make FLAG-tagged full-length
KIR2DL5, the cDNA was amplified by PCR using the sense primer
(EcoRI), 5
Ј-AT CGT ACG AAT TCA CAT GAG GGT GGT CAG GA-3Ј,
and the antisense primer (NotI), 5
Ј-ATGCAG CGG CCG CTC AGA TTC
CAG CTG CTG GTA-3
Ј. The EcoRI-EcoRI fragment containing a leader
peptide and N-terminal FLAG epitope tag (DYKDDDK) was cloned from
the pFLAG-CMV3 vector (Sigma-Aldrich). The EcoRI-NotI fragment of
2DL5.1 and EcoRI-EcoRI fragment of the leader plus FLAG sequences
were cloned into the pBMN-IRES-EGFP vector. SAP was amplified by
PCR from a Jurkat cDNA library using the sense primer (SalI), 5
Ј-AC-
GACA GTC GAC GAT GGA CGC AGT GGC TGT-3
Ј, and the antisense
primer containing Myc-tag (NotI), 5
Ј-ACG ACA GCG GCC GCT AAA
GAT CTT CTT CAG AAA TAA GTT TCT GTT CTG GGG CTT TCA
GGC AGA-3
Ј. The SalI-NotI fragment of SAP was cloned into the pBMN-
IRES-EGFP vector. All PCR was performed using Platinum Pfx DNA
polymerase (Life Technologies, Rockville, MD), and the integrity of all
constructs was confirmed by automated sequencing in the Fox Chase Can-
cer Center DNA sequence facility.
Retroviral transduction into NK-92 cell line
The retrovirus-mediated transduction was performed, as previously de-
scribed (20). The transduced NK cells were sorted for expression of either
GFP or 3DL1 using the 3DL1-specific PE-conjugated DX9 mAb (BD
PharMingen, San Diego, CA) in the Fox Chase Cancer Center Cell Sorting
Facility. These sorted cells stably expressed the receptors for at least 1 mo.
Uniform exogenous 3DL1 expression in
Ͼ98% of the transduced cell pop-
ulation was confirmed at the time of every experiment using PE-conjugated
DX9 mAb by flow cytometric analysis on a FACScan analyzer (BD Bio-
sciences, Mountain View, CA). SAP and 3DL1 were coexpressed in se-
quentially transduced NK-92 cells by sorting for EGFP and DX9-PE stain-
ing, respectively.
Redirected cytotoxicity assay
NK-92 cells were cultured with fresh IL-2-containing medium on the day
before assay, and redirected cytolytic activity was tested against the
Fc
␥RII/III
ϩ
P815 murine mastocytoma cell line in a 4-h
51
Cr release assay,
as previously described (31). HLA-B*2702-expressing 721.221 cells could
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KIR2DL5 IS AN INHIBITORY RECEPTOR
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