and SHP-2 (20). This result suggests that conjugation may be in-
hibited better by SHP-1 (or both phosphatases together) than by
SHP-2 alone.
The SAP-binding ITSM sequence has recently been described in
several human immunoreceptors, including 2B4, SLAM, CD84,
Ly-9, and NTB-A (32, 33). Although these CD2 family members
mainly exhibit activating functions, evidence indicates that en-
gagement of some of them can inhibit cell activation in the absence
of SAP expression. However, the exact consensus sequence of the
ITSM is not yet established, because another receptor, CRACC/
CSI, does not bind SAP despite the existence of two ITSM-like
sequences (44). Similarly, our experiments failed to detect SAP
binding to 3DL1/L5 by immunoprecipitation, and overexpression
of SAP did not affect the inhibitory function of 3DL1/L5 in NK-92
cells, indicating that SAP does not contribute to the unique inhib-
itory function of 2DL5. Another important incidental finding is
that we could not detect SHP-2 binding to 2B4 when it was phos-
phorylated in NK-92 cells (Fig. 5A). Although 2B4 has been
shown to bind SHP-2 in the absence of SAP in COS-7 or BaF3
cells (35, 45, 46), SHP-2 recruitment to 2B4 has never been dem-
onstrated in human NK cells, including primary human NK cells
(36, 37), YT cells (35), and NK-92 cells (Fig. 5A), or even in the
absence of SAP in NK cells derived from X-linked lymphoprolif-
erative disease patients (37).
The function of 2DL4 was originally thought to be inhibitory in
NK cells, because it has an ITIM in the cytoplasmic domain and
binds the nonclassical MHC-I molecule, HLA-G (47, 48). How-
ever, it has become apparent that 2DL4 is an activating receptor,
which strongly induces IFN-
␥ production when engaged (28–30).
In contrast, when only the cytoplasmic domain of 2DL4 was tested
in isolation, it inhibited NK cell cytotoxicity (30, 31). Because of
the high homology between the two members of the type II KIR
subfamily (79% amino acid identity) (49), 2DL5 and 2DL4, we
examined the inhibitory capacity of the full-length 2DL5 appended
with an N-terminal FLAG epitope tag in NK-92 cells. The full-
length 2DL5 suppressed cytotoxicity, demonstrating that 2DL5 is
an inhibitory receptor. Although we cannot exclude binding of
2DL5 to an activating accessory protein that is not expressed in
NK-92 cells, the absence of a charged amino acid in the 2DL5
transmembrane domain makes this possibility unlikely. Thus, ev-
idence indicates that 2DL5 is an inhibitory receptor, while 2DL4 is
an activating receptor within the type II KIR2D subgroup of hu-
man NK cells. The weak inhibitory capacity of the full-length
2DL5 compared with the chimera, 3DL1/L5, may be due to
weaker affinity of the FLAG-specific Ab compared with that of
DX9, anti-KIR3DL1 mAb. Alternatively, unique D0 –D2 struc-
tural orientations of 2DL4 and 2DL5 or mAb-binding orientation
of the N-FLAG motif may prevent optimal engagement in the
redirected cytotoxicity conjugates when compared with engage-
ment of the 3DL1 extracellular domain by DX9 mAb. In accor-
dance with our results, hemogglutinin-tagged 2DL4 with a muta-
tion in the transmembrane arginine demonstrated only weak
inhibition when engaged with anti-hemagglutinin-tag Ab (30).
Human NK cells use inhibitory lectin-like and Ig-like receptors,
including CD94/NKG2A and KIRs, to recognize MHC-I mole-
cules and block responses toward normal cells. CD94/NKG2A en-
gages with the nonclassical HLA-E, while the majority of KIR
family members are specific for subsets of HLA-A, -B, or -C al-
lotypes (50). KIR genes are differentially inherited among individ-
uals in the human population (51). A model for KIR gene expres-
sion has been proposed, in which two major expression profiles
were defined as A and B haplotypes. Individuals exhibiting the
group A haplotype have six KIR genes, including 2DL1, 2DL3,
3DL1, and 2DS4, while the group B KIR haplotypes are more
variable in their organization, including at least one additional
gene (51). Although the 2DL4 gene is present in almost all indi-
viduals and is reportedly transcribed in all NK cells, 2DL5 belongs
to the B haplotype and is clonally expressed by a subset of NK
cells in those individuals (22). It is tempting to speculate that the
2DL5 gene derives from 2DL4, because the exon-intron genomic
DNA organization of 2DL5 is similar to that of 2DL4 (49), they
share 79% amino acid homology, and they have the same config-
uration of extracellular Ig-like domains and a long cytoplasmic tail
(49). Furthermore, it is interesting that the C-terminal tyrosine in
the cytoplasmic domain of 2DL4 of pygmy chimpanzee, common
chimpanzee, and rhesus monkey is in the context of SxYxxL, sim-
ilar to the TxYxxL of 2DL5, suggesting related evolution and/or
functional importance. Our results demonstrate that the lack of the
charged transmembrane residue in 2DL5 allows it to function as an
inhibitory member of the type II KIR2D subfamily. Our data in
this report and our previous analysis of 2DL4 (31), however, dem-
onstrate that the atypical nature of or complete loss of the C-ter-
minal ITIM of type II KIR2D receptors establishes a SHP-2-se-
lective docking site. Thus, it is tempting to speculate that the
classical inhibitory KIRs, such as 3DL1, seems to have acquired
SHP-1-binding capacity to enhance or insure signaling in NK cells
that express both SHP-1 and SHP-2 when they obtained two typ-
ical ITIMs in the cytoplasmic domain. Whether KIRs use distinct
PTPs to inhibit different activation signaling pathways or whether
SHP-1 and SHP-2 have redundant roles in inhibition are still un-
known and await further analysis.
The ligand specificities of the type II KIR2D receptors remain to
be clarified. The ligand specificity of 2DL5 has not been reported
to date. Although two groups have reported that HLA-G serves as
a ligand for 2DL4 (47, 48, 52), two other groups have disputed this
ligand specificity (53, 54). Nonetheless, the type II KIR2D recep-
tors exhibit a unique Ig domain architecture (D0 and D2) that
borrows from the conserved Ig domains of the KIR3D subfamily
(possessing D0, D1, and D2), but is distinct from those of the type
I KIR2D subfamily (consisting of D1 and D2 domains). Because
D1 and D2 domains contribute the structural elements that recog-
nize the classical HLA-A, -B, and -C molecules (55–57), the
D0 –D2 orientation implies distinct ligand specificity for 2DL4 and
2DL5. Although it is unclear whether they recognize the same
ligand, our results demonstrate that 2DL5 transduces negative sig-
nals, while 2DL4 is an activating member of this type II KIR2D
subfamily.
Acknowledgments
We thank Drs. M. Colonna, C. Vilches, E. Long, B. Perussia, C. Lutz, and
G. Nolan for reagents. We also thank Drs. Glenn Rall, David Wiest, and
Akiko Kikuchi-Maki for critical reading of this manuscript and the following
research facilities at Fox Chase Cancer Center for reagents and technical sup-
port: DNA sequencing, DNA synthesis, cell culture, and cell sorting.
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