normal (data not shown), but this high SAP level in NK-92 cells
did not affect the inhibition by 3DL1/L5, when compared with
cells transduced with retrovirus containing the empty pBMN-
IRES-EGFP vector (Fig. 5B). These results indicate that SAP can-
not associate with the ITSM-like sequence in the cytoplasmic do-
main of 2DL5, and therefore, SAP does not influence 3DL1/L5-
mediated inhibition of NK-92 cells.
The full-length KIR2DL5 can inhibit NK cell activation
Despite the strong inhibitory capacity of the 2DL4 cytoplasmic
domain in isolation (30, 31), substantial evidence indicates that
2DL4 is an activating receptor due to a charged transmembrane
residue that appears to mediate association with a transmembrane
accessory protein (28 –30). Therefore, it was important to address
the function of the full-length 2DL5 protein. For these experi-
ments, the full-length 2DL5 was modified with an N-terminal
FLAG epitope tag and introduced into NK-92 cells by retroviral
transduction (Fig. 6, A and B). FLAG-2DL4 expression was al-
ways lower than FLAG-2DL5, as demonstrated in Fig. 6B. En-
gagement of the full-length 2DL5 with anti-FLAG mAb modestly,
but reproducibly suppressed redirected cytotoxicity (Fig. 6C),
while engagement of KIR2DL4 weakly potentiated cytotoxicity
(Fig. 6C), as has been previously reported (28 –30). Collectively,
our results indicate that 2DL5 is an inhibitory receptor in human
NK cells.
Discussion
It is well established that SHP-1 is recruited to phosphorylated
ITIMs on the cytoplasmic domains of classical type I KIRs. A
number of studies confirm that phosphorylation of both ITIMs is
required for SHP-1 recruitment (8, 9, 13–16, 18, 31, 43). In con-
trast, our results (20, 31) and the work of Bruhns et al. (43) show
that phosphorylation of the N-terminal ITIM of KIRs is essential
and sufficient for strong inhibitory function that correlates with
SHP-2 recruitment. It is of interest in the present study that 2DL5
showed weaker inhibitory capacity than 3DL1 (Table I), especially
in conjugation assays (Fig. 2), even though both receptors re-
cruited both SHP-1 and SHP-2 (Fig. 3). Surprisingly, we further
showed that DN-SHP-1 failed to block the inhibition by 3DL1/L5
(Fig. 4), while DN-SHP-2 did block, indicating that 2DL5 pre-
dominantly uses a SHP-2-dependent inhibitory pathway. Our re-
sults further demonstrate that two canonical ITIMs are critical for
SHP-1-dependent inhibition through KIRs. Thus, the atypical
ITSM-like sequence in 2DL5 seems to reduce the avidity of SHP-1
binding that is necessary for competent inhibitory function. Evi-
dence of an inhibitory role for SHP-2 catalytic activity in 2DL5
function is further supported by our recent demonstration that di-
rect fusion of the SHP-2 catalytic domain to the intracytoplasmic
interface of 3DL1 results in a functional inhibitory receptor (20).
Interestingly, as shown in Fig. 2C, the inhibitory KIR cytoplasmic
domains that function predominantly through SHP-2 (2DL5,
2DL4, and mutant forms of 3DL1; Figs. 3 and 4) (20, 31) exhibit
substantially reduced capacity to disrupt target cell conjugation
when compared with 3DL1, which functions through both SHP-1
FIGURE 5.
The ITSM-like sequence of KIR2DL5 does not recruit
SAP. A, 3DL1/L5-transduced NK-92 cells were treated with pervanadate
for 10 min and lysed with 1% Triton X-100, and sequential immunopre-
cipitates were prepared with anti-CD56, anti-2B4, and then anti-3DL1
mAbs. Each lane represents immunoprecipitation from a lysate of 40 mil-
lion cells. Samples were analyzed on 15% SDS-PAGE gels under reducing
conditions. Sequential immunoblot analysis was performed with anti-
phosphotyrosine, anti-SHP-2, and anti-SAP. B, Redirected cytotoxicity as-
say using 3DL1/L5-NK-92 cells secondarily transduced with either vector
alone (pBMN-IRES-EGFP (GFP); right panel) or SAP in the same vector
(middle panel). The stable double-transduced NK-92 cells were tested for
their abilities to lyse P815 in the absence of mAb (E) or in the presence of 1
g/ml anti-3DL1 mAb (DX9; F) or the anti-CD56 mAb (B159.5.2; open
cross) as a control. Results are representative of six independent experiments.
FIGURE 6.
Engagement of FLAG-tagged full-length KIR2DL5 can in-
hibit NK cell cytotoxicity. A, Schematic representations of the full-length
KIR2DL5 constructs. FLAG-tag is shown as o. D0 and D2 are highly
conserved Ig-like domains in the KIR family. TM and CY represent trans-
membrane and cytoplasmic domains, respectively. Tyrosine-based ITIM
motif (VTYAQL) and ITSM-like sequence of 2DL5 (TMYMEL) are
marked in the cytoplasmic domains. B, Flow cytometric analysis of 2DL5-
transduced cells. The NK-92 cell line was transduced with the constructs
shown as in A with retrovirus. The data show staining with anti-FLAG
(M2; filled line) and PE-labeled anti-mouse Ig
as a secondary alone
(dashed line). C, Redirected cytotoxicity assay using NK-92 cells trans-
duced with the KIR2DL5 receptor. The stable transduced NK-92 cells were
tested for their abilities to lyse P815 in the absence of mAb (E) or in the
presence of 1
g/ml anti-FLAG mAb (M2; F) or the anti-CD56 mAb
(B159.5.2; open cross) as a control. Results are representative of three
independent experiments, showing identical patterns of inhibition.
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KIR2DL5 IS AN INHIBITORY RECEPTOR
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