Vol 436|4 August 2005|doi:10.
1038/nature03847
Licensing of natural killer cells by host major
histocompatibility complex class I molecules
Sungjin Kim1, Jennifer Poursine-Laurent1, Steven M. Truscott2, Lonnie Lybarger2†, Yun-Jeong Song1,
Liping Yang1, Anthony R. French1,3, John B. Sunwoo1,4, Suzanne Lemieux5, Ted H. Hansen2
& Wayne M. Yokoyama1,2
Self versus non-self discrimination is a central theme in biology
from plants1 to vertebrates, and is particularly relevant for
lymphocytes that express receptors capable of recognizing self-
tissues and foreign invaders. Comprising the third largest lym-
phocyte population, natural killer (NK) cells recognize and kill
cellular targets and produce pro-inflammatory cytokines. These
potentially self-destructive effector functions can be controlled
by inhibitory receptors for the polymorphic major histocompati-
bility complex (MHC) class I molecules that are ubiquitously
expressed on target cells2–4. However, inhibitory receptors are
not uniformly expressed on NK cells, and are germline-encoded
by a set of polymorphic genes that segregate independently from
MHC genes5,6. Therefore, how NK-cell self-tolerance arises in vivo
is poorly understood. Here we demonstrate that NK cells acquire
functional competence through ‘licensing’ by self-MHC mol-
ecules. Licensing involves a positive role for MHC-specific inhibi-
tory receptors and requires the cytoplasmic inhibitory motif
originally identified in effector responses. This process results in
two types of self-tolerant NK cells—licensed or unlicensed—and
may provide new insights for exploiting NK cells in immuno-
therapy. This self-tolerance mechanism may be more broadly
applicable within the vertebrate immune system because related
germline-encoded inhibitory receptors are widely expressed on
other immune cells.
NK cells from MHC-deficient mice and humans are defective in
target killing7,8. These defects could be due to effects on NK cell
subpopulations, or altered expression or function of activation or
inhibitory receptors9,10. These issues are difficult to assess with
cellular targets because the repertoire of receptors on NK cells that
are involved in target recognition is incompletely defined. Here we
describe the use of a target cell-free system (see Methods) to analyse
the activation of individual resting NK cells by stimulation through
the NK1.1 molecule, also known as NK-cell receptor protein 1c
(Nkrp1c) or killer-cell lectin-like receptor subfamily B member 1c
(Klrb1c). NK1.1 is an activation receptor expressed by all mature and
developing CD32 NK cells in C57BL/6J (B6) mice11. NK1.1 cross-
linking led to the production of interferon (IFN)-g by large numbers
of NK cells from wild-type B6 mice, but not from MHC class Ia- and
Ib-deficient mice that lack b2-microglobulin (b 2m 2 /2 ), or from
mice that lack MHC class Ia alleles only (H2-K b2 /2 ;D b2 /2 )
(Fig. 1a). Even with high anti-NK1.1 antibody concentrations,
many fewer b 2m 2 /2 or K b2 /2 ;D b2 /2 NK cells produced IFN-g
(Supplementary Fig. 1). Similar effects were observed with stimula-
tion through other activation receptors and by tumour targets,
although no differences were noted when the NK cells were stimu-
lated with phorbol 12-myristate 13-acetate (PMA) and ionomycin
1
Howard Hughes Medical Institute, Rheumatology Division, Departments of Medicine, 2Pathology and Immunology, 3Pediatrics and 4Otolaryngology, Washington University
School of Medicine, St. Louis, Missouri 63110, USA. 5Institut national de la recherche scientifique, INRS-Institut Armand-Frappier, Université du Québec, Laval, Québec H7V 1B7,
Canada. †Present address: Department of Cell Biology and Anatomy, College of Medicine, University of Arizona, Tuscon, Arizona 85724, USA.
© 2005 Nature Publishing Group
LETTERS
(Supplementary Figs 2 and 3). However, the differential activation of
NK cells from MHC-sufficient and MHC-deficient hosts was less
pronounced when NK cells were pre-stimulated with interleukin-2
(IL-2) or polyinosinic-polycytidylic acid (poly-I:C) (Supplementary
Fig. 4). Finally, co-incubation of wild-type and b 2m 2 /2 splenocytes
during NK1.1 crosslinking led to IFN-g production selectively from
wild-type NK cells (data not shown), indicating a cell-autonomous
effect. Thus, the intrinsic defects of NK cells from MHC class
Ia-deficient hosts suggest that NK cell functional maturation requires
specific interaction with host MHC class Ia molecules. We term this
process MHC class I-dependent ‘licensing’ to distinguish it from
MHC class I-dependent ‘education’, which implies different events
occurring during T-cell development.
The Ly49 C-type lectin family of receptors (now also known as
killer cell lectin-like receptors subfamily A or Klra) that are expressed
on NK cells may be involved in licensing because they recognize
MHC class Ia molecules in effector responses2,12,13. However, licensed
NK cells were found in mice deficient in DAP12 (DNAX activating
protein of 12 kDa; also known as killer-cell activating receptor-
associated protein or KARAP) or other signalling-chain molecules
(DAP10, Fc1RIg or CD3z) (Supplementary Fig. 2), indicating that
Ly49 (ref. 14) and other activation receptors that are associated with
these adaptor molecules are not critical for licensing. As for Ly49
inhibitory receptors, we analysed NK cells expressing Ly49A and
Ly49C because of their well-defined MHC class I specificities and
the availability of monoclonal antibodies12. Ly49Cþ NK cells from
wild-type B6 (H2b) mice readily produced IFN-g in response to
NK1.1 stimulation, but not when they were derived from b 2m 2 /2 or
K b2 /2 ;D b2 /2 mice (Fig. 1a). This effect was not due to differences
in NK1.1 expression between Ly49Cþ and Ly49C2 subsets, or
between wild-type and MHC-deficient NK cells (data not shown).
In contrast, Ly49Aþ NK cells from wild-type B6 mice showed poor
IFN-g production. We therefore postulated that licensing was related
to self-H2 ligands for specific Ly49 receptors because Ly49A does not
have an H2b ligand, whereas Ly49C can recognize many MHC class I
ligands including H2-Kb (refs 2, 12). Indeed, stimulated Ly49Aþ NK
cells strongly produced IFN-g only when they were derived from
MHC-congenic B10 mouse strains that express the Ly49A ligand
H2-Dd (Fig. 1b). Production of tumour necrosis factor (TNF)-a
followed a similar pattern (Fig. 1b). Likewise, resting Ly49Aþ NK
cells from B10.D2 mice deficient in recombination-activating gene 2
(B10.D2 Rag2 2 /2 ) killed YAC-1 and CHO target cells better than
Ly49A2 NK cells from the same animals (Fig. 1c). In contrast,
Ly49Aþ NK cells from B10 Rag2 2 /2 mice killed with similar
efficiency to Ly49A2 NK cells from the same animals. Taken together,
these data indicate that NK-cell competence for cytokine production
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LETTERS
and cytolysis is dependent on host MHC class I molecules. This
positive effect is associated with the expression of self-MHC class
I-specific receptors on NK cells that were originally identified as
inhibitory receptors in effector responses2.
We studied NK cells derived from H2-D d transgenic K b2 /2 ;D b2 /2
mice that express only one MHC class Ia molecule. Ly49Aþ NK cells
from these mice were readily triggered by anti-NK1.1, whereas cells
from K b2 /2 ;D b2 /2 mice were not (Fig. 2a). Similar effects were
observed with NK cells derived from B6 (H2b) mice that were
transgenic for H2-D d (data not shown). The effect of transgenic
H2-Dd molecules in K b2 /2 ;D b2 /2 mice was also manifested in the
Ly49A2 population (Fig. 2a), and was perhaps due to the presence of
other Ly49 receptors with H2-Dd specificity12, recognition by the
NKG2/CD94 heterodimer of the MHC class I molecule Qa-1 (which
Figure 1 | Host MHC class Ia molecules alter IFN-g production and cytotoxic
activity of NK subsets. a, Splenocytes from the indicated mouse strains
were incubated with immobilized anti-NK1.1 antibody and analysed for
intracellular IFN-g. Gated CD32 TCR-b2 DX5þ cells are shown, and the
numbers represent the percentages of IFN-gþ cells among the Ly49Aþ or
Ly49Cþ cell populations. b, Splenocytes from the indicated mouse strains
were incubated with immobilized anti-NK1.1 antibody and analysed for
intracellular IFN-g and TNF-a. The H2 MHC haplotypes are indicated
under each strain name. Gated CD32 TCR-b2 DX5þ cells are shown, and
the numbers represent the percentages of cytokine-producing cells among
the Ly49Aþ cell population. c, Ly49Aþ NK1.1þ and Ly49A2 NK1.1þ
cells were sorted from the spleen cells of Rag2-deficient B10.D2 and
Rag2-deficient B10 mice, and used in standard 4-h killing assays against the
target cells indicated. Effector:target (E:T) ratio is indicated on the x axis.
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© 2005 Nature Publishing Group
NATURE|Vol 436|4 August 2005
can present the H2-Dd leader peptide to NK cells)15, or an unknown
receptor on NK cells for MHC class I molecules. Nevertheless, the
data indicate that NK cells expressing a cognate inhibitory receptor
for a self-MHC class I molecule possess enhanced functional
activities.
To more precisely investigate the functional relationship between
Ly49 inhibitory receptors and host MHC class I molecules, we used a
single-chain trimer (SCT) MHC class I molecule consisting of
peptide, b2m and H2-Kb heavy chain connected by Gly-Ser linkers16.
SCT–Kb tetramers bound a subset of b 2m 2 /2 NK cells that co-
stained with the monoclonal antibody 4LO3311 (Fig. 2b), which is
monospecific for Ly49C (ref. 17). This result was similar to the
staining of NK cells from MHC class I-disparate mice with conven-
tional MHC class I tetramers18. In contrast, staining with other Ly49
monoclonal antibodies did not correlate with tetramer binding.
Furthermore, SCT–Kb tetramer binding was specifically blocked by
pre-incubation with anti-Ly49C (Fig. 2c). Similar data were obtained
with conventional (wild-type) Kb tetramers (data not shown) that
bind Ly49C transfectants12.
To generate an SCT–Kb transgenic mouse that does not express any
other b2m-associated MHC class Ia or Ib molecules, we produced
transgenic mice expressing SCT–Kb ubiquitously (data not
shown), and backcrossed the founder mouse to triple-knockout
b 2m 2 /2 ;K b2 /2 ;D b2 /2 animals. The cis interactions between an
NK-cell receptor and its cognate MHC ligand were recently reported
to block MHC class I tetramer binding, providing evidence for in vivo
self-MHC ligand–receptor engagement18. In SCT–Kb-transgenic
and wild-type B6 mice, binding of both SCT–Kb and conventional
(wild-type) H2-Kb tetramers was similarly absent, indicating
that this biological property was intact with the transgenic
SCT–Kb molecules (Fig. 2d). Finally, anti-Ly49C reactivity was
lower in SCT–Kb-transgenic b 2m 2 /2 ;K b2 /2 ;D b2 /2 mice com-
pared with non-transgenic b 2m 2 /2 ;K b2 /2 ;D b2 /2 mice (Fig. 2e),
and is consistent with decreased antibody reactivity to NK-cell
receptors in the presence of self-MHC class I ligands9,19. Thus, the
SCT–Kb molecules react specifically with Ly49C on primary NK cells
and exhibit biological properties similar to wild-type H2-Kb
molecules.
Ly49Cþ NK cells from the SCT–Kb-transgenic b 2m 2 /2 ; K b2 /2 ;
b2 /2
D mice gained the ability to produce IFN-g, compared with
Ly49Cþ NK cells from non-transgenic triple-knockout mice (Fig. 2e),
whereas Ly49C2 NK cells, even with higher anti-NK1.1 stimulation,
were comparable between transgenic and non-transgenic triple-
knockout mice. Moreover, there was no specific enhancement of
Ly49Aþ NK-cell activity in SCT–Kb-transgenic triple-knockout mice
(data not shown). Thus, the expression of a single MHC class I
molecule selectively licenses functional activity of NK cells with a
cognate inhibitory receptor.
To further study the action of NK-cell receptors in licensing, we
transduced haematopoietic stem cells with bicistronic retroviral
vectors containing green fluorescent protein (GFP) to distinguish
expression of transduced Ly49A from endogenous Ly49A. As
expected, there was correlated expression of Ly49A and GFP (Sup-
plementary Fig. 5). In B10.D2 mice, GFPþ NK cells were more potent
at producing IFN-g compared with GFP2 NK cells (Fig. 3a). The
relative frequencies of IFN-g-producing cells were about twofold
higher in GFPþ cells compared with GFP2 cells (Fig. 3c). In contrast,
this effect was not seen in B10 hosts (Fig. 3a). Thus, transduced
Ly49A expression conferred a positive effect on NK cell licensing that
exceeded the existing NK-cell function, but only in hosts with a
cognate MHC class I ligand for Ly49A.
Licensing by Ly49A could be due to a signal from Ly49A itself.
Alternatively, Ly49A could activate stromal cells through MHC
crosslinking, or facilitate cell–cell contact between developing NK
cells and stromal elements. To address these possibilities, we
expressed a Ly49A molecule that lacked the cytoplasmic domain
(designated Ly49AcytoD) in B10.D2 mice. Although this molecule was
NATURE|Vol 436|4 August 2005 LETTERS

Figure 2 | Licensing of NK cells expressing

inhibitory Ly49 receptors specific for self-MHC
class I molecules. a, Splenocytes from the
indicated mouse strains were incubated with
immobilized anti-NK1.1 antibody and analysed
for intracellular IFN-g. Gated CD32 TCR-b2
DX5þ cells are shown, and the numbers represent
the percentages of IFN-gþ cells among the
Ly49Aþ cell population. b, b 2m 2 /2 splenocytes
were stained with the indicated monoclonal
antibodies and SCT–Kb tetramer together.
Gated CD32 CD192 NK1.1þ cells are shown.
c, b 2m 2 /2 splenocytes were first incubated with
the indicated monoclonal antibodies and
subsequently stained with SCT–Kb tetramer.
Gated CD32 CD192 NK1.1þ cells are shown.
d, Splenocytes from the indicated mouse strains
were stained with SCT–Kb or WT–Kb tetramer.
Gated CD32 CD192 NK1.1þ cells are shown.
e, Splenocytes from the indicated mouse strains
were incubated with immobilized anti-NK1.1
antibody (1:30,000 and 1:10,000 dilution) and
analysed for intracellular IFN-g. Gated CD32
TCR-b2 DX5þ cells are shown, and the numbers
represent the percentages of IFN-gþ cells among
the Ly49Cþ or Ly49C2 cell populations. Tg,
transgenic.

a Ly49A–GFP Ly49Acyto∆–GFP expressed at comparable levels (Supplementary Fig. 5), we did not
B10.D2 B10 B10.D2
observe enhanced NK-cell activity (Fig. 3a, c). The only known
signalling motif in the Ly49A cytoplasmic domain is the immuno-
receptor tyrosine-based inhibitory motif, or ITIM (ref. 20). A mutant
Ly49A (designated Ly49AYtoF) with a Tyr-to-Phe change in the ITIM,
15.8% 2.7% 7.1% known to abrogate inhibitory signalling in effector responses20, was
GFP

expressed by retroviral transduction at levels comparable to intact

8.8% 3.0% 7.9%
Ly49A (Supplementary Fig. 5). The ITIM mutant failed to confer
licensing in B10.D2 mice (Fig. 3b, c). Taken together, these results
show that the ITIM in the cytoplasmic domain of the inhibitory
IFN-γ receptor is critical for the licensing effect, and that the receptor itself
is responsible for the signals that direct NK-cell licensing.
b Ly49A–GFP Ly49AYtoF–GFP c
The ITIM of Ly49A recruits the tyrosine phosphatase SHP1 (src
Relative IFN-γ productivity
of GFP+ to GFP– NK cells

3 homology region 2 domain-containing phosphatase 1; also known as

26.9% 16.4% protein tyrosine phosphatase non-receptor type 6 or PTPN6), which
2
is required for inhibitory signalling in effector responses20. To
GFP

1 examine its role in licensing, we analysed NK-cell function in

16.3% 17.8% SHP1-defective motheaten-viable (me-v) mice (Ptpn6 me-v). NK1.1
0
crosslinking led to IFN-g production from me-v-NK cells at levels
FP

FP

FP
G

that were intermediate between b 2m-deficient and wild-type NK

–G

–G
A–

IFN-γ
∆

oF
to
49

Yt
A cy
Ly

cells (Fig. 4a). To address whether the results were influenced by the
9A
49

4
Ly
Ly

pleiotropic abnormalities found in me-v mice, we studied mixed-

Figure 3 | Gene transfer of intact Ly49A, but not cytoplasmic domain-
deleted (Ly49AcytoD) or ITIM-mutated (Ly49AYtoF) Ly49A, licenses NK
cells in the presence of its ligand. a, Mice were reconstituted with syngeneic
BM cells after retroviral transduction of the indicated constructs. Spleen
cells from the indicated mouse strains were incubated with immobilized
anti-NK1.1 antibody and analysed for intracellular IFN-g. Gated CD32
TCR-b2 DX5þ cells are shown, and the numbers represent the percentages
of IFN-gþ cells among the GFPþ or GFP2 cell populations. b, Spleen cells
from B10.D2 mice reconstituted with syngeneic BM cells after retroviral
transduction of the indicated constructs were incubated with immobilized
anti-NK1.1 antibody and analysed for intracellular IFN-g. Gated CD32
TCR-b2 CD192 NK1.1þ cells are shown, and the numbers represent the
percentages of IFN-gþ cells among GFPþ or GFP2 cells. c, Relative IFN-g
productivity is depicted as the ratio of the percentage of IFN-gþ cells among
the GFPþ NK cells to the percentage of IFN-gþ cells among the GFP2 NK
cells. Each symbol represents an individual mouse and the bars represent the
mean ratios of the indicated groups; wild-type Ly49A (mean ^ s.d.,
1.73 ^ 0.42, n ¼ 20), Ly49AcytoD (1.09 ^ 0.17, n ¼ 11) and Ly49AYtoF
(0.96 ^ 0.19, n ¼ 17). The differences between wild-type Ly49A–GFP and
either Ly49AcytoD–GFP or Ly49AYtoF–GFP were statistically significant
(P , 0.0001).
© 2005 Nature Publishing Group
bone-marrow (BM) chimaeras, which use different alleles of Ly5
(also known as protein tyrosine phosphatase receptor type C or
PTPRC) to distinguish BM origin. BM derived from me-v mice
(carrying the Ly5.2 allele) and wild-type mice (carrying the Ly5.2 or
the Ly5.1 allele) was transplanted into irradiated wild-type recipient
mice (carrying Ly5.1). In the mixed-chimaeric mice, me-v-derived
and wild-type-derived NK cells displayed similar levels and patterns
of IFN-g production (Fig. 4b), indicating that the originally observed
reduced IFN-g production by me-v NK cells (Fig. 4a) was due to
indirect effects. ITIMs can potentially recruit other molecules, but
preliminary data do not implicate known interacting molecules in
licensing (Supplementary Note). Nevertheless, these data suggest
that although licensing is mediated through an ITIM-dependent
mechanism, it is distinct from ITIM-mediated, SHP1-dependent
inhibition during effector responses.
Here we show that NK cells, like other lymphocytes, undergo a
process that results in self-tolerance. Consistent with a positive effect
during development, BM-derived NK cells show evidence of
enhanced proliferation if they express an inhibitory receptor for a
711
LETTERS
Figure 4 | Licensing is preserved in SHP1-deficient NK cells. a, Splenocytes
from the indicated mouse strains were incubated with immobilized anti-
NK1.1 antibody and analysed for intracellular IFN-g. Gated CD32 TCR-b2
CD192 NK1.1þ cells are shown, and the numbers represent the percentages
of IFN-gþ cells among the Ly49Cþ or Ly49C2 cells. b, Splenocytes from
mixed BM chimaeric mice or from b 2m 2 /2 control mice were incubated
with immobilized anti-NK1.1 antibody and analysed for intracellular IFN-g.
Mixed BM chimaeric mice were produced by injection of Ly5.2þ B6 plus
Ly5.1þ B6 or Ly5.2þ me-v plus Ly5.1þ B6 BM into irradiated Ly5.1þ B6 mice
and analysed after 9 weeks. Donor-derived wild-type or me-v NK cells were
distinguished by the presence of the different Ly5 alleles. Gated Ly5.12
CD32 TCR-b2 CD192 NK1.1þ (upper panel) and Ly5.22 CD32 TCR-b2
CD192 NK1.1þ (lower panel) cells are shown, and the numbers represent
the percentages of cytokine-producing cells among the Ly49Cþ or Ly49C2
cell populations. Data shown are representative of five mice per group
analysed in two separate experiments.
self-MHC class I molecule (Supplementary Fig. 6). In some respects,
licensing resembles the process of positive selection that occurs in the
thymus for T cells with appropriate self-reactive T-cell antigen
receptors21. However, NK cells use a distinctly different process that
is ITIM-dependent, and there is no evidence, as yet, for negative
Figure 5 | Model for NK cell licensing in the H2-Kb1 Dd– mouse. For clarity,
this diagram depicts the situation for developing NK cells bearing either
Ly49C alone (top) or Ly49A alone (bottom). Licensing results in two types of
self-tolerant NK cells. The Ly49Cþ NK cell becomes licensed and is inhibited
by H2-Kb (self), whereas cells without self-specific receptors are not licensed
even though they can express receptors for other MHC alleles; that is, the
Ly49Aþ NK cell is not licensed.
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NATURE|Vol 436|4 August 2005
selection or clonal deletion of NK cells. In contrast to T cells, which
use rearranged receptors that activate effector functions in adaptive
immunity, NK cells use non-rearranged receptors that are, para-
doxically, involved in effector inhibition to find self antigens for
functional maturation in innate immunity (Fig. 5). Therefore,
licensing results in two types of self-tolerant NK cells—licensed NK
cells with self-MHC class I inhibitory receptors, and unlicensed NK
cells.
Licensing pairs an inhibitory receptor with its cognate self-
MHC class I ligand for functional development, thus providing a
mechanism for how independently segregating genes for non-
rearranged receptors and ligands give rise to tolerance of self
antigens. NK cells that do not express self-MHC class I specific
receptors do not become licensed, and have no need to be
inhibited by MHC class I because they are not functionally
competent. Therefore, MHC class I-dependent effector inhibition
is relevant only for competent NK cells; they are inhibited by the same
self-MHC-specific receptors that originally conferred licensing.
Finally, germline-encoded inhibitory receptors with ITIMs, related
to the NK cell receptors, are broadly expressed on many leukocytes22,
suggesting that they might also be involved in self-tolerance
mechanisms.
It should be noted that each individual NK cell is potentially
licensed separately; in hosts with heterozygous MHC alleles, there
should be individual NK cells that are licensed and thus inhibited by
different MHC molecules. In this respect, licensing should be
relevant to understanding the function of NK cells in other contexts,
such as BM transplantation, and the vexing problem of ‘hybrid
resistance’ in which host NK cells in F1-hybrid mice reject BM from
inbred parental strains23. Only normal tissues expressing the full
complement of self-MHC class I molecules should be resistant to all
licensed NK cells in the F1 hybrid, whereas tissues bearing only one
haplotype would be resistant to some, but not all, NK cells. On the
other hand, tissues expressing non-self MHC molecules that are
promiscuously recognized by the inhibitory receptors on licensed
NK cells should be protected—an important issue in interpreting
results from BM transplant studies in mice and humans24. In related
studies, donor-derived NK cells have an anti-tumour effect in BM
transplantation therapy for leukaemia25. Although recipient HLA
class I alleles are clearly important, the absence of the appropriate
recipient HLA ligands for the donor killer cell immunoglobulin-like
inhibitory receptors (KIRs; functionally similar to murine Ly49
receptors) does not always impart an effective outcome26. Licensing
indicates that donor HLA class I alleles will influence the functional
capacity of transplanted NK cells.
Notably, the licensing effect was less prominent in pre-activated
NK cells, suggesting that licensing requirements could be bypassed
under certain circumstances. Perhaps unlicensed NK cells become
activated in response to pro-inflammatory cytokines, which are
known to activate most, if not all, NK cells during infections27.
Such effects may aid anti-pathogen defences by potentially breaking
NK-cell tolerance and allowing the participation of larger numbers of
NK cells at local sites of inflammation, suggesting a potentially
important function of unlicensed NK cells.
METHODS
Mice. C57BL/6J mice (wild-type B6 mice), B6 mice carrying the Ly5.1 antigen,
and C57BL/10J (B10) congenic mice (including B10.D2, B10.A, B10.A(2R) and
B10.D2(R107)) were purchased from The Jackson Laboratory. In addition,
b 2m2 /2 , Fc1RIg2 /2 , CD3z2 /2 and motheaten-viable Ptpn6 me-v mice, bred
onto the B6 background, were also purchased from The Jackson Laboratory. B10
Rag22 /2 and B10.D2 Rag22 /2 mice were purchased from Taconic Farms.
D d-transgenic B6 mice were provided by D. Margulies, and crossed to the
K b2 /2 ;D b2 /2 double-knockout mice provided by F. Lemonier. DAP12 2 /2
and DAP10 2 /2 mice were provided by T. Takai and M. Colonna, respectively,
and were completely backcrossed to the B6 background using a marker-assisted
breeding strategy. All mice were used in accordance with institutional guide-
lines for animal experimentation.
NATURE|Vol 436|4 August 2005
SCT transgenic mice. A transgene construct for the expression of the oval-
bumin-peptide (OVA)–Kb SCT (see ref. 28 for a detailed description of the SCT)
was generated from an H2-Kb genomic clone. The first and second exons of the
H2-Kb gene were replaced with a hybrid exon encoding the leader sequence of
murine b2m, followed by the OVA-derived SIINFEKL peptide, a 15-mer Gly–Ser
linker, the mature coding sequence of murine b 2m, a 20-mer Gly–Ser
linker and the a1 domain of H2-Kb. SCT–Kb is recognized by antigen-specific,
H2-Kb-restricted cytotoxic T lymphocytes, indicating that its three-dimensional
topology is preserved16. Expression of the OVA–Kb transgene was driven by a
2-kilobase fragment encompassing the H2-Ld promoter, and microinjection of
the purified expression cassette into B6 embryos was performed by
the Mouse Genetics Core at Washington University. Transgene-positive
founders were crossed to B6 mice with targeted disruptions of the b 2m and
K b;D b genes29.
Cytokine assays. Spleen cells were collected from mice that did not receive any
prior treatment,unless otherwise indicated. For the stimulation of NK cells,
spleen cells (107 cells ml2 1) were incubated with an equal volume of tumour
targets (106 cells ml2 1) for 30–60 min, and then further incubated in the
presence of brefeldin A for an additional 5–8 h. Alternatively, spleen cells
(107 cells ml2 1) were incubated in 6-well plates coated with anti-NK1.1 mono-
clonal antibody ascites at a 1:30,000 dilution (chosen for most experiments
because its effects were comparable to tumour stimulation; see Supplementary
Fig. 2), unless otherwise indicated. Similar observations were made with
purified monoclonal antibody (data not shown). Intracellular IFN-g was
detected with either phycoerythrin (PE)-conjugated or fluorescein isothio-
cyanate (FITC)-conjugated anti-IFN-g monoclonal antibody (XMG1.2) as
described previously11. Alternatively, intracellular TNF-a was detected with
PE-conjugated anti-TNF-a antibody on cells stimulated with anti-NK1.1 at
1:10,000 dilution. To gate on NK cells, cells were stained for either the universal
NK cell marker DX5 or NK1.1, which yielded essentially identical results. To
exclude T cells from analysis, both CD3þ and T-cell antigen receptor (TCR)-bþ
cells were gated out in all experiments, and CD19þ cells were gated out in some
experiments.
Cytotoxicity assay. Ly49Aþ and Ly49A2 NK cells were sorted from the spleen
cells of unmanipulated Rag2-deficient B10 and Rag2-deficient B10.D2 mice to
prevent inadvertent contamination with NKT cells. Sorted cells (with a com-
parable purity of .90%) were tested directly in standard 51Cr-release assays
using 96-well V-bottom plates.
Retroviral gene transduction. Retroviral vectors pMX–Ly49A–IRES–EGFP,
pMX–Ly49AcytoD–IRES–EGFP and pMX–Ly49AYtoF–IRES–EGFP were con-
structed by inserting the Ly49A complementary DNA, its cytoplasmic tail-
deleted mutant cDNA, or the Ly49AYtoF mutant cDNA into the pMX–IRES–GFP
vector (a gift from T. Kitamura), respectively. The mutants were generated by
polymerase chain reaction mutagenesis and verified by sequence analysis. The
cytoplasmic deletion mutant had an amino-terminal deletion (amino acids 2 to
32), whereas the Ly49AYtoF mutant had a codon mutation (TAT to TTC at codon
8). The vectors were transfected into the Plat-E packaging cell line with the
FuGENE 6 transfection system (Roche) to produce recombinant virus. The
viral supernatants were used to infect BM haematopoietic precursors
as described previously30. Infected BM cells were injected into the tail vein
of 9.5-Gy-irradiated B10 or B10.D2 mice. The spleen cells were analysed at
least 6 weeks after transplantation.
Mixed bone marrow chimaeras. BM cells were prepared from femora and tibiae
of wild-type B6 (Ly5.2), me-v (Ly5.2) and wild-type B6 (Ly5.1) mice. A total of
2 £ 106 mixed BM cells (Ly5.2þ B6 plus Ly5.1þ B6; or Ly5.2þ me-v plus
Ly5.1þ B6), mixed at a 4:1 (Ly5.2:Ly5.1) ratio, were injected via the tail vein of
9.5-Gy-irradiated wild-type B6 (Ly5.1) mice. The spleen cells were analysed at
least 9 weeks after transplantation.
Received 27 March; accepted 19 May 2005.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements Work in the Yokoyama laboratory is supported by the
Howard Hughes Medical Institute, the Barnes-Jewish Hospital Foundation and
grants from the National Institutes of Health. Transgenic production and
genotyping were supported by the Rheumatic Diseases Core Center grant. This
study was also supported by an NIH grant to the Hansen laboratory. The
authors thank M. Miley and D. Fremont for initial production of the SCT–Kb
tetramers, E. Holroyd, J. Mohan, D. Higuchi and R. Rodrigues for technical
assistance, and P. Allen, M. Colonna, J. Loh and E. Unanue for critical comments
on the manuscript.
Author Information Reprints and permissions information is available at
npg.nature.com/reprintsandpermissions. The authors declare no competing
financial interests. Correspondence and requests for materials should be
addressed to W.M.Y. (yokoyama@wustl.edu).
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