* Laboratory of Experimental Immunology, Division of Basic Sciences, and
Intramural Research Support Program, SAIC Frederick, National Cancer Institute-FCRDC, Frederick, Maryland
Correspondence: Stephen K. Anderson, SAIC-Frederick, NCI-FCRDC, Bldg. 560, Rm. 31-93, Frederick, MD 21702-1201. E-mail: andersn{at}mail.ncifcrf.gov
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Key Words: NK cells • Ly49 • cell surface receptors • signal transduction
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Activating Ly49s lack an ITIM and instead contain an arginine residue in their transmembrane region that associates with the signal-transducing protein DAP12 [12 , 13 ]. DAP12 contains an immunoreceptor tyrosine-based activation motif (ITAM) and transmits activating signals resulting in phosphorylation of various substrates [14 15 16 17 ]. Furthermore, the activating receptors induce intracellular calcium release and cytokine production in response to antibody-mediated cross-linking or ligation by specific MHC class I ligands. When expressed on NK cells, activating Ly49s enhance the killing of target cells bearing the appropriate MHC class I molecule [11 , 18 ]. Presently, the known activating Ly49 molecules include Ly49D, H, and P. Three additional ITIM-lacking, potential activating Ly49 candidates (k, l, and n) are known from C57BL/6 (B6) genomic sequence fragments [3 ].
Ly49d has been cloned from B6 mice, and the Ly49D protein delivers stimulatory signals leading to the lysis of target cells bearing H2-Dd, Dr, or Dsp2 MHC class I [1 , 11 , 18 ]. In agreement with the noted MHC specificity, depletion of Ly49D+ NK cells abrogates the ability of H-2b mice to reject H-2d bone marrow cells [19 ]. Ly49D has been shown also to mediate xenogeneic lysis of Chinese hamster ovary cells and rat lymphoblasts in an MHC-dependent fashion [20 , 21 ]. Ly49h cDNAs have been cloned from B6 and B6/CBA F1 hybrids, and Ly49H signals via DAP12, leading to the phosphorylation of various substrates such as DAP12 and Syk [2 , 15 , 16 ]. As shown for Ly49D, stimulation through Ly49H leads to intracellular calcium mobilization and cytokine secretion [16 ]. Ly49P was identified in 129/J mice, and it shares with the other activating Ly49s the ability to associate with DAP12 as well as the induction of DAP12 phosphorylation and intracellular calcium flux in response to antibody-mediated cross-linking [4 ].
A previous study has shown that the currently available Ly49-specific monoclonal antibodies (mAbs) display strain-specific differences by flow cytometric analysis [22 ]. In a screen of 12 strains of inbred mice with the Ly49D-specific mAb 4E5, only half of the strains tested showed a positively staining NK population. Immunoprecipitation experiments designed to identify activating and inhibitory Ly49 molecules have shown that NK cells from non-B6 mouse strains express cross-reactive yet functionally opposed Ly49 molecules. The mAb YE1/48 immunoprecipitates an inhibitory Ly49 in B6 mice (Ly49A), whereas an activating Ly49 is detected by this antibody in 129/J mice (Ly49P). The nature of a novel Ly49G-related activating receptor (Ly49L) detected by mAb 4D11 in CBA/J NK cells is the subject of the current study.
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Mouse NK cells and tumor cell lines
NK cells from 129/J, B6, C3H/He, and CBA/J mice were enriched
from spleens of 12- to 16-week-old animals by collecting nylon wool
nonadherent cells and depleting T cells using mAbs to CD8 and CD4 plus
complement. Routinely, 75–80% of the resulting cells were
DX5+. Mouse NK cells were cultured for 7–10 days in RPMI
1640 medium supplemented with 1000 cetus units/ml interleukin (IL)-2
(Hoffman LaRoche, Nutley, NJ), 10% fetal calf serum (FCS),
nonessential amino acids, sodium pyruvate, HEPES, ß-mercaptoethanol,
2 mM L-glutamine, and 100 units/ml penicillin plus 100
µg/ml streptomycin. The human fetal liver cell line CP
[16
], which is DAP12+, CD56+,
CD16-, and CD3-, was maintained in RPMI 1640
medium containing 10% fetal bovine serum (FBS), glutamine, and
antibiotics.
Polymerase chain reaction (PCR) cloning of CBA/J Ly49 cDNA
CBA/J NK cell poly-A mRNA was isolated using the QuickPrep
Micro mRNA purification kit (Amersham Pharmacia Biotech, Piscataway,
NJ). A total of 1 µg mRNA was used to generate cDNA with the
TimeSaver cDNA synthesis kit (Amersham Pharmacia). Degenerate Ly49
primers were used to amplify 50 ng cDNA. The 5' primer used was:
5'-YACTCCCAMGATGAGTGAGC-3', and the 3' primer used was:
5'-RTTGGARARTYAATSAGGR-3'. Thirty cycles of amplification were
performed with High Fidelity PCR Supermix (Gibco BRL, Grand Island, NY)
at temperatures of 94°C (denaturation) for 30 s, 57°C
(annealing) for 30 s, and 73°C (extension) for 1 min. PCR
products were cloned into pcDNA3.1/V5/His-TOPO expression vector using
the Eukaryotic TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA). Clones
were sequenced with vector-specific primers using the Sequenase
quick-denature kit (Amersham, Cleveland, OH) or by automated DNA
sequencing using the Dye Terminator Cycle Sequencing Ready Reaction Kit
(ABI, Columbia, MD). Identical Ly49l clones were isolated
from two separate PCR reactions and sequenced. The Ly49l1-4
sequences are available from GenBank under the following accession
numbers: (Ly49l1) AF204265, (Ly49l2) AF204266,
(Ly49l3) AF204267, and (Ly49l4) AF204268.
Phylogenetic analysis
Sequence similarities were ascertained using the GrowTree
software available on the Web-based University of Wisconsin (Madison,
WI) SeqWeb sequence analysis service. Specifically, tree construction
was performed using the unweighted pairgroup method with arithmetic
mean method, according to the Jukes-Cantor algorithm. The bootstrap
values in 1000 replications were estimated using the Phylogeny
Inference Package (PHYLIP) version 3.5c. The extracellular (beginning
of exon 4 to the stop codon in exon 7) nucleotide-coding regions of the
following sequences were used in tree construction:
Ly49cB6 (U56404),
Ly49cBALB/c (U49868),
Ly49iB6 (U49865; referred to as Ly49c
in the description), Ly49jB6 (AF110492),
Ly49hB6 (L78253), Ly49eB6
(U10091), Ly49fB6 (U10092),
Ly49aB6 (M25812),
Ly49aBALB/c (U34890),
Ly49p129/J (AF146570),
Ly49o129/J (AF146571),
Ly49dB6 (L78247), Ly49gB6
(U10094), and Ly49bB6 (U10304).
Ly49-specific reverse transcription (RT)-PCR analysis
Synthesis of cDNA from NK cells of 129/J, B6, and C3H/He
mice was done essentially as described above for CBA/J mice.
Ly49l-specific PCR analysis was conducted with forward
primer 5'-GGTGAGGCTTGAGGAGACAG-3' and (B6-specific) reverse primer
5'-CTTCCCACAAATACAGATGAATGATTTATCACATTTATC-3' plus (CBA/J-specific)
reverse primer 5'-CTTCCCACAAATACAGATGAATGATTTATCACAGTTATC-3'
corresponding to peptides (forward) 22-28 and (reverse) 264-274 of the
Ly49L1 aa sequence (see Fig. 2A
). PCR was performed with 35 cycles of
94°C for 30 s, 65°C for 15 s, and 72°C for 30 s.
Ly49g-specific primers used were forward primer
5'-GTTGCAGAAACTAGTGAGGAC-3' and reverse primer
5'-GATGTATGATTTACCACAGTCC-3', corresponding to nucleotides (forward)
18-25 and (reverse) 261-268 of the Ly49G1 aa sequence. PCR was
performed with 35 cycles of 94°C for 30 s, 55°C for 15 s,
and 72°C for 30 s. PCR controls to show the specificity of the
Ly49l and g primers were performed with 1 ng of
cloned Ly49 cDNA.
 View larger version (44K): [in a new window] |
Figure 2. (A) Proposed amino acid sequence of Ly49L1-4. The amino acid sequences
of Ly49L1-4 is compared with Ly49G1 and D1. Dashes indicate regions of
identity with Ly49L1. Asterisks represent sequence gaps. The
transmembrane region is underlined. L1 has both sets of boxed amino
acids, L2 is missing Box-2, L3 is missing Box-1, and L4 is missing
Box-1 and -2. cDNAs were isolated from at least two separate cloning
experiments to eliminate PCR error. These sequences can be found under
GenBank accession numbers (Ly49l1) AF204265,
(Ly49l2) AF204266, (Ly49l3) AF204267, and
(Ly49l4) AF204268. (B) Phylogenetic tree of the nucleotide
sequence coding for the extracellular region (exon 4 to termination
codon in exon 7) of the various known Ly49 molecules. The
Ly49a and c alleles of CBA/J and BALB/c mice are
identical. The isoforms of Ly49g and l without
the large insert in the stalk region (Ly49g2 and
Ly49l2) were used to decrease bias in pairing.
Ly49b was included as an outgroup to artificially root the
tree. Numbers at branch nodes represent bootstrap confidence-level
percentages of 1000 bootstrap replications. Unlabeled branch nodes had
a bootstrap value of 100%. Tree structure was produced using the UPGMA
neighbor-joining method of the Web-based University of Wisconsin SeqWeb
sequence analysis service.
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Detection of phosphorylated proteins and DAP12
Transiently transfected CP cells or cultured mouse NK cells were
stimulated with 0.1 mM pervanadate as previously described
[25
] for 15 min at 37°C or with specific antibody
followed by an isotype-specific cross-linker for 2 min at 37°C. Cells
were disrupted in lysis buffer (1.0% Triton X-100, 300 mM NaCl, 50 mM
Tris, 2 mM EDTA, 0.4 mM sodium orthovanadate, plus protease inhibitors)
and then centrifuged at 15,000 rpm for 30 min at 4°C. Lysates were
immunoprecipitated overnight with specific mAb cross-linked to rProtein
G Agarose (Gibco BRL). Beads were washed in wash buffer containing
0.2% Triton X-100, and proteins eluted in nonreducing Laemmli buffer
and separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) on precast 10% or 16% Tris-glycine gels
(Novex, San Diego, CA). Proteins were transferred to Immobilon-P
(Millipore, Bedford, MA), and the blots were blocked with 5% bovine
serum albumin (BSA), 0.1% Tween/20 in phosphate-buffered saline (PBS).
Biotinylated 4G10 was used to detect phosphoproteins followed by
streptavidin-horseradish peroxidase (HRP; UBI). Alternatively, DX37
(mouse antihuman DAP12) was used to detect DAP12 followed by sheep
antimouse IgG-HRP (Amersham, Piscataway, NJ). Blots were treated with a
chemiluminescent reagent (Amersham, Arlington Heights, IL) and
then exposed to BioMax ML film (Kodak, Rochester, NY).
Flow cytometric analysis
Cells were stained using Ly49-specific primary mAb followed by
an fluorescein isothiocyanate (FITC)-conjugated goat antirat IgG (H+L)
secondary (FGART; Kirkegaard & Perry Lab., Gaithersburg, MD) and
analyzed on a FACSort flow cytometer (Becton Dickinson, Mountain View,
CA).
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100 kDa, but activating Ly49 molecules lack an ITIM,
and instead the DAP12 homodimer is detected at 24 kDa
[4
]. Ly49D (4E5)- and Ly49H (1F8)-reactive mAb showed
the expected receptor-associated DAP12 in B6 mice, but neither detected
any phosphoproteins in CBA/J mice (Fig. 1
). This indicates that Ly49D and Ly49H are not present in the NK
cells of this strain or that these mAbs are strain-specific.
Surprisingly, DAP12-association in CBA/J NK cells was seen in
immunoprecipitations with 4D11 and to a lesser extent, YE1/48. Both of
these antibodies only recognize inhibitory Ly49s (G and A,
respectively) in B6 NK cells. An activating Ly49 has been detected
previously with YE1/48 in 129/J mice (Ly49P) [4
], but a
4D11 cross-reacting activating Ly49 has not yet been described.
 View larger version (98K): [in a new window] |
Figure 1. Detection of mDAP12 coimmunoprecipitated with Ly49-specific mAbs in B6
vs. CBA/J NK cells. NK cells from C57BL/6 (left) and CBA/J (right) mice
expanded with IL-2 for 7–10 days were washed and stimulated with
pervanadate. Cells were lysed and immunoprecipitated with the indicated
anti-Ly49 antibodies. YE1/48 binds to Ly49A, and 4E5 binds to Ly49D in
C57BL/6 mice. The immune complexes were run on a nonreducing 10% gel,
transferred, and blotted with 4G10 (antiphosphotyrosine antibody). A
representative blot of two experiments is shown.
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The remaining nine cDNA clones were of a novel activating Ly49 that most closely matched the partially characterized Ly49l gene from B6 mice. The B6-Ly49l gene is known from 77 bp and 113 bp fragments of the putative exons 2 and 7, respectively [3 ]. The CBA/J clone was identical to the known Ly49l sequence except for a single, nonconservative nucleotide substitution in exon 7. Therefore, we believe we have isolated the CBA/J allele of Ly49l. Ly49L lacks the tyrosine residue in the ITIM motif but has an arginine residue in the transmembrane domain that could potentially bind to DAP12 (Fig. 2A ). Ly49L is extremely similar to Ly49G in the extracellular region. Four different isoforms of Ly49L were discovered. Shown in Figure 2A is the full length Ly49L1 protein sequence which contains a large insertion in the stalk-coding domain (Box-2) that so far has been described only for Ly49G1 [1 ]. Ly49L2 lacks this large insertion, Ly49L3 lacks three amino acids in the intracellular domain (Box-1), which has also been described for two other activating Ly49s, D and H [26 ], and Ly49L4 lacks both of these sequences. Finally, Ly49L has an extra amino acid (position 90; threonine) not shared by any other Ly49. To eliminate possible PCR-related sequence errors, each Ly49 described was isolated from two separate PCR reactions and sequenced.
Clustering of inhibitory and activating Ly49 extracellular domains
The striking homology of Ly49l to Ly49g in
the region coding for the extracellular domain was reminiscent of
another activating/inhibitory pair, Ly49a and p
[4
]. To see if this "domain swapping" is used by
other members of the Ly49 family, a phylogenetic tree was constructed
for the intracellular and extracellular nucleotide-coding regions. When
possible, CBA/J sequences were used; otherwise, an alternative strain
was used to provide a complete tree. An interesting clustering effect
was seen with this analysis. Comparison of the intracellular domains
showed that the activating and inhibitory Ly49 genes formed separate
clusters as expected (unpublished results). However, when the
extracellular domains were analyzed in this fashion, each of the known
activating Ly49 molecules clustered with an inhibitory Ly49 (Fig. 2B)
.
Ly49l was grouped with Ly49g (this clustering
took place even when using the Ly49L2 isoform that lacks the
Ly49G1-like insertion), Ly49p clustered with Ly49a,
Ly49d clustered with Ly49o, and Ly49h
clustered with the Ly49i/j/c subfamily. This suggests that
diversity within the Ly49 family is not only because of gene
duplication but is also a result of gene recombination. Although
Ly49e and f are not grouped with activating
counterparts, there are two potential activating
Ly49c-related genes in B6 mice [3
],
suggesting that the majority of Ly49 family members will have
activating/inhibitory counterparts.
Ly49L is expressed in CBA/J but not B6 NK cells
The B6-Ly49l sequences were originally identified from
genomic fragments [3
]. To our knowledge, no expression
of this gene has been described for B6 mice. To determine if Ly49L is
expressed in B6 and other strains of mice, an RT-PCR-based approach was
developed. Primers were designed that could bind and amplify B6 and
CBA/J allelic forms of Ly49l-derived cDNA, and similar
primers were designed for Ly49g as a positive control.
Previous studies have shown that Ly49G is expressed on many different
strains of mice [22
]. The specificity of this assay is
shown when both sets of primers are used on cloned Ly49 cDNAs
(Fig. 3
, left panels). To test for Ly49l and g
transcripts in primary cells, NK cell cDNA from various mouse strains
was produced and then analyzed by this approach. As expected,
Ly49g was found in all four strains tested. It is
interesting that Ly49l transcripts were only detected in
CBA/J and C3H/He but not 129/J or B6 NK cells (Fig. 3
, right panels).
The PCR products obtained in this analysis were cloned and sequenced to
confirm their identity with Ly49l. These results suggest
that Ly49L expression is restricted to certain strains of inbred mice.
 View larger version (26K): [in a new window] |
Figure 3. Detection of Ly49l mRNA in various mouse strains. Poly-A RNA
was isolated from the listed strains of mice, reverse-transcribed, and
then amplified in the presence of Ly49l- or
Ly49g-specific primers (right). These primers were designed
to be capable of binding and amplifying all known alleles of these
genes. As a control to show primer specificity, 1 ng of various
vector-contained Ly49 cDNAs was amplified using the same primers
(left).
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Figure 4. Reactivity of Ly49L2 to a panel of anti-Ly49 mAb. (A)
Fluorescein-activated cell sorter (FACS) analysis. CP cells were
transfected with Ly49L2, Ly49A, Ly49G2, or Ly49D. Cells were stained
with the indicated mAb followed by an FITC-labeled secondary. YE1/48 is
specific for Ly49A, 4E5 is specific for Ly49D, and 4D11 reacts with
Ly49G. (B) Immunoprecipitation analysis. CP cells were transfected with
Ly49L2 by electroporation. Cell lysates were immunoprecipitated with
the indicated mAb. Immune complexes were run under nonreducing
conditions, transferred, and blotted with DX37 (antihuman DAP12).
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(IFN-) secretion in CBA/J NK cells
(unpublished results).
Antibody-mediated cross-linking of Ly49L2 leads to DAP12
phosphorylation
Cross-linking of Ly49D by specific antibody leads to DAP12
phosphorylation [16
]. To see if Ly49L was a functional
activating receptor also, Ly49L2-transfected CP cells were subjected to
cross-linking with YE1/48 mAb. Cross-linking of Ly49D with 4E5,
followed by immunoprecipitation and detection of phosphorylated
proteins, reveals a "ladder" of the various DAP12 phosphorylation
states. Similarly, cross-linking Ly49L2 with YE1/48 yielded such a
ladder also (Fig. 5
). A western blot run in parallel shows that equal levels of DAP12
are present in cells cross-linked with specific or irrelevant mAb.
These data suggest that Ly49L2 is a functional activating receptor in
CBA/J mice. As expected, a band at 100 kDa is not present because of
the lack of tyrosine residues in the intracellular domain of Ly49L.
 View larger version (56K): [in a new window] |
Figure 5. Antibody-mediated cross-linking of Ly49L2 results in mDAP12
phosphorylation. CP cells were transfected with the indicated cDNAs in
expression vectors by electroporation. Cells were treated with specific
mAb or irrelevant rat IgG followed by a 2 min incubation with an
isotype-specific secondary antibody at 37°C. Cell lysates were
immunoprecipitated with 4E5 or 4D11, specific for Ly49D and L2,
respectively (by immunoprecipitation only for Ly49L2). Immune complexes
were run under nonreducing conditions, transferred, and blotted with
4G10 (antiphosphotyrosine) or DX37 (antihuman DAP12).
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In a recent study of MHC class I binding by Ly49s, no binding of Ly49D or H to MHC class I was detected using cell-cell or MHC-tetramer binding assays [27 ]. This shows that the affinity of the activating receptors for MHC class I is considerably lower than the inhibitory Ly49s. This may suggest that class I MHC is not the real in vivo ligand for activating Ly49 receptors, or that the affinity is lower for activating receptors to prevent autoimmunity. Another alternative is that activating receptors bind to an MHC class I-related ligand and possess weak cross-reactivity with MHC class I. Perhaps the activating receptors evolved from the inhibitory receptors to provide a mechanism whereby NK cells could eliminate NK-resistant viruses expressing molecules that mimic MHC class I [28 ]. Although inhibitory receptors bind to constitutively expressed ligands to suppress NK cell activation, activating receptors would respond to a newly synthesized molecule, a product of a native stress-induced gene or originating from a pathogen.
The study of Ly49 expression in various mouse strains has lead to an accumulation of evidence indicating that Ly49 expression is strain-specific. The current study demonstrates that Ly49L is expressed in CBA/J and C3H/He but not 129/J or B6 NK cells. Similarly, previous studies have shown that Ly49P is expressed in 129/J but not B6 mice [4 ]. Ly49P is cross-reactive with YE1/48, and so the DAP12-associating Ly49 seen in Figure 1 could be Ly49L and/or Ly49P. However, Ly49p was not detected in CBA/J NK cells by PCR cloning using primers capable of isolating Ly49p from 129/J mice. Ly49A and D are expressed in B6 mice, but there is no evidence from biochemical studies for the presence of Ly49A in 129 strains of mice [4 , 29 , 30 ] or for Ly49D expression in CBA/J NK cells (Fig. 1 4 E5 immunoprecipitation lane). No Ly49H protein was detected in CBA/J immunoprecipitation experiments (Fig. 1) , and no Ly49h cDNAs were cloned. Therefore, the Ly49h cDNA cloned from B6-CBA F1 mice [2 ] is probably of B6 origin.
The observed differences in Ly49 repertoire between inbred mouse strains raises questions relating to the mechanism of selective Ly49-gene expression. It is possible that the genes of the unexpressed Ly49s in the various strains are completely missing. A Southern blotting study of Ly49c and a-related genes in a panel of mouse strains has indicated significant differences in gene number between strains [3 ]. Another possibility is gene silencing, as evidenced by the presence of the Ly49l gene in B6 mice, but no detectable Ly49l mRNA. The B6 Ly49l gene has been only partially characterized, so the mechanism of silencing is unknown [3 ]. Transcripts of the Ly49k and n genes in B6 mice were shown to contain incomplete open-reading frames as a result of missing exons or premature stop codons [31 ]. In addition, an improperly spliced transcript of Ly49m containing a stop codon at the beginning of exon 4 was isolated from a B6 NK cell cDNA library (unpublished results). Therefore, it appears that inactivation by mutation has silenced several of the B6 Ly49 genes. The inability to detect Ly49l transcripts using PCR primers corresponding to known exons suggests that the Ly49l promoter activity may be altered in B6 mice because of mutation of cis-acting elements or transcription factors. The TCF-1 transcription factor has been implicated in Ly49a transcription, however it does not appear to be required for the transcription of other Ly49s [30 ]. Recently, we have found that unlike the Ly49a promoter, the Ly49i promoter does not contain TCF-1 binding sites (unpublished results). The observation that TCF-1 knockout mice show no difference in the level of Ly49C/I expression supports this finding [30 ]. Perhaps there are specific transcription factors required for the expression of individual Ly49s, and the B6 mouse is missing an Ly49l-specific transcription factor. An understanding of the Ly49l gene defect in B6 mice will require the complete characterization of the Ly49l locus in B6 and CBA/J mice.
Another possible mechanism governing the expression of various Ly49 molecules is the MHC haplotype environment of the NK cells, if one assumes that NK cell selection occurs somewhat like thymic selection for T cells. There is considerable evidence indicating that an MHC-dependent education process operates during NK cell Ly49 repertoire development [32 ]. Ly49L was found to be expressed in CBA/J and C3H/He mice, which are of the H-2k background, but there was no expression in B6 or 129/J mice, both of the H-2b background. We cannot discount the possibility that the Ly49 expression pattern and the MHC haplotype are the product of the two strains sharing a common ancestor and, therefore, genotypes. The observation that a highly related inhibitory Ly49 exists for each activating Ly49 may bear on this argument (Fig. 2B) . If one assumes that the MHC specificities are similar for each activating and inhibitory partner, then this could serve as a basis for selection through MHC. If an NK cell expresses a self-reactive activating Ly49, then the survival of that NK cell may depend on the coexpression of its inhibitory partner. Alternatively, other inhibitory receptors with similar specificities could provide a negative signal, preventing the development of self-reactive NK cells. The generation of mice lacking activating Ly49 receptors may provide insight into the true functional role of activating Ly49 family members.
Received March 12, 2000; revised June 8, 2000; accepted June 14, 2000.
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