* Laboratory of Experimental Immunology, DBS, National Cancer Institute-FCRDC, and
SAIC-Frederick, NCI-FCRDC, Frederick, Maryland
Correspondence: Dr. John R. Ortaldo, NCI-FCRDC, Bldg. 560, Rm. 31-93, Frederick, MD 21702-1201. E-mail: Ortaldo{at}mail.ncifcrf.gov
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Key Words: Ly-49 • NK • ontogeny • product rule
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The inhibitory Ly-49 receptors, Ly-49A, C, G, and I, inhibit NK cell function upon binding of class I ligands on target cells [1 2 3 ]. These Ly-49 inhibitory receptors as well as inhibitory KIRs contain cytoplasmic immune receptor tyrosine-based inhibitory motifs (ITIMs) that are phosphorylated upon stimulation leading to the recruitment of SHP-1 phosphatase and attenuation of intracellular signals [1 , 4 , 5 ]. Receptors exist in both species that can activate NK cells [6 7 8 9 ]. The predicted amino acid sequences for the activating Ly-49D and Ly-49H do not contain any ITIMs in their cytoplasmic domains, confirming that these are not inhibitory receptors. Studies have shown that activating receptors like Ly-49D do not become phosphorylated after pervanadate treatment or receptor cross-linking and do not recruit SHP-1 [10 ]. In contrast, Ly-49D has been shown to mobilize intracellular 2+Ca and mediate reverse antibody-dependent cellular cytotoxicity (ADCC) in the presence of specific monoclonal antibody (mAb) [10 , 11 ].
These activating Ly-49 and KIR molecules have been shown to associate with a 12 kD homodimeric protein, DAP12, that contains an immunoreceptor tyrosine-based activation motif (ITAM), which is critical for positive signaling by these receptors [10 , 11 ]. The Ly-49D- and Ly-49H-activating receptors contain an arginine residue in their transmembrane domain that serves as a required docking element for DAP12 binding to the receptor [9 10 11 ]. Association of the homodimeric DAP12 with these activating receptors has been shown in transfected cells to be essential for phosphorylation of DAP12 following receptor triggering, intracellular calcium mobilization, and cytokine secretion [8 , 9 , 11 , 12 ].
Most studies to date have examined the mature NK cell repertoire from human or murine systems to analyze functional receptors. It is well known that NK cells from newborns lack Ly-49 receptors. These newborn NK cells express NK1.1 and demonstrate lytic ability to prototypic NK targets like YAC-1 [13 ]. These NK cells develop and increase their Ly-49 inhibitory receptor repertoire through the first 5 weeks of life. The exact nature of the signals that are required for expression of Ly-49s is currently under study by several laboratories and involves many cytokines as well as stromal interaction. Regardless of the requisite signals for Ly-49 expression, previous studies have shown that as mice age, expression of their inhibitory Ly-49 receptors occurs with maximal levels reached by about 4 weeks of age [13 , 14 ]. Little has been done to examine the functional status of inhibitory and activating Ly-49s during maturation. By using five- and six-color flow cytometric analyses, we wanted to determine if the developmental expression of activating and/or inhibitory receptors indicated a regulated vs. random pattern. Previous studies from Raulet’s laboratory [14 ] have proposed that Ly-49A and G2 develop based on mathematical probabilities, e.g., coexpression of receptors can be calculated by the product rule for receptor frequency. We chose to examine if the activating Ly-49 receptor repertoire preceded inhibitory receptors, especially those recognizing the same major histocompatibility complex (MHC) ligand. Additionally, we wanted to determine if subsets containing activating Ly-49 receptors expressed different reportorial combinations of inhibitory receptors than the general NK population.
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Antibodies used
The following mAbs to Ly-49 receptors were used: YE148 (Ly-49A),
4D11 (Ly-49G2), and 4E5 (Ly-49D), as previously described
[15
]. 1F8 rat mAb was provided by Dr. V. Kumar (Dallas,
TX). NK1.1, DX-5, and CD3
(Becton Dickinson/Pharmingen, San Jose,
CA) were used for flow cytometric analysis. Rat immunoglobulin (IgG)
was used as a control for immunoprecipitations and was purchased from
Becton Dickinson/Pharmingen. Antirat IgG was used as a cross-linking
reagent. 4G10 antibody recognizes phosphotyrosine (Pty) and was
purchased from UBI (Lake Placid, NY) in its biotinylated form.
Antihuman DAP12 (DX37) was a gift from Dr. Lewis Lanier (DNAX Corp.,
San Francisco, CA). Rabbit antimouse DAP12 was generated from immune
complexes of Ly-49D and DAP12 (unpublished results).
Flow cytometry analysis (FCA)
Cells were stained as previously described [12
],
analyzed on a FACSort flow cytometer (Becton Dickinson), and analyzed
or sorted on a MoFlo cytometer (Cytomation Inc., Ft. Collins, CO).
Cells were stained directly using phycoerythrin (PE) and fluorescein
isothiocyanate (FITC)-labeled primary abs or stained indirectly using a
primary ab followed by an isotype-specific FITC- or PE-conjugated
secondary or a biotinylated primary ab followed by Streptavidin Per-CP
(Becton Dickinson). Five- and six-color analysis used Alexa 350
and Alexa 594 (Molecular Probes, Eugene, OR) laser-activated dyes that
were directly linked to anti-Ly-49D and anti-Ly-49G2. In some
experiments, biotin-labeled antibodies were used with allophycocyanin
(APC).
Cytokine measurement
Cytokines were measured using interferon-
(IFN-), IL-3,
and granulocyte-macrophage colony-stimulating factor (GM-CSF)
enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems,
Minneapolis, MN). Cell stimulations were performed with cells at 1 x 106/ml. Antibodies were added at a concentration of 2
µg/106 cells for 30 min on ice. Cells were then washed
and plated on 24-well Costar (Corning, NY) plates that were precoated
with 2 µg/well rabbit antirat IgG and blocked with media containing
10% fetal calf serum. All samples were collected after 18 h
incubation (37°C, 5% CO2) and were measured in duplicate
against the standard curve of the assay and described as pg/ml. In all
assays, the SD of the cytokine measurement was <25 pg/ml.
Calcium mobilization
Analyses of the changes in intracellular
Ca+2 concentration
([Ca+2]i) were done using a
FACSort flow cytometer (Becton Dickinson, Mountain View, CA) and the
calcium-sensitive fluorochrome, Flou-3 (Molecular Probes). Briefly,
cells (2x106/ml) were incubated at 25°C in Dulbecco’s
phosphate-buffered saline (DPBS) without Ca+2
or Mg+2 containing 15 µg/ml Flou-3. After 30
min, cells were washed in DPBS and held at room temperature in the dark
until analysis. The [Ca+2]i was
monitored with the loaded cells (40 µl) diluted to 500 µl with
37°C DPBS with Ca+2 and
Mg+2, glucose, and sodium pyruvate. Flou-3 was
excited by the argon laser at 488 nm, and the levels of fluorescence
were monitored. The cells were kept at 37°C during analysis. Baseline
data were collected for 20–30 sec, then the cells were stimulated with
primary (10 µg/ml) mAb, followed 20–30 sec later by rabbit antirat
antibody (10 µg/ml) or goat antirat antibody (10 µg/ml). Data were
analyzed using the MultiTime Kinetic Experiment Analysis Software
(Phoenix Flow Systems, San Diego CA) and were expressed as the
percent-responding cells relative to unstimulated baseline
measurements.
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Figure 1. Expression of NK, NK/T, and T cells in mouse spleen. Fresh, uncultured
C57BL/6 splenocytes from newborn to 8-week-old mice were analyzed.
Values represent the % of mononuclear cells expressing CD3 and/or
NK1.1.
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) expressions parallel each other. It
should be noted that all activating and inhibitory forms of the Ly-49s
were expressed at a similar percentage, except that Ly-49A was not
expressed at high levels. It should be noted that NK cell expression of
Ly-49A was low because of high levels of coexpression on NK/T cells
(see below). However, the limited number of NK cells that are present
in 0- to 3-week-old mice makes the performance of detailed phenotype
and functional analyses difficult, if not impossible. Therefore we
expanded the NK cells in culture with IL-2 using a standard protocol to
obtain adherent NK (ANK). Based on previous studies
[19
20
21
] with fetal- and bone marrow-derived NK cells,
Ly-49 phenotypic changes did not occur in IL-2 expansion. These cells
remained Ly-49-negative once they were removed and expanded in
vivo. To verify the validity of this expansion, mouse splenic
lymphocytes from 1 to 9 weeks old mice were harvested and expanded with
IL-2. These cells were analyzed for expression of Ly-49G2 before
culture on day 0 and after 7 days of expansion in IL-2. The results of
one of three representative experiments examining Ly-49G2 are shown in
Figure 3
. As can be seen, the Ly-49G2 expression of NK1.1+ NK
cells on day 0 or 7 remained very similar after culture and expansion.
This same effect was seen with the other Ly-49s tested. The small Ly-49
increases in cultures from 5- to 8-week-old mice were not seen in all
experiments. However, the day 7 IL-2 ANK cultures from mice that were
newborn to 9 weeks of age were considerably enriched for NK cells
(ranging from 50–85% CD3-, NK1.1+ cells) and
expanded from 20–1000-fold (unpublished results). Therefore, the
in vitro IL-2 expansion allowed the phenotypic and
functional analysis of NK cells from young mice without changing their
intrinsic Ly-49 phenotype. Thus, all further functional studies used
IL-2-cultured ANK cells.
 View larger version (16K): [in a new window] |
Figure 2. Graphic representation of Ly-49 expression in young mice. The
percentage of Ly-49A, C/I, D, G2, and H was evaluated on
CD3-, NK1.1+ splenocytes by flow cytometric
analysis from Figure 2
.
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Figure 3. Expression of Ly-49s at 0 and 7 days of IL-2 culture in young mice.
Ly-49G2 expression was evaluated by flow cytometric analysis on
CD3-, NK1.1+ splenocytes from fresh or day 7
ANK cultures with IL-2. Chart is representative of five experiments.
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Functional analysis of Ly-49 cytokine production
Although these data suggest that the newly developed and expressed
Ly-49s are functional, we directly examined ligand cross-linking of
sorted NK1.1+, Ly-49D+ NK cells for cytokine
production. Studies from our laboratory have shown that cross-linking
Ly-49D induces cytokine production [8
] of IFN- and
GM-CSF. As shown in Figure 4
, IFN- production and GM-CSF (unpublished results) were strongly
induced by anti-Ly-49D antibody but not by control IgG and not in
Ly-49D- NK cells. When Ly- 49H cross-linking was examined
in these populations (expressing 60–75% 1F8), significant production
of IFN- also was observed. The levels of anti-Ly-49H were lower,
presumably because of the cross-reaction of the 1F8 and its ability to
trigger the inhibitory Ly-49C/I also present on these cells. In
experiments not shown, 1F8+ 596- NK cells from
8-week-old mice (which lacked the inhibitory Ly-49C/I) produced levels
of IFN- similar to Ly-49D when cross-linked with 1F8 (Ly-49H). Thus,
these data indicate collectively that the activating Ly-49s expressed
in young mice are functional.
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Figure 4. Evaluation of cytokine production. Ly-49D+ or
D- NK cells were sorted, and IL-2 was cultured for 7 days
from 2-, 3-, and 8-week-old mice. IL-2-grown NK cells were examined for
their ability to secrete IFN- after cross-linking cells with
anti-Ly-49D (4E5), anti-Ly-49H (1F8), or rat IgG. Similar results were
obtained with 3-week Ly-49D- NK cells and 8-week NK cells
(unpublished results).
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Figure 5. Evaluation of functional status of Ly-49s by calcium mobilization with
IL-2-cultured NK cells. ANK (CD3-, Ly-49D+)
cells sorted from 8-, 3-, and 2-week-old mice and then cultured for
5–7 days with IL-2 were evaluated for their ability to mobilize
calcium after treatment with anti-Ly-49D or Ly-49G2 alone (A) or in
combination (B–D). Calcium mobilization was examined flow
cytometrically by detection of Flou-3 following stimulation with
primary antibody (event arrow 1°) and cross-linking with rabbit
antirat antibody after 30 sec (event arrow 2°). The percent
responding cells is shown as determined on the FACSort.
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Figure 8. Kinetics of coexpression of H-2Dd-activating and
-inhibitory NK receptors. Values represent typical percentages seen on
indicated subset NK cells (bulk CD3- NK1.1+
cells or CD3- NK1.1+ Ly-49D+)
expressing Ly-49G2 and Ly-49A, as evaluated by five- and/or six-color
analysis at 1 and 8 weeks of age. Values are representative of four
experiments.
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Figure 6. Evaluation of Ly-49s using five-color analysis. Ly-49A, C/I, D, G2, and
CD3 were evaluated on splenic ANK cells by flow cytometric analyses.
Histograms are representative of four experiments. FITC and PE dyes
were activated by 488 nm Argon laser, Alexa350 by UV laser,
and Alexa594 and APC by dye laser.
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Figure 7. Flow expression of H-2Dd inhibitory (Ly-49G2 and Ly-49A)
receptors coexpression and activating Ly-49D NK receptors.
Representative dotplot of Ly-49G2 vs. Ly-49A expression on
CD3-, NK1.1+, or CD3-,
NK1.1+, Ly-49D+ NK cells from 1- or 8-week-old
ANK cells. Values represent typical percentages seen on subsets as
evaluated by five- and/or six-color analysis. Values are representative
of four experiments.
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Table 1. Comparison of Observed and Expected Frequencies of Ly-49 Receptor
Coexpression with Activating Ly-49s
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Table 2. Expression of Ly-49s on T cells
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Examination of sorted Ly-49D+ NK cells demonstrated that receptor cross-linking with anti-Ly-49D (4E5) resulted in similar and rapid calcium mobilization (Fig. 6) in young (1- to 2-week) and 8-week-old mice.
Examination of cytokine induction demonstrated that receptor
cross-linking with anti-Ly-49D (4E5) resulted in IFN- (Fig. 5)
and
GM-CSF production. Thus, using these established parameters for Ly-49D
activation, the activating receptors on newborn NK cells are totally
functional like those on mature NK cells. Fourth, are the inhibitory
receptors on NK cells functional immediately after birth? To determine
this, we again used similar assumptions that inhibitory receptors 1)
would become tyrosine-phosphorylated and 2) would block activating
receptors when simultaneously cross-linked. By examining tyrosine
phosphorylation of the inhibitory receptors (Fig. 6)
and by
demonstrating that Ly-49G2 was able to potently block the calcium
mobilization triggered by Ly-49D in sorted Ly49D+,
Ly-49G2+ NK cells, we were able to conclude that NK
inhibitory receptors are completely functional in newborn as well as
adult mice.
Ontogeny of Ly-49 repertoire
Because the repertoire of activating receptors has not been
examined thoroughly, we sought to determine if they followed the
proposed product rule [14
]. The expression patterns of
activating and inhibitory receptors were determined using simultaneous
five- and six-color analysis. The product rule proposes that Ly-49
receptors are put on NK cells randomly with no select pattern observed.
Only recently [22
, 23
] have we begun to
elucidate some additional information about the binding ligands for the
inhibitory Ly-49s; e.g., Ly-49A recognizes H-2Dd, and
Ly-49G2 recognizes H-2Dd strongly and recognizes
H-2Ld weakly. Ly-49C and Ly-49I have a broad recognition of
H-2b. Recent studies by George et al.
[28
] indicated that Ly-49D recognized H-2Dd
and that high levels of coexpression of inhibitory H-2Dd
recognizing Ly-49s were evident. By analysis of combinations of
inhibitory and activating receptors on NK cells from newborn through
8-week-old animals, some striking patterns of expression were seen. NK
cells that expressed the activating receptor Ly-49D, which binds
H-2Dd simultaneously, expressed high levels of inhibitory
receptor for that same class I molecule. This was detectable as early
as week 1 and seen through adulthood (>8 weeks). This high level of
expression of Ly-49G2 and Ly-49A was not seen on total
NK1.1+ cells or on other examined subsets and did not
follow the product rule (see Table 1
). This strongly indicated that
directed expression or selection was occurring during development of NK
cells. As our culture and expansion studies showed, this did not occur
during day 7 in vitro culture with IL-2 but presumably
during in vivo activation and interaction, which may require
stromal elements. This high level of expression of Ly-49G2/A (>75%)
would not be consistent with Raulet’s previous hypothesis
[14
] of random product-rule expression. The class I
ligand recognition of Ly-49H is not known presently, but our studies
would suggest that Ly-49H recognizes aspects of H-2b based
on its activating nature and coexpression on Ly-49C/I subsets. Our data
would strongly suggest that a direct coexpression of activating and
selected inhibitory Ly-49s is observed and may be dependent on having
similar ligand-binding specificities.
Ontogeny of T cell Ly-49 repertoire
The T cell expression of Ly-49s, limited to inhibitory receptors,
appeared to have different kinetics than NK cells. T cell expression of
Ly-49s was maximal by 1 week of age and remained constant through 8
weeks of age. Thus, unlike NK cells where newborn mice lack Ly-49s and
then develop exponentially over 4–5 weeks, T cells appear to express
Ly-49 inhibitory receptors rapidly, especially on the
NK1.1+ subset, and this expression is not altered with age.
Understanding this difference of kinetics in expression between T cells
and NK cells might help explain how transcription of Ly-49s is
regulated.
Summary
In summary, our studies demonstrate that activating and inhibitory
Ly-49s develop in newborns through 8 weeks of age and that these
receptors are functional upon expression on NK cells. In addition,
activating and inhibitory Ly-49s develop simultaneously. However, the
distribution of inhibitory Ly-49s is selected in NK cells expressing
activating Ly-49s recognizing the same MHC Class I and does not follow
the product rule. Our studies also raise important questions related to
the mechanism by which selected Ly-49s are expressed. How do some NK
cells coexpress Ly-49D and Ly-49G2/A at such a high level on the same
subset, whereas other NK cells do not? Is there an active selection
process in NK cells, or is the promoter for the Ly-49s somehow linked
so that expression of Ly-49D increases coexpression of Ly-49G2/A? These
are important questions to resolve in the overall understanding of how
Ly-49s function in NK cells and in vivo.
Received March 23, 2000; revised June 29, 2000; accepted June 30, 2000.
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