(Journal of Leukocyte Biology. 2000;68:748-756.)
© 2000
by Society for Leukocyte Biology
Activating Ly-49D NK receptors: expression and function in relation to ontogeny and Ly-49 inhibitor receptors
John R. Ortaldo*,
Robin Winkler-Pickett* and
Gordon Wiegand
* 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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ABSTRACT
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Developmental changes in the repertoire of activating Ly-49 family
members have not been examined previously. In the present study, we
have examined the expression and function of the activating Ly-49s (D
and H) from birth through 8 weeks of age. We demonstrate that 1)
activating Ly-49s are expressed early, 2) their expression intensity is
not different from adult NK cells, and 3) activating receptors are
functional. Examination of the inhibitory Ly-49s also demonstrated
functional capacity immediately upon expression. To examine the
kinetics of expression of the repertoire of activating Ly-49 members,
we utilized five- and six-color flow cytometric analyses of NK cells
from birth through adulthood. Previous studies examining the inhibitory
Ly-49 repertoire have proposed that expression is regulated by the
product rule. Our results indicated that Ly-49D, which recognizes
H-2Dd, had a discordantly high coexpression of the
inhibitory Ly-49s that recognized H-2Dd (Ly-49A and
Ly-49G2). The product rule of Ly-49 expression does not explain the
coexpression of selected activating and inhibitory receptors. This high
level of coexpression of H-2Dd recognizing activating and
inhibitory Ly- 49s suggests an in vivo selection or
regulated coexpression.
Key Words: Ly-49 • NK • ontogeny • product rule
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INTRODUCTION
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Murine natural killer (NK) cells express multiple Ly-49 receptors
that are type II transmembrane receptors. These receptors inhibit or
activate NK cell functions such as cytolysis or cytokine secretion. A
functionally similar family of molecules exists on human NK cells, the
killer cell immunoglobulin-like receptors (KIRs). In human and mouse,
there exist inhibitory and activating forms; however, the human KIRs
are structurally dissimilar to the murine Ly-49 family of receptors,
because they belong to the superimmunoglobulin family of receptors.
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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MATERIALS AND METHODS
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NK cell isolation
Splenic NK cells were isolated from C57BL/6 (B6) mice and grown
for 7–10 days in 1000 U/ml Cetus recombinant interleukin (IL)-2, as
previously described [12
].
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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RESULTS
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Subset development
The analysis of NK cell subsets for their Ly-49 expression and
function is determined by the use of specific NK (NK1.1 and DX-5) and T
(CD3) markers. In addition, the use of these markers allows the
simultaneous examination of Ly-49s on the unique T cell subset that
expresses NK1.1 and CD3, termed the NK/T cell by many authors
[16
17
18
]. Using these parameters, we examined the
presence of these subsets from birth through 8 weeks of age. A
representative experiment in C57Bl/6 mice is shown in Figure 1
. In this and all experiments, age and subset evaluations were
obtained from a pool of five mice. Splenic lymphocytes from newborn
mice did not express CD3 at high levels; however, this subset of
CD3+, NK1.1- T cells was quite evident and
expanded through 8 weeks. NK cells appeared at birth and rose gradually
from 1–3%. The NK/T subset remained quite low and was evident at
1.5–2% after 3 weeks of age.
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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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Flow cytometric analysis of Ly-49
To perform a detailed survey of Ly-49 expression on NK cells, mice
from birth through 8 weeks of age were analyzed by three-color flow
cytometry. The Ly-49 expression or NK cells is summarized in
Figure 2
. Although NK1.1-positive cells are present at birth, very few, if
any, activating or inhibitory Ly-49s were present. During maturation,
all Ly-49s rose in percentage expression from 1 week through 8 weeks,
generally reaching maximal expression between 4–5 weeks (average of
four experiments). The analysis of activating and inhibitory receptors
indicated that simultaneous expression occurred during development. It
can be observed in Figure 2
that from 1–5 weeks of age, the Ly-49D
(•) and Ly-49G2 (
) 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.
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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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Biochemical analysis of Ly-49 expression
To determine whether the Ly-49 receptors on young mice were
biochemically functional, we evaluated Ly-49 receptors on ANK cells
from 1- to 8-week-old mice. Results (unpublished results) demonstrated
a strong phosphorylation of the homodimeric Ly-49G2 protein,
characteristic of the inhibitory Ly-49s. However, the activating Ly-49s
(D and H) demonstrated the typical phosphorylation of DAP12, the
signaling moiety associated with these activating Ly-49s. Sorted
NK1.1+, Ly-49D+, and NK1.1+,
Ly-49H+ cells that were cultured with IL-2 and expanded
from 1- to 5-week-old mice also demonstrated phosphorylation of DAP12.
In addition, when the Ly-49C/I/H+ subset was examined,
immunoprecipitation with 1F8 (anti-Ly-49H) demonstrated Ly-49 (94 kDa)
receptor and DAP12 (30 kDa) bands. This indicates the 1F8 antibodies’
cross-reactivity with Ly-49C/I (inhibitory) receptors and the
coexpression of activating Ly-49H receptors on these Ly-49C/I subsets.
These data suggest that these young NK cells have functional Ly-49s at
1 week of age.
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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Calcium mobilization
Characteristically, Ly-49s recognize class I molecules
and regulate lysis of targets. Activating Ly-49s, like Ly-49D
[22
, 23
], have been shown to be involved as
a stimulus for lysis of H-2Dd-expressing targets, whereas
Ly-49G2 has been shown to strongly inhibit killing of
H-2Dd-expressing targets [12
,
24
, 25
], which includes blocking or
dominating the activating receptors like Ly-49D. One of the earliest
events in killing [26
] is a calcium-dependent
activation. Thus, to evaluate a second functional aspect of activating
Ly-49s as well as the inhibitory Ly-49s, we examined CD3-
NK1.1+ IL-2-cultured cells that coexpressed Ly-49D and
Ly-49G2. We examined this subpopulation’s ability to mount and
regulate calcium mobilization after receptor cross-linking. As
shown in Figure 5A
, Ly-49D+ NK cells from 8-, 3-, or 2-week-old mice
mediated a strong calcium mobilization after anti-Ly-49D (4E5)
cross-linking. This response was not seen in Ly-49D- NK
cells. To evaluate the ability of inhibitory receptors to modulate this
NK cell activation, we cross-linked Ly-49D and Ly-49G2 simultaneously
and compared the resultant calcium mobilization. As shown in Figure 5A 5B
5C
8
-, 3-, or 2-week Ly-49D+,G+ NK cells
were able to mediate a strong calcium mobilization to Ly-49D alone, but
this response was oblated by simultaneous cross-linking of the
inhibitory Ly-49G2. Thus, the activating and inhibitory Ly-49s are
functional in young mice immediately after expression.
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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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Ontogeny of Ly-49 repertoire
The above studies indicate that Ly-49s are placed on
the surface of NK cells immediately after birth and are functional.
However, our studies do not speak to the relationship of each receptor.
Previous studies from Raulet’s laboratory [14
]
examining inhibitory receptors have suggested that Ly-49 repertorial
expression is random, and expression patterns reflect strict
mathematical probabilities based on overall expression. To examine this
hypothesis more directly, we examined activating Ly-49s (D and H) for
coexpression of inhibitory ligands. It is known that Ly-49G2 and Ly-49D
recognize H-2Dd. Thus, as a result of Raulet’s model
[14
], the coexpression of these receptors should follow
the product rule. Therefore, we examined the expression of the
inhibitory Ly-49G2 on Ly-49D subsets using a five- and six-color flow
cytometric analysis. Using the UV-activated dye Alexa350 and a dye
laser-activated reagent Alexa594, we could examine five and six (using
tandem reagents) fluorescence parameters. To perform this analysis, we
used sets of reagents indicated in Figure 6
. We show the Ly-49 expression levels on lymphocytes from 1- and
8-week-old mice compared with a control IgG on 8-week-old mice (control
for 1 week not shown but identical to 8 weeks). Similar to what was
seen in Figure 2
, expression of Ly-49s is minimal or negative at 1 week
but present at 8 weeks. When coexpression was examined on
Ly-49D+ or total NK cells (Fig. 7
), an interesting finding was made. The sum of Ly-49G2 and Ly-49A
(quadrants R1, R2, and R4), which are inhibitory NK receptors with
H-2Dd as their ligand, is expressed on >70% of the
Ly-49D+ NK cells at 1 week of age and remained constant
through 8 weeks. When this is compared with Ly-49G2 and Ly-49A
expression on all CD3- NK cells, the pattern showed a
strong increase with age, parallel to that shown in Figure 2
. A
representative chart of Ly-49G2- and Ly-49A-summed expression on total
NK (CD3- NK1.1+) vs. Ly-49D+ NK
cells is shown in Figure 8
. The examination of total NK cells does not reveal this high level
of expression of Ly-49G2 and Ly-49A that is seen in the
Ly-49D+ NK cells as early as 1 week of age. Further
examination of cells expressing the Ly-49H-activating receptor
indicates that these cells have significant levels of inhibitory
receptors also, namely Ly-49C/I based on 5E6 expression, which
recognize H-2Db. This is not seen when Ly-49G2 or
Ly-49A receptors are analyzed. The levels are 50% at week 1 and remain
>50% through 8 weeks (unpublished results). This analysis is
complicated by the cross-reactivity of 1F8 with Ly-49C/I and must await
further analysis with Ly-49H-specific reagents. However, analysis of
1F8-expressing subsets indicated that activating receptor (based on
DAP12 phosphorylation) was expressed on Ly-49C/I (5E6)-positive and
-negative subsets (unpublished results).
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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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The product rule analysis of activating Ly-49 receptors is shown in
Table 1
. Depicted are NK cells from 1- and 3-week-old mice. The frequency
of expression that was expected for coexpression of activating and
inhibitory Ly-49s is compared with the actual frequency obtained based
on five-color analysis. As can be seen with Ly-49D, a significant
deviation (based on paired-T test) was observed with Ly-49G2 and Ly-49A
(bold values) at 1 and 3 weeks of age. This high level of coexpression
was not observed with Ly-49H. Because Ly-49G, Ly-49D, and Ly-49A
recognize H-2Dd, this would indicate a nonrandom process
rather than a product-rule expression. When Ly-49H was examined, no
similar elevated expression was observed. However, this analysis is
difficult because 1F8 reacts with the Ly-49C/I molecules also, and the
activating Ly-49H+ subset is known to contain these
inhibitory moieties (see above). Further analysis with Ly-49H-specific
antibodies is required.
Analysis of Ly-49 expression in NK/T cells
Finally, the analysis allowed examination of the Ly-49 expression
in T cells. We have shown previously [27
] that Ly-49s
are present and functional on mature T cells (>8 weeks). In addition,
our studies have demonstrated [27
] that T cells lacked
Ly-49D, the activating Ly-49 receptor. The present analysis on
NK1.1+ T cells (NK/T) has shown similarly that Ly-49D is
lacking at all ages tested and that true Ly-49H [1F8-positive cells
that do not express Ly-49C/I (5E6)] expression is also lacking (see
Table 2 ). Further examination of the Ly-49H-activating receptor with a
Ly-49H-specific reagent is needed. It should be noted that the percent
of NK1.1+ T cells is quite low, <3% for all ages of mice
that were studied (see Fig. 1
). Interestingly, during the examination
of T cells expressing the inhibitory Ly-49s (A, C/I, and G2), we saw
that the kinetics of expression were quite different from that found
with CD3-, NK1.1+ cells. Another factor is
that a significant percentage of Ly-49A and Ly-49C expression is
contributed by CD3+ NK1.1+ NK/T cells. This can
only be seen if three-color analysis is used. In NK/T cells, all Ly-49s
were very low at birth but appeared near maximal by 1 week of age, with
little variance seen after this time point.
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DISCUSSION
|
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Ontogeny of Ly-49 function
In the present study, we addressed several hypotheses related to
the expression of activating and/or inhibitory receptors. Previous
studies [14
, 19
20
21
] have examined
inhibitory Ly-49 expression during ontogeny but failed to consider the
coexpression of the newly described activating Ly-49D and H receptors
or their functional status. Thus, we examined similar issues related to
ontogeny of Ly-49D and function. First, do NK cells develop activating
Ly-49 receptors before, during, or after expression of inhibitory
receptors? By examining Ly-49D and Ly-49H (on Ly-49C/I-negative subsets
of NK cells), we could determine that at birth and 1 week of age,
activating and inhibitory receptors seemed to develop at the same time.
Although the absolute percentage of the receptors differed, their
expression and kinetics of expression through 4 weeks were quite
similar. Second, is there a delay or difference in the expression of
inhibitory Ly-49 receptors based on the H-2 expression of the host? By
comparing Ly-49G2 and Ly-49C/I, we conclude there was no difference.
Ly-49C and I have been shown to recognize aspects of H-2b,
the class I phenotype of C57Bl/6 mice; however, Ly-49D and Ly-49G2
(which recognize H-2d) were expressed early and increased
their expressions at a similar rate. Thus, the presence of
H-2b did not delay Ly-49C/I expression compared with
Ly-49G2. Third, are the expression levels and functional status of the
activating receptors variant early after birth? One could hypothesize
that the level of receptor expression could be low or that the
receptors may be nonfunctional at birth. By examining the level of
Ly-49D and Ly-49H (Fig. 2) , we saw no difference in the mean
fluorescence intensity of activating or inhibitory Ly-49s. To examine
the function of the activating receptor, we examined several parameters
that have been associated with Ly-49D: 1) phosphorylation and
association of DAP12, 2) calcium mobilization after receptor
cross-linking, and 3) induction of cytokines after receptor
cross-linking. We were able to show that Ly-49D and Ly-49H did
associate with DAP12, leading to tyrosine phosphorylation of DAP12 at a
similar level in young and old mice.
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.
|
ACKNOWLEDGEMENTS
|
|---|
This project has been funded in whole or part with Federal funds
from the National Cancer Institute, National Institutes of Health,
under Contract No. NO1-CO-56000. The content of this publication does
not necessarily reflect the views or policies of the Department of
Health and Human Services, nor does mention of trade names, commercial
products, or organizations imply endorsement by the U.S. Government.
The publisher or recipient acknowledges the right of the U.S.
Government to retain a nonexclusive, royalty-free license in and to any
copying covering the article.
Received March 23, 2000;
revised June 29, 2000;
accepted June 30, 2000.
|
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TOP
ABSTRACT
INTRODUCTION
