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*
Division of Hematologic Malignancies, Dana-Farber Cancer Institute; Department of Medicine, Harvard Medical School;
Department of Radiology, Beth Israel Deaconess Medical Center, Boston, MA 02115;
Bone Marrow Transplant Program, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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70-kDa heterodimeric complexes consisting of a 25-kDa
glycoprotein and 40- to 45-kDa molecules. Analysis by
two-dimensional isoelectric focusing/SDS-PAGE suggests that several
different 40- to 45-kDa species are present in the CD94 receptor
complex in human NK cells. NKH3 reacted with essentially all resting NK
cells, although CD94 is expressed at higher levels on the
CD56bright (i.e., high level of CD56)
CD16dim/neg (i.e., low level of or absent CD16)
subpopulation than on the more abundant
CD56dimCD16bright NK cell subset. Moreover, the
Z199 mAb, which appears to recognize NKG2-A species that can form
heterodimers with CD94, stained virtually all CD56bright NK
cells, but only a subset of CD56dim NK cells. Ligation of
CD94 augmented the proliferation of CD56bright NK cells in
response to IL-2 or IL-15 by as much as 10-fold. Secretion of IFN-
by CD56bright NK cells stimulated with IL-2 or IL-15 was
also enhanced up to 10-fold after CD94 ligation. CD94 mAb did not
consistently costimulate proliferation of or IFN- production by
CD56dim NK cells cultured with IL-2 or IL-15. In contrast,
irradiated K562 cells costimulated proliferation of both
CD56bright and CD56dim NK cells. These results
indicate that CD56bright and CD56dim NK cells
can be costimulated through different receptors, which may allow these
distinct NK cell subsets to be independently regulated in vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(3, 4, 5), TNF (3, 6),
granulocyte-macrophage CSF (7), and IL-5 (8), that can regulate the
function of T cells, B cells, monocyte/macrophages, and eosinophils.
Indeed, cytokines produced by NK cells during innate immune responses
can modulate the differentiation of activated T cells into effector
cells and thereby influence Ag-specific, adaptive T cell responses
(9).
NK cells have potent cytolytic activity and can lyse Ab-coated target
cells through Ab-dependent cell-mediated cytotoxicity
(ADCC)3 and certain
neoplastic or infected cells through an Ab-independent,
MHC-unrestricted process known as natural killing. The receptor on NK
cells that triggers ADCC is an oligomeric complex composed of CD16
(FcRIIIA) noncovalently associated with homodimers and heterodimers
of the 
family of signaling molecules (10, 11). Although several
candidate NK receptors have been described, the receptors that trigger
natural killing have not been unequivocally defined. The sensitivity of
target cells to natural killing appears to reflect the expression of
various ligands engaging NK cell receptors that can deliver either
activatory or inhibitory signals. Several inhibitory receptors
recognizing specific MHC class I allotypes have been described in the
mouse and in man (12, 13, 14, 15).
Despite recent advances elucidating the mechanisms that control NK cytolytic activity, the regulation of NK cell proliferation remains poorly understood. Several cytokines, including IL-2, IL-4, IL-6, IL-7, IL-12, and IL-15, can induce the proliferation of resting NK cells; IL-2 and IL-15 appear to be the most potent primary NK cell mitogens (16, 17, 18, 19). Optimal proliferation of NK cells, however, requires cell contact-dependent signals in addition to primary mitogenic stimuli (16, 17, 19, 20). The CD28/B7 receptor/ligand system has been implicated in the costimulation of murine NK cells (21). Costimulation of human NK cells, in contrast, does not appear to involve receptors, including CD2, CD27, CD28, CD29, or LFA-1 (16, 17), that have been shown to costimulate T lymphocytes.
Resting human NK cells can be divided into phenotypically and
functionally distinct subpopulations based on expression of the CD56
and CD16 molecules (22, 23, 24, 25). The great majority of peripheral blood NK
cells expresses low levels of CD56 but abundant CD16. These
CD56dim CD16bright NK cells have potent
cytolytic activity but proliferate poorly in response to primary
mitogenic cytokines. In contrast, CD56bright
CD16neg/dim lymphocytes constitute 10% of peripheral
blood NK cells and exhibit relatively poor cytolytic activity in the
absence of exogenous cytokines. Nevertheless, CD56bright NK
cells proliferate vigorously in response to IL-2 or IL-15 alone
(16, 17, 18, 23, 24, 25). The developmental relationship between the
CD56bright and CD56dim NK cell subsets has not
been fully elucidated.
In efforts to identify and characterize receptors mediating cell
contact-dependent costimulation we have produced new mAb reactive with
human NK cells. NKH3, a murine mAb of the IgA class, was found to
recognize the CD94 Ag. NKH3 immunoprecipitates heterodimers composed of
CD94 and multiple 40- to 45-kDa species from human NK cells. Ligation
of the CD94 receptor complex by NKH3 costimulates proliferation and
IFN- secretion by CD56bright NK cells cultured with IL-2
or IL-15. In contrast, NKH3 had no consistent effect on proliferation
or cytokine production by resting CD56dim NK cells. These
studies indicate that the CD94 receptor complex mediates costimulation
of CD56bright NK cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Highly purified human NK cells were isolated by flow cytometry and cultured with 20% LCM and 1 µM ionomycin as previously described (16, 17). After growth in culture for 14 to 28 days, polyclonal expanded NK cells were pelleted, resuspended in sterile PBS, and injected i.p. into female BALB/c mice. Splenocytes from immunized mice were fused with NS-1/1-Ag4-1 myeloma cells using polyethylene glycol by methods previously described (26). Supernatants from several of the resulting hybridomas were strongly reactive with activated NK cells, but weakly reactive or nonreactive with resting T cells. The supernatant from one of these hybridomas, 3B8-0991-1, was subcloned twice by limiting dilution; the mAb produced by the subcloned hybridoma was named NKH3. Indirect staining of reactive cells using NKH3 and FITC-conjugated class- and subclass-specific goat anti-mouse Ig demonstrated NKH3 to be of the IgA class. Ascites was produced by i.p. injection of subcloned hybridoma cells into BALB/c mice.
Polyclonal expanded NK cells were radiolabeled with
125I, lysed, and immunoprecipitated using NKH3. NKH3
immunoprecipitates contained radiolabeled species migrating at 70
kDa under nonreducing conditions and 40 to 45 kDa under reducing
conditions, suggesting that NKH3 recognizes the CD94 receptor complex.
This was confirmed using the CD94 mAb HP-3B1 and Jurkat cells
transfected with a cDNA encoding human CD94 (both reagents provided by
Dr. Miguel Lopez-Botet, Madrid, Spain). NKH3 and HP-3B1 both react with
CD94-transfected Jurkat cells but not parental Jurkat cells. Reciprocal
competition of NK cell staining by NKH3 and HP-3B1 was observed in flow
cytometry experiments, indicating that these mAb recognize the same or
closely related epitopes. Furthermore, preclearing of NK cell lysates
with NKH3, but not control IgA mAb, removed radiolabeled species that
could be immunoprecipitated by HP-3B1. NKH3 was formally assigned to
the CD94 cluster at the Sixth International Workshop on Human Leukocyte
Differentiation Ags in November 1996.
Other mAb
Fluorochrome-conjugated murine mAb, including CD3 (IgG1), NKH1
(CD56, IgG1), and CD16 (IgG1) as well as purified MY4 (CD14, IgG2b)
were obtained from Coulter Immunology (Hialeah, FL). FITC- and
phycoerythrin-conjugated class- and subclass-specific goat
anti-mouse Ig secondary Ab were purchased from Southern
Biotechnology (Birmingham, AL). T1/24T6G12 (CD5, IgG2a) and T3/RW2
(CD3, IgG1) were used as dilutions of ascites. Murine mAb HP-3B1 (CD94,
IgG2a) (27) was provided by Dr. Miguel Lopez-Botet (Madrid, Spain).
Murine mAb Z199 (anti-NKG2-A/B, IgG2b) (28, 29) was provided by
Drs. Lorenzo Moretta and Alessandro Moretta (Genova, Italy). Control
murine IgA 
ascites (TEPC 15) was purchased from Sigma Chemical Co.
(St. Louis, MO).
Cytokines and reagents
LCM was prepared as previously described (16, 17). Recombinant
human IL-2 (sp. act., 1.05 x 107 U/mg) was
provided by Amgen (Thousand Oaks, CA), and recombinant human IL-12 (sp.
act., 5.2 x 106 U/mg) was supplied by Genetics
Institute (Cambridge, MA). Recombinant simian IL-15 was purchased from
Genzyme (Cambridge, MA), and ionomycin was obtained from
Calbiochem-Behring (La Jolla, CA). IFN- ELISA kits were purchased
from Endogen (Cambridge, MA) and used according to the manufacturer’s
instructions. Culture supernatants to be assayed for IFN- were
collected at either 72 or 96 h of culture and stored at -80°C
before use.
Isolation and culture of human NK cells
PBMC were isolated by Ficoll-diatrizoate density gradient centrifugation from cytopheresis buffy coats obtained from normal volunteer donors. Adherent mononuclear cells were depleted by incubation on sterile scrubbed nylon wool columns for 60 min at 37°C. Enriched NK cells were obtained by negative selection as described previously (30), using immunomagnetic beads and T3/RW2 (CD3) with or without T1/24T6G12 (CD5) and MY4 (CD14). Highly purified CD56bright and CD56dim NK subsets were isolated from populations of enriched NK cells by cell sorting as previously described (25). Basal culture medium was RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 µg/ml streptomycin, 100 µg/ml gentamicin, and 15% heat-inactivated FCS. Activation and in vitro expansion of NK cells using LCM and ionomycin were performed as previously described (16, 17). Polyclonal NK cell cultures were maintained at cell concentrations of 1 to 2 x 106/ml by addition of basal medium supplemented with LCM.
Cell lines
K562 cells were obtained from the American Type Culture Collection (Rockville, MD) and maintained in basal culture medium. NKL, a neoplastic human NK cell line (31), was maintained in basal culture medium supplemented with 50 U/ml of IL-2.
Immunofluorescence studies
Cells (0.5–1 x 106 cells/sample) were stained either directly or indirectly with fluorochrome-conjugated mAb, washed, fixed in 1% formalin, and analyzed by flow cytometry as previously described (32).
Proliferation assays
Sorted NK cells were plated at 30,000 cells/well (1.5 x
105 cells/ml) in 96-well microtiter plates (Flow
Laboratories, McLean, VA) with the indicated cytokines and/or mAb. The
final dilution of mAb-containing ascites added to the cultures was
1/400. In some experiments sorted NK cells were cultured with
irradiated (10,000 cGy) K562 stimulator cells at an NK cell:stimulator
cell ratio of 5:1. Unless otherwise stated, freshly sorted NK cells
were cultured for a total of 5 days, and [3H]TdR (1
µCi/well) was added for the last 16 h of culture. In some
experiments sorted CD56dim and CD56bright NK
cells were expanded in vitro for 6 to 8 days using LCM and ionomycin,
washed, plated at 30,000 cells/well in 96-well microtiter plates, and
incubated in proliferation assays for 48 to 96 h.
[3H]TdR incorporation was measured using a 1205 Betaplate
liquid scintillation counter (Pharmacia, Turku, Finland) after
collecting samples with a 96 Mach II harvester (Tomtec, Orange, CA).
The results presented in Figures 3 to 5
are the mean ± SD counts
per minute from triplicate wells.
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Iodinations were performed by the lactoperoxidase method using 1.0 mCi of 125I/107 cells. Whole cell lysates were prepared from radiolabeled cells using 1% Nonidet P-40 lysis buffer as previously described (33). Immunoprecipitations and gel electrophoresis experiments were performed essentially as previously described (33). Two-dimensional IEF/SDS-PAGE gels were run with an IEF gradient of pH 4 to 8. For glycanase treatments, immunoprecipitates were treated with N-glycanase (Genzyme) according to the manufacturer’s instructions. Control treatments were performed under identical conditions without addition of enzyme.
Statistical analysis
Calculations of the means, SEs, and p values from paired Student’s t test were performed on a Macintosh Duo Dock computer (Apple, Cupertino, CA) using the StatView software program (Brainpower, Calabasas, CA) according to the manufacturer’s instructions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NKH3 is a murine mAb of the IgA class that reacts with the human
CD94 Ag (see Materials and Methods). NKH3
immunoprecipitates from NK cell lysates treated without glycanases
(Fig. 1) contain heterodimeric complexes
composed of a 25-kDa glycoprotein associated with several 40- to
45-kDa molecules. Slightly different patterns of electrophoretic
mobility were seen when comparing the monoclonal leukemic cell line NKL
(Fig. 1A) with polyclonal preparations of NK cells
from normal donors (Fig. 1B). The 25-kDa
glycoprotein corresponds to the protein encoded by the CD94 cDNA,
whereas at least some of the 40- to 45-kDa molecules appear to be
members of the NKG2 family of C-type lectins (29, 34, 35).
N-glycanase treatment of NKH3 immunoprecipitates from NKL
cell lysates yielded a 21-kDa protein and predominant 26- to
30-kDa species; the results of O-glycanase treatment were
not significantly different from those of control treatment (data not
shown). Persistence after deglycosylation of several species with
higher apparent molecular masses than the predominant 26- to 30-kDa
species (data not shown) suggests that different NKG2 family members
(or related molecules) may form heterodimers with CD94 in a clonal NK
cell population. The multiple species revealed by two-dimensional
IEF/SDS-PAGE may thus reflect both variable glycosylation and different
peptide sequences of the CD94-associated molecules.
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B). Furthermore, the
level of CD94 expression on CD56bright
CD16dim/neg NK cells is clearly higher than that detected
on most CD56dim CD16bright NK cells (Fig. 2).
Following in vitro activation, however, both the
CD56bright and CD56dim NK subsets express high
levels of CD94 (data not shown).
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C). These results suggest that virtually
all CD56bright NK cells, but only subsets of
CD56dim NK cells, express CD94/NKG2-A (p43) receptor
complexes. Moreover, it seems likely that some Z199-negative
CD56dim NK cells express previously described CD94/p39
receptor complexes, which appear to deliver activatory signals for NK
cytolysis (36). Costimulation of CD56bright NK cell proliferation by ligation of CD94
Effects of CD94 mAb on the cytolytic function of activated
populations of NK cells, including NK cell clones, have been
extensively examined (27, 28, 29, 37, 38). However, published studies do
not describe the effects of CD94 ligation on the proliferation of
resting NK cells. The phenotypically distinct
CD56bright and CD56dim NK cell subsets can
be distinguished functionally by their disparate proliferative
responses to cytokines. To address the potential role of CD94 in the
regulation of NK cell proliferation, resting NK cells were sorted into
pure CD56bright and CD56dim subsets and
cultured with various concentrations of IL-2 or IL-15 in the presence
or the absence of the NKH3 mAb. Ligation of CD94 augmented the
proliferation of CD56bright NK cells in response to IL-2 or
IL-15 by as much as 10-fold (Fig. 3).
CD94-mediated costimulation of CD56bright NK cells was very
reproducible: NKH3 enhanced IL-2-induced proliferation of
CD56bright NK cells by 4 ± 1-fold (mean ± SE;
range, 2- to 10-fold) in 15 separate experiments. The greatest degree
of costimulation was typically seen at suboptimal concentrations of
IL-2 (Fig. 3B and data not shown). Nevertheless, the
increase in proliferation of CD56bright NK cells in the
presence of NKH3 compared with control IgA mAb was highly statistically
significant for all concentrations of IL-2 and IL-15 tested
(p < 0.0005 for 10, 100, or 1000 pM IL-2;
p 
0.01 for 2.5 ng/ml; and p 0.025
for 5 and 10 ng/ml IL-15). Costimulation of CD56bright NK
cell proliferation was also observed using the HP-3B1 mAb (data not
shown), confirming that costimulation was due to CD94 ligation and was
not an idiosyncratic effect of the NKH3 mAb.
As previously described (16, 23, 24, 25), the CD56dim NK cell
subset proliferates poorly in response to IL-2 alone; the maximum
proliferative response of CD56dim NK cells is, on the
average, 10% of that seen with the CD56bright subset.
Ligation of CD94 by NKH3 had no consistent costimulatory effect on
proliferation of CD56dim NK cells in response to IL-2
(Table I). Indeed, in 8 of 15 normal
donors tested, NKH3 modestly inhibited (by 40 ± 10%) the
proliferation of CD56dim NK cells in response to 1000 pM
IL-2. This inhibitory effect was inconsistent, however; it varied among
different donors (Table I) and according to the concentration of IL-2
used in the proliferation assay (data not shown). Moreover, in four
separate experiments CD94 mAb had no significant stimulatory or
inhibitory effect on proliferation of CD56dim NK cells in
response to IL-15 (p > 0.1 for 2.5 ng/ml,
p > 0.05 for 5 ng/ml, and p > 0.4 for
10 ng/ml IL-15).
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We have previously reported that the optimal proliferation of
resting CD56dim NK cells requires both mitogenic
cytokines and cell contact-dependent costimulatory signals. In our
experimental system K562 cells most consistently provide
contact-dependent costimulation, although activated T lymphocytes are
also effective (16, 17). In contrast to CD94 mAb, irradiated K562 cells
strongly costimulated the proliferation of CD56dim NK cells
in response to IL-2 (Fig. 4A). K562-mediated
costimulation of CD56bright NK cells is also observed (Fig. 4B). CD94 mAb appeared to have either no major effect
(Fig. 4B) or a partial inhibitory effect (Fig. 4A) on proliferation of NK cells costimulated with
K562. The modest inhibitory effect (30–40% inhibition) of CD94 mAb
on IL-2-induced proliferation of CD56dim NK cells proved to
be statistically significant in multiple experiments (Table II and data not shown). Taken together,
our data indicate that the costimulation mediated by CD94 ligation is
distinct from that mediated by contact with K562.
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) and
CD56dim (data not shown) NK cells, extending previous
findings. In multiple experiments CD94 mAb significantly inhibited the
proliferation of preactivated NK cells in response to 10 pM
(p 0.025), 100 pM (p
0.005), and 1000 pM (p 0.01) IL-2.
Ligation of CD16 triggered the apoptosis of IL-2-activated, but not
resting, NK cells (39, 40). Similarly, ligation of CD94 selectively
induced the apoptosis of preactivated NK cells (41). Further
investigation is required to determine the relative contributions of
apoptosis, autolysis, and other processes to CD94-mediated inhibition
of activated NK cell proliferation. CD94-mediated costimulation of cytokine secretion by CD56bright NK cells
In addition to mediating ADCC and natural killing, NK cells can
regulate other cell types through the production of proinflammatory and
immunoregulatory cytokines (3, 4, 5, 6, 7, 8). To determine whether ligation of
CD94 could costimulate NK cell cytokine production, sorted
CD56bright and CD56dim NK cells were
cultured with IL-2 or IL-15 in the presence or the absence of CD94 mAb.
CD56bright NK cells cultured with IL-2 and NKH3 secreted 2-
to 10-fold more IFN- than CD56bright NK cells cultured
with IL-2 alone (Fig. 6). In contrast,
CD94 mAb had no consistent effect on IFN- production by
CD56dim cells (data not shown). Similar results were
obtained with IL-15, although in repeated experiments IL-15 was
reproducibly less potent than IL-2 in stimulating IFN- secretion
(data not shown). IL-2 and IL-12 synergistically stimulated IFN-
secretion by CD56bright NK cells, as expected; modestly
higher levels of IFN- were produced in the presence of CD94 mAb
(data not shown). In contrast, IL-12 potently inhibited IL-2-induced
proliferation of CD56bright NK cells, as previously
described (30). Ligation of CD94 did not significantly affect
CD56bright NK cell proliferation in response to IL-2 plus
IL-12 (p > 0.1 in three separate experiments).
Thus, augmented IFN- secretion in the presence of CD94 mAb is
not simply due to increased NK cell number as a result of
costimulation of proliferation. Moreover, ligation of CD94 does not
appear to overcome the inhibitory effect of IL-12 on IL-2-induced
proliferation of resting human NK cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Avoidance of autoimmune tissue damage by NK cells would appear to require positive selection, to assure that each NK cell expresses at least one inhibitory receptor for an autologous MHC class I allotype, and/or negative selection, to eliminate NK cells expressing activatory receptors for the products of syngeneic class I alleles. Nevertheless, the mechanisms by which the distinct NK cell repertoires of different individuals are generated in vivo remain obscure. In one plausible model of positive NK cell selection, ligation of class I receptors that are destined to inhibit the cytolytic activity of mature NK cells delivers positive signals for viability and/or proliferation of immature NK cell precursors. Thus, NK cell progenitors expressing inhibitory receptors for self class I allotypes would preferentially survive and expand in vivo. Experimental data to support this hypothesis, however, have been lacking.
Our studies provide direct evidence that putative MHC class I receptors can deliver positive signals for NK cell proliferation. Ligation of CD94 strongly augmented the proliferation of CD56bright NK cells, but not that of CD56dim NK cells, in response to either IL-2 or IL-15. It has been speculated that CD56bright CD16dim/neg lymphocytes are immature NK progenitors that differentiate into mature CD56dim CD16bright NK cells in vivo (22, 23). The high proliferative potential of CD56bright NK cells as well as their lack of cytoplasmic granules and poor cytolytic activity when freshly isolated tend to support this hypothesis. Class I molecules on adjacent cells could selectively costimulate CD56bright NK cells expressing CD94 receptors specific for syngeneic class I allotypes, allowing preferential expansion of self MHC-specific NK cells in vivo. IL-15 is produced by bone marrow and thymic epithelial stromal cells and has been implicated in the proliferation and differentiation of NK cell progenitors (51, 52). In the presence of limiting concentrations of IL-15, ligation of class I receptors might be required to preserve the viability of immature NK cells, leading to the death of NK progenitors lacking at least one receptor for a self class I allotype.
Bottino et al. (53) have identified another potential costimulatory receptor, p50.3/PAX, on resting human NK cells. p50.3 appears to be closely related to p50.1 and p50.2, previously characterized MHC class I receptors that can activate the cytolytic activity of human NK cells. Unlike CD94, p50.3 appears to be expressed on only a subset of NK cells in a minority of healthy donors. Substantial increases in the percentage of p50.3-positive cells were observed after unfractionated PBMC were cultured with anti-p50.3 mAb and IL-2, suggesting that ligation of p50.3 can costimulate IL-2-induced proliferation of some NK cells. However, the effects of anti-p50.3 mAb on the proliferation of highly purified NK cells have not been reported (53). Moreover, the ligand for p50.3 has not been defined; data supporting a role for p50.3 in the recognition of class I molecules were not found. Thus, there is currently little evidence to suggest a similar role for p50.3 and the CD94 receptor complex in the biology of human NK cells.
Ligation of CD94 has been reported to inhibit, activate, or have no
effect on the cytolytic activity of different NK clones (28, 36, 38, 50). Furthermore, CD94 has been implicated in the recognition of
different HLA-A, -B, and -C allotypes (28, 48, 49). The CD94 gene
encodes an invariant protein with an extremely short cytoplasmic
domain, however, belying the pleiotropic functional effects of CD94
ligation (54). The CD94 receptor complex has been found to include
43- and 39-kDa glycoproteins covalently linked to invariant CD94
molecules of 25 kDa (34, 36, 49). The cytolytic activity of NK
clones expressing predominantly CD94/p43 heterodimers is inhibited by
CD94 ligation, whereas NK clones expressing predominantly CD94/p39
heterodimers are activated (36). NKG2-A/B glycoproteins have recently
been shown to constitute at least some of the 43-kDa molecules that are
covalently associated with CD94 (29, 34, 35). NKG-2A/B, NKG2-C, NKG2-D,
and NKG2-E are related members of a family of C-type lectins (34, 55).
Like the p58 and p70 killer inhibitory receptor molecules, NKG2-A/B
possess cytoplasmic ITIM; NKG2-C, NKG2-D, and NKG2-E lack ITIM in their
cytoplasmic domains. Ligation of chimeric NKR-P1C/NKG2-A receptors
expressed in a rat NK cell line inhibited cytolytic activity, whereas
ligation of chimeric NKR-P1C/NKG2-C receptors activated cytotoxicity
(56). Thus, CD94/NKG2-A heterodimers may constitute inhibitory
receptors, and CD94 molecules disulfide-linked to NKG2-C, -D, and/or -E
may constitute activatory receptors for human NK cell cytotoxicity.
Nevertheless, association of NKG2-C, -D, or -E molecules with CD94 on
the surface of human NK cells has yet to be directly demonstrated.
Moreover, participation of 43-kDa species other than NKG2-A/B in
functional CD94 receptor complexes has not been excluded. Our
biochemical analysis of NKH3 immunoprecipitates suggests that several
different 40- to 45-kDa species may form heterodimers with CD94 in
NK cells from an individual donor. Whether any of these molecules
differs from NKG2-A or- B in amino acid sequence remains to be
determined.
Investigation of the effects of NKH3 on resting CD56bright and CD56dim NK cells was essential for elucidating the role of CD94 as a costimulatory receptor. Although virtually all human NK cells express CD94, NKH3 has no major effect on the proliferation of unfractionated resting NK cells (our unpublished data). This is probably because the CD56dim subset generally constitutes >90% of peripheral blood NK cells, and ligation of CD94 does not consistently affect the proliferation of polyclonal, resting CD56dim NK cells. Expression of different CD94/NKG2 heterodimers and/or associated signaling molecules by CD56bright and CD56dim NK cells could be responsible for the differential responses of these subsets to CD94 ligation. Our flow cytometry results using the Z199 mAb suggest that CD56bright NK cells uniformly express CD94/NKG2-A receptors, whereas the latter are expressed by only a subset of CD56dim NK cells. Since virtually all CD56dim NK cells express CD94, Z199-negative CD56dim NK cells may express other NKG2 species in covalent association with CD94. Expression of different CD94/NKG2-A, -B, -C, -D, or -E receptors by individual NK cells within the CD56dim population might underlie the inconsistent effects of CD94 mAb on the proliferation of CD56dim NK cells that we observed. Moreover, we cannot exclude the possibility that some CD56dim NK cells, perhaps those expressing a CD94/NKG2 phenotype identical with that of CD56bright NK cells, can be strongly costimulated by CD94 ligation. However, in two preliminary experiments we observed no substantial difference in response to CD94 ligation of sorted CD56dim Z199-positive vs CD56dim Z199-negative NK cells (our unpublished data). Further studies will be required to address these issues. Such studies will be complicated by the fact that CD94 mAb strongly inhibit the proliferation of CD56bright or CD56dim NK cells that have been preactivated in vitro. NK clones have been crucial reagents for studying the complex role of CD94 in the regulation of NK cell cytotoxicity (28, 36, 38, 50). Since NK clones are activated in vitro by the conditions required for their long term maintenance, however, it seems unlikely that NK clones will prove as useful in investigations of CD94-mediated costimulation.
Ligation of CD94 also costimulates IFN- secretion by
CD56bright NK cells cultured with IL-2 or IL-15. CD94
ligation has been reported to augment the secretion of IFN- and
TNF-
by polyclonal expanded NK cells (8, 38). Nevertheless,
selective CD94-mediated costimulation of IFN- secretion by resting
CD56bright NK cells has not been previously described.
IFN- production appears to be an important function of NK cells
during early immune responses to cells infected with obligate
intracellular pathogens (1, 5). Paracrine stimulation of NK cells by
monocyte-derived IL-12 and IL-15, and reciprocal monocyte stimulation
by IFN-, TNF-, and granulocyte-macrophage CSF secreted by
activated NK cells may augment the innate immune response to these
pathogens as well as regulate the differentiation of activated CD4 T
cells into helper effector cells. Furthermore, IL-2 secreted by
activated CD4 T cells may further augment IFN- production by NK
cells, especially in concert with IL-12 and/or CD94 ligation. Thus,
CD56bright NK cells may play an immunoregulatory role
during innate and adaptive immune responses to infected or neoplastic
cells.
Proliferation of CD56dim NK cells in response to IL-2 or IL-15 is costimulated by the presence of irradiated K562 cells, but is not consistently costimulated by ligation of CD94. Thus, CD94 does not appear to mediate the contact-dependent NK cell costimulation that we have previously described (16, 17). Further work is necessary to identify the putative costimulatory receptors expressed by CD56dim NK cells. Nevertheless, our data indicate that CD56bright and CD56dim NK cells can be costimulated through different receptors, which may allow these distinct NK cell subsets to be independently regulated in vivo.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael J. Robertson, Bone Marrow Transplant Program, Division of Hematology/Oncology, Indiana University Cancer Research Institute, 1044 W. Walnut St., Room R4–202, Indianapolis, IN 46202.
3 Abbreviations used in this paper: ADCC, Ab-dependent cell-mediated cytotoxicity; dim, low level; bright, high level; neg, absent; LCM, leukocyte-conditioned medium; IEF, isoelectric focusing; ITIM, immunoreceptor tyrosine-based inhibitory motif.
Received for publication March 28, 1997. Accepted for publication October 24, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by NK cells: a role for IL-1ß in the T cell-independent mechanism of resistance against intracellular pathogens. J. Immunol. 155:4347.[Abstract]
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