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The Journal of Immunology, 1998, 160: 1618-1626.
Copyright © 1998 by The American Association of Immunologists

Participation of the CD94 Receptor Complex in Costimulation of Human Natural Killer Cells1

Stephan D. Voss{dagger}, John Daley*, Jerome Ritz* and Michael J. Robertson2,*,{ddagger}

* Division of Hematologic Malignancies, Dana-Farber Cancer Institute; Department of Medicine, Harvard Medical School; {dagger} Department of Radiology, Beth Israel Deaconess Medical Center, Boston, MA 02115; {ddagger} Bone Marrow Transplant Program, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Optimal proliferation and expansion of human NK cells require mitogenic cytokines together with cell contact-dependent costimulation. Production of mAb that can modulate human NK cell proliferation yielded NKH3, which recognizes the CD94 Ag. NKH3 immunoprecipitates contain ~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-{gamma} 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-{gamma} 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
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NK cells are a distinct subpopulation of lymphocytes that plays an important role in natural immunity against viruses and other obligate intracellular pathogens (1, 2). Activated NK cells secrete several immunomodulatory cytokines, including IFN-{gamma} (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 (Fc{gamma}RIIIA) noncovalently associated with homodimers and heterodimers of the {zeta} 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-{gamma} 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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation and characterization of NKH3 mAb

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 {kappa} 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-{gamma} ELISA kits were purchased from Endogen (Cambridge, MA) and used according to the manufacturer’s instructions. Culture supernatants to be assayed for IFN-{gamma} 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 5GoGoGo are the mean ± SD counts per minute from triplicate wells.



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  		FIGURE 3. Costimulatory effect of CD94 ligation on proliferation of CD56bright NK cells. Resting cell-sorted CD56bright NK cells were cultured in various concentrations of IL-15 (A) or IL-2 (B) in the presence of NKH3 (solid bars) or control IgA mAb (hatched bars). The results shown are representative of data obtained in five (A) or 15 (B) separate experiments. Similar relative results with lower absolute levels of proliferation were obtained after 24, 48, and 72 h of culture with IL-2 (data not shown).

 


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  		FIGURE 4. Effect of CD94 ligation on K562-mediated costimulation of NK cell proliferation. Resting cell-sorted CD56dim (A) and CD56bright (B) NK cells were cultured in various concentrations of IL-2 in the presence of control IgA mAb only (open bars), control IgA mAb plus irradiated K562 cells (hatched bars), or NKH3 and irradiated K562 cells (solid bars).

 


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  		FIGURE 5. Inhibitory effect of CD94 ligation on proliferation of previously activated CD56bright NK cells. Resting cell-sorted CD56bright NK cells were expanded in vitro for 8 days with LCM and ionomycin as previously described (16, 17). Preactivated cells were then cultured for 96 h in various concentrations of IL-2 in the presence of NKH3 (solid bars) or control IgA mAb (hatched bars). Results shown are representative of data obtained in four separate experiments. Similar results were obtained using preactivated CD56dim NK cells.

 
Iodination, immunoprecipitation, and gel electrophoresis

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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Differential expression of CD94 receptor complexes on CD56bright and CD56dim NK cells

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. 1Go) 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. 1GoA) with polyclonal preparations of NK cells from normal donors (Fig. 1GoB). 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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  		FIGURE 1. Biochemical characterization of the CD94 receptor complex on human NK cells. Cell surface iodinated NKL cells (A) and polyclonal normal NK cells (B) were solubilized, immunoprecipitated with NKH3, and subjected to two-dimensional IEF/SDS-PAGE. One-dimensional IEF gels are oriented acidic to basic (left to right). Standard m.w. markers are indicated for the second dimension. The small arrow at the top indicates the position of an internal standard, bovine muscle actin (pI 5.3–5.4). Identical results were obtained with the CD94 mAb HP-3B1 (data not shown). No radiolabeled species were identified on autoradiographs of gels from control IgA mAb immunoprecipitates (data not shown). Gels shown are representative of six separate experiments.

 
Previous studies suggested that CD94 is expressed on only a fraction of resting human NK cells (27). However, published two-color immunofluorescence histograms do not reveal clearly distinct CD94-positive and -negative subpopulations of CD56+ or CD16+ PBL. It is therefore possible that CD94 is actually expressed by all NK cells, but that the intensity of CD94 staining for some resting NK cells is too low to resolve from background fluorescence by routine flow cytometry. Indeed, we found that NKH3 reacts with virtually all resting peripheral blood NK cells from healthy donors (Fig. 2GoB). 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. 2Go). Following in vitro activation, however, both the CD56bright and CD56dim NK subsets express high levels of CD94 (data not shown).



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  		FIGURE 2. Expression of CD94 receptor complexes on resting NK cell subsets. Enriched NK cells prepared from PBMC of normal donors, as described in Materials and Methods, were stained with FITC-conjugated CD16 (A), unconjugated NKH3 followed by secondary staining with FITC-labeled goat anti-mouse IgA (B), or unconjugated Z199 followed by secondary staining with FITC-labeled goat anti-mouse IgG2b and phycoerythrin-conjugated NKH1 (CD56). Logarithm of red fluorescence is displayed on the abscissa, and logarithm of green fluorescence is presented on the ordinate. The results shown are representative of data from 15 separate experiments (A and B) or 9 separate experiments (C).

 
The Moretta group has developed murine mAb, Z199 and Z270, that preferentially react with NK clones bearing CD94/p43 receptor complexes that can inhibit NK cell cytolytic activity upon ligation by CD94 mAb (28). Z199 appears to react with the human NKG2-A molecule regardless of whether the latter is associated with CD94 (29). We found that Z199 reacts strongly with resting CD56bright NK cells, whereas CD56dim NK cells are heterogeneously stained by Z199 (Fig. 2GoC). 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. 3Go). 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. 3GoB 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 IGo). 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 IGo) 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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  		Table I. Effects of CD94 ligation on proliferation of CD56bright and CD56dim NK cells from different healthy donors

 
Costimulation mediated by CD94 ligation and by contact with K562 are distinct

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. 4GoA). K562-mediated costimulation of CD56bright NK cells is also observed (Fig. 4GoB). CD94 mAb appeared to have either no major effect (Fig. 4GoB) or a partial inhibitory effect (Fig. 4GoA) 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 IIGo 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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  		Table II. Effects of K562 and CD94 ligation on proliferation of CD56dim NK cellsa

 
CD94 ligation has been shown to inhibit the proliferation of unsorted NK cells that were previously activated and then recultured in presence of IL-2 (27). This was postulated to be the result of an autolytic or "fratricidal" process, whereby NK cells binding CD94 mAb were induced to lyse adjacent autologous NK cells (37). Resting NK cells were sorted into CD56bright and CD56dim populations, activated and expanded in vitro with LCM and ionomycin, and then recultured in the presence of IL-2. Ligation of CD94 strongly inhibited the IL-2-induced proliferation of expanded CD56bright (Fig. 5Go) 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-{gamma} than CD56bright NK cells cultured with IL-2 alone (Fig. 6Go). In contrast, CD94 mAb had no consistent effect on IFN-{gamma} 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-{gamma} secretion (data not shown). IL-2 and IL-12 synergistically stimulated IFN-{gamma} secretion by CD56bright NK cells, as expected; modestly higher levels of IFN-{gamma} 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-{gamma} 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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  		FIGURE 6. Costimulatory effect of CD94 ligation on IFN-{gamma} secretion by CD56bright NK cells. Resting cell-sorted CD56bright NK cells were cultured for 72 to 96 h in various concentrations of IL-2 in the presence of NKH3 (solid bars) or control IgA mAb (hatched bars). Aliquots of supernatant were removed and tested for IFN-{gamma} as described in Materials and Methods. Results from two separate experiments are shown. Negligible levels of IFN-{gamma} were detected in supernatants of CD56dim NK cells cultured in parallel (data not shown). Proliferative responses were comparable to those shown in Figure 3Go.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Human NK cells express several receptors that can recognize specific MHC class I allotypes. The killer cell inhibitory receptors, type I glycoproteins belonging to the Ig superfamily, have been the most extensively characterized of these receptors (15). p58.1 (EB6, CD158a), and p58.2 (GL183, CD158b) possess immunoreceptor tyrosine-based inhibitory motifs (ITIM) in the cytoplasmic region and transmit dominant inhibitory signals after ligation by certain HLA-C molecules (15, 42, 43). p70 (NKB1) and p140 (Q66/NKAT4) also have cytoplasmic ITIM and inhibit lysis of target cells expressing specific HLA-B and HLA-A alleles, respectively (44, 45). Activatory variants of several human NK cell receptors for MHC class I molecules have also been described (46, 47). The extracellular domains of p50.1 and p50.2 are almost identical with p58.1 and p58.2, respectively, but the p50 receptors lack cytoplasmic ITIM and trigger, rather than inhibit, NK cytolytic function. The CD94 receptor complex, which has been implicated in the recognition of certain HLA-A, -B, and -C allotypes (28, 48, 49), can also transduce either positive or negative signals for NK cytotoxicity (28, 36, 38, 50).

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-{gamma} secretion by CD56bright NK cells cultured with IL-2 or IL-15. CD94 ligation has been reported to augment the secretion of IFN-{gamma} and TNF-{alpha} by polyclonal expanded NK cells (8, 38). Nevertheless, selective CD94-mediated costimulation of IFN-{gamma} secretion by resting CD56bright NK cells has not been previously described. IFN-{gamma} 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-{gamma}, TNF-{alpha}, 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-{gamma} 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.


   Acknowledgments
 
We thank Dr. Miguel Lopez-Botet for providing CD94 transfectants and the HP-3B1 mAb, Drs. Alessandro and Lorenzo Moretta for providing the Z199 mAb, Brian Williams, Christopher Pickett, and Christine Cameron for technical assistance, and Jared Gollob for helpful discussions.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Radiologic Oncology Training Grant T32CA59367–01A1 (to S.D.V.), National Institutes of Health Grant CA41619 (to J.R.), and a grant from the Coulter Corp. (to M.J.R.). Back

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. Back

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. Back

Received for publication March 28, 1997. Accepted for publication October 24, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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