Heterogeneity in the ability of cytotoxic murine NK cell clones to enhance Ig secretion in vitro

Quirijn Vos, Clifford M. Snapper1 and James J. Mond

1 Departments of Medicine and Pathology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, USA

Correspondence to: Q. Vos


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We recently described a panel of cytotoxic murine NK cell clones that also enhanced Ig secretion by B cells activated in an in vitro model of T cell-independent type 2 (TI-2) responses. We employed dextran-conjugated anti-IgD ({alpha}{delta}-dex) as a model antigen. Here we study the mechanism of Ig induction by these clones. Addition of the various NK clones to sort-purified B cells stimulated with {alpha}{delta}-dex and IL-2 resulted in a markedly heterogeneous increase in Ig secretion, which varied from 3-fold, as mediated by clone PKO 56, to 15-fold, as induced by clone PKO 101. The other NK cells showed intermediate levels of Ig induction. Furthermore, while addition of as few as 0.04% of PKO 101 cells stimulated significant increases and 1% induced near maximum Ig production, a 3% addition of PKO 56 cells was required for significant enhancement of Ig secretion. Supernatant material collected from the NK clones mediated Ig production at levels that mirrored the induction by the corresponding cells. Cytokine analysis showed that while all members of the NK panel produced IFN-{gamma}, only two secreted granulocyte macrophage colony stimulating factor and that the levels of Ig induction mediated by the NK clones correlated only with their levels of IFN-{gamma} secretion. Culture of B and NK cells in the presence of anti-IFN-{gamma} demonstrated that IFN-{gamma} was the critical cytokine in NK-induced Ig production. These findings establish heterogeneity in the ability of NK cells to increase Ig secretion in vitro and show that NK-produced IFN-{gamma} is an important factor in determining this heterogeneity.

Keywords: B cells, IFN-{gamma}, Ig secretion, NK cells, TI-2 response


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
T cell-independent type 2 (TI-2) antigens induce Ig secretion by mechanisms which are only partly understood. These antigens possess multiple repeating antigenic epitopes that mediate extensive cross-linking of antigen receptors and can thereby activate B cells in the absence of T cells (13). TI-2 antigen induces specific antibodies in nude mice (4) and in mice that lack MHC class II antigens (5), demonstrating a pathway of induction of Ig secretion that does not require T cells or other sources of MHC class II-restricted help. Studies employing dextran-conjugated anti-IgD mAb ({alpha}{delta}-dex) as an in vitro TI-2 model antigen have demonstrated that Ig secretion could be induced in the absence of T cells, but did require cytokine-mediated help (6). These findings suggested that in vivo responses to TI-2 antigens might also require some form of ancillary non-T cell-derived help, that did not follow MHC class II-restricted pathways. A role for such help is further suggested by the observed induction of Ig class switching during in vivo TI-2 responses (4,7). IFN-{gamma} is among the cytokines that may regulate in vivo responses to TI-2 antigens, since it has been shown to induce both Ig secretion (8) and isotype switching (9) in {alpha}{delta}-dex-activated B cells. NK cells are a population of non-T cells that can produce IFN-{gamma} during the onset of bacterial infection (10,11) and upon in vitro activation with IL-2 (12), and that have been implicated in the induction of Ig secretion in vivo (13,14). While involvement of NK cells in immune responses to bacterial (15,16), protozoan (17,18) and viral infections (19,20) has been described, the antigens inducing these NK responses and the mechanism of their recognition by NK cells are largely unknown. Furthermore, the description of phenotypically distinct subsets (2124), that display functional diversity, may reflect a level of heterogeneity among NK cells similar to the ones observed for other lymphocyte populations.

In a previous study of NK cell-mediated B cell activation in an in vitro model of TI-2 responses we have shown that the elimination of asialo-GM1+ cells from T cell-depleted B cell populations resulted in the abrogation of Ig secretion induced by {alpha}{delta}-dex in the presence of IL-2 (25). Moreover, we showed that addition of cells from short-term cultures of NK cells, obtained from scid splenocytes that were activated in vitro with high doses of IL-2, induced Ig secretion in sort-purified B cells (25,26). To extend these findings and to address functional heterogeneity within the NK cell population, we recently established a murine NK cell panel (27). Culture of B and T cell-depleted splenocytes from mice that were homozygous for a mutation in the p53 tumor suppressor gene in the presence of IL-2 and poly(IC) resulted in one long-term parental line, PKO, and four clones, PKO 2, PKO 34, PKO 56 and PKO 101. Culture of spleen cells from normal C57BL/6 mice under similar conditions resulted in one long-term NK line, B6 NK. All NK cells expressed high levels of perforin and a panel of granzymes, and, with the exception of PKO 101, displayed NK cytotoxicity. Here we use this NK panel to analyze the mechanism of NK-mediated enhancement of Ig secretion in B cells in our in vitro TI-2 model. We demonstrate a marked heterogeneity in the ability of the NK clones to enhance Ig secretion in vitro and found that this heterogeneity is reflected in the differences in the production of IFN-{gamma} by these NK cells.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
CBA.C57BL/6 F1 mice were obtained from the Jackson Laboratories (Bar Harbor, ME) and were used at 8–10 weeks of age. The experiments were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals, Institute of Animal Resources, National Research Council, Department of Health, Education and Welfare publication (National Institutes of Health) 78-23.

Culture medium
RPMI 1640 (Biowhittaker, Walkersville, MD) supplemented with 10% heat-inactivated FCS (Gibco, Grand Island, NY), L-glutamine (2 mM), HEPES (25 mM), 2-mercaptoethanol (50 mM), non-essential amino acids (100 mM), penicillin (100 U/ml), pyruvate (1 mM) and streptomycin (100 µg/ml) was used for culturing cells.

Reagents
{alpha}{delta}-dex, of anti-IgD a allotype, H{delta}a/1 (28), and anti-IgD b allotype, AF3.33 (29), specificity were prepared by conjugation of the respective mAb to a high mol. wt dextran (2x106 Da) as previously described (30). For both conjugates approximately six mAb were conjugated to each dextran molecule. The concentration of {alpha}{delta}-dex reflects the amount of mAb and not the entire dextran conjugate.

Murine recombinant IL-2 was a kind gift from Dr Maurice Gately (Hoffmann-La Roche, Nutley, NJ). BSA, FITC and Tween 20 were purchased from Sigma (St Louis, MO). XMG-6 rat anti-mouse IFN-{gamma} of IgG1 isotype (31), J4-1 rat IgG1 isotype-matched control mAb (32) and RA3-6B2 rat anti-mouse B220 (33) were purified from ascites. RA3-6B2 was conjugated to FITC using a standard protocol. Purified mAb and biotinylated mAb specific for IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, granulocyte macrophage colony stimulating factor (GM-CSF), IFN-{gamma} and tumor necrosis factor (TNF)-{alpha}, and corresponding cytokine standards were purchased from PharMingen (San Diego, CA).

Establishment of a panel of murine NK cell clones
The PKO long-term NK line, its clones PKO 2, PKO 34, PKO 56 and PKO 101, and the B6 NK line were generated and maintained as previously described (27).

Preparation and culture of highly purified splenic B cells
CBA.C57BL/6 F1 spleen cells were treated with rat anti-Thy-1.2 (34), followed by complement. Cells were then fractionated by centrifugation over a discontinuous Percoll gradient (Pharmacia Fine Chemicals, Piscataway, NJ) consisting of 70, 50 and 40% Percoll solutions (with densities of 1.086, 1.062 and 1.050 g/ml respectively). The cells were collected from the 70/50% interface, washed in cold clear HBSS staining buffer containing 3% heat-inactivated FCS, penicillin (100 U/ml) and streptomycin (100 µg/ml), and incubated at a density of 107 cells/ml in a 1 µg/ml solution of anti-B220–FITC in staining buffer for 30 min on ice. After one wash the cells were resuspended at a density of 107 cells/ml in staining buffer and electronically sorted on an Epics Elite cytometer (Coulter, Hialeah, FL) for a B220bright phenotype. Reanalysis of sorted cells, that were subsequently stained with anti-IgD–phycoerythrin (PE), consistently revealed the presence of >99% IgD+ B220bright B cells.

Stimulation of highly purified B cells in the presence of NK cells
The highly purified B cells were cultured for 5 days in flat-bottom 96-well plates (Costar). Cultured cells were incubated at 105 cells/ml in a total volume of 200 µl culture media containing IL-2 (50 U/ml), {alpha}{delta}-dex against both a and b haplotype (10 ng/ml), and the indicated percentage of NK cells at 37°C in a humidified atmosphere containing 5% CO2. For the experiments studying the role of IFN-{gamma} in NK-mediated enhancement of Ig secretion, cells were incubated in the presence of 10 µg/ml of XMG-6 anti-IFN-{gamma} mAb or an equal amount of J4-1 isotype-matched control mAb. All experimental groups were established in triplicate and pooled before the quantitation of IgM-secreting cells.

Analysis of the requirement of cellular interaction in NK-mediated induction of Ig secretion in cultures of highly purified B cells
To assess the requirement of cellular interaction in NK-mediated induction of Ig secretion in sort-purified B cells, cell culture was performed in 24-well plates containing inserts with a protein-permeable membrane (Transwell, 0.4 µm pore size; Costar). The lower compartment of these wells received 4x104 sort-purified B cells. NK cells were added either to the lower or the upper compartment as indicated. Culture was performed in a total volume of 800 µl culture media containing IL-2 (50 U/ml) and {alpha}{delta}-dex against both a and b haplotype (10 ng/ml).

Quantitation of secreted cytokine concentrations in murine NK cell culture supernatants
Supernatants were generated by culturing NK cells at a concentration of 106 cells/ml in 24-well plates in a final volume of 1 ml, in the presence of either IL-2 (50 U/ml) or phorbol myristate acetate (PMA; 20 ng/ml) plus ionomycin (1 µM). Concentrations of IL-1, IL-2, IL-3, IL-4, IL-4, IL-6, IL-10, GM-CSF, IFN-{gamma} and TNF-{alpha} were measured by ELISA according to the manufacturer's instructions.

Stimulation of highly purified B cells in the presence of NK culture supernatants
To generate NK cell supernatants under culture conditions closely resembling those encountered in B cell stimulation cultures, NK cells were cultured in 96-well plates containing 200 µl of culture medium supplemented with {alpha}{delta}-dex (10 ng/ml) and IL-2 (50 U/ml). Cells were cultured at the following densities: 10% (2000 cells/well), 3% (600 cells/well), 1% (200 cells/well), 0.3% (60 cells/well) and a control without NK cells. Supernatants were harvested after 24, 48, 72 and 96 h of incubation, and were subsequently centrifuged, sterile filtered and stored at –70°C. These supernatants were added to B cell cultures at a 9/10 dilution (90%). All experimental groups were established in triplicate and pooled before the quantitation of IgM-secreting cells.

Quantitation of IgM-secreting cells in the cultures of stimulated B cells
IgM-secreting cells were detected by spot-ELISA according to a modified version of a previously described protocol (35). Cellulose ester membrane 96-well plates (Millipore Multiscreen; Millipore, Molsheim, France) were coated with 50 µl of a 5 µg/ml solution of goat anti-mouse {kappa} light chain antiserum in PBS by incubating overnight at 4°C. Plates were postcoated using culture medium, and cell suspensions from the stimulated B cell cultures were added and incubated for 6 h in a humidified atmosphere containing 5% CO2 at 37°C. Goat anti-mouse IgM alkaline phosphatase was added and incubated overnight at 4°C. Next, the plates were developed using BCIP/NBT substrate solution (Kirkegaard & Perry, Gaithersburg, MD) and spots representing individual IgM-secreting cells were enumerated using an inverted microscope. All data shown represent the mean of three individual measurements and had standard errors that were consistently <10% of the mean.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cloned NK cells show heterogeneity in their enhancement of Ig secretion in an in vitro model of TI-2 antigen responses
Using an in vitro model for TI-2 antigens employing {alpha}{delta}-dex plus IL-2, we have previously demonstrated that addition of NK cells from short-term cultures to highly purified B cells obtained by electronic sorting induced an Ig secretory response (25,26). We recently described a novel panel of murine NK clones derived from spleens of mice that were homozygous for a mutation of the p53 tumor suppressor gene (27). This panel consists of one parental line, PKO, and four clones, PKO 2, PKO 34, PKO 56 and PKO 101. Moreover, one additional line, B6 NK, was isolated from spleens of normal C57BL/6 mice. To study the mechanism of NK-mediated enhancement of Ig secretion in more detail, sort-purified B cells were cultured in the presence of various concentrations of PKO cells (Fig. 1Go). As previously reported, B cells activated with {alpha}{delta}-dex and/or IL-2 secrete at best only moderate amounts of Ig. Addition of NK cells to cultures stimulated with medium, {alpha}{delta}-dex or IL-2 resulted in little if any further induction of Ig secretion. In contrast, addition of NK cells to cultures of B cells activated with {alpha}{delta}-dex plus IL-2 resulted in 9-fold enhancement of Ig secretion, when NK cells constituted 5% of the total cell population. This pattern of induction of Ig secretion was similar for all the members of the NK panel (data not shown). Analysis of the corresponding B cell culture supernatants for secreted Ig showed consistent correlation with the number of Ig-secreting cells detected by spot-ELISA (data not shown). To study possible heterogeneity in the capacity of individual members of our panel to induce Ig secretion, various concentrations of each of the clones were added to cultures of highly purified B cells stimulated with {alpha}{delta}-dex plus IL-2. The data shown in Table 1Go demonstrate that all members of our NK panel mediated significant enhancement of Ig secretion over background levels, which was optimal at a concentration of 3% NK cells (600 NK cells per 20,000 B cells). However, the absolute level of enhancement showed significant heterogeneity, varying between ~3-fold for PKO 56 to ~15-fold for PKO 101. Further, the PKO 2 and PKO 101 clones enhanced Ig secretion at concentrations as low as 0.3%, where PKO 34, PKO 56 and B6 NK were relatively ineffective. To further test the sensitivity of our TI-2 in vitro system to NK-mediated enhancement of Ig secretion, the PKO line and the PKO 101 clone were titrated to even lower concentrations. Figure 2Go shows that PKO 101 induced a significant increase in Ig even at a concentration as low as 0.04% (eight NK cells per 20,000 B cells).



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Fig. 1. The PKO parent NK line enhances Ig secretion in sort-purified B cells. Highly purified B cells were isolated from populations of T cell-depleted, Percoll gradient-fractionated CBA.C57BL/6 F1 splenocytes by electronic cell sorting after staining with anti-B220–FITC. The B cells were cultured in 96-well plates at a concentration of 105 cells/ml in a final volume of 200 µl, without addition, in the presence of {alpha}{delta}-dex (10 ng/ml), with IL-2 (50 U/ml), or in the presence of a combination of both stimuli with the indicated percentage of cells of the PKO parent NK line. Cells were analyzed for IgM-secreting cells by spot-ELISA after 5 days of culture. These data are representative of three individual experiments.

 

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Table 1. A panel of NK clones displays heterogeneity in the level of enhancement of Ig production in sort-purified B cells stimulated with {alpha}{delta}-dex plus IL-2
 


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Fig. 2. As few as eight NK cells per 20,000 B cells enhance Ig secretion in sort-purified B cells cultured with {alpha}{delta}-dex plus IL-2. Highly purified B cells were isolated from populations of T cell-depleted, Percoll gradient-fractionated CBA.C57BL/6 F1 splenocytes by electronic cell sorting after staining with anti-B220–FITC. The B cells were cultured in 96-well plates at a concentration of 105 cells/ml in a final volume of 200 µl with {alpha}{delta}-dex (10 ng/ml) and IL-2 (50 U/ml) and the indicated percentage of cells of the PKO parent line or the PKO 101 NK clone. Cells were analyzed for IgM-secreting cells by spot-ELISA after 5 days of culture. These data are representative of three individual experiments.

 
Delayed addition of cloned NK cells still results in enhanced Ig secretion in {alpha}{delta}-dex plus IL-2-activated highly purified B cells
To determine the optimal timing for NK-mediated induction of Ig secretion, we studied the effects of delayed addition of NK cells to cultures of sort-purified B cells stimulated with {alpha}{delta}-dex plus IL-2. Thus titrated amounts of PKO 101 cells were added at the onset of the B cell cultures or after delays of 16 and 40 h. The data shown in Fig. 3Go demonstrate that a delay in PKO 101 addition did result in a decrease in Ig production, but that addition of NK cells as late as 40 h after the onset of the B cell culture still mediated a highly significant increase over background Ig production.



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Fig. 3. Delayed addition of PKO 101 cloned NK cells to cultures of sort-purified B cells stimulated with {alpha}{delta}-dex plus IL-2 still induces enhanced Ig secretion. Highly purified B cells were isolated from populations of T cell-depleted, Percoll gradient-fractionated CBA.C57BL/6 F1 splenocytes by electronic cell sorting after staining with anti-B220–FITC. The B cells were cultured in 96-well plates at a concentration of 105 cells/ml in a final volume of 200 µl with {alpha}{delta}-dex (10 ng/ml) and IL-2 (50 U/ml) and the indicated percentages of cells of the PKO 101 NK cells, that were added at the onset of the B cell stimulation or after the indicated number of hours. Cells were analyzed for IgM-secreting cells by spot-ELISA after 5 days of culture. These data are representative of three individual experiments.

 
NK-mediated enhancement of Ig secretion in highly purified B cells stimulated with {alpha}{delta}-dex plus IL-2 does not require B–NK interactions
To study the mechanism of NK-mediated Ig production, we analyzed the requirement for physical interactions between B and NK cells. To this end, B and NK cells were cultured in a system consisting of two compartments separated by a protein permeable membrane. The data shown in Fig. 4Go demonstrate that culture of {alpha}{delta}-dex plus IL-2-stimulated sort-purified B cells with PKO NK cells, that were added to either the same compartment as that of the B cells or to the compartment on the other side of the membrane, resulted in comparable levels of Ig secretion. Similar results were obtained for the other members of the NK panel (data not shown).



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Fig. 4. NK-mediated enhancement of Ig secretion in sort-purified B cells stimulated with {alpha}{delta}-dex plus IL-2 does not require B–NK interaction. Highly purified B cells were isolated from populations of T cell-depleted, Percoll gradient-fractionated CBA.C57BL/6 F1 splenocytes by electronic cell sorting after staining with anti-B220–FITC. The B cells were cultured in 24-well plates at a concentration of 5x104 cells/ml in a final volume of 800 µl with {alpha}{delta}-dex (10 ng/ml) and IL-2 (50 U/ml). To these cultures cells of the PKO NK parent line were added in the same compartment (mixed) or in a compartment separated by a protein permeable membrane (separated). Cells were analyzed for IgM-secreting cells by spot-ELISA after 5 days of culture. These data are representative of three individual experiments.

 
Murine NK cell clones display heterogeneity in cytokine secretion
The finding that the NK-mediated induction of Ig secretion did not require physical interaction with the B cells indicates that the NK cells mediate their stimulatory activity by soluble factors. To identify cytokines that may be involved in the NK-mediated Ig induction, and to analyze the relationship between the cytokine profiles of each of the clones and their respective level of enhancement of Ig secretion, each of the members of our NK panel was cultured overnight in the presence of either IL-2, in order to investigate the cytokines secreted under similar conditions used for B cell stimulation, or with PMA plus ionomycin to further analyze the capacity of cytokine secretion of each of the NK clones at optimal levels of gene expression. After harvesting of the cultures and isolation of the supernatants each was analyzed by ELISA for the presence of a panel of cytokines including IL-1, IL-2, IL-3, IL-4, IL-5, IL-10, GM-CSF, IFN-{gamma} and TNF-{alpha}. The data shown in Table 2Go demonstrate that each of the lines secreted IFN-{gamma}, and that both PKO and PKO 56 also secreted GM-CSF. There were no detectable levels of any of the other cytokines that were assayed (data not shown). The addition of {alpha}{delta}-dex at the concentration that was used for B cell stimulation, 10 ng/ml, did not influence the NK cytokine secretion induced by IL-2 (data not shown). A comparison of the levels of IFN-{gamma} secretion in the presence of IL-2 with the optimal level of Ig induction (Table 1Go) for each of the members of the NK panel in five sets of individual experiments revealed a reproducible correlation between these two parameters. PKO 101 consistently gave the greatest degree of help and was the highest producer of IFN-{gamma}; PKO 56 was the lowest in both, with the other members of the NK panel being intermediates.


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Table 2. A panel of NK clones displays heterogeneity in the level of IFN-{gamma} and GM-CSF production after culture in IL-2 or PMA plus ionomycin
 
NK-induced enhancement of Ig production in B cells stimulated with {alpha}{delta}-dex and IL-2 is mediated by IFN-{gamma}
The previous data showed that the members of the NK panel displayed heterogeneity in IFN-{gamma} and GM-CSF production. Furthermore, the amounts of IFN-{gamma} produced in the presence of IL-2 correlated with the levels of induction of Ig secretion in the presence of the corresponding NK cells. We have previously reported that IFN-{gamma} can induce strong IgM secretion in {alpha}{delta}-dex plus IL-2-stimulated highly purified B cells (8) and that addition of GM-CSF can further enhance the effects of IFN-{gamma} (36). Thus, we determined whether either of these two cytokines mediated the observed Ig induction. All of the clones were titrated in cultures of highly purified B cells stimulated with {alpha}{delta}-dex and IL-2 in the presence of anti-cytokine mAb. Figure 5Go shows the effect of the addition of anti-IFN-{gamma} mAb to cultures of B cell that were stimulated in the presence of each of the individual members of our NK panel. A crucial role for IFN-{gamma} in NK-mediated enhancement of Ig induction is reflected by the finding that addition of the anti-IFN-{gamma} mAb completely abrogated Ig induction by all of the members of our NK panel. Culture in the presence of anti-GM-CSF mAb did not result in a significant reduction of Ig production and had no influence on the effects of the IFN-{gamma} mAb (data not shown). This data is consistent with our earlier report that IFN-{gamma} can stimulate IgM secretion by B cells activated with {alpha}{delta}-dex plus IL-2 (8). A comparison of the number of viable B and NK cells after 5 days of culture in the presence of either anti-IFN-{gamma} or isotype-matched mAb did not reveal any significant differences in the number of each of these cell types, suggesting that IFN-{gamma} was required to stimulate B cells to Ig secretion (data not shown). Addition of anti-IFN-{gamma} mAb to cultures of B cells physically separated from NK cells by a protein permeable membrane resulted in a similar abrogation of NK-induced Ig secretion (data not shown).



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Fig. 5. NK-mediated enhancement of Ig secretion in sort-purified B cells stimulated with {alpha}{delta}-dex plus IL-2 is inhibited by anti-IFN-{gamma} mAb. Highly purified B cells were isolated from populations of T cell-depleted, Percoll gradient-fractionated CBA.C57BL/6 F1 splenocytes by electronic cell sorting after staining with anti-B220–FITC. The B cells were cultured in 96-well plates at a concentration of 105 cells/ml in a final volume of 200 µl with {alpha}{delta}-dex (10 ng/ml) and IL-2 (50 U/ml) and the indicated percentages of cells of each of the members of the NK panel. Culture was performed in the presence of 10 µg/ml of anti-IFN-{gamma} or isotype-matched control mAb. Cells were analyzed for IgM-secreting cells by spot-ELISA after 5 days of culture. These data are representative of three individual experiments.

 
NK supernatants induce enhancement of Ig secretion with the same level of heterogeneity as the corresponding NK clones
To determine whether the heterogeneity in Ig production resulted from differences in IFN-{gamma} concentrations in cultures of B cells with the individual members of the NK panel, the corresponding supernatants were harvested at different time points and analyzed for IFN-{gamma} content. The levels of IFN-{gamma} induced under these circumstances were below the detection limit of our ELISA (data not shown). However, we have shown that IFN-{gamma} can induce IgM secretion at concentrations that were below the detection limit of our IFN-{gamma}-specific ELISA (8). Thus, we assayed the IFN-{gamma} concentration of the NK supernatants indirectly, by testing their effect on {alpha}{delta}-dex plus IL-2 stimulated sort-purified B cells, that displayed a high sensitivity for NK-mediated stimulation (Fig. 2Go). To this end, titrated amounts of NK cells were cultured under identical conditions as used for B cell stimulation culture, but in the absence of B cells that might consume the secreted IFN-{gamma}. Supernatants of all of the cultures were harvested after 1, 2, 3 and 4 days of incubation, and were added to highly purified B cells. In parallel, B cells were cultured with the same concentrations of NK cells used in the supernatant production in the presence of {alpha}{delta}-dex and IL-2. The results obtained for PKO 2, PKO 56 and PKO 101 are shown in Fig. 6Go. The data demonstrate a correlation between the levels of Ig production induced by the NK cells and those induced by their supernatants. Addition of anti-IFN-{gamma} to the NK culture supernatants resulted in a complete absence of Ig induction (data not shown). A comparison of the data obtained for PKO 56, a poor inducer of Ig secretion, with those of PKO 101, the strongest inducer in the NK panel, show that the supernatants of the latter clones that were harvested after 1 day of NK culture were already able to increase secretion over background level. In contrast, PKO 56 supernatants harvested after 4 days of NK culture still only induced a minor increase in Ig secretion over background levels. To address the possibility that PKO 56 might, in addition to IFN-{gamma}, produce another factor that could inhibit the induced Ig secretion, we performed experiments in which cells and supernatants of PKO 56 and PKO 101 were mixed. These experiments showed no inhibitory effects of either PKO 56 cells or supernatants on PKO 101-induced Ig production (data not shown). Similar to the data obtained with PKO 101 cells, the data in Fig. 6Go demonstrate that PKO 101 supernatants harvested after 4 days of culture induced optimal Ig production at 3% and a decline at 10% NK cells. We cannot exclude the possibility that factors other than cellular overcrowding and depletion of culture medium were responsible for the observed kinetics. We tested the effect of anti-transforming growth factor-ß mAb on the induction of Ig-secreting cells by PKO 101 cells and supernatants, but observed no effect on the NK dose–response curve (data not shown). Thus, the data shown in Fig. 6Go strongly indicate that the amount of IFN-{gamma} secreted by NK cells is a crucial parameter in the level of Ig induction. Differences in IFN-{gamma} production account for the observed heterogeneity in NK-helper activity, and are a determining and constant characteristic of each NK clone.



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Fig. 6. Correlation between the levels of Ig secretion induced by NK clones and by their supernatants from timed cultures of corresponding numbers of NK cells. Highly purified B cells were isolated from populations of T cell-depleted, Percoll gradient-fractionated CBA.C57BL/6 F1 splenocytes by electronic cell sorting after staining with anti-B220–FITC. The B cells were cultured in 96-well plates at a concentration of 105 cells/ml in a final volume of 200 µl with {alpha}{delta}-dex (10 ng/ml) and IL-2 (50 U/ml) and the indicated percentages of cells of each of the three NK clones. In addition B cells were stimulated in the presence of supernatants resulting from cultures of equivalent numbers of the corresponding NK cells harvested after the indicated number of days. Cells were analyzed for IgM-secreting cells by spot-ELISA after 5 days of culture. These data are representative of three individual experiments.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The present study determines the effect of cloned murine NK cells on the Ig secretory response of highly purified B cells stimulated in vitro by a prototypic TI-2 antigen. In this model B cells are activated with {alpha}{delta}-dex and IL-2, and require the addition of a source of additional B cell stimulation to produce Ig (6). We have previously shown that this stimulation can be provided by non-B cells present in T cell-depleted spleens from normal mice and that addition of freshly explanted or in vitro activated scid NK cells to {alpha}{delta}-dex plus IL-2-stimulated B cells induced high levels of Ig secretion in sort-purified B cells (25,26). To study the mechanism of NK-mediated B cell activation in detail, while avoiding the possible influence of even small numbers of contaminating cells that share phenotypic characteristics with NK cells (37), and to address functional heterogeneity among individual NK cells with regard to this process, the present communication employed a panel of cloned NK cells. The experiments presented in this study demonstrate that while all NK clones could enhance Ig secretion, there was significant heterogeneity in their helper activity, both in the level of Ig induction and in the number of NK cells required for significant B cell stimulation. Furthermore, the differences in the dose–response relationship of Ig induction observed among the clones were mirrored by the ones observed for the corresponding supernatants. Analysis of the cytokines produced by the NK clones revealed that the level of IFN-{gamma} secretion was an important parameter in the observed heterogeneity. Furthermore, addition of anti-IFN-{gamma} mAb blocked the Ig enhancement mediated by all members of our panel. GM-CSF, which was produced by two members of the NK panel, did not have a significant effect on NK-mediated Ig induction. Although these findings show the importance of IFN-{gamma} as a mediator of NK help to B cells and correlate the functional heterogeneity among the NK clones with differences in the level of IFN-{gamma} production, we cannot exclude the possibility that differences in the secretion of other unidentified cytokines play a part in the described heterogeneity. Employing a variety of assay systems, others have reported that NK cells can either suppress (3840) or support (4144) B cell differentiation. However, it has been suggested that NK-mediated suppression is an indirect effect requiring T cells in the presence of accessory cells, whereas enhancement of Ig production is a direct effect of the NK cell (45). Our data using murine NK clones support the notion of a direct stimulatory effect of NK cells on IgM production by B cells activated with {alpha}{delta}-dex in the presence of IL-2. Since we have previously demonstrated that IFN-{gamma} can induce IgG2a in this system (8), we are currently studying NK induction of Ig isotypes other than IgM. While the findings of these studies are still inconclusive, our preliminary data suggest that the level of IgG2a induction by the individual clones also correlates with their level of IFN-{gamma} production and that IgG2a production does not require physical interaction between B and NK cells (data not shown). The dose–response curve of NK-induced Ig secretion consistently showed an optimum at a concentration of 3% NK cells for each of the clones studied, with higher concentrations of NK cells being inhibitory. Since the presence of IL-2 in the B cell cultures induces the NK cells to proliferate, it is possible that this `suppressive' effect is due to cellular overcrowding and associated depletion of the culture medium. Nevertheless, we cannot exclude the possibility that the presence of NK-produced inhibitory factors that are only present at functional levels in the cultures containing the highest NK concentrations also influence the observed NK dose–response relationship. Both the experiments employing the culture of B and NK cells in separate compartments and the ones using NK cell supernatants showed that the NK stimulation did not require physical interaction between both cell types. Whereas these findings are in agreement with data from our previous studies (25,26), they do not support the mechanism of NK cell-mediated B cell activation proposed by Gray and Horwitz (44). According to their hypothesis NK cell-mediated Ig induction depends on two processes: one that requires cellular interaction, the other being contact independent. Since Gray and Horwitz employed human peripheral lymphocytes, a different agonist to stimulate the B cell and non-clonal derived NK cells, it is difficult to compare these data with the ones presented in this study. Thus we demonstrate that clonal NK cells are heterogeneous in the enhancement of Ig secretion in B cells stimulated by multivalent Ig cross-linking. In addition, our data emphasize that very small numbers of NK cells can induce Ig secretion if they are stimulated to produce IFN-{gamma}. Since many TI antigens stimulate B cells via multivalent Ig cross-linking and display variation in their ability to stimulate NK cells, we provide another example of heterogeneity within the innate immune system and it's effect on Ig secretion.


    Acknowledgments
 
The authors thank Drs Fred Finkelman and Andrew Lees for selected reagents, and Mark Moorman and Karen Wollcott for expert assistance in electronic cell sorting. This work was supported by grants from the National Institutes of Health (AI 33411 and AI 32560). Opinions and assertions herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences.


    Abbreviations
 
{alpha}{delta}-dexdextran-conjugated anti-IgD mAb
GM-CSFgranulocyte macrophage colony stimulating factor
pKO-53mice homozygous for a mutation in the p53 tumor suppressor gene
PMAphorbol myristate acetate
TI-2T cell-independent type 2
TNFtumor necrosis factor

    Notes
 
Transmitting editor: Z. Ovary

Received 30 July 1998, accepted 13 October 1998.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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