© 1992 Oxford University Press
International Immunology, Vol. 4, No. 12, pp. 1373 - 1380
Activation of B lymphocytes by NK cells Dorothy Yuan, Julie Wilder, Tarn Dang, Michael Bennett, and Vinay Kumar Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA Key words. B lymphocytes, helper factor, T-IFN, IgM secretion, NK cells, proliferation, tumor necrosis factor-a
The ability of NK calls to Induce differentiation of B lymphocytes to IgM secretion In vttro has been Investigated. Homogeneous preparations of NK cells obtained from IL-2 propagated splenocytes from SCID mice were found to have the ability to Induce resting B lymphocytes to proliferate and secrete significant amounts of IgM. The induction Is greatly enhanced by the presence of both IL-2 and IL-5 and does not require T lymphocytes or adherent cells in the responding population. Cell contact between the two populations Is not necessary suggesting that the effect is mediated by soluble factors) which can be produced even by irradiated NK cells. Because the activity cannot be replaced by either r-r-IFN or tumor necrosis factor-a or inhibited by antibodies to these lymphokines, a novel NK cell-derived factor(s) may be involved. The Implications of this Interaction between NK cells and B lymphocytes are discussed. Introduction Complex intercellular interactions form an integral part of the immune response. Much progress has been made towards understanding the interactions that need to occur between T, B, and antigen presenting cells for the initiation, as well as the regulation, of antibody production. An additional cell type which has been implicated in immunoregulation is the NK cell. There is considerable evidence that NK cells regulate B cell functions in that depletion of NK cells either in vivo or in vitro results in alterations in levels of antibody production (1 - 3 ) . However, the mechanisms underlying NK mediated regulation are not clear. The ability to purify NK cells and propagate them in vitro in the presence of IL-2 has permitted a much more direct approach to the analysis of the biology of NK cells. These studies have been further facilitated by the use of cells from SCID mice which, because of the virtual absence of T or B cells (4), have significantly simplified the analysis. Thus, utilizing this system, we have previously shown that co-culture of purified B and NK cells resulted in measurable alterations in the responses of both cell types which are dependent on the activation status of the B lymphocytes. Low density B lymphocytes (presumably in vivo pre-activated) can enhance the production of HFN by NK cells (5), which in turn induces these B cells to switch to lgG2a production after mitogenic stimulation (6). In the course of these experiments it came to our attention that, in the absence of mitogens, NK cells sometimes increased the basal level of IgM secretion. We therefore proceeded to investigate this phenomenon more systematically. We have now shown that NK
cells can elaborate a factor(s) which induces B lymphocytes to proliferate and secrete IgM. The activity of this factor is not dependent on the production of dFN or tumor necrosis factor TNF-a but requires the presence of IL-2 and IL-5. Thus, in contrast to the effect of B lymphocytes on NK cells, which is contact dependent, the factor(s) elaborated by NK cells, which can polyclonally stimulate B lymphocytes, appears to be constitutively produced.
Methods Animals Female BALB/c and Swiss NUDE mice (obtained from Harian Sprague Dawley, Inc., Indianapolis, IN) and C57BL/6 x DBA/2 (B6D2F1) F, mice (obtained from Jackson Laboratories, Bar Harbor, ME) were kept in specific pathogen-free conditions. C.B-17-sc/d mice (7) were raised in our own colony from a breeding pair originally obtained from Dr Mel Bosma (Fox Chase Cancer Center, Philadelphia, PA). All mice were used at age 6 - 1 0 weeks. Cell preparations T lymphocytes were depleted from spleen cell preparations by treating the cells with an anti-Thy-1.2 mAb (H013.4; 8) and baby rabbit complement (Pel-freez, Brown Deer, Wl) as previously described (9). High density ( > 1.095 mg/ml) resting
Correspondence to: D. Yuan Transmitting editor: L. Lanier
Received 27 July 1992, accepted 4 September 1992
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Abstract
1374 Activation of B lymphocytes by NK cells B lymphocytes were separated from low density (< 1.076 mg/ml) lymphocytes by centrifugation in discontinuous density gradients of Percoll (Pharmacia Fine Chemicals, Piscalaway, NJ) prepared as previously described (10). In some experiments B lymphocytes were further purified by panning on goat anti-IgM plates (CappelCooper Biomedical, Malvern, PA) by the method of Wysockj and Sato (11). Where indicated, cells were irradiated with 2500 cGy of (137Cs) 7-radiation. This dose reproducibly reduced the IL-2 proliferate response of SCID splenocytes to background levels. In other cases cells were fixed with glutaraldehyde (0.13%) and washed before use.
After T cell depletion and Percoll density sedimentation, high density lymphocytes were stained with an optimal amount of biotinylated goat anti-lgD and phycoerythrin - avidm as previously described (12). Positive cells were isolated from non-staining cells by sorting on the FACS Star Plus (Becton-Dickinson, San Jose, CA). Both sorted and control cells (non-sorted or non-stained) were cultured overnight before use to allow regeneration of capped receptors. Propagation of SCID NK cells SCID splenic NK cells were propagated as previously described (13). Cells harvested after 6 - 1 4 days of culture were pooled, washed three times with Balanced Salt Solution and used in the co-culture experiments. These cells have been previously shown to be virtually 100% NK-2.1 + , CD3", Lyt2", and L3T4" and have no rearranged Ig or TCR genes (13). Cell culture B lymphocytes were cultured in RPMI supplemented with 10% FCS with low lipopolysaccharide (LPS) content (Gibco, Grand Island, NY), glutamine, sodium pyruvate, non-essential amino acids, penicillin-streptomycin, and 2-mercaptoethanol. Cell concentrations varied between 0.5 and 1 x 106 cells/ml. Each culture condition was performed in triplicate in 96-well polyvinyl microtiter plates (Costar, Cambridge, MA) at 0.2 ml/well. Plates were incubated in a humidity controlled incubator maintaned with 7% CO2 at 37°C. For the membrane partition experiments, 3 x 106 NK cells (in 100^1) were added to chamber wells constructed with microporous, 0.4 ^m pore size, polycarbonate membrane (Transwells, Costar, Cambridge, MA). The lower portions of the wells contained 5 x 105 cells/ml (in 650 jd) high density B cells. Lymphokines and reagents Recombinant human IL-2 was a gift from Cetus Corporation (Emeryville, CA). Recombinant murine IL-1 and IL-4 were obtained from Dr Steven Gillis (Immunex, Seattle, WA). Recombinant murine IL-5 was obtained from Dr Colin Sanderson (MRC, Mill Hill, UK). Recombinant mouse TIFN was a gift from Shering Corporation, USA, distributed by the American Cancer Society (New York, NY). Recombinant human TNF-a was purchased from Genzyme Corporation (Boston, MA). LPS (Difco Laboratories, Detroit, Ml) was used at a final concentration of 20/tg/ml. Antj-rlFN (XMG-1; 14) was obtained by affinity purification of hybridoma supernatants. It was added to cultures at a concentration (3 /ig/ml) shown to be effective in neutralizing at
Proliferation assay Triplicate culture wells were pulsed with 2 ^Ci of [3H]thymidme (25 Ci/mmol, Amersham Corporation, Arlington Heights, IL) for 6 h and harvested using an automated cell harvester. [3H]Thymidine uptake was determined by counting in a Beckman (Irvine, CA) L53801 liquid scintillation counter. Measurement of IgM concentrations At the end of the incubation period, supernatants from triplicate wells were pooled and IgM concentrations were measured by radioimmunoassay as described (15). Briefly, total antibodies were capured in 96-well polyvinyl microtiter plates coated with 5 ng of affinity-purified goat anti-IgM (Cappel-Cooper Biomedical). Unbound sites were saturated with 50% FCS - PBS. The amount of bound antibodies was determined with affinity-purified goat anti-IgM labeled with Na12Sl. If duplicate determinations showed > 10% variation the assay was repeated. Concentrations were determined by comparison with standard curves using purrfied hybridoma IgM in each assay. Test for mycoplasma DNA Various supernatants from NK cells growing in culture were tested for the presence of mycoplasma RNA sequences using the Mycoplasma T.C. Rapid Detection System (Gen-Probe, San Diego, CA). The assay was performed according to the manufacturer's directions. Results Induction of IgM secretion by IL-2 propagated NK cells As a source of highly purified NK cells, splenocytes from SCID mice were cultured in the presence of 500 U/ml IL-2 for 6 - 1 4 days. Previous experiments have shown that cells obtained from these cultures are virtually 100% NK-2.1+, CD3", Lyt2~, and L3T4~, and have no re-arranged Ig or TCR genes (13). B lymphocytes consisted of T-depleted BALB/c or B6D2F1 splenocytes which were fractionated into high and low density subpopulations by Percoll gradient sedimentation. Figure 1 presents the results of an experiment representative of five independent experiments. The high density B lymphocytes cultured either with IL-2 alone or in combination with IL-4 or IL-5 resulted in only a slight increase in the production of IgM when measured on day 5. Further, these cells did not respond to a supernatant obtained from phorbol-12-myristate-13-acetate (PMA)-stimulated EL4 thymoma cells (10) which induced secretion of IgM from the low density activated B lymphocytes (data not shown). Upon addition of NK cells, both with and without exogenous lymphokines, enhancement of IgM secretion can be observed. However, the most significant increase was dependent on the presence of both IL-2 and IL-5 in the medium. The addition of only IL-5 to NK cells was insufficient for the induction of IgM secretion (data not shown). Furthermore, the extent of help
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FACS
least 20 U/ml of TIFN and can completely prevent the TIFN mediated inhibition of WEHI279 proliferation (5). Anti-murine TNF-a was purchased from R & D Systems (Minneapolis, MN) and used at 3/ig/ml, which is 10-fold the concentration shown to be effective for 50% inhibition of their indicator cell line (0.05 ng/ml TNFor).
Activation of B lymphocytes by NK cells 1375
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Fig. 1 . Enhancement of IgM secretion by NK cells. High density BALB/c B lymphocytes at a concentration of 5 x 105 cells/ml were cultured with or without NK cells which had been propagated for 14 days at the indicated ratios. IL-2 and IL-4 were each added at 100 U/ml and IL-5 was added at 5 U/ml. Duplicate samples from pooled triplicate cultures were assayed for IgM production 5 days after initiation of culture. Duplicate determinations did not vary by > 1 0 % in all values presented.
was dependent on the number of NK cells added in the cocultures. At ratios of NK:B cells > 1 : 1 , IgM secretion did not further increase and tended to be inhibitory (data not shown). In comparison, identical cultures stimulated with LPS resulted in ~ 10-fold higher responses (data not shown). Addition of NK cells to low density B lymphocytes resulted in a similar enhancement of IgM secretion (data not shown); however, due to the highly variable activation status of these cells, the extent of augmentation was less consistent. Therefore only high density resting B lymphocytes were used in subsequent experiments. Help by NK cells is not mediated via other lymphocytes Although the fractionation of B lymphocytes was preceded by depletion of T cells by anti-Thy-1.2 antibodies plus C , residual T cells were found to remain in the preparation to variable extents as determined by response to concanavalin A (Con A) stimulation. To ascertain that B lymphocytes can respond in the total absence of conventional T cells, high density cells were prepared from spleens obtained from NUDE mice and co-cultured with IL-2 propagated SCID NK cells. Figure 2 shows that although the response was somewhat lower, these B cells could be induced by NK cells to secrete IgM in the presence of IL-2 and IL-5. To further insure that T cells do not play a role, small resting B cells were purified by allowing them to bind to plates coated with anti-lgM. The Con A response (5 /ig/ml) of cells removed from the plates was reduced to background levels, but FACS analysis of the cells showed that they were only 95% lgM + suggesting that some adherent cells may have co-purified during the panning procedure. This population was then compared with non-panned B cells for their ability to respond to NK cells. Figure 3 shows that there was no significant difference in the responses of the two populations. Furthermore, examination of the IgM accumulation on both days 5 and 7 showed no significant difference in the kinetics of the response of the two populations. Finally, to obtain a population of B cells which is not
Fig. 2. Response of NUDE B lymphocytes to NK stimulation. High density splenocytes from a NUDE mouse were cultured with NK cells (propagated for 12 days) at a ratio of 1.1 (each at 5 x 10 s cells/ml) in microtiter wells with or without lymphokines (IL-2 at 100 U/ml, IL-5 at 5 U/ml) as indicated. In addition, transwell cultures were set up by diluting B lymphocytes in 0.6 ml at 5 x 105 cells/ml in bottom wells with NK cells added to transwells at 3 x 106 cells/100 jd. Culture supernatants were assayed for IgM content on day 6 as in Rg 1. Duplicate determinations did not vary by > 10% in all values presented.
contaminated by NK or adherent cells which may co-purify during panning, Percoll fractionated T cell depleted B lymphocytes were stained with anti-lgD and isolated by cell sorting. From a starting population which was 94% lgD + , sorting resulted in a population which was 99.9% lgD + . The cells were cultured overnight to regenerate cell surface Ig and subsequently cultured with NK cells with or without lymphokines. In addition to IL-2 and IL-5, IL-1 was also added to the cultures to insure optimal responses in the absence of adherent cells. Comparison of the response of stained versus non-stained lymphocytes (Figure 4) showed that stained cells gave a lower response. Because the reduction was apparent also in the background response to lymphokines as well as to LPS, this effect may be attributed to the ligation of cell surface Ig. Nevertheless, when sorted cells are compared with non-sorted but stained B lymphocytes, it is dear that the response to NK cells was not affected by the further purification step. The further reduction in LPS response of the sorted B cells is probably due to depletion of adherent cells. Indeed it was found that the responses of all cultures containing IL-1 were increased - 2 0 % when compared with those containing only IL-2 and IL-5 (data not shown). From these experiments it can be concluded that neither T lymphocytes nor adherent cells are required for the help provided by NK cells. Help from NK cells is resistant to irradiation but is eliminated by fixation To determine if replication of NK cells during co-culture with B lymphocytes was necessary for the induction of IgM secretion, NK cells were first irradiated before initiation of culture. Figure 5 shows the results of one of two experiments performed. Clearly irradiation did not significantly alter this activity. However, mild fixation of NK cells with glutaraldehyde completely eliminated their ability to induce IgM secretion (data not shown).
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Fig. 3. Comparison of the response of anti-IgM panned B lymphocytes with non-panned B cells. After anti-Thy-1.2 treatment, high density B cells (B6D2F1) were panned on anti-IgM plates. After removal from plates, the cells were cultured overnight in bulk before they were aliquoted into mere-culture plates at 5 x 105 cells/ml in the presence or absence of equal numbers erf NK cells (propagated 7 days) with or without lymphokines (IL-2 at 100 U/ml, IL-5 at 5 U/ml) as indicated. Control cultures consisted of non-panned B lymphocytes treated identically. Culture supernatants were harvested on day 5 and day 7 and assayed for IgM accumulation as indicated in Fig. 1. Duplicate determinations did not by vary by > 10% in all values presented.
Help for B lymphocytes can be mediated by a soluble tactor(s) To determine whether the ability of NK cells to induce IgM secretion is mediated by a secreted factor, the two cell types were separated from each other by a Millipore membrane during culture. As shown by a representative experiment (total of three) in Fig. 2, B lymphocytes could be induced to secrete IgM even if NK cells were placed in a transwell. Again, the activity was dependent on the presence of IL-2 and IL-5. It is interesting that the resultant IgM secretion was higher in cultures where the two cell types were separated. The significance of this finding is not clear because cell density conditions were different owing to conditions imposed by the architecture of the transwell set up. However, it is also possible that the membrane can prevent inhibitory interactions which depend upon cell contact. In other experiments (not shown) it was found that upon removal of the transwell containing NK cells either on day 1 or 2 after initiation of culture, B cells could still be induced to secrete IgM (not shown), although at a reduced level, suggesting that the activity may be unstable during culture. If the NK help is mediated by a soluble factor, culture supernatants of NK cells should contain the active factor. Media from propagated NK cells were collected on day 6 of culture, 3 days after the last feeding with IL-2. The supernatant was added in two different concentrations to resting B cells in the presence of various lymphokines. Figure 6 shows the results obtained from supernatants collected from two different NK cultures. When 20 - 25% of the culture media was made up with NK supernatant, a significant enhancement of IgM production can be obtained in the presence of IL-2 and IL-5. However, the increase is relatively low compared with experiments in which NK cells were present. Similar results were obtained from at least six other independent culture supernatants (not shown). Nonetheless, because the NK cell supernatant could enhance IgM secretion, it was possible to test its effect on inducing B cell proliferation without having to assess the contribution of each component if we were to measure the NK:B cell co-cultures.
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Fig. 4. NK cells can help sorted B lymphocytes. FACS sorted lgD + T cell-depleted high density B lymphocytes (B6D2F1) were first cultured overnight and then replated at 2.5 x 105 cells/ml with an equal number of NK cells (propagated for 6 days). Control cultures consisting of non-stained or non-sorted cells were treated identically. Lymphokines when added were at the concentrations indicated in Rg. 1. IL-1 was added at 5 ng/ml and LPS was added at 20 fig/ml. Culture supernatants were collected at day 6 and assayed for IgM production as in Rg. 1. Duplicate determinations did not vary by > 1 0 % in all values presented.
Figure 7 (representative data from frve independent expenments) shows the day 3 proliferative response of small B cells cultured in the presence of NK supernatants. Interestingly, despite the fact that the supernatant contained residual IL-2 from the NK cultures, addition of fresh IL-2 enhanced the proliferative response. Further, the most extensive enhancement occurred in cultures containing IL-4. Statistical evaluation using the two sample T test showed that the proliferation of cells cultured in the presence of NK supernatants differed significantly from those
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Fig. 5. Effect of irradiation on the B ceil stimulatory activity of NK. High density B lymphocytes (B6D2F1) were cultured at 2.5 x 105 cells/ml at a ratio of 1.1 with irradiated or non-irradiated NK cells (propagated for 6 days). Day 6 IgM accumulation was measured as described in Fig. 1. Duplicate determinations did not by vary by > 1 0 % in all values presented.
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Fig. 7. B cell proliferation in response to NK supernatants Supernatants collected, as in Fig. 6, were added to triplicate B cell cultures at the concentrations indicated. Where indicated, IL-2 was added at 100 U/ml and IL-4 at 25 U/ml. [3H]Thymidine was added for 6 h on day 3 before the cells were harvested. Data represent the extent of incorporation in mean c.p.m. ± SE of the mean.
selected experiments, were found not to contain mycoplasma RNA sequences. ISO--
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Fig. 6. B cell stimulatory activity in NK culture supernatants. Supernatants collected from 6 day NK cultures were centnfuged at 10,000 g and then added to B cell (B6O2F1) cultures (5 x 10s cells/ml) at the concentrations indicated. IgM accumulation was measured on day 6. Duplicate determinations did not by vary by > 1 0 % in all values presented.
cultured only in IL-2 plus IL-4 (P < 0.166 for 12.5% supernatant and P s 0.076 for 25% supernatant). However, IL-5 added together with IL-2 did not significantly increase the level of proliferation induced by IL-2 alone (data not shown). The response in these cultures was much decreased 6 days after initiation of culture (data not shown). Comparison of the proliferative response with that of LPS-activated culture suggests that - 1 0 % as many cells were stimulated. Because both NK cells and their supernatants were obtained from relatively long term cultures, we tested for the inadvertent presence of mycoplasma bacteria which may affect B cell differentiation. However, the supernatants used in the experiments shown in Figs 6 and 7, as well as other randomly
NK cells have been shown to secrete a number of known lymphokines. However, the only lymphokines documented to have effects on mature B lymphocytes are T-IFN and TNF-a. Furthermore, most of these effects have been restricted to the proliferative response. Nevertheless, to determine if these two lymphokines could be responsible for the help mediated by NK cells, they were added in various concentrations to B cell cultures in the presence of IL-2 and IL-5. Addition of r-HFN, alone or in combination with r-TNF-a, did not increase the stimulatory effect of IL-2 and IL-5 on either high or low density B lymphocytes (Fig. 8). r-TNF-a was somewhat suppressive for the response which was more evident for low density B lymphocytes. In addition, antibodies to T-IFN and TNF-a were included in the NK - B cell cultures to test whether the lymphokines play a role only in the presence of other NK factors. Figure 9 (representative of two independent experiments) shows that these antibodies exerted no demonstrable effect on the response of B cells, either in the presence or absence of NK cells. Thus, these two lymphokines are apparently not directly responsible for the increase in Ig secretion induced by NK cells. Discussion Using purified NK cells, obtained by in vitro propagation of SCID splenocytes, we have shown that they are able to induce highly purified small resting B lymphocytes to differentiate to IgM secretion (Fig. 4). It is important to note that the effect of NK cells can be exerted on high density resting B cells. The resting status of these B cells is based on the criterion that, when compared with large B lymphocytes, they secrete only minimal amounts of IgM in response to IL-2, IL-4, IL-5, or combinations of these lymphokines (Fig. 8). They also do not respond to supernatants
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Fig. 8. Effect of T-IFN and TNF-a on the B lymphocyte stimulatory activity of IL-2 plus IL-5. Various concentrations of T-IFN and TNF-a were added as indicated to cultures of high density (A) or low density (B) lymphocytes (BALB/c) in the presence of IL-2 (100 U/ml) and IL-5 (5 U/ml) Day 6 IgM accumulation was measured as described in Fig. 1. Duplicate determinations did not by vary by > 1 0 % in all values presented.
from PMA-stimulated EL-4 lymphoma cells which can induce IgM secretion by low density B lymphocytes (10, and data not shown). Unlike the effect of NK cells on human B lymphocytes (16,17), cross-linking of cell surface determinants is not required. On the other hand, we have noted that low density B lymphocytes will also respond to NK cells. However, despite the much greater absolute response, the fold increase in IgM secretion in the presence of NK cells is not as dramatic. In this respect, it should be noted that there is a report of NK cell activation of Ig secretion after stimulation of human B cells by Staphyiococcus aureus (SAC) in the presence of rlL-2 (16). It is possible, therefore, that the pre-activation of human B cells by SAC mimics in vivo activation of B lymphocytes and brings Ig secretion up to a detectable level. The use of NK cells propagated from the spleens of SCID mice gives assurance that the helper factor(s) is not produced by T cells. Moreover, the activity does not require T lymphocytes in the responding populations as was clearly shown by the use of highly purified B cells obtained by either panning or FACS. Furthermore, lymphocytes obtained from NUDE mice are also responsive. It is also clear that adherent cells play a minor role in the response of B lymphocytes to NK cells. The IL-2 propagated NK
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Fig. 9. Effect of antibodies to T-IFN and TNF-a on response of B lymphocytes to NK help. High density B lymphocytes (B6D2F1) were cultured with or without NK cells, which had been propagated for 7 days in the presence or absence of 3 /ig/ml anti-T-IFN or 3 (ig/ml anti-TNF-a as indicated. IgM accumulation on day 5 was assessed as described in Rg. 1. Duplicate determinations did not by vary by > 10% in all values presented.
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D
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Activation of B lymphocytes by NK cells 1379
Because NK cells produce some lymphokines also made by T cells, it is possible that the NK product(s) described here are not unique to NK cells. It should be noted, however, that there are very few T cell factors which can induce small resting B lymphocytes to secrete Ig without direct physical interaction (22 - 25). In contrast, NK cells can effectively help B lymphocytes when placed across a Millipore membrane. These results also suggest that cell -cell interactions are not necessary for induction of factor(s) secretion by NK cells. However, because the supernatants of NK cells have much reduced activity when compared with the cells themselves, the factor(s) may not be stable. Finally, the capacity to help B lymphocytes is not restricted to propagated NK cells from SCID splenocytes. We have found that fresh SCID splenocytes (manuscript in preparation) as well as adherent LAK cells from B6 splenocytes (J. Wilder and D. Yuan, unpublished observations), can also induce IgM secretion by resting B cells. Therefore, it is unlikely that production of this factor(s) is either restricted to cells from SCID animals or dependent on high concentrations of IL-2. In this regard, it will be important to assess the in vivo significance of the documented NK - B cell interaction. Most of the available in vivo data relating to NK cell effects on B lymphocyte differentiation are based on utilizing antigen-specific systems and they show that NK cells
down-regulate the responses (1 - 3). The in vivo effect of NK cells on polyclonal secretory response or bystander effects has not been extensively explored. Given the dependence of the NK help on T cell derived lymphokines (IL-2 and IL-5), it is conceivable that NK cells serve to magnify T-dependent responses in the appropriate microenvironment and/or pathological condition. For example, the continued production of antibodies in bone marrow transplant patients with depressed T cell numbers has been attributed to help mediated by the presence of activated large granular lymphocytes (26).
Acknowledgements We thank Jing Tan for help in preparation of NK cells, Pat Collins for expert assistance in ceD sorting, and Marilyn Mims for cheerful secretarial assistance. Supported by National Institutes of Health Grants CA51426, AI-20451, and CA09082.
Abbreviations (Don A LPS PMA SAC TNF
concanavalin A lipopolysacchande phorbol-12-myristate-13-acetate Staphylococcus aureus tumor necrosis factor
References 1 Abruzzo, L V. and Rowley, D. A. 1983. Homeostasis of the antibody responses, immunoregulation by NK cells. Science 222:581. 2 Khater, M., Macai, J., Genyea, C , and Kaplan. J. 1986 Natural killer cell regulation of age-related and type-specific variations in antibody responses to pneumococcal polysaccharides. J. Exp. Med. 164:1505. 3 Robles, C. P. and Pollack, S. B. 1986 Regulation of the secondary in vrtro antibody response by endogenous natural killer cells: kinetics, isotype preference, and non-identity with T suppressor cells. J. Immunol. 1372418. 4 Schuler, W., Weiler, I. J., Schuler, A., Phillips, R. A , Rosenberg, N., Mak, T. W., Kearney, J. F., and Perry, R. P. 1986. Rearrangement of antigen receptor genes is defective in mice with severe combined immune deficiency. Cell 46:963. 5 Michael, A., Hackett, J. J , Bennett, M., Kumar, V., and Yuan, D. 1989. Regulation of B lymphocytes by natural killer cells: role of gamma interferon. J. Immunol. 142:1095 6 Mikhael, A., Shao, A., and Yuan, D. 1991. Productive interactions between B and NK cells. Nat. Immun. Cell. Growth. Regul., 218:71. 7 Bosma, G. C , Custer, R. P., and Bosma, M. J. 1983. A severe combined immunodeficiency mutation in the mouse. Nature 301:527. 8 Marsnak-Rothstein, A , Fink, P., Gridtey, T, Raufet, D. H., Bevan, M J., and Getter, M. L. 1979. Properties and applications ol monoclonal antibodies directed against determinants of the Thy-1 locus. J. Immunol. 122.2491. 9 Yuan, D., vitetta, E. S., and Kettman, J. 1977. Cell surface immunoglobulin. II. Antibody responsiveness of subpopulations of B lymphocytes bearing different isotypes. J. Exp. Med. 145:1421. 10 Layton, J. E., Krammer, P. H., Hamaoka, T., Uhr, J. W., and Vitetta, E. S. 1985. Small and large B cell subsets respond differently to T cell-derived B cell growth and differentiation factors. J. Moll. Cell Immunol. 2:155. 11 Wysocki, L. J. and Sato, V. L. 1978. 'Panning' for lymphocytes: a method for cell selection. Proc. Natl Acad. Sci. USA 75:2844. 12 Yuan, D. and Tucker, P. W. 1984. Transcriptional regulation of the ii-6 gene in normal murine B lymphocytes. J. Exp. Med. 160:564. 13 Tutt, M. M., Kuziel, W. A., Hackett, J., Jr, Bennett, M., Tucker, P. W., and Kumar, V. 1986. Murine natural killer cells do not express functional transcripts of the or-, (3-, or r-chain genes of the T cell receptor. J. Immunol. 137:2998.
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preparations contain very few, if any, cells which stain with the macrophage marker F4/80 or MAC 1 (18), and they were able to induce Ig secretion by a population of B lymphocytes which was 99.9% pure. The activation of B lymphocytes by NK cells appears to be mediated by a soluble product(s) which can continue to be secreted by NK cells after irradiation. Because the helper activity is eliminated by mild fixation, the secreted product may not have a cell surface phase. Although we have not as yet characterized the active soluble factor(s) because of the apparent instability, increased IgM secretion is probably not mediated by two of the lymphokines known to be secreted by NK cells, T-IFN and TNF-a. We have shown that, under our conditions of B cell culture, these lymphokines do not increase Ig secretion. In addition, inclusion of anti-T-IFN or anti-TNF-a in the co-cultures does not affect the induction of B cell differentiation. Therefore, these lymphokines may not be implicated even indirectly. Although production of T-IFN by NK cells is not enhanced by high density B lymphocytes (5,6), we have previously reported secretion of a low basal level of T-IFN by NK cells (5). This production is obviously insufficient to affect either B cell proliferation or differentiation. Of the other lymphokines reported to be produced by NK cells, only TNF-a has been shown to stimulate mature B lymphocytes, and the effect has been documented only for human B cells (16,19). In this report we have shown this lymphokine to have little impact on murme B cell differentiation either in combination with r-IFN or with other lymphokines. Inasmuch as low density B lymphocytes can respond to IL-2 and IL-5 alone, presumably because of the presence of high affinity receptors (20,21), it is possible that induction by the NK factor is mediated by the alteration of lymphokine receptor expression on resting B lymphocytes. Experiments are in progress to test this possibility. In this respect it should be noted that the number of responding B cells in this system is relatively low, both in terms of proliferation and differentiation. Therefore only a subpopulation may be induced.
1380 Activation of B lymphocytes by NK cells 169 1693 21 Loughnan, M. S. and Nossal, G. J. V. 1989. Interleukin 4 and 5 control expression of IL-2 receptor on murine B cells through independent induction of its two chains. Nature 340:76. 22 Hodgkin, P. E., Yamashita, L. C, Coffman, R L, and Kehry, M R. 1990. Separation of events mediating B cell proliferation and Ig production by using T cell membranes and lymphokines. J Immunol. 145:2025. 23 Noelle, R J., Daum, J., BartJett, W. C, McCann, J., and Shepherd. D M. 1991. Cognate interactions between helper T cells and B cells. J. Immunol. 146:1118. 24 Abehsira-Amar, O., Leclercq, L, Chastagner, P., and Theze, J. 1987. B cell activating factor(s) (BCAF) containing supernatant can induce all classes of immunoglobulin and IgG switches in resting B cells. Lymph. Res. 6:169. 25 Saffran, D. C. and Singhal, S. K 1990. Further characterization of murine bone marrow-derived natural suppressor cells Potential relationships between NS and NK/LAK activities. Cell. Immunol 128.301. 26 Brenner, M. K , Vyakarnam, A., Reittie, J E., Wimpens, J. Z., Grab, J. P., Hoffbrand, A. V , and Prentice, H G. 1987. Human large granular lymphocytes induce immunoglobulin synthesis after bone marrow transplantation. Eur. J. Immunol. 1743.
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14 Cherwinski, H. C , Schumacher, J. H., Brown, K. D., and Mosmann, T. R. 1987. Two types of mouse helper T cell clone: further differences in lymphokine synthesis between TH1 and TH2 clones revealed by RNA hybridization, functionally monospecific bioassays and monoclonal antibodies. J. Exp. Med. 166:1229. 15 Yuan, D. and Vrtetta, E. S. 1984. Biosynthetic, surface labeling, and isolation of membrane immunoglobulin. Methods Enzymol. 108:426. 16 Katz, P., Whalen, G., Cupps, T. R , Mitchell, S. Ft., and Evans, M. 1989. Natural killer cells can enhance the proliferative response of B lymphocytes. Cell. Immunol. 120:270. 17 Becker, J. C , Kolanus, W., Lonnemann, C , and Schmidt, R. E. 1990. Human natural killer clones enhance in vitro antibody production by tumor necrosis factor alpha and gamma interferon. Scand. J. Immunol. 32:153. 18 Murphy, W. J , Kumar, V., and Bennett, M. 1990. Natural killer cells activated with interleukin 2 in vitro can be adoptively transferred and mediate hematopoietic histocompatibility-1 antigen-specific bone marrow rejection in vivo. Eur. J. Immunol. 20'1729. 19 Jelinek, D F and Lipsky, P. E. 1987. Enhancement of human B cell proliferation and differentiation by tumor necrosis factor-alpha and interleukin 1. J. Immunol. 139:2970 20 Rolink, A. G., Melchers, F, and Palacios, R 1989. Monoclonal antibocfies reactive with the mouse interleukin 5 receptor. J. Exp. Med.
International Immunology, Vol. 4, No. 12, pp. 1373 - 1380
Activation of B lymphocytes by NK cells Dorothy Yuan, Julie Wilder, Tarn Dang, Michael Bennett, and Vinay Kumar Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA Key words. B lymphocytes, helper factor, T-IFN, IgM secretion, NK cells, proliferation, tumor necrosis factor-a
The ability of NK calls to Induce differentiation of B lymphocytes to IgM secretion In vttro has been Investigated. Homogeneous preparations of NK cells obtained from IL-2 propagated splenocytes from SCID mice were found to have the ability to Induce resting B lymphocytes to proliferate and secrete significant amounts of IgM. The induction Is greatly enhanced by the presence of both IL-2 and IL-5 and does not require T lymphocytes or adherent cells in the responding population. Cell contact between the two populations Is not necessary suggesting that the effect is mediated by soluble factors) which can be produced even by irradiated NK cells. Because the activity cannot be replaced by either r-r-IFN or tumor necrosis factor-a or inhibited by antibodies to these lymphokines, a novel NK cell-derived factor(s) may be involved. The Implications of this Interaction between NK cells and B lymphocytes are discussed. Introduction Complex intercellular interactions form an integral part of the immune response. Much progress has been made towards understanding the interactions that need to occur between T, B, and antigen presenting cells for the initiation, as well as the regulation, of antibody production. An additional cell type which has been implicated in immunoregulation is the NK cell. There is considerable evidence that NK cells regulate B cell functions in that depletion of NK cells either in vivo or in vitro results in alterations in levels of antibody production (1 - 3 ) . However, the mechanisms underlying NK mediated regulation are not clear. The ability to purify NK cells and propagate them in vitro in the presence of IL-2 has permitted a much more direct approach to the analysis of the biology of NK cells. These studies have been further facilitated by the use of cells from SCID mice which, because of the virtual absence of T or B cells (4), have significantly simplified the analysis. Thus, utilizing this system, we have previously shown that co-culture of purified B and NK cells resulted in measurable alterations in the responses of both cell types which are dependent on the activation status of the B lymphocytes. Low density B lymphocytes (presumably in vivo pre-activated) can enhance the production of HFN by NK cells (5), which in turn induces these B cells to switch to lgG2a production after mitogenic stimulation (6). In the course of these experiments it came to our attention that, in the absence of mitogens, NK cells sometimes increased the basal level of IgM secretion. We therefore proceeded to investigate this phenomenon more systematically. We have now shown that NK
cells can elaborate a factor(s) which induces B lymphocytes to proliferate and secrete IgM. The activity of this factor is not dependent on the production of dFN or tumor necrosis factor TNF-a but requires the presence of IL-2 and IL-5. Thus, in contrast to the effect of B lymphocytes on NK cells, which is contact dependent, the factor(s) elaborated by NK cells, which can polyclonally stimulate B lymphocytes, appears to be constitutively produced.
Methods Animals Female BALB/c and Swiss NUDE mice (obtained from Harian Sprague Dawley, Inc., Indianapolis, IN) and C57BL/6 x DBA/2 (B6D2F1) F, mice (obtained from Jackson Laboratories, Bar Harbor, ME) were kept in specific pathogen-free conditions. C.B-17-sc/d mice (7) were raised in our own colony from a breeding pair originally obtained from Dr Mel Bosma (Fox Chase Cancer Center, Philadelphia, PA). All mice were used at age 6 - 1 0 weeks. Cell preparations T lymphocytes were depleted from spleen cell preparations by treating the cells with an anti-Thy-1.2 mAb (H013.4; 8) and baby rabbit complement (Pel-freez, Brown Deer, Wl) as previously described (9). High density ( > 1.095 mg/ml) resting
Correspondence to: D. Yuan Transmitting editor: L. Lanier
Received 27 July 1992, accepted 4 September 1992
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Abstract
1374 Activation of B lymphocytes by NK cells B lymphocytes were separated from low density (< 1.076 mg/ml) lymphocytes by centrifugation in discontinuous density gradients of Percoll (Pharmacia Fine Chemicals, Piscalaway, NJ) prepared as previously described (10). In some experiments B lymphocytes were further purified by panning on goat anti-IgM plates (CappelCooper Biomedical, Malvern, PA) by the method of Wysockj and Sato (11). Where indicated, cells were irradiated with 2500 cGy of (137Cs) 7-radiation. This dose reproducibly reduced the IL-2 proliferate response of SCID splenocytes to background levels. In other cases cells were fixed with glutaraldehyde (0.13%) and washed before use.
After T cell depletion and Percoll density sedimentation, high density lymphocytes were stained with an optimal amount of biotinylated goat anti-lgD and phycoerythrin - avidm as previously described (12). Positive cells were isolated from non-staining cells by sorting on the FACS Star Plus (Becton-Dickinson, San Jose, CA). Both sorted and control cells (non-sorted or non-stained) were cultured overnight before use to allow regeneration of capped receptors. Propagation of SCID NK cells SCID splenic NK cells were propagated as previously described (13). Cells harvested after 6 - 1 4 days of culture were pooled, washed three times with Balanced Salt Solution and used in the co-culture experiments. These cells have been previously shown to be virtually 100% NK-2.1 + , CD3", Lyt2", and L3T4" and have no rearranged Ig or TCR genes (13). Cell culture B lymphocytes were cultured in RPMI supplemented with 10% FCS with low lipopolysaccharide (LPS) content (Gibco, Grand Island, NY), glutamine, sodium pyruvate, non-essential amino acids, penicillin-streptomycin, and 2-mercaptoethanol. Cell concentrations varied between 0.5 and 1 x 106 cells/ml. Each culture condition was performed in triplicate in 96-well polyvinyl microtiter plates (Costar, Cambridge, MA) at 0.2 ml/well. Plates were incubated in a humidity controlled incubator maintaned with 7% CO2 at 37°C. For the membrane partition experiments, 3 x 106 NK cells (in 100^1) were added to chamber wells constructed with microporous, 0.4 ^m pore size, polycarbonate membrane (Transwells, Costar, Cambridge, MA). The lower portions of the wells contained 5 x 105 cells/ml (in 650 jd) high density B cells. Lymphokines and reagents Recombinant human IL-2 was a gift from Cetus Corporation (Emeryville, CA). Recombinant murine IL-1 and IL-4 were obtained from Dr Steven Gillis (Immunex, Seattle, WA). Recombinant murine IL-5 was obtained from Dr Colin Sanderson (MRC, Mill Hill, UK). Recombinant mouse TIFN was a gift from Shering Corporation, USA, distributed by the American Cancer Society (New York, NY). Recombinant human TNF-a was purchased from Genzyme Corporation (Boston, MA). LPS (Difco Laboratories, Detroit, Ml) was used at a final concentration of 20/tg/ml. Antj-rlFN (XMG-1; 14) was obtained by affinity purification of hybridoma supernatants. It was added to cultures at a concentration (3 /ig/ml) shown to be effective in neutralizing at
Proliferation assay Triplicate culture wells were pulsed with 2 ^Ci of [3H]thymidme (25 Ci/mmol, Amersham Corporation, Arlington Heights, IL) for 6 h and harvested using an automated cell harvester. [3H]Thymidine uptake was determined by counting in a Beckman (Irvine, CA) L53801 liquid scintillation counter. Measurement of IgM concentrations At the end of the incubation period, supernatants from triplicate wells were pooled and IgM concentrations were measured by radioimmunoassay as described (15). Briefly, total antibodies were capured in 96-well polyvinyl microtiter plates coated with 5 ng of affinity-purified goat anti-IgM (Cappel-Cooper Biomedical). Unbound sites were saturated with 50% FCS - PBS. The amount of bound antibodies was determined with affinity-purified goat anti-IgM labeled with Na12Sl. If duplicate determinations showed > 10% variation the assay was repeated. Concentrations were determined by comparison with standard curves using purrfied hybridoma IgM in each assay. Test for mycoplasma DNA Various supernatants from NK cells growing in culture were tested for the presence of mycoplasma RNA sequences using the Mycoplasma T.C. Rapid Detection System (Gen-Probe, San Diego, CA). The assay was performed according to the manufacturer's directions. Results Induction of IgM secretion by IL-2 propagated NK cells As a source of highly purified NK cells, splenocytes from SCID mice were cultured in the presence of 500 U/ml IL-2 for 6 - 1 4 days. Previous experiments have shown that cells obtained from these cultures are virtually 100% NK-2.1+, CD3", Lyt2~, and L3T4~, and have no re-arranged Ig or TCR genes (13). B lymphocytes consisted of T-depleted BALB/c or B6D2F1 splenocytes which were fractionated into high and low density subpopulations by Percoll gradient sedimentation. Figure 1 presents the results of an experiment representative of five independent experiments. The high density B lymphocytes cultured either with IL-2 alone or in combination with IL-4 or IL-5 resulted in only a slight increase in the production of IgM when measured on day 5. Further, these cells did not respond to a supernatant obtained from phorbol-12-myristate-13-acetate (PMA)-stimulated EL4 thymoma cells (10) which induced secretion of IgM from the low density activated B lymphocytes (data not shown). Upon addition of NK cells, both with and without exogenous lymphokines, enhancement of IgM secretion can be observed. However, the most significant increase was dependent on the presence of both IL-2 and IL-5 in the medium. The addition of only IL-5 to NK cells was insufficient for the induction of IgM secretion (data not shown). Furthermore, the extent of help
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FACS
least 20 U/ml of TIFN and can completely prevent the TIFN mediated inhibition of WEHI279 proliferation (5). Anti-murine TNF-a was purchased from R & D Systems (Minneapolis, MN) and used at 3/ig/ml, which is 10-fold the concentration shown to be effective for 50% inhibition of their indicator cell line (0.05 ng/ml TNFor).
Activation of B lymphocytes by NK cells 1375
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Fig. 1 . Enhancement of IgM secretion by NK cells. High density BALB/c B lymphocytes at a concentration of 5 x 105 cells/ml were cultured with or without NK cells which had been propagated for 14 days at the indicated ratios. IL-2 and IL-4 were each added at 100 U/ml and IL-5 was added at 5 U/ml. Duplicate samples from pooled triplicate cultures were assayed for IgM production 5 days after initiation of culture. Duplicate determinations did not vary by > 1 0 % in all values presented.
was dependent on the number of NK cells added in the cocultures. At ratios of NK:B cells > 1 : 1 , IgM secretion did not further increase and tended to be inhibitory (data not shown). In comparison, identical cultures stimulated with LPS resulted in ~ 10-fold higher responses (data not shown). Addition of NK cells to low density B lymphocytes resulted in a similar enhancement of IgM secretion (data not shown); however, due to the highly variable activation status of these cells, the extent of augmentation was less consistent. Therefore only high density resting B lymphocytes were used in subsequent experiments. Help by NK cells is not mediated via other lymphocytes Although the fractionation of B lymphocytes was preceded by depletion of T cells by anti-Thy-1.2 antibodies plus C , residual T cells were found to remain in the preparation to variable extents as determined by response to concanavalin A (Con A) stimulation. To ascertain that B lymphocytes can respond in the total absence of conventional T cells, high density cells were prepared from spleens obtained from NUDE mice and co-cultured with IL-2 propagated SCID NK cells. Figure 2 shows that although the response was somewhat lower, these B cells could be induced by NK cells to secrete IgM in the presence of IL-2 and IL-5. To further insure that T cells do not play a role, small resting B cells were purified by allowing them to bind to plates coated with anti-lgM. The Con A response (5 /ig/ml) of cells removed from the plates was reduced to background levels, but FACS analysis of the cells showed that they were only 95% lgM + suggesting that some adherent cells may have co-purified during the panning procedure. This population was then compared with non-panned B cells for their ability to respond to NK cells. Figure 3 shows that there was no significant difference in the responses of the two populations. Furthermore, examination of the IgM accumulation on both days 5 and 7 showed no significant difference in the kinetics of the response of the two populations. Finally, to obtain a population of B cells which is not
Fig. 2. Response of NUDE B lymphocytes to NK stimulation. High density splenocytes from a NUDE mouse were cultured with NK cells (propagated for 12 days) at a ratio of 1.1 (each at 5 x 10 s cells/ml) in microtiter wells with or without lymphokines (IL-2 at 100 U/ml, IL-5 at 5 U/ml) as indicated. In addition, transwell cultures were set up by diluting B lymphocytes in 0.6 ml at 5 x 105 cells/ml in bottom wells with NK cells added to transwells at 3 x 106 cells/100 jd. Culture supernatants were assayed for IgM content on day 6 as in Rg 1. Duplicate determinations did not vary by > 10% in all values presented.
contaminated by NK or adherent cells which may co-purify during panning, Percoll fractionated T cell depleted B lymphocytes were stained with anti-lgD and isolated by cell sorting. From a starting population which was 94% lgD + , sorting resulted in a population which was 99.9% lgD + . The cells were cultured overnight to regenerate cell surface Ig and subsequently cultured with NK cells with or without lymphokines. In addition to IL-2 and IL-5, IL-1 was also added to the cultures to insure optimal responses in the absence of adherent cells. Comparison of the response of stained versus non-stained lymphocytes (Figure 4) showed that stained cells gave a lower response. Because the reduction was apparent also in the background response to lymphokines as well as to LPS, this effect may be attributed to the ligation of cell surface Ig. Nevertheless, when sorted cells are compared with non-sorted but stained B lymphocytes, it is dear that the response to NK cells was not affected by the further purification step. The further reduction in LPS response of the sorted B cells is probably due to depletion of adherent cells. Indeed it was found that the responses of all cultures containing IL-1 were increased - 2 0 % when compared with those containing only IL-2 and IL-5 (data not shown). From these experiments it can be concluded that neither T lymphocytes nor adherent cells are required for the help provided by NK cells. Help from NK cells is resistant to irradiation but is eliminated by fixation To determine if replication of NK cells during co-culture with B lymphocytes was necessary for the induction of IgM secretion, NK cells were first irradiated before initiation of culture. Figure 5 shows the results of one of two experiments performed. Clearly irradiation did not significantly alter this activity. However, mild fixation of NK cells with glutaraldehyde completely eliminated their ability to induce IgM secretion (data not shown).
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Fig. 3. Comparison of the response of anti-IgM panned B lymphocytes with non-panned B cells. After anti-Thy-1.2 treatment, high density B cells (B6D2F1) were panned on anti-IgM plates. After removal from plates, the cells were cultured overnight in bulk before they were aliquoted into mere-culture plates at 5 x 105 cells/ml in the presence or absence of equal numbers erf NK cells (propagated 7 days) with or without lymphokines (IL-2 at 100 U/ml, IL-5 at 5 U/ml) as indicated. Control cultures consisted of non-panned B lymphocytes treated identically. Culture supernatants were harvested on day 5 and day 7 and assayed for IgM accumulation as indicated in Fig. 1. Duplicate determinations did not by vary by > 10% in all values presented.
Help for B lymphocytes can be mediated by a soluble tactor(s) To determine whether the ability of NK cells to induce IgM secretion is mediated by a secreted factor, the two cell types were separated from each other by a Millipore membrane during culture. As shown by a representative experiment (total of three) in Fig. 2, B lymphocytes could be induced to secrete IgM even if NK cells were placed in a transwell. Again, the activity was dependent on the presence of IL-2 and IL-5. It is interesting that the resultant IgM secretion was higher in cultures where the two cell types were separated. The significance of this finding is not clear because cell density conditions were different owing to conditions imposed by the architecture of the transwell set up. However, it is also possible that the membrane can prevent inhibitory interactions which depend upon cell contact. In other experiments (not shown) it was found that upon removal of the transwell containing NK cells either on day 1 or 2 after initiation of culture, B cells could still be induced to secrete IgM (not shown), although at a reduced level, suggesting that the activity may be unstable during culture. If the NK help is mediated by a soluble factor, culture supernatants of NK cells should contain the active factor. Media from propagated NK cells were collected on day 6 of culture, 3 days after the last feeding with IL-2. The supernatant was added in two different concentrations to resting B cells in the presence of various lymphokines. Figure 6 shows the results obtained from supernatants collected from two different NK cultures. When 20 - 25% of the culture media was made up with NK supernatant, a significant enhancement of IgM production can be obtained in the presence of IL-2 and IL-5. However, the increase is relatively low compared with experiments in which NK cells were present. Similar results were obtained from at least six other independent culture supernatants (not shown). Nonetheless, because the NK cell supernatant could enhance IgM secretion, it was possible to test its effect on inducing B cell proliferation without having to assess the contribution of each component if we were to measure the NK:B cell co-cultures.
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Figure 7 (representative data from frve independent expenments) shows the day 3 proliferative response of small B cells cultured in the presence of NK supernatants. Interestingly, despite the fact that the supernatant contained residual IL-2 from the NK cultures, addition of fresh IL-2 enhanced the proliferative response. Further, the most extensive enhancement occurred in cultures containing IL-4. Statistical evaluation using the two sample T test showed that the proliferation of cells cultured in the presence of NK supernatants differed significantly from those
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Fig. 7. B cell proliferation in response to NK supernatants Supernatants collected, as in Fig. 6, were added to triplicate B cell cultures at the concentrations indicated. Where indicated, IL-2 was added at 100 U/ml and IL-4 at 25 U/ml. [3H]Thymidine was added for 6 h on day 3 before the cells were harvested. Data represent the extent of incorporation in mean c.p.m. ± SE of the mean.
selected experiments, were found not to contain mycoplasma RNA sequences. ISO--
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cultured only in IL-2 plus IL-4 (P < 0.166 for 12.5% supernatant and P s 0.076 for 25% supernatant). However, IL-5 added together with IL-2 did not significantly increase the level of proliferation induced by IL-2 alone (data not shown). The response in these cultures was much decreased 6 days after initiation of culture (data not shown). Comparison of the proliferative response with that of LPS-activated culture suggests that - 1 0 % as many cells were stimulated. Because both NK cells and their supernatants were obtained from relatively long term cultures, we tested for the inadvertent presence of mycoplasma bacteria which may affect B cell differentiation. However, the supernatants used in the experiments shown in Figs 6 and 7, as well as other randomly
NK cells have been shown to secrete a number of known lymphokines. However, the only lymphokines documented to have effects on mature B lymphocytes are T-IFN and TNF-a. Furthermore, most of these effects have been restricted to the proliferative response. Nevertheless, to determine if these two lymphokines could be responsible for the help mediated by NK cells, they were added in various concentrations to B cell cultures in the presence of IL-2 and IL-5. Addition of r-HFN, alone or in combination with r-TNF-a, did not increase the stimulatory effect of IL-2 and IL-5 on either high or low density B lymphocytes (Fig. 8). r-TNF-a was somewhat suppressive for the response which was more evident for low density B lymphocytes. In addition, antibodies to T-IFN and TNF-a were included in the NK - B cell cultures to test whether the lymphokines play a role only in the presence of other NK factors. Figure 9 (representative of two independent experiments) shows that these antibodies exerted no demonstrable effect on the response of B cells, either in the presence or absence of NK cells. Thus, these two lymphokines are apparently not directly responsible for the increase in Ig secretion induced by NK cells. Discussion Using purified NK cells, obtained by in vitro propagation of SCID splenocytes, we have shown that they are able to induce highly purified small resting B lymphocytes to differentiate to IgM secretion (Fig. 4). It is important to note that the effect of NK cells can be exerted on high density resting B cells. The resting status of these B cells is based on the criterion that, when compared with large B lymphocytes, they secrete only minimal amounts of IgM in response to IL-2, IL-4, IL-5, or combinations of these lymphokines (Fig. 8). They also do not respond to supernatants
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Fig. 8. Effect of T-IFN and TNF-a on the B lymphocyte stimulatory activity of IL-2 plus IL-5. Various concentrations of T-IFN and TNF-a were added as indicated to cultures of high density (A) or low density (B) lymphocytes (BALB/c) in the presence of IL-2 (100 U/ml) and IL-5 (5 U/ml) Day 6 IgM accumulation was measured as described in Fig. 1. Duplicate determinations did not by vary by > 1 0 % in all values presented.
from PMA-stimulated EL-4 lymphoma cells which can induce IgM secretion by low density B lymphocytes (10, and data not shown). Unlike the effect of NK cells on human B lymphocytes (16,17), cross-linking of cell surface determinants is not required. On the other hand, we have noted that low density B lymphocytes will also respond to NK cells. However, despite the much greater absolute response, the fold increase in IgM secretion in the presence of NK cells is not as dramatic. In this respect, it should be noted that there is a report of NK cell activation of Ig secretion after stimulation of human B cells by Staphyiococcus aureus (SAC) in the presence of rlL-2 (16). It is possible, therefore, that the pre-activation of human B cells by SAC mimics in vivo activation of B lymphocytes and brings Ig secretion up to a detectable level. The use of NK cells propagated from the spleens of SCID mice gives assurance that the helper factor(s) is not produced by T cells. Moreover, the activity does not require T lymphocytes in the responding populations as was clearly shown by the use of highly purified B cells obtained by either panning or FACS. Furthermore, lymphocytes obtained from NUDE mice are also responsive. It is also clear that adherent cells play a minor role in the response of B lymphocytes to NK cells. The IL-2 propagated NK
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Fig. 9. Effect of antibodies to T-IFN and TNF-a on response of B lymphocytes to NK help. High density B lymphocytes (B6D2F1) were cultured with or without NK cells, which had been propagated for 7 days in the presence or absence of 3 /ig/ml anti-T-IFN or 3 (ig/ml anti-TNF-a as indicated. IgM accumulation on day 5 was assessed as described in Rg. 1. Duplicate determinations did not by vary by > 10% in all values presented.
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D
0
Activation of B lymphocytes by NK cells 1379
Because NK cells produce some lymphokines also made by T cells, it is possible that the NK product(s) described here are not unique to NK cells. It should be noted, however, that there are very few T cell factors which can induce small resting B lymphocytes to secrete Ig without direct physical interaction (22 - 25). In contrast, NK cells can effectively help B lymphocytes when placed across a Millipore membrane. These results also suggest that cell -cell interactions are not necessary for induction of factor(s) secretion by NK cells. However, because the supernatants of NK cells have much reduced activity when compared with the cells themselves, the factor(s) may not be stable. Finally, the capacity to help B lymphocytes is not restricted to propagated NK cells from SCID splenocytes. We have found that fresh SCID splenocytes (manuscript in preparation) as well as adherent LAK cells from B6 splenocytes (J. Wilder and D. Yuan, unpublished observations), can also induce IgM secretion by resting B cells. Therefore, it is unlikely that production of this factor(s) is either restricted to cells from SCID animals or dependent on high concentrations of IL-2. In this regard, it will be important to assess the in vivo significance of the documented NK - B cell interaction. Most of the available in vivo data relating to NK cell effects on B lymphocyte differentiation are based on utilizing antigen-specific systems and they show that NK cells
down-regulate the responses (1 - 3). The in vivo effect of NK cells on polyclonal secretory response or bystander effects has not been extensively explored. Given the dependence of the NK help on T cell derived lymphokines (IL-2 and IL-5), it is conceivable that NK cells serve to magnify T-dependent responses in the appropriate microenvironment and/or pathological condition. For example, the continued production of antibodies in bone marrow transplant patients with depressed T cell numbers has been attributed to help mediated by the presence of activated large granular lymphocytes (26).
Acknowledgements We thank Jing Tan for help in preparation of NK cells, Pat Collins for expert assistance in ceD sorting, and Marilyn Mims for cheerful secretarial assistance. Supported by National Institutes of Health Grants CA51426, AI-20451, and CA09082.
Abbreviations (Don A LPS PMA SAC TNF
concanavalin A lipopolysacchande phorbol-12-myristate-13-acetate Staphylococcus aureus tumor necrosis factor
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preparations contain very few, if any, cells which stain with the macrophage marker F4/80 or MAC 1 (18), and they were able to induce Ig secretion by a population of B lymphocytes which was 99.9% pure. The activation of B lymphocytes by NK cells appears to be mediated by a soluble product(s) which can continue to be secreted by NK cells after irradiation. Because the helper activity is eliminated by mild fixation, the secreted product may not have a cell surface phase. Although we have not as yet characterized the active soluble factor(s) because of the apparent instability, increased IgM secretion is probably not mediated by two of the lymphokines known to be secreted by NK cells, T-IFN and TNF-a. We have shown that, under our conditions of B cell culture, these lymphokines do not increase Ig secretion. In addition, inclusion of anti-T-IFN or anti-TNF-a in the co-cultures does not affect the induction of B cell differentiation. Therefore, these lymphokines may not be implicated even indirectly. Although production of T-IFN by NK cells is not enhanced by high density B lymphocytes (5,6), we have previously reported secretion of a low basal level of T-IFN by NK cells (5). This production is obviously insufficient to affect either B cell proliferation or differentiation. Of the other lymphokines reported to be produced by NK cells, only TNF-a has been shown to stimulate mature B lymphocytes, and the effect has been documented only for human B cells (16,19). In this report we have shown this lymphokine to have little impact on murme B cell differentiation either in combination with r-IFN or with other lymphokines. Inasmuch as low density B lymphocytes can respond to IL-2 and IL-5 alone, presumably because of the presence of high affinity receptors (20,21), it is possible that induction by the NK factor is mediated by the alteration of lymphokine receptor expression on resting B lymphocytes. Experiments are in progress to test this possibility. In this respect it should be noted that the number of responding B cells in this system is relatively low, both in terms of proliferation and differentiation. Therefore only a subpopulation may be induced.
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14 Cherwinski, H. C , Schumacher, J. H., Brown, K. D., and Mosmann, T. R. 1987. Two types of mouse helper T cell clone: further differences in lymphokine synthesis between TH1 and TH2 clones revealed by RNA hybridization, functionally monospecific bioassays and monoclonal antibodies. J. Exp. Med. 166:1229. 15 Yuan, D. and Vrtetta, E. S. 1984. Biosynthetic, surface labeling, and isolation of membrane immunoglobulin. Methods Enzymol. 108:426. 16 Katz, P., Whalen, G., Cupps, T. R , Mitchell, S. Ft., and Evans, M. 1989. Natural killer cells can enhance the proliferative response of B lymphocytes. Cell. Immunol. 120:270. 17 Becker, J. C , Kolanus, W., Lonnemann, C , and Schmidt, R. E. 1990. Human natural killer clones enhance in vitro antibody production by tumor necrosis factor alpha and gamma interferon. Scand. J. Immunol. 32:153. 18 Murphy, W. J , Kumar, V., and Bennett, M. 1990. Natural killer cells activated with interleukin 2 in vitro can be adoptively transferred and mediate hematopoietic histocompatibility-1 antigen-specific bone marrow rejection in vivo. Eur. J. Immunol. 20'1729. 19 Jelinek, D F and Lipsky, P. E. 1987. Enhancement of human B cell proliferation and differentiation by tumor necrosis factor-alpha and interleukin 1. J. Immunol. 139:2970 20 Rolink, A. G., Melchers, F, and Palacios, R 1989. Monoclonal antibocfies reactive with the mouse interleukin 5 receptor. J. Exp. Med.
