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Natural killer cells can enhance the proliferative responses of B lymphocytes
CELLULAR
120,270-276 (1989)
IMMUNOLOGY
SHORT COMMUNICATIONS Natural Killer Cells Can Enhance the Proliferative Responses of 6 Lymphocytes PAUL KATZ, GAIL WHALEN, THOMAS R. CUPPS, S. RAY MITCHELL, AND MISHELL EVANS Division ofRheumatology,
Immunology and Allergy, Department OfMedicine, School ofMedicine, Washington, D.C. 20007
Georgetown University
Received November 14,1988; accepted December 30, 1988
In addition to lytic activity against malignant and virally transformed target cells, recent evidence has suggestedthat natural killer (NK) cells can modulate immune activities such as the suppression of B cell responsesthrough noncytotoxic means. Using human B cells and highly purified autologous NK cells, we have demonstrated that NK cells can substantially augment the proliferative responsesof B cells stimulated with the surface immunoglobulin crosslinking agents anti-IgM or Staphylococcus aureus Cowan strain I (SAC). This “enhancer” activity of NK cells was quite potent and was observed at an NK:B cell ratio as low as0.05. Peak blastogenic responsesof B cells cocultured with NK cells in the presence of B cell activators were observed at 2-3 days, similar to the responsesof B cells in the absence of NK cells. Using the inhibitor of DNA synthesis mitomycin C, we determined that B cells and not NK cells were proliferating in cocultures of these lymphocytes stimulated with SAC. Activated B cells neither prevented the lysis of the isotope-labeled NK-sensitive target cell line K562 nor formed conjugates with NK cells, suggestingthat cell contact was not a prerequisite for the effect. These studies have further expanded the functional repertoire of NK cells to include enhancer as well as suppressor and lyk
aCtiVitk3.
0 1989 Academic
Press, Inc.
INTRODUCTION
Considerable information has accrued regarding the activation, proliferation, and differentiation of B lymphocytes ultimately leading to the production of antibody (1, 2). Although early studies in man addressedthese issuesin systemswhich were largely dependent upon the interaction of B cells with T cells or T cell products and which reflected only a relatively “late” phase of B cell function, it is now possible to examine these events in vitro using polyclonal signals which can activate B lymphocytes by interacting with surface immunoglobulin (Ig) (I), analogous to the binding of antigen which in turn initiates the transition of resting cells into antibody-producing and -secreting lymphocytes. Positive and negative signals transmitted by non-B cells can profoundly influence B cell function (3,4). In this regard, studies in animal models (5,6) and in man (79) have documented that natural loller (NK) cells can act as potent suppressors of B cell function and antibody production in vitro and in vivo. Those studies in man have largely employed mitogen-induced, T cell-dependent systemsof antibody production which assessthe activity of only a small population of B cells at a relatively “late” stage of activation and differentiation. Since B cell activation and proliferation can 270 OOO8-8749189 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights ofreproduction in any form reserved
SHORT
COMMUNICATIONS
271
be induced and measured by agents which can crosslink surface Ig (I), we have investigated the effects of NK cells on these responses.We now report that NK cells can act as potent “enhancers” of B cell blastogenic responses. METHODS Isolation of cells. For these studies, highly purified NK cells devoid of contamination with non-NK cells were necessary.Nylon wool passedperipheral blood lymphocytes ( 106) obtained from adult donors were treated with 0.45 ~1 anti-Leu- 1 (CD5) (Becton-Dickinson, Mountain View, CA), 0.2 ~1anti-T3 (CD3) (Ortho Pharmaceuticals, Raritan, NJ), 0.1 ~1 anti-T4 (CD4) (Ortho Pharmaceuticals), and 0.1 ~1 antiCR1 (Becton-Dickinson) for 1 hr at 4°C. Cells were rosetted with bovine erythrocytes coated with goat anti-mouse IgG (Boehringer-Mannheim Biochemicals, Indianapolis, IN) and centrifuged on Ficoll-Hypaque cushions; nonrosetted cells were ~95% NK cells as determined by fluorescent staining with the NK-reactive monoclonal antibodies anti-Leu- 11c (CD 16) ( 10) (Becton-Dickinson) or anti-NKH- 1 ( 11) (Coulter Immunology, Hialeah, FL). NK cells were free of contaminating T and B cells as determined by staining with anti-T3 and anti-Leu-16 (Becton-Dickinson), respectively, and lacked monocytes by esterasestaining. Additionally, NK cells did not proliferate to the T cell mitogen phytohemagglutinin and did not contain CD4 mRNA by Northern blot analysis (data not shown). No NK cell proliferative responsesto the B cell activators anti-p or SAC were observed (see below). These NK cells contained all the lytic activity in our lymphocyte populations; no cytotoxicity was observed with rosetted (non-NK) cells (data not shown). Purified B cells were obtained by treatment of peripheral blood mononuclear cells with 25 mML-leucine methyl ester (Sigma Chemical Co, St. Louis, MO) to kill monocytes and NK cells. Cells were rosetted with aminoethylisothiouronium bromidetreated sheep erythrocytes to deplete T lymphocytes. B cells so obtained were >90% pure as determined by fluorescent staining with anti&u- 16 and were free of T cells, monocytes, and NK cells when reacted with fluoresceinated anti-T3, esterase,and fluoresceinated anti&u- 11c, respectively. B cell proliferation assays. B cell proliferative responsesto the surface Ig crosslinking agents anti-IgM or Staphylococcus aureus Cowan strain I (SAC) were determined as previously described ( 12). Briefly, lo5 peripheral blood B cells were cultured with 100 pg/ml F(ab)‘z goat anti-human IgM (p chain specific) (anti-p) (Cappel Laboratories, Cochranville, PA) or a 1:12,500 dilution of SAC (Bethesda Research Laboratories, Bethesda, MD) in 10%fetal calf serum (FCS) in RPM1 1640 media in 96-well flat-bottom microtiter plates. After 3 days in culture at 37°C in 5% COz in air at 100% humidity, wells were pulse-labeled with 1 &i [3H]thymidine (New England Nuclear, Boston, MA) for 18 hr and the isotope incorporation was determined. Results are ‘expressedas the means of triplicate wells in counts per minute (cpm) X 10m3. In some experiments, DNA synthesis by lymphocytes was blocked by treatment with 40 pg/ml mitomycin C (mito) (Sigma Chemical Co.) for 1 hr at 37°C. Cells so treated were washed four times prior to use in the above assays. Cytotoxicity assuys. A standard chromium-5 1 (5’Cr)-release microcytotoxicity assay for determination of NK activity was employed ( 13). Briefly, lo6 K562 target cells were labeled for 1 hr with 300 &i “Cr (ICN, Cleveland, OH), washed three times, and resuspended in 10%FCS in RPM1 1640 media. Ten thousand labeled target cells (100 ~1) were mixed with an equal volume of effector cells in V-bottom microtiter
SHORT COMMUNICATIONS 1 ocIANTI-p
A
SAC
B
6b
iI,
)-
4
1I_ B
B
B+NK
B+NK
FIG. 1. The effect of NK cells on anti-p- and SAC-induced B cell proliferation. B cells were stimulated with anti-r ( 100 pg/mI) (A) (n = 20) or SAC ( 1: 12,500) (B) (n = 15) in the presence or absence of autologous NK cells. Circles represent the average of triplicate samples and bars represent mean responses.
wells. Spontaneous release of “Cr by target cells was determined by placing labeled target cells in wells in the absence of effector cells. Plates were centrifuged and incubated for 4 hr under tissue culture conditions. Supematant was removed and counted in a gamma counter; percentage cytotoxicity (or percentage “Cr release) was determined by supematant cpm minus spontaneous release cpm divided by total target cell cpm minus spontaneous release cpm X 100. In some experiments, cold target inhibition studies were performed in which unlabeled K562 cells or B cells stimulated with SAC for 3 days were added in increasing number to mixtures of labeled K562 cells and effector cells and cytotoxicity was determined as described above. Statistical analysis. Data were compared with the Student’s t test for paired samples. RESULTS We initially examined the ability of highly purified human NK cells devoid of contamination with non-NK cells to affect the proliferative responses of autologous B cells to the surface Ig crosslinkers anti-p or SAC. Anti-p or SAC induced substantial B lymphocyte proliferation as measured by thymidine incorporation which peaked at 3 days (Fig. 1). NK cells significantly increased B cell DNA synthesis when added at the initiation of the culture period (P < 0.00003 for anti-p; P < 0.0003 for SAC). NK cells alone did not proliferate to either agent and neither B cells, NK cells, nor NK cells plus B cells proliferated in the absence of anti-p or SAC (data not shown).
273
SHORT COMMUNICATIONS
5
1.0
0.25
0.5
0.1
, 0.05
NK:B
FIG. 2. The effect of decreasednumbers of NK cells on B cell blastogenesis.Proliferative responsesof B
cells alone are shown at the lower left. Data points represent means of triplicate samples.
These results were noted when the ratio of NK:B cells was 1:1; however, decreasing the number of NK cells still resulted in substantial enhancement of B cell blastogenesis (Fig. 2). Thus, an NK:B cell ratio as low as 0.05 still produced significant increments in proliferative responsesof B cells to both anti-p and SAC, indicative of the potent helper activity of NK cells. It was necessaryto ascertain if NK cells altered the kinetics of B cell proliferative responsesto polyclonal signals. To determine this, B cells were cocultured with antip with or without autologous NK cells and thymidine incorporation was determined on Days 2-6. The proliferation of B cells to anti-p peaked at Days 2-3 of the assay period and similar kinetic responseswere noted for NK cell-induced augmentation, suggesting that NK cells did not exert helper effects by merely shifting the time to maximal responsiveness(Fig. 3). Similar results were observed when SAC rather than anti-p was used (data not shown). Although DNA synthesis was increased when B cells were cultured with NK cells in the presence of known B cell stimulators, we could not be certain which cell type was responsive on the basis of these experiments. Despite the lack of NK cell blastogenesisto these signals, it was conceivable that NK cells were proliferating in response to signals delivered by activated B cells. To test this, we used mitomycin C (mito), an inhibitor of DNA synthesis, to determine the nature of the increased proliferative responses we had observed. Pretreatment of B cells with mito prior to stimulation with SAC abrogated DNA synthesis (Fig. 4). When mito-treated B cells were cocultured with NK cells, the augmented blastogenesisobserved with untreated B cells was eliminated, indicating that B cells were proliferating to SAC in the presence of NK cells. Mito treatment of NK cells prior to coculture with B cells did not prevent increasedB cell DNA synthesis induced by NK cells. Therefore, B cells were the proliferating cells in this system and NK cells had enhancer activity for these B lymphocytes. Given these results, it was important to determine if NK cells could bind activated B cells, which would indicate that cell-to-cell contact might be relevant to the ob-
274
SHORT COMMUNICATIONS 30
I
c---a
25-I
a cws
+--O~+NK
b z t l5 4 10
5 0: o^ 20 0
2
3
4
5
Day
FIG. 3. The effectsof NK cells on the kinetics of B cell proliferation to anti-p. B cells were cultured with anti-p with or without autologous NK cells (1: 1 ratio) and blastogenic responsesdetermined on days 2-6. Data points represent the means of triplicate samples. Similar results were noted in three separate experiments.
served enhancer activity. For these studies, we employed a standard assay used for the assessmentof NK cell lytic function against the NK-sensitive target cell line, K562 (Fig. 5). When added to standard NK cytotoxicity assays,unlabeled or “cold” K562 cells or other NK-sensitive target cells abrogate NK-dependent lysis of labeled K562 cells by binding to NK cell surface receptors thereby sterically inhibiting the binding and subsequent lolling of labeled targets. With this technique, we used B cells acti-
CPM (x10-3) 4 I
6 1
I
6 !
1
10 I
B+NK
Blnno+NK
B+NKmno
FIG. 4. Delineation of the proliferating cell in B-NK cell interactions. DNA synthesis was blocked by pretreating B or NK cells with mito. Bars represent means of triplicate responses. Mito treatment of B cells (Bmito) inhibited SAC-induced blastogenesis.NK cells significantly increased B cell DNA synthesis; however, this was not observed when Bmito cells were employed. Pretreatment of NK cells with mito (NKmito) still resulted in NK cell-associated augmentation of SAC-induced B cell blastogenesis.Similar results were observed in four separate experiments and were also noted when DNA synthesis was blocked by y irradiation (data not shown).
275
SHORT COMMUNICATIONS
01
I
0 I 510
20
50
100
Cold Targets (~10.~)
FIG. 5. Activated B cells do not bind to NK surface receptors required for lytic activity. A cold target inhibition assaywas utilized in which unlabeled SAC-activated B cells or unlabeled K562 cells were added in increasing number to mixtures of labeled K562 cells and purified NK cells (5: 1 effector ratio). Data points represent the mean percentage cytotoxicity of triplicate wells. The dotted line representsthe percentage cytotoxicity (40%) in the absenceof cold target cells.
vated with SAC for 3 days as cold targets and compared this inhibitory activity with that of cold K562 cells (Fig. 5). Although unlabeled K562 cells inhibited NK cell killing of isotope-labeled K562 cells, unlabeled SAC-stimulated B cells did not prevent target cell lysis. Therefore, activated B cells did not bind to the same recognition structures on NK cells as susceptible target cells. In experiments not shown, we have not observed SAC-activated B cell binding to NK cells in a direct visualization, single cell-in-agarose assay (14). These data suggest that NK cell augmentation of B cell responsesdoes not require direct NK-B cell interactions. DISCUSSION The present study has demonstrated a heretofore unrecognized role for NK cells in the regulation of normal B cell function. Although previous studies have demonstrated the ability of NK cells to act as suppressor cells of antibody production (5-9), we now believe that the functional repertoire of these cells should be expanded to include helper activity aswell. It is now evident that B cells can be activated to proliferate in vitro by agents which can crosslink surface Ig (I), analogous to the binding of antigen in vivo. We have demonstrated that NK cells can substantially augment the blastogenesis of B cells induced by SAC or anti-p, even at a ratio to B cells which is quite low. These data would suggestthat NK cells are quite potent in the ability to enhance these responses.Additionally, we have demonstrated that NK cells do not merely alter the kinetics of B cell proliferation; the time to peak thymidine incorporation was similar for B cells in the presence or absence of NK cells. Furthermore, our data show that the proliferating cell in cocultures of B cells plus NK cells is clearly the B cell since inhibition of B cell DNA synthesis abrogated NK cell-induced boosting of B cell blastogenesis while similar treatment of NK cells did not prevent this effect. Last, it does not appear that direct NK-B cell contact is a prerequisite for this effect as activated B cells neither bound to target cell receptors on NK cells nor formed conjugates with these lytic cells in direct visualization assays.
276
SHORT COMMUNICATIONS
Further studies will be needed to determine whether NK cells can be separatedinto definable subsetsmediating enhancer or suppressor activities, as has been done for T lymphocytes. Similarly, it will be important to determine the mechanism(s) whereby NK cells can augment B cell responses. In data reported here, we have determined that NK-B cell contact is not a prerequisite to NK cell-dependent augmentation of B lymphocyte blastogenesis. Two studies have shown that populations of lymphocytes containing NK cells are capable of producing a B cell growth factor (BCGF) when stimulated with the T cell mitogen phytohemagglutinin (15, 16). However, these were heterogeneous lymphocytes in which contamination with non-NK cells may have contributed to the observed BCGF activity. Furthermore, removal of NK cells with the NK cell-reactive monoclonal antibody anti-Leu- 11 eliminated lytic function but did not affect BCGF production (16), suggesting that this factor was derived from cells lacking the NK cell phenotype. In preliminary studies, we have demonstrated that stimulation of NK cells with SAC did not induce BCGF production although phytohemagglutinin does (data not shown). However, the addition of NK cells to cultures of B cells plus SAC resulted in substantial BCGF production above that of SAC-stimulated B cells alone. Studies are now in progress to determine if NK cells are producing BCGF in response to stimulation by SAC-activated B cells or if NK cells are inducing BCGF production by activated B cells. Therefore, although the mode of NK cell-induced enhancement of B cell blastogenesis is as yet unknown, the spectrum of NK cell activity appears to encompass not only cytotoxicity but significant and diverse immunoregulatory functions as well. Just as the precise mechanism of NK cell killing awaits precise clarification, the elucidation of these nonlytic activities warrants further investigation. ACKNOWLEDGMENTS The authors thank Andrew Pachner for critical review and Carol Moran for expert editorial assistance. This work was supported by grants from the National Institutes of Health (A124636) and from the Metropolitan Washington Chapter of the Arthritis Foundation.
REFERENCES 1. Muraguchi, A., Kehrl, J. H., Butler, J. L., and Fauci, A. S., J. C/in. Immunol. 4,337, 1984. 2. Singer, A., and Hodes, R. J., Annu. Rev. Immunol. I,21 1, 1983. 3. Howard, M., and Paul, W. E., Annu. Rev. Immunol. 1,307, 1983. 4. Moller, G., Immunol. Rev. 63, 1, 1984. 5. Nabel, G., Allard, W. J., and Cantor, H., J. Exp. Med. 156,658, 1982. 6. Abruzzo, L. V., and Rowley, D. A., Science 222,58 1, 1983. 7. Tilden, A. B., Abo, T., and Balch, C. M., J. Immunol. 130, 1171, 1983. 8. Arai, S., Yamamoto, H., Itoh, K., and Kumagai, K., J. Immunol. 131,65 1, 1983. 9. Brieva, J. A., Targan, S., and Stevens, R. H., J. Immunol. 132,611, 1984. 10. Lanier, L. L., Phillips, J. H., Warner, N. L., and Babcock, G. F., J. Leuk. Biol. 35, 11, 1984. 1I. Griffin, J. D., Hercend, T., Beveridge, R., and Schlossman, S. F., J. Immunol. 130,2947, 1983. 12. Muraguchi, A., Kehrl, J. H., Butler, J. L., and Fauci, A. S., J. Immunol. 132, 176, 1984. 13. Katz, P., Zaytoun, A. M., Lee, J. H., Jr., Panush, R. S., and Longley, S., J. Immunol. 129, 1966, 1983. 14. Katz, P., Zaytoun, A. M., and Fauci, A. S., J. Clin. Invest. 70, 1231, 1982. 15. Procopio, A. D. G., Allavena, P., and Ortaldo, J. R., J Immunol. 1353264, 1985. 16. Pistoia, V., Cozzolino, F., Torcia, M., Castigli, E., and Ferrarini, M., J. Immunol. 134,3179, 1985.
120,270-276 (1989)
IMMUNOLOGY
SHORT COMMUNICATIONS Natural Killer Cells Can Enhance the Proliferative Responses of 6 Lymphocytes PAUL KATZ, GAIL WHALEN, THOMAS R. CUPPS, S. RAY MITCHELL, AND MISHELL EVANS Division ofRheumatology,
Immunology and Allergy, Department OfMedicine, School ofMedicine, Washington, D.C. 20007
Georgetown University
Received November 14,1988; accepted December 30, 1988
In addition to lytic activity against malignant and virally transformed target cells, recent evidence has suggestedthat natural killer (NK) cells can modulate immune activities such as the suppression of B cell responsesthrough noncytotoxic means. Using human B cells and highly purified autologous NK cells, we have demonstrated that NK cells can substantially augment the proliferative responsesof B cells stimulated with the surface immunoglobulin crosslinking agents anti-IgM or Staphylococcus aureus Cowan strain I (SAC). This “enhancer” activity of NK cells was quite potent and was observed at an NK:B cell ratio as low as0.05. Peak blastogenic responsesof B cells cocultured with NK cells in the presence of B cell activators were observed at 2-3 days, similar to the responsesof B cells in the absence of NK cells. Using the inhibitor of DNA synthesis mitomycin C, we determined that B cells and not NK cells were proliferating in cocultures of these lymphocytes stimulated with SAC. Activated B cells neither prevented the lysis of the isotope-labeled NK-sensitive target cell line K562 nor formed conjugates with NK cells, suggestingthat cell contact was not a prerequisite for the effect. These studies have further expanded the functional repertoire of NK cells to include enhancer as well as suppressor and lyk
aCtiVitk3.
0 1989 Academic
Press, Inc.
INTRODUCTION
Considerable information has accrued regarding the activation, proliferation, and differentiation of B lymphocytes ultimately leading to the production of antibody (1, 2). Although early studies in man addressedthese issuesin systemswhich were largely dependent upon the interaction of B cells with T cells or T cell products and which reflected only a relatively “late” phase of B cell function, it is now possible to examine these events in vitro using polyclonal signals which can activate B lymphocytes by interacting with surface immunoglobulin (Ig) (I), analogous to the binding of antigen which in turn initiates the transition of resting cells into antibody-producing and -secreting lymphocytes. Positive and negative signals transmitted by non-B cells can profoundly influence B cell function (3,4). In this regard, studies in animal models (5,6) and in man (79) have documented that natural loller (NK) cells can act as potent suppressors of B cell function and antibody production in vitro and in vivo. Those studies in man have largely employed mitogen-induced, T cell-dependent systemsof antibody production which assessthe activity of only a small population of B cells at a relatively “late” stage of activation and differentiation. Since B cell activation and proliferation can 270 OOO8-8749189 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights ofreproduction in any form reserved
SHORT
COMMUNICATIONS
271
be induced and measured by agents which can crosslink surface Ig (I), we have investigated the effects of NK cells on these responses.We now report that NK cells can act as potent “enhancers” of B cell blastogenic responses. METHODS Isolation of cells. For these studies, highly purified NK cells devoid of contamination with non-NK cells were necessary.Nylon wool passedperipheral blood lymphocytes ( 106) obtained from adult donors were treated with 0.45 ~1 anti-Leu- 1 (CD5) (Becton-Dickinson, Mountain View, CA), 0.2 ~1anti-T3 (CD3) (Ortho Pharmaceuticals, Raritan, NJ), 0.1 ~1 anti-T4 (CD4) (Ortho Pharmaceuticals), and 0.1 ~1 antiCR1 (Becton-Dickinson) for 1 hr at 4°C. Cells were rosetted with bovine erythrocytes coated with goat anti-mouse IgG (Boehringer-Mannheim Biochemicals, Indianapolis, IN) and centrifuged on Ficoll-Hypaque cushions; nonrosetted cells were ~95% NK cells as determined by fluorescent staining with the NK-reactive monoclonal antibodies anti-Leu- 11c (CD 16) ( 10) (Becton-Dickinson) or anti-NKH- 1 ( 11) (Coulter Immunology, Hialeah, FL). NK cells were free of contaminating T and B cells as determined by staining with anti-T3 and anti-Leu-16 (Becton-Dickinson), respectively, and lacked monocytes by esterasestaining. Additionally, NK cells did not proliferate to the T cell mitogen phytohemagglutinin and did not contain CD4 mRNA by Northern blot analysis (data not shown). No NK cell proliferative responsesto the B cell activators anti-p or SAC were observed (see below). These NK cells contained all the lytic activity in our lymphocyte populations; no cytotoxicity was observed with rosetted (non-NK) cells (data not shown). Purified B cells were obtained by treatment of peripheral blood mononuclear cells with 25 mML-leucine methyl ester (Sigma Chemical Co, St. Louis, MO) to kill monocytes and NK cells. Cells were rosetted with aminoethylisothiouronium bromidetreated sheep erythrocytes to deplete T lymphocytes. B cells so obtained were >90% pure as determined by fluorescent staining with anti&u- 16 and were free of T cells, monocytes, and NK cells when reacted with fluoresceinated anti-T3, esterase,and fluoresceinated anti&u- 11c, respectively. B cell proliferation assays. B cell proliferative responsesto the surface Ig crosslinking agents anti-IgM or Staphylococcus aureus Cowan strain I (SAC) were determined as previously described ( 12). Briefly, lo5 peripheral blood B cells were cultured with 100 pg/ml F(ab)‘z goat anti-human IgM (p chain specific) (anti-p) (Cappel Laboratories, Cochranville, PA) or a 1:12,500 dilution of SAC (Bethesda Research Laboratories, Bethesda, MD) in 10%fetal calf serum (FCS) in RPM1 1640 media in 96-well flat-bottom microtiter plates. After 3 days in culture at 37°C in 5% COz in air at 100% humidity, wells were pulse-labeled with 1 &i [3H]thymidine (New England Nuclear, Boston, MA) for 18 hr and the isotope incorporation was determined. Results are ‘expressedas the means of triplicate wells in counts per minute (cpm) X 10m3. In some experiments, DNA synthesis by lymphocytes was blocked by treatment with 40 pg/ml mitomycin C (mito) (Sigma Chemical Co.) for 1 hr at 37°C. Cells so treated were washed four times prior to use in the above assays. Cytotoxicity assuys. A standard chromium-5 1 (5’Cr)-release microcytotoxicity assay for determination of NK activity was employed ( 13). Briefly, lo6 K562 target cells were labeled for 1 hr with 300 &i “Cr (ICN, Cleveland, OH), washed three times, and resuspended in 10%FCS in RPM1 1640 media. Ten thousand labeled target cells (100 ~1) were mixed with an equal volume of effector cells in V-bottom microtiter
SHORT COMMUNICATIONS 1 ocIANTI-p
A
SAC
B
6b
iI,
)-
4
1I_ B
B
B+NK
B+NK
FIG. 1. The effect of NK cells on anti-p- and SAC-induced B cell proliferation. B cells were stimulated with anti-r ( 100 pg/mI) (A) (n = 20) or SAC ( 1: 12,500) (B) (n = 15) in the presence or absence of autologous NK cells. Circles represent the average of triplicate samples and bars represent mean responses.
wells. Spontaneous release of “Cr by target cells was determined by placing labeled target cells in wells in the absence of effector cells. Plates were centrifuged and incubated for 4 hr under tissue culture conditions. Supematant was removed and counted in a gamma counter; percentage cytotoxicity (or percentage “Cr release) was determined by supematant cpm minus spontaneous release cpm divided by total target cell cpm minus spontaneous release cpm X 100. In some experiments, cold target inhibition studies were performed in which unlabeled K562 cells or B cells stimulated with SAC for 3 days were added in increasing number to mixtures of labeled K562 cells and effector cells and cytotoxicity was determined as described above. Statistical analysis. Data were compared with the Student’s t test for paired samples. RESULTS We initially examined the ability of highly purified human NK cells devoid of contamination with non-NK cells to affect the proliferative responses of autologous B cells to the surface Ig crosslinkers anti-p or SAC. Anti-p or SAC induced substantial B lymphocyte proliferation as measured by thymidine incorporation which peaked at 3 days (Fig. 1). NK cells significantly increased B cell DNA synthesis when added at the initiation of the culture period (P < 0.00003 for anti-p; P < 0.0003 for SAC). NK cells alone did not proliferate to either agent and neither B cells, NK cells, nor NK cells plus B cells proliferated in the absence of anti-p or SAC (data not shown).
273
SHORT COMMUNICATIONS
5
1.0
0.25
0.5
0.1
, 0.05
NK:B
FIG. 2. The effect of decreasednumbers of NK cells on B cell blastogenesis.Proliferative responsesof B
cells alone are shown at the lower left. Data points represent means of triplicate samples.
These results were noted when the ratio of NK:B cells was 1:1; however, decreasing the number of NK cells still resulted in substantial enhancement of B cell blastogenesis (Fig. 2). Thus, an NK:B cell ratio as low as 0.05 still produced significant increments in proliferative responsesof B cells to both anti-p and SAC, indicative of the potent helper activity of NK cells. It was necessaryto ascertain if NK cells altered the kinetics of B cell proliferative responsesto polyclonal signals. To determine this, B cells were cocultured with antip with or without autologous NK cells and thymidine incorporation was determined on Days 2-6. The proliferation of B cells to anti-p peaked at Days 2-3 of the assay period and similar kinetic responseswere noted for NK cell-induced augmentation, suggesting that NK cells did not exert helper effects by merely shifting the time to maximal responsiveness(Fig. 3). Similar results were observed when SAC rather than anti-p was used (data not shown). Although DNA synthesis was increased when B cells were cultured with NK cells in the presence of known B cell stimulators, we could not be certain which cell type was responsive on the basis of these experiments. Despite the lack of NK cell blastogenesisto these signals, it was conceivable that NK cells were proliferating in response to signals delivered by activated B cells. To test this, we used mitomycin C (mito), an inhibitor of DNA synthesis, to determine the nature of the increased proliferative responses we had observed. Pretreatment of B cells with mito prior to stimulation with SAC abrogated DNA synthesis (Fig. 4). When mito-treated B cells were cocultured with NK cells, the augmented blastogenesisobserved with untreated B cells was eliminated, indicating that B cells were proliferating to SAC in the presence of NK cells. Mito treatment of NK cells prior to coculture with B cells did not prevent increasedB cell DNA synthesis induced by NK cells. Therefore, B cells were the proliferating cells in this system and NK cells had enhancer activity for these B lymphocytes. Given these results, it was important to determine if NK cells could bind activated B cells, which would indicate that cell-to-cell contact might be relevant to the ob-
274
SHORT COMMUNICATIONS 30
I
c---a
25-I
a cws
+--O~+NK
b z t l5 4 10
5 0: o^ 20 0
2
3
4
5
Day
FIG. 3. The effectsof NK cells on the kinetics of B cell proliferation to anti-p. B cells were cultured with anti-p with or without autologous NK cells (1: 1 ratio) and blastogenic responsesdetermined on days 2-6. Data points represent the means of triplicate samples. Similar results were noted in three separate experiments.
served enhancer activity. For these studies, we employed a standard assay used for the assessmentof NK cell lytic function against the NK-sensitive target cell line, K562 (Fig. 5). When added to standard NK cytotoxicity assays,unlabeled or “cold” K562 cells or other NK-sensitive target cells abrogate NK-dependent lysis of labeled K562 cells by binding to NK cell surface receptors thereby sterically inhibiting the binding and subsequent lolling of labeled targets. With this technique, we used B cells acti-
CPM (x10-3) 4 I
6 1
I
6 !
1
10 I
B+NK
Blnno+NK
B+NKmno
FIG. 4. Delineation of the proliferating cell in B-NK cell interactions. DNA synthesis was blocked by pretreating B or NK cells with mito. Bars represent means of triplicate responses. Mito treatment of B cells (Bmito) inhibited SAC-induced blastogenesis.NK cells significantly increased B cell DNA synthesis; however, this was not observed when Bmito cells were employed. Pretreatment of NK cells with mito (NKmito) still resulted in NK cell-associated augmentation of SAC-induced B cell blastogenesis.Similar results were observed in four separate experiments and were also noted when DNA synthesis was blocked by y irradiation (data not shown).
275
SHORT COMMUNICATIONS
01
I
0 I 510
20
50
100
Cold Targets (~10.~)
FIG. 5. Activated B cells do not bind to NK surface receptors required for lytic activity. A cold target inhibition assaywas utilized in which unlabeled SAC-activated B cells or unlabeled K562 cells were added in increasing number to mixtures of labeled K562 cells and purified NK cells (5: 1 effector ratio). Data points represent the mean percentage cytotoxicity of triplicate wells. The dotted line representsthe percentage cytotoxicity (40%) in the absenceof cold target cells.
vated with SAC for 3 days as cold targets and compared this inhibitory activity with that of cold K562 cells (Fig. 5). Although unlabeled K562 cells inhibited NK cell killing of isotope-labeled K562 cells, unlabeled SAC-stimulated B cells did not prevent target cell lysis. Therefore, activated B cells did not bind to the same recognition structures on NK cells as susceptible target cells. In experiments not shown, we have not observed SAC-activated B cell binding to NK cells in a direct visualization, single cell-in-agarose assay (14). These data suggest that NK cell augmentation of B cell responsesdoes not require direct NK-B cell interactions. DISCUSSION The present study has demonstrated a heretofore unrecognized role for NK cells in the regulation of normal B cell function. Although previous studies have demonstrated the ability of NK cells to act as suppressor cells of antibody production (5-9), we now believe that the functional repertoire of these cells should be expanded to include helper activity aswell. It is now evident that B cells can be activated to proliferate in vitro by agents which can crosslink surface Ig (I), analogous to the binding of antigen in vivo. We have demonstrated that NK cells can substantially augment the blastogenesis of B cells induced by SAC or anti-p, even at a ratio to B cells which is quite low. These data would suggestthat NK cells are quite potent in the ability to enhance these responses.Additionally, we have demonstrated that NK cells do not merely alter the kinetics of B cell proliferation; the time to peak thymidine incorporation was similar for B cells in the presence or absence of NK cells. Furthermore, our data show that the proliferating cell in cocultures of B cells plus NK cells is clearly the B cell since inhibition of B cell DNA synthesis abrogated NK cell-induced boosting of B cell blastogenesis while similar treatment of NK cells did not prevent this effect. Last, it does not appear that direct NK-B cell contact is a prerequisite for this effect as activated B cells neither bound to target cell receptors on NK cells nor formed conjugates with these lytic cells in direct visualization assays.
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Further studies will be needed to determine whether NK cells can be separatedinto definable subsetsmediating enhancer or suppressor activities, as has been done for T lymphocytes. Similarly, it will be important to determine the mechanism(s) whereby NK cells can augment B cell responses. In data reported here, we have determined that NK-B cell contact is not a prerequisite to NK cell-dependent augmentation of B lymphocyte blastogenesis. Two studies have shown that populations of lymphocytes containing NK cells are capable of producing a B cell growth factor (BCGF) when stimulated with the T cell mitogen phytohemagglutinin (15, 16). However, these were heterogeneous lymphocytes in which contamination with non-NK cells may have contributed to the observed BCGF activity. Furthermore, removal of NK cells with the NK cell-reactive monoclonal antibody anti-Leu- 11 eliminated lytic function but did not affect BCGF production (16), suggesting that this factor was derived from cells lacking the NK cell phenotype. In preliminary studies, we have demonstrated that stimulation of NK cells with SAC did not induce BCGF production although phytohemagglutinin does (data not shown). However, the addition of NK cells to cultures of B cells plus SAC resulted in substantial BCGF production above that of SAC-stimulated B cells alone. Studies are now in progress to determine if NK cells are producing BCGF in response to stimulation by SAC-activated B cells or if NK cells are inducing BCGF production by activated B cells. Therefore, although the mode of NK cell-induced enhancement of B cell blastogenesis is as yet unknown, the spectrum of NK cell activity appears to encompass not only cytotoxicity but significant and diverse immunoregulatory functions as well. Just as the precise mechanism of NK cell killing awaits precise clarification, the elucidation of these nonlytic activities warrants further investigation. ACKNOWLEDGMENTS The authors thank Andrew Pachner for critical review and Carol Moran for expert editorial assistance. This work was supported by grants from the National Institutes of Health (A124636) and from the Metropolitan Washington Chapter of the Arthritis Foundation.
REFERENCES 1. Muraguchi, A., Kehrl, J. H., Butler, J. L., and Fauci, A. S., J. C/in. Immunol. 4,337, 1984. 2. Singer, A., and Hodes, R. J., Annu. Rev. Immunol. I,21 1, 1983. 3. Howard, M., and Paul, W. E., Annu. Rev. Immunol. 1,307, 1983. 4. Moller, G., Immunol. Rev. 63, 1, 1984. 5. Nabel, G., Allard, W. J., and Cantor, H., J. Exp. Med. 156,658, 1982. 6. Abruzzo, L. V., and Rowley, D. A., Science 222,58 1, 1983. 7. Tilden, A. B., Abo, T., and Balch, C. M., J. Immunol. 130, 1171, 1983. 8. Arai, S., Yamamoto, H., Itoh, K., and Kumagai, K., J. Immunol. 131,65 1, 1983. 9. Brieva, J. A., Targan, S., and Stevens, R. H., J. Immunol. 132,611, 1984. 10. Lanier, L. L., Phillips, J. H., Warner, N. L., and Babcock, G. F., J. Leuk. Biol. 35, 11, 1984. 1I. Griffin, J. D., Hercend, T., Beveridge, R., and Schlossman, S. F., J. Immunol. 130,2947, 1983. 12. Muraguchi, A., Kehrl, J. H., Butler, J. L., and Fauci, A. S., J. Immunol. 132, 176, 1984. 13. Katz, P., Zaytoun, A. M., Lee, J. H., Jr., Panush, R. S., and Longley, S., J. Immunol. 129, 1966, 1983. 14. Katz, P., Zaytoun, A. M., and Fauci, A. S., J. Clin. Invest. 70, 1231, 1982. 15. Procopio, A. D. G., Allavena, P., and Ortaldo, J. R., J Immunol. 1353264, 1985. 16. Pistoia, V., Cozzolino, F., Torcia, M., Castigli, E., and Ferrarini, M., J. Immunol. 134,3179, 1985.