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Cultures of purified human natural killer cells: Growth in the presence of interleukin 2
CELLULARIMMUNOLOGY72, 178-185(1982)
SHORT COMMUNICATIONS Cultures of Purified Human Natural Killer Cells: Growth in the Presence of lnterleukin 2 TUOMOTIMONEN,**' JOHN R. ORTALDO,**~BEDAM. STADLER,~ GuuD. BONNARD,* SUSANO.SHARROW,$ ANDRONALD B. HERBERMAN* *Laboratory of Immunodiagnosis and Slmmunology Branch, National Cancer Institute, and TLaboratory of Microbiology and Immunology. National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20205 Received March 8. 1982: accepted May 23. 1982 We have isolated highly enriched populations of LGL, which are virtually devoid of mature typical lymphocytes (as enumerated by morphological and surface antigen analysis using monoclonal antibodies, e.g., OKT3) in comparison to T cells which contain greater than 95% sheep erythrocyte-forming cells and are devoid of LGL and NK/K activities. Both types of cells grew in the presence of crude or partially purified IL-2. Cultures of LGL could be initiated consistently even in the absence of lectins and the cultured LGL retained their characteristic morphology and cytotoxic activity. However, within 7-10 days after initiation, the cultured LGL changed in surface phenotype to become antigenically indistinguishable from cultured T cells.
INTRODUCTION Natural killer (NK) cells are lymphoid cells endowed with spontaneous cytolytic activity preferentially directed toward malignant and virus-infected target cells (1, 2). There are data that support the hypotheses that NK cells may be involved in resistance against neoplasia (3, 4) and against various infections (5-7) and in regulation of normal hematopoiesis (8, 9). The lineage of NK cells has remained controversial, with evidence for associations with both myelomonocytic (10, 11) and T cells (12, 15). We have recently identified human NK cells as a morphological subpopulation of lymphoid cells, termed large granular lymphocytes (LGL), which represent 2-6’S of peripheral blood white cells (13, 15). LGL can be enriched to >90% purity by discontinuous Percoll density gradient centrifugation and subsequent depletion of high-affinity sheep erythrocytes rosette-forming cells from NK cell-enriched low-density fractions (13). In this communication we show for the first time that purified human LGL (NK cells) can be cultured in the presence of T-cell growth factor (interleukin 2, IL-2) and that these cultured LGL retain their characteristic morphology and NK activity. In contrast, similar cultures of T cells devoid of LGL had a different morphology and exerted an entirely different pattern ’ Supported by grant lRO1 CA 23809-l from National Cancer Institute, National Institutes of Health, Bethesda, Md. *To whom all correspondence should be addressed: BRTB, BRMP, NCI-FCRF, Frederick, Md. 21701. 178
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of cytotoxicity. These results suggest a possible relationship of NK cells to T cells and indicate a new approach to the dissection of cytotoxic responses of cultured human lymphoid cells. MATERIALS
AND METHODS
Isolation of LGL and T cells. The purity of isolated LGL preparations was determined by morphological analysis (which revealed that consistently more than 90% of the cells were LGL, with no detectable contamination by small or mediumsized lymphocytes [without granules]) and by phenotype analysis where an undetectable number of cells (~1%) expressed the “pan-T” antigen detected by the monoclonal antibody OKT3 ( 16), as determined by a Staphylococcus aureus binding assay (17). The T cells were purified from the high-density Percoll fractions and were >90% sheep erythrocyte rosette-forming and >85% OKT3-positive small or medium-sized lymphocytes (with no detectable LGL contamination). Culture of cells. Both LGL and T cells were cultured in the presence of 10% crude, 4~ concentrated conditional medium (CM) derived from phytohemagglutinin-stimulated cultures of human peripheral blood lymphocytes ( 18) (Associated Biomedics Systems, Buffalo, N.Y.) in RPM1 1640 medium (Biofluids Inc., Rockville, Md.) supplemented with 10% heat-inactivated human AB serum (Gibco, Grand Island, N.Y.), 0.29 mg/ml glutamine, 100 IU/ml penicillin, and 10 pg/ml streptomycin. All cultures were performed in 24-well tissue culture plates (Costar, Cambridge, Mass.) and were initiated (approximately 5 X lo5 cells/ml) with addition of 10% (5 X lo4 cells/ml) 4000 R-irradiated peripheral blood mononuclear cells as feeder cells; no feeder cells were used thereafter. By this method it was possible to increase the number of LGL 20- to 40-fold and the number of T cells 50- to go-fold during the 4-week culture period used in this study.
RESULTS Morphologically, in 10 consecutive cultures, the cultured LGL retained their typical weakly basophilic cytoplasm and kidney-shaped nuclei in >90% of the cells and the characteristic azurophilic cytoplasmic granules in approximately 78% of the cells. Cultured T cells exhibited a typical blast cell morphology with strongly basophilic cytoplasm, and ~10% of the cells had cytoplasmic granules (Fig. 1). To verify that IL-2 was responsible for the growth of LGL, the proliferative capacities of peripheral blood lymphocytes (PBL), LGL, and T cells were tested in the presence of human IL-2 partially purified by sequential chromatography on phenyl Sepharose, DEAE Sephacel, and AcA 54 gel filtration (19). This material was subjected to isoelectrofocusing, yielding three peaks of IL-2 activity at pZ’s of 6.5 (IL-~-(U), 7.2 (IL-2-p), and 8.2 (IL-2-y). This charge heterogeneity is due to different degrees of glycosylation of the IL-2 molecule, while the IL-2 activity at a pZ of 8.2 has been shown to be due to a nonglycosylated form of the lymphokine (20). Crude and purified IL-2 preparations have been adjusted to the same biological activity using the murine IL-2-dependent cell line CT6, as described earlier (21). As shown in Fig. 2, the level of thymidine incorporation of cultured LGL in the presence of the purified IL-2 preparations was similar to or higher than that obtained in the presence of crude CM, indicating that the growth factor needed
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c FIG. 1. Fresh and cultured subpopulations of lymphoid cells (X975). (A) Fresh large granular lymphocytes. (B) Cultured large granular lymphocytes, Day 20. (C) Fresh T cells. (D) Cultured T cells, Day 20. Similar morphology was seen at Day 10 and as late as Day 30, when the cultures were routinely discontinued.
for the proliferation of LGL was indeed IL-2 and that lectins or other factors in crude CM were not required. The cytotoxic activity of the cultured cells was determined 10 and 20 days after the initiation of the cultures. The cells were incubated in complete growth medium in the absence of IL-2 for 24 hr prior to testing in order to minimize irrelevant or
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FIG. 2. [3H]Thymidine incorporation of (left) cultured peripheral blood lymphocytes (PBL), (middle) large granular lymphocytes (LGL), and (right) T cells in the presence of crude conditioned medium (CM) containing various amounts of IL-2 (0) or various species of partially purified IL-2 differing in their isoelectric points: IL-2-a (pH 6.5) (0) IL-24 (pH 7.2) (X), and IL-29 (pH 8.2) (A). Ten units of IL-2 correspond to an IL-2 activity needed for the long-term growth of human and murine IL-2dependent cell lines (21). Data are expressed as incorporation of [)H]thymidine (cpm X 10m4)after pulse labeling of 5 X IO4 cells for 6 hr with I pCi of [‘Hlthymidine. The cells had been previously cultured for IO days in the presence of crude CM and were then further cultured for 2 days in the presence of growth factors or medium alone (for background incorporation).
lectin-induced cytolysis detected in rapidly growing cells (22). As shown in Fig. 3, cultured LGL exerted a pattern of spontaneous and interferon-augmentable NK cell activities similar to the pattern of fresh LGL against the susceptible target cell lines K562, MOLT4, and G-l 1 (23). They also exhibited antibody-dependent and lectin-induced cellular cytotoxicities (ADCC and LICC, respectively) against murine lymphoma cells (RLbl), whereas they did not significantly lyse NK-insensitive allogenic blasts or RLBl alone. As previously reported (24), fresh, purified T cells were cytotoxic only in LICC. In contrast, cultured T cells were reactive in LICC, against allogeneic blasts, and against the monolayer target cell G- 11, but they did not lyse NK-sensitive leukemia target cells K.562 and MOLT4. Furthermore, the cultured T cells did not demonstrate ADCC activity, indicating the absence of detectable Fc receptors for IgG on these cells. We have previously reported the fresh LGL are predominantly Fc,R+, OKT 1O+, OKMl+, and OKT3- (5,7), in contrast to small T lymphocytes (Fc,R-, OKTlO-, OKMl-, OKT3+). A major proportion of the cultured LGLs (60-75’S) (Fig. 4) reacted with OKT3. This increase in expression of OKT3 on LGL cultures was seen in every culture. In cultured LGL and T cells, as previously demonstrated (27), Ia expression was seen on most (85-100s) on IL-2 expanded cells (data not shown).
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DISCUSSION Thus, we have demonstrated that cultures of highly purified NK cells and T cells are both morphologically and functionally different. These results, in combination with the observed rapid proliferation of both LGL and T cells in the presence of IL-2, seemto rule out the possibilities either (i) that minute amounts of conventional T cells among LGL would be responsible for the observed growth of NK cell fractions or (ii) that a minor contamination of T cells by LGL would be the explanation for the detected cytotoxicities among cultured T cells. Although apparently nonselective cytotoxicities among cultured T cells were detectable, there are major indications that these reactivities were not due to classic NK cells: the nonreactivity of cultured T cells with NK cell-sensitive target cells K562 and MOLT4, the lack of ADCC activity, and the inability of interferon to augment cytotoxicity in these target cell systems. The reactivity of cultured T cells against
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FIG. 3. Cytotoxic activities of fresh and cultured (top) large granular lymphocytes (LGL) and (bottom) T cells in a 4-hr 5’Cr-release assay (effecter/target cell ratio 2O:l). K562, target cell derived from a patient with chronic myelogenous leukemia; MOLT4, a T-cell leukemia line; RLbl, a murine T-cell lymphoma; ADCC, cytotoxicity against RL6 1 coated with rabbit anti-mouse T-cell serum (13); LICC, cytotoxic activity against RLdl in the presence of 1 rg/ml phytohemagglutinin; G-l 1, a cell line derived from a patient with breast carcinoma; BLASTS, allogeneic phytohemagglutinin-induced blasts derived from peripheral blood mononuclear cells. Solid bars: 3-hr pretreatment of effector cells with 800 IU of human fibroblast interferon (specific activity 2 X 10’ IU/mg protein, HEM Research, Rockville, Md.). Mean values of the results obtained from three separate experiments are shown.
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FIG. 4. Flow microfluorometry analysis of large granular lymphocytes (LGL) and T cells with monoclonal OKT3. Fresh [(a) and (c)] and cultured [(b) and (d)] cells with monoclonal OKT3 (---) are compared to background without monoclonal (OKT3) (- - - ). T cells are shown at Day 0 (a) and Day I4 (b). LGL are shown at Day 0 (c) and Day I4 (d).
allogeneic blasts is consistent with the previously demonstrated capacity of polyclonally activated T cells to lyse NK-insensitive phytohemagglutinin blasts (22). As previously reported (25), cultured T cells lyse a variety of solid tumor target cells but not various leukemia or lymphoma cells. It remains to be established whether this interferon-augmentable reactivity, also detected in this study (25), is due to a subpopulation of natural effector cells that are inactive in fresh blood, to the generation during culture of a subpopulation of NK cells with restricted specificity, or to polyclonal activation of T cells due to the phytohemagglutinin content of the CM. The ability to culture both purified NK cells and T cells is of interest for several reasons. First, the proliferation of LGL in the presence of IL-2 suggests a possible relation to the T-cell lineage. The contention that NK cells may in fact belong to the T-cell lineage is further supported by observations that the majority of LGL acquired reactivity with T-cell-specific OKT3 antibodies after 7- 10 days in culture (26). One cannot dismiss the possibility that although this growth factor has been considered to be entirely selective for T cells (27), it may also promote the prolif-
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eration of NK cells that actually belong to a separate lineage. Second, the present study is the first to demonstrate that cultures with NK and ADCC activities can be initiated from purified populations of effector cells. Previous studies indicated that cultures of human (22) or mouse (28, 29) mononuclear leukocytes in the presence of IL-2 had NK-like activity, but they failed to discriminate between the alternative possibilities of development in vitro of NK cells from other cell types or of initial responsiveness of NK cells to IL-2. Third, the technology for the expansion of human NK cells should facilitate characterization and biochemical analysis of NK cells. Cloning of human NK cells should become feasible, so that such controversial issues as (i) target-cell specificity of NK cells (30, 3 1), (ii) the relationship and possible overlapping of NK cells and K cells responsible for ADCC (32, 33), and (iii) interferon production versus cytolytic capacity of individual NK cells (34) can be studied in detail. Fourth, the use of T cells devoid of LGL in autologous mixed lymphocyte-tumor cultures, which are then propagated with IL2, may yield more selectively cytotoxic cells than those obtained from cultures initiated from unfractionated, LGL-containing peripheral blood mononuclear cells. This may help to resolve the question of the frequency of occurrence of human T cells with specifically immune anti-tumor cytolytic activity. REFERENCES 1. Herberman, R. B., and Holden, H. T., Advan. Cancer Res. 27, 305, 1978. 2. Herberman, R. B. (Ed.), “Natural Cell-Mediated Immunity Against Tumors.” Academic Press, New York, 1980. 3. Haller, O., Hansson, M., Kiessling, R., and Wigzell, H., Nature (London) 270, 609, 1977. 4. Riccardi, C., Santoni, A., Barlozzari, T., Puccetti, P., and Herberman, R. B., In?. J. Cancer 25, 475, 1980. 5. Brooks, C. G., Rees, R. G., and Leach, R. H., Eur. J. Immunol. 9, 159, 1979. 6. Trischmann, T., Tanowitz, H., Wittner, M., and Bloom, B., Exp. Parasirol. 45, 160, 1978. 7. Santoli, D., Trinchieri, G., and Leifn, F. S., J. Immunol. 121, 526, 1978. 8. Hansson, M., Kiirre, K., Kiessling, R., Roder, J., Andersson, B., and Hiiyry, P., J. Immunol. 123, 756, 1979. 9. Hansson, M., Kiessling, R., and Andersson, B., J. Clin. Invest. 11, 8, 1981. 10. Kay, H. D., and Horwitz, D. A., J. Clin. Invest. 66, 847, 1980. 11. Lohmann-Matthes, M.-L., Domzig, W., and Roder, J. C., J. Immunol. 123, 1883, 1979. 12. West, W. H., Cannon, G. B., Kay, H. D., Bonnard, G. D., and Herberman, R. B., J. Immunol. 118, 355, 1977. 13. Timonen, T., Ortaldo, J. R., and Herberman, R. B., J. Exp. Med. 153, 569, 1981. 14. Mattes, M. J., Sharrow, S. O., Herberman, R. B., and Holden, H. T., J. Immunol. 123, 2851, 1979. 15. Timonen, T., Saksela, E., Ranki, A., and Hayry, P., Cell. Immunol. 48, 133, 1979. 16. Kung, P. C., Goldstein, G., Reinherz, E. L., and Schlossman, S. F., Science 206, 347, 1979. 17. Ranki, A.,Totterman, T. H., and Hiiyry, P., Scund. J. Immunol. 5, 1129, 1976. 18. Bonnard, G. D., Yasaka, K., and Maca, R. D., Cell. Immunol. 51, 390, 1980. 19. Stadler, B. M., Dougherty, S. F., Carter, C., Berenstein, E. H., Fox, P. C., Siraganian, R. P., and Oppenheim, J. J., In “Proceedings of the International Workshop on Lymphokines and Thymic Factors” (A. Goldstein, Ed.). Raven Press, New York, in press. 20. Robb, R. J., and Smith, K. A., Mol. Immunol., in press. 21. Stadler, B. M., Dougherty, S. F., Farrar, J. J., and Oppenheim, J. J., Submitted for publication. 22. Ortaldo, J. R., Timonen, T., and Bonnard, G. D., Behring Inst. Mitt. 67, 258, 1980. 23. Timonen, T., Ortaldo, J. R., and Herberman, R. B., Submitted for publication. 24. Lang, N., Ortaldo, J. R., Bonnard, G. D., and Herberman, R. B., JNCI 69, 3684, 1982. 25. Kedar, E., Herberman, R. B., Gorelik, E., Sredni, B., Bonnard, G. D., and Navarro, N., In “The
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Potential Role of T Cell Subpopulations in Cancer Therapy” (A. Fefer, Ed.), pp. 173- 19 I. Raven Press, New York. Ortaldo, J. R., Timonen, T., Vose, B. M., and Alvarez, J. A., In “The Potential Role of T Cell Subpopulations in Cancer Therapy” (A. Fefer, Ed.), pp. 197-201. Raven Press, New York. Smith, K. A., Baker, P. E., Gillis, S., and Ruscetti, F. W., Mol. Immunol. 17, 579, 1980. Dennart, G., Nature (London) 287, 47, 1980. Kuribayashi, K., Gillis, S., Kern, D. E., and Henry, C. S., /. Immunol. 126, 2321, 1981. Phillips, W. H., Ortaldo, J. R., and Herberman, R. B., L Immunol. 125, 2322, 1980. Trinchieri, G., Granato, D., and Perussia, B., J. Immunol. 126, 335, 1981. Kay, H. D., Bonnard, G. D., West, W. H., and Herberman, R. B., J. Immunol. 118, 2058, 1977. Neville, M. E., J. Immunol. 125, 2604, 1980. Timonen T., Saksela, E., Virtanen, I., and Cantell, K., Eur. J. Immunol. 10, 422, 1980.
SHORT COMMUNICATIONS Cultures of Purified Human Natural Killer Cells: Growth in the Presence of lnterleukin 2 TUOMOTIMONEN,**' JOHN R. ORTALDO,**~BEDAM. STADLER,~ GuuD. BONNARD,* SUSANO.SHARROW,$ ANDRONALD B. HERBERMAN* *Laboratory of Immunodiagnosis and Slmmunology Branch, National Cancer Institute, and TLaboratory of Microbiology and Immunology. National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20205 Received March 8. 1982: accepted May 23. 1982 We have isolated highly enriched populations of LGL, which are virtually devoid of mature typical lymphocytes (as enumerated by morphological and surface antigen analysis using monoclonal antibodies, e.g., OKT3) in comparison to T cells which contain greater than 95% sheep erythrocyte-forming cells and are devoid of LGL and NK/K activities. Both types of cells grew in the presence of crude or partially purified IL-2. Cultures of LGL could be initiated consistently even in the absence of lectins and the cultured LGL retained their characteristic morphology and cytotoxic activity. However, within 7-10 days after initiation, the cultured LGL changed in surface phenotype to become antigenically indistinguishable from cultured T cells.
INTRODUCTION Natural killer (NK) cells are lymphoid cells endowed with spontaneous cytolytic activity preferentially directed toward malignant and virus-infected target cells (1, 2). There are data that support the hypotheses that NK cells may be involved in resistance against neoplasia (3, 4) and against various infections (5-7) and in regulation of normal hematopoiesis (8, 9). The lineage of NK cells has remained controversial, with evidence for associations with both myelomonocytic (10, 11) and T cells (12, 15). We have recently identified human NK cells as a morphological subpopulation of lymphoid cells, termed large granular lymphocytes (LGL), which represent 2-6’S of peripheral blood white cells (13, 15). LGL can be enriched to >90% purity by discontinuous Percoll density gradient centrifugation and subsequent depletion of high-affinity sheep erythrocytes rosette-forming cells from NK cell-enriched low-density fractions (13). In this communication we show for the first time that purified human LGL (NK cells) can be cultured in the presence of T-cell growth factor (interleukin 2, IL-2) and that these cultured LGL retain their characteristic morphology and NK activity. In contrast, similar cultures of T cells devoid of LGL had a different morphology and exerted an entirely different pattern ’ Supported by grant lRO1 CA 23809-l from National Cancer Institute, National Institutes of Health, Bethesda, Md. *To whom all correspondence should be addressed: BRTB, BRMP, NCI-FCRF, Frederick, Md. 21701. 178
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of cytotoxicity. These results suggest a possible relationship of NK cells to T cells and indicate a new approach to the dissection of cytotoxic responses of cultured human lymphoid cells. MATERIALS
AND METHODS
Isolation of LGL and T cells. The purity of isolated LGL preparations was determined by morphological analysis (which revealed that consistently more than 90% of the cells were LGL, with no detectable contamination by small or mediumsized lymphocytes [without granules]) and by phenotype analysis where an undetectable number of cells (~1%) expressed the “pan-T” antigen detected by the monoclonal antibody OKT3 ( 16), as determined by a Staphylococcus aureus binding assay (17). The T cells were purified from the high-density Percoll fractions and were >90% sheep erythrocyte rosette-forming and >85% OKT3-positive small or medium-sized lymphocytes (with no detectable LGL contamination). Culture of cells. Both LGL and T cells were cultured in the presence of 10% crude, 4~ concentrated conditional medium (CM) derived from phytohemagglutinin-stimulated cultures of human peripheral blood lymphocytes ( 18) (Associated Biomedics Systems, Buffalo, N.Y.) in RPM1 1640 medium (Biofluids Inc., Rockville, Md.) supplemented with 10% heat-inactivated human AB serum (Gibco, Grand Island, N.Y.), 0.29 mg/ml glutamine, 100 IU/ml penicillin, and 10 pg/ml streptomycin. All cultures were performed in 24-well tissue culture plates (Costar, Cambridge, Mass.) and were initiated (approximately 5 X lo5 cells/ml) with addition of 10% (5 X lo4 cells/ml) 4000 R-irradiated peripheral blood mononuclear cells as feeder cells; no feeder cells were used thereafter. By this method it was possible to increase the number of LGL 20- to 40-fold and the number of T cells 50- to go-fold during the 4-week culture period used in this study.
RESULTS Morphologically, in 10 consecutive cultures, the cultured LGL retained their typical weakly basophilic cytoplasm and kidney-shaped nuclei in >90% of the cells and the characteristic azurophilic cytoplasmic granules in approximately 78% of the cells. Cultured T cells exhibited a typical blast cell morphology with strongly basophilic cytoplasm, and ~10% of the cells had cytoplasmic granules (Fig. 1). To verify that IL-2 was responsible for the growth of LGL, the proliferative capacities of peripheral blood lymphocytes (PBL), LGL, and T cells were tested in the presence of human IL-2 partially purified by sequential chromatography on phenyl Sepharose, DEAE Sephacel, and AcA 54 gel filtration (19). This material was subjected to isoelectrofocusing, yielding three peaks of IL-2 activity at pZ’s of 6.5 (IL-~-(U), 7.2 (IL-2-p), and 8.2 (IL-2-y). This charge heterogeneity is due to different degrees of glycosylation of the IL-2 molecule, while the IL-2 activity at a pZ of 8.2 has been shown to be due to a nonglycosylated form of the lymphokine (20). Crude and purified IL-2 preparations have been adjusted to the same biological activity using the murine IL-2-dependent cell line CT6, as described earlier (21). As shown in Fig. 2, the level of thymidine incorporation of cultured LGL in the presence of the purified IL-2 preparations was similar to or higher than that obtained in the presence of crude CM, indicating that the growth factor needed
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c FIG. 1. Fresh and cultured subpopulations of lymphoid cells (X975). (A) Fresh large granular lymphocytes. (B) Cultured large granular lymphocytes, Day 20. (C) Fresh T cells. (D) Cultured T cells, Day 20. Similar morphology was seen at Day 10 and as late as Day 30, when the cultures were routinely discontinued.
for the proliferation of LGL was indeed IL-2 and that lectins or other factors in crude CM were not required. The cytotoxic activity of the cultured cells was determined 10 and 20 days after the initiation of the cultures. The cells were incubated in complete growth medium in the absence of IL-2 for 24 hr prior to testing in order to minimize irrelevant or
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FIG. 2. [3H]Thymidine incorporation of (left) cultured peripheral blood lymphocytes (PBL), (middle) large granular lymphocytes (LGL), and (right) T cells in the presence of crude conditioned medium (CM) containing various amounts of IL-2 (0) or various species of partially purified IL-2 differing in their isoelectric points: IL-2-a (pH 6.5) (0) IL-24 (pH 7.2) (X), and IL-29 (pH 8.2) (A). Ten units of IL-2 correspond to an IL-2 activity needed for the long-term growth of human and murine IL-2dependent cell lines (21). Data are expressed as incorporation of [)H]thymidine (cpm X 10m4)after pulse labeling of 5 X IO4 cells for 6 hr with I pCi of [‘Hlthymidine. The cells had been previously cultured for IO days in the presence of crude CM and were then further cultured for 2 days in the presence of growth factors or medium alone (for background incorporation).
lectin-induced cytolysis detected in rapidly growing cells (22). As shown in Fig. 3, cultured LGL exerted a pattern of spontaneous and interferon-augmentable NK cell activities similar to the pattern of fresh LGL against the susceptible target cell lines K562, MOLT4, and G-l 1 (23). They also exhibited antibody-dependent and lectin-induced cellular cytotoxicities (ADCC and LICC, respectively) against murine lymphoma cells (RLbl), whereas they did not significantly lyse NK-insensitive allogenic blasts or RLBl alone. As previously reported (24), fresh, purified T cells were cytotoxic only in LICC. In contrast, cultured T cells were reactive in LICC, against allogeneic blasts, and against the monolayer target cell G- 11, but they did not lyse NK-sensitive leukemia target cells K.562 and MOLT4. Furthermore, the cultured T cells did not demonstrate ADCC activity, indicating the absence of detectable Fc receptors for IgG on these cells. We have previously reported the fresh LGL are predominantly Fc,R+, OKT 1O+, OKMl+, and OKT3- (5,7), in contrast to small T lymphocytes (Fc,R-, OKTlO-, OKMl-, OKT3+). A major proportion of the cultured LGLs (60-75’S) (Fig. 4) reacted with OKT3. This increase in expression of OKT3 on LGL cultures was seen in every culture. In cultured LGL and T cells, as previously demonstrated (27), Ia expression was seen on most (85-100s) on IL-2 expanded cells (data not shown).
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DISCUSSION Thus, we have demonstrated that cultures of highly purified NK cells and T cells are both morphologically and functionally different. These results, in combination with the observed rapid proliferation of both LGL and T cells in the presence of IL-2, seemto rule out the possibilities either (i) that minute amounts of conventional T cells among LGL would be responsible for the observed growth of NK cell fractions or (ii) that a minor contamination of T cells by LGL would be the explanation for the detected cytotoxicities among cultured T cells. Although apparently nonselective cytotoxicities among cultured T cells were detectable, there are major indications that these reactivities were not due to classic NK cells: the nonreactivity of cultured T cells with NK cell-sensitive target cells K562 and MOLT4, the lack of ADCC activity, and the inability of interferon to augment cytotoxicity in these target cell systems. The reactivity of cultured T cells against
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FIG. 3. Cytotoxic activities of fresh and cultured (top) large granular lymphocytes (LGL) and (bottom) T cells in a 4-hr 5’Cr-release assay (effecter/target cell ratio 2O:l). K562, target cell derived from a patient with chronic myelogenous leukemia; MOLT4, a T-cell leukemia line; RLbl, a murine T-cell lymphoma; ADCC, cytotoxicity against RL6 1 coated with rabbit anti-mouse T-cell serum (13); LICC, cytotoxic activity against RLdl in the presence of 1 rg/ml phytohemagglutinin; G-l 1, a cell line derived from a patient with breast carcinoma; BLASTS, allogeneic phytohemagglutinin-induced blasts derived from peripheral blood mononuclear cells. Solid bars: 3-hr pretreatment of effector cells with 800 IU of human fibroblast interferon (specific activity 2 X 10’ IU/mg protein, HEM Research, Rockville, Md.). Mean values of the results obtained from three separate experiments are shown.
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FIG. 4. Flow microfluorometry analysis of large granular lymphocytes (LGL) and T cells with monoclonal OKT3. Fresh [(a) and (c)] and cultured [(b) and (d)] cells with monoclonal OKT3 (---) are compared to background without monoclonal (OKT3) (- - - ). T cells are shown at Day 0 (a) and Day I4 (b). LGL are shown at Day 0 (c) and Day I4 (d).
allogeneic blasts is consistent with the previously demonstrated capacity of polyclonally activated T cells to lyse NK-insensitive phytohemagglutinin blasts (22). As previously reported (25), cultured T cells lyse a variety of solid tumor target cells but not various leukemia or lymphoma cells. It remains to be established whether this interferon-augmentable reactivity, also detected in this study (25), is due to a subpopulation of natural effector cells that are inactive in fresh blood, to the generation during culture of a subpopulation of NK cells with restricted specificity, or to polyclonal activation of T cells due to the phytohemagglutinin content of the CM. The ability to culture both purified NK cells and T cells is of interest for several reasons. First, the proliferation of LGL in the presence of IL-2 suggests a possible relation to the T-cell lineage. The contention that NK cells may in fact belong to the T-cell lineage is further supported by observations that the majority of LGL acquired reactivity with T-cell-specific OKT3 antibodies after 7- 10 days in culture (26). One cannot dismiss the possibility that although this growth factor has been considered to be entirely selective for T cells (27), it may also promote the prolif-
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