[CANCER RESEARCH 49, 940-944, February 15, 1989] Gene Expression during the Generation of Human Lymphokine-activated Killer Cells: Early Induction of Inter leukin IßbyInter leukin 2 '

Elizabeth J. Kovacs,2 Suzanne K. Beckner, Dan L. Longo, Luigi Varesio, and Howard A. Young

Department of Anatomy, Loyola University, Maywood, Illinois [E. J. K.]; Program Resources, Inc., National Cancer Institute-Frederick Cancer Research Facility, Frederick, Maryland [S. K. B.]; and Laboratory of Molecular Immunoregulation, Biological Response Modifiers Program, Division of Cancer Treatment, National Cancer Institute-Frederick Cancer Research Facility, Frederick, Maryland [D. L. L., L. V., H. A. Y.J

ABSTRACT great as that which is generated from mixed populations of PBL (8). This observation suggests either that other cells in the Culture of human peripheral blood leukocytes with 2 (II,- mixed PBL population are directly responsible for the lytic 2) stimulates their differentiation into lymphokine-activated killer (LAK) activity or that mediators produced by these cells in response cells, with a broad range of cytotoxicity against fresh tumor cells and to IL-2 indirectly enhance lytic activity by triggering LGL or tumor cell lines (Grimm et al., J. Exp. Med., 155: 1823-1841, 1982). We chose to utilize a molecular approach to determine whether II -2 cytotoxic T-cells to become activated. stimulates the expression of cytokine genes by the mixed cell population In order to determine whether secretory products of cells in which may be involved in the generation or regulation of lytic activity. the mixed PBL population are involved in the generation of Northern blot analysis performed with total cellular RNA from LAK lytic activity, we chose to utilize a molecular approach to cells cultured for varying periods of time with II,-2 revealed that the examine the expression of cytokine mRNAs produced by the genes which code for jinterleukin 1 (II -1 )«and ß,7-, mixed population of PBLs during culture with . a, and ] were not spontaneously ex Our studies reveal that mRNAs encoding cytokines which mod pressed. As soon as 2 h after II -2 treatment, II,-1«and \\.-\t1 mRNAs ulate the activities of immune cells, IL-la, IL-1/3, and IFN-7, were expressed. Both nonadherent and adherent populations of LAK cells express II.-I/ÕmRNA; however, the adherent population produced and cytokines which are cytotoxic for certain tumor cells, TNF more IL-1/3 mRNA and maintained its expression for a prolonged period and LT, are expressed during the generation of LAK cells. of time. Other cytokine mRNAs (f-interferon, tumor necrosis factor a, These cytokines may be important in the expression of lytic and lymphotoxin) were expressed later than the 1I.-1 mRNAs with activity. maximal levels between Days 2 through 7. Our results indicate that LAK cell populations can generate a variety of cytokines which may be involved MATERIALS AND METHODS in the generation of lytic activity. Reagents. Human recombinant IL-2 was generously supplied by the Cetus Corporation, Emeryville, CA. The IL-2 has a specific activity of 3x10'' units/mg of protein. The IL-2 was diluted so that a unit INTRODUCTION corresponds to a Biological Response Modifiers Program unit meas The IL-23-induced differentiation of PBL into LAK cells has ured as previously described (11). Generation of LAK activity was been well described. LAK cells exhibit a broad range of in vitro maximal when cells were cultured with 1000 units/ml of IL-2. cytotoxicity against fresh tumor cells and tumor cell lines with Generation of LAK Cells. Normal healthy volunteers or cancer pa tients undergoing LAK and IL-2 therapy were leukophoresed using a no activity against normal cells (1). Numerous studies with 2-needle leukophoresis procedure on a Celltrifuge II leukophoresis established murine tumor models have demonstrated the anti- apparatus (Fenwal Laboratories, Deerfield, IL) as described elsewhere tumor efficacy of LAK cells when administered with high doses (12). The final volume of the leukophoresis pack was 400 to 500 ml. of IL-2 (2-4). These observations form the basis for ongoing An equal volume of HBSS without calcium and magnesium was added cancer clinical trials in patients with advanced metastatic dis to the bag, and 35 ml were layered over 15 ml of separation ease who have demonstrated significant tumor regression (2). medium (Litton Bionetics, Kensington, MD); dilution and gradient While it has been demonstrated that LAK cells consist of a preparation took less than 10 min. Gradients were cent rifugoti at 900 heterogeneous population of cells which includes natural killer x g for 15 min at room temperature, and the separated mononuclear and cytotoxic T-cells (5-8), little is known about the mecha cells were removed, pooled, and washed twice in HBSS. Cells were incubated at a density of K)'1cells/ml in RPMI 1640 containing 2% nising) by which lytic activity is induced. Recent reports have heat-inactivated human AB serum, 50 units/ml of penicillin sulfate, 50 described the effects of administration of immune mediators on Mg/ml of streptomycin, 50 Mg/ml of gentamicin sulfate, and 2 RIM the generation of LAK cells (9, 10); however, the role of glutamine (GIBCO, Grand Island, NY) with 1000 units/ml of IL-2 cytokines in the induction of lytic activity has not been deter (Cetus Corporation) as described (13). Cells were grown in roller bottles mined. and harvested at the indicated times for measurement of LAK activity Although evidence indicates that highly purified populations and cytokine mRNA production. Adherent cells were harvested for of LGL can be induced to differentiate into LAK cells following RNA isolation as described below. IL-2 treatment, the lytic activity of such a population is not as Cell Counts. Nonadherent cells were harvested and resuspended in HBSS containing trypan blue. Cells were counted with a hemocytom- Received 7/12/88; revised 9/6/88; accepted 11/18/88. eter. The concentration of effector cells in cytotoxicity measurements The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in (see below) thus reflects only viable mononuclear cells. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Preparation of Human Monocytes, T-Cells, and LGLs. Peripheral ' Research sponsored, at least in part, by the National Cancer Institute, blood mononuclear leukocytes were separated on Ficoll-Hypaque as Department of Health and Human Services, under Contract NOl-Co-23910 with previously described (14), and monocytes were obtained after incubation Program Resources, Inc. 2To whom requests for reprints should be addressed, at Department of for 2 h, followed by removal of nonadherent cells. For preparation of Anatomy, Loyola University Sir itch School of Medicine, 2160 South First human LGL and T-cells, adherent cells were removed by incubation on Avenue, Maywood, IL 60153. plastic, followed by nylon wool. Highly enriched populations of LGL 3The abbreviations used are: IL-2, interleukin 2; II I. interleukin 1; LAK, and T-cells were obtained by centrifugation through Percoli density lymphokine-activated killer; TNF, tumor necrosis factor; LT, lymphotoxin; IFN, interferon; PBL, peripheral blood ; LGL, large granular lymphocytes; gradients (Pharmacia Chemicals, Upsalla, Sweden) (14). Morphologi HBSS, Hanks' balanced salt solution; CSF-1, colony-stimulating factor 1; ELISA, cal differentials were determined by examination of cytocentrifuge enzyme-linked immunosorbent assay; cDNA, complementary DNA. preparations stained with Giemsa. 940

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Measurement of Cytotoxicity. LAK activity was measured against the Daudi cell line in a 4-h "Cr release assay. The target cell used were in the logarithmic phase of growth and labeled for l h at 37°Casdescribed O

cpm released by cells during incubation % of release x 100 total cpm incorporated into cells

LAK activity is expressed in lytic units which are defined as the number of cells which cause 20% lysis of 5 x IO3cells over the range of effectontarget ratios used, and calculated according to the method Fig. 1. Kinetics of IL-1/3 mRNA expression in LAK cells. The mixed popu of Prossetal. (16). lation of PBL from a patient was cultured for various periods of time with IL-2. Detection of IL-1/3 by ELISA. IL-1/îprotein determinations were Northern blot analysis was performed on 20 ¿

RESULTS IL-1/8 mRNA Expression by LAK Cells. Studies performed with freshly isolated PBL revealed that cytokine mRNAs were •¿IL-1beta not spontaneously expressed in cells from either patients who received IL-2 for 3 days or normal volunteers; however, as soon as 2 h after incubation with IL-2, the expression of a 1.8- kilobase IL-10 mRNA was detected (Fig. 1). There was no consistent difference between the kinetics or magnitude of expression of cytokine mRNAs in samples from patients and volunteers (Table 1). Peak levels of IL-IßmRNA expression in the nonadherent population of LAK cells were reached in all Fig. 2. Kinetics of IL-1/3 mRNA expression in adherent LAK cells. The mixed population of PBL from a patient was cultured for various periods of time in the samples (9 of 9) between 2 and 18 h of culture and usually absence or presence of IL-2. Total cellular RNA was isolated from the adherent returned to baseline levels by 48 h. cells, and Northern blot analysis was performed on 20 n%of RNA. The filter was Since adherence of cells to the walls of the culture vessels hybridized with a cDNA probe for human IL-10. could have accounted for the decrease in the amount of IL-Iß mRNA detected in the nonadherent population with time, of IL-1/3 mRNA in the latter population remained elevated for parallel studies were performed in adherent LAK cells. The a prolonged period of time (>48 h). As expected, there was kinetics of IL-IßmRNA expression in adherent LAK cells transient expression of IL-1/îmRNA in untreated adherent cultured in the absence or presence of IL-2 was examined (Fig. cells (detected at 2 h of culture and absent by 18 h). Presumably 2). Like the nonadherent PBL, adherent PBL did not sponta this early transient rise was induced by attachment of cells to neously express cytokine mRNAs, but were capable of express the culture vessel. The level of IL-1/3 mRNA after 2 h of culture ing IL-1/3 mRNA rapidly (within 2 h of treatment with IL-2). in the presence of IL-2 is greater than in the absence of IL-2, The peak level of IL-IßmRNA expressed in nonadherent cells further demonstrating the induction of IL-1/3 mRNA by IL-2. was at 2 h and in adherent cells at 18 h. Furthermore, the level These data suggest either that the nonadherent cells which 941

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1989 American Association for Cancer Research. CYTOKINE GENE EXPRESSION IN LAK CELLS express IL-1/8 mRNA attach to plastic with time, or, alterna adherent LAK in an 18-h period was greater than that which tively, that two distinct populations of cells, one adherent and was produced by nonadherent or unfractionated LAK cells on the other nonadherent, express IL-1/3 mRNA at different rates a per cell basis. These data suggest that the adherent PBL after treatment with IL-2. population was responsible for the bulk of the production of Prior to culture the mixed population of PBL included 7% IL-1/8 in response to IL-2. monocytes (Table 1), and following culture with IL-2, a fraction To attempt to determine the role of IL-1/9 in the generation of the cells adheres to the culture vessel. To confirm the of LAK activity, we added exogenous IL-1 to PBL on Days 0 presence of monocytes in this heterogeneous population of to 5 of culture in the presence or absence of IL-2. The addition adherent LAK cells, we fractionated PBL after 18 h of culture of IL-1/3 had no effect on the magnitude of or kinetics of with IL-2 into adherent and nonadherent cells, isolated RNA, generation of lytic activity (data not shown). and compared the steady-state levels of the -specific Expression of IL-la, IFN-% TNF-o, LT mRNAs by LAK CSF-1 receptor mRNA using the \-fms probe. CSF-1 receptor Cells. The expression of IL-la mRNA, like that of IL-1/8, was mRNA was expressed in the adherent LAK cell population usually detected during the first day of culture (5 of 8 samples; Fig. 4; Table 2). In all cases, the level of a 2.2-kilobase IL-la (Fig. 3), suggesting the presence of monocytes in that popula tion. After 18 h of culture, CSF-1 receptor mRNA was not mRNA was 5- to 50-fold lower than that of IL-1/3 mRNA. In one sample IL-la mRNA could not be detected, and in two detectable in the nonadherent population, presumably due to adherence of monocytes to the culture vessel. The level of CSF- others, one patient and one volunteer, IL-la mRNA was ex pressed later than usual, at 3 and 7 days, respectively, after IL- 1 receptor expression in adherent PBLs is less than 10% that of a pure population of blood monocytes, suggesting that the population of cells attaching to tissue culture vessels differs in Patlent A Patent B the absence and presence of IL-2 with a higher proportion of monocytes in the former case. A significant portion of the adherent cells which attach in the presence of IL-2 is probably «vv activated LGL (20). Production of IL-1/3 Protein by IL-2-stimulated PBL. IL-1/3 protein was not detectable in supernatant fluids from unstimu- IL 1 lated PBL; however, after 18 h of stimulation with IL-2, IL-1/3 (400 pg/ml) was produced. The level of IL-1/3 induced by IL-2 was comparable to that which was induced by lipopolysaccha- ride (10 //g/ml). As expected, the amount of IL-l/j produced by t •¿â€¢yit* IFN

I TNF A°v

¥*» LT

Fig. 4. Kinetics of expression of IL-la, IFN-y, TNF-a, and LT mRNAs. Northern blot analysis was performed on RNA (20 fig) isolated from nonadherent PBL from two patients who were stimulated with IL-2 for various periods of time. The filter was first hybridized with a probe for IL-la and then sequentially stripped and rehybridized with probes for IFN-7, TNF-a, and LT. v - fms i Table 2 Summary ofcytokine mRNA expression mRNA expression by IL-2-treated PBL

Patients Volunteers ~G H~~

Lytic activity*

Cytokine mRNAs IL-la ++ ND* + + + + + - IL-1/3TNF-a

Fig. 3. The expression of \-fms in adherent and nonadherent LAK cells. PBL were cultured with IL-2 for 18 h and separated into adherent and nonadherent LT+ + + ND ND + ND + + cells. Total cellular RNA was isolated from each subpopulation of PBL, and " Lytic units per IO1cells. Lytic activities below 200 were considered low lytic Northern blot analysis was performed on 20 MSof RNA. The filter was hybridized units. with a cDNA probe for v-fms. * ND, not done. 942

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2 treatment. The latter samples, coincidentally, yielded low bound), rather than the secreted, form of IL-1 is required for levels of LAK activity (Table 2); however, no correlation could synergistic activation of PBL (with IL-2). Alternatively, antag be made between the delayed expression of IL-1 mRNA and onistic mediators such as prostaglandins, which are induced by low LAK activity. IL-1 (24), may play a role in masking any positive effects of IL- In addition to IL-la and ßmRNAs, other cytokine mRNAs 1. (IFN-7, TNF, and LT) were also expressed by LAK cells (Table While attachment to plastic and/or incubation in tissue cul 2). IFN-7 mRNA (1.3 kilobases) was expressed in 8 of the 9 ture medium has a positive effect on IL-l/J mRNA expression samples tested. Like other cytokines, IFN-7 mRNA was never (Fig. 2; Ref. 25), our data indicate that IL-2 also has a pro detected in untreated cells. However, unlike IL-1^ mRNA, the nounced effect on the induction of IL-1/3 mRNA expression by time course of expression of IFN-7 mRNA was quite variable. adherent PBL (Fig. 2). The adherent PBL cultured in the In general, IFN-7 mRNA was expressed by the nonadherent presence of IL-2 express IL-lßmRNA at high levels and for a cell population between 2 and 5 days after treatment with IL-2 more prolonged period of time than cultured cells in the absence (Fig. 4, Patient A). However, some samples had maximal levels of IL-2. Whether this difference is due to an alteration in the of expression as early as 18 h (Fig. 4, Patient B) and others as half-life of IL-1/3 mRNA in the two populations of cells remains late as 7 days. Often, but not always, IFN-7 mRNA was to be determined. expressed prior to the appearance of lytic activity. Monocytes are among the cell types in the mixed population All of the LAK cell preparations tested were positive for a of PBL that are capable of producing IL-1/3 mRNA; however, 1.4-kilobase LT mRNA (6 of 6) and for a 1.6-kilobase TNF-a murine T-cell lines (21), human B-lymphocytes and B-cell lines mRNA (9 of 9) (Table 1). However, the kinetics of expression (26, 27), and primary cultures of human LGL (28) secrete IL- of mRNAs which code for these cytokines in LAK cells was 1. Because of the high level of IL-1/3 mRNA expression in quite variable (Fig. 4). Neither cytokine mRNA was sponta stimulated monocytes, IL-1/3 mRNA can be detected in a mixed neously expressed by untreated PBL, and the maximal expres population of cells containing as little as 1% monocytes,4 sug sion of both mRNAs was generally between 3 and 5 days. gesting that in a mixed population of PBL containing 7% monocytes (Table 1), the percentage of monocytes is above that which is required for the detection of IL-1/3 mRNA. DISCUSSION The induction of IL-1/3 mRNA expression by IL-2 in any cell Our analysis of the expression of cytokine mRNA production type is against the current dogma of which states in LAK cells indicates that several cytokines (IL-la, IL-lß, that IL-1 induces IL-2 (29, 30). However, with the recent IFN-7, TNF-a, and LT) are expressed during the 3- to 5-day reports of IL-2 receptor expression by human peripheral blood incubation period required for the in vitro generation of LAK monocytes (31), it was reasonable to predict that induction of activity. As expected, cytokine mRNAs were not produced IL-1 gene expression could also be triggered by IL-2 treatment. spontaneously by PBLs from normal healthy volunteers, but An analysis of purified human monocytes revealed that the cells were induced following IL-2 stimulation. Surprisingly, however, do not spontaneously express IL-1/3 mRNA, but after culture cells from cancer patients to whom i.v. IL-2 had been admin with IL-2 for 5 h produce high levels of the 1.8-kilobase IL-lß istered prior to leukophoresis also failed to spontaneously ex mRNA (Ref. 32 and 33; Footnote 5). press cytokine mRNAs. Like LAK cells from normal volun The role of monocytes and in the induction of teers, cells from these patients were capable of producing cy lytic activity by IL-2-treated PBL is questionable. Hoyer and tokine mRNAs following culture with IL-2. A likely reason for coworkers (10) reported that the addition of monocytes to IL- this observation is that the cells activated by IL-2 in vivo are 2 stimulated PBL-inhibited LAK killing. More recently, trans quickly removed from the circulation; hence the rebound in forming ßwasshown to inhibit the generation of lymphocytes after IL-2 stimulation results from recruitment of LAK cells (34) and TNF-a to enhance lytic activity induced by newly generated cells from unstimulated precursors. suboptimal doses of IL-2 (34, 35). Our data demonstrating that It has not been established whether the elaboration of lytic the addition of exogenous IL-1 has no effect on the generation activity by 11,2 stimulated PBLs is due to direct effects of IL- of lytic activity (data not shown) suggest that the inhibitory 2 on certain populations of cells in the mixed culture or an effect of monocytes was not a result of release of this mediator. indirect result of another immunomodulatory "secondary" cy In summary, a number of cytokine mRNAs are expressed tokine expressed by a subpopulation of PBL, such as IFN-7 during the generation of LAK cell activity. Further studies and IL-1. Because of the rapid kinetics of induction of expres utilizing methods which block or enhance production of these sion of IL-1 mRNAs (peaking at 2 h after IL-2 treatment in molecules will be required to determine the role of these pro some samples; Figs. 1 and 2), and the absence of production of teins in the generation of LAK activity. Some of the severe side another immunomodulatory cytokine, such as IFN-7 protein, effects of LAK therapy are identical with those induced follow in the first 6 h of culture with IL-2 (data not shown), we believe ing systemic administration of IL-1 and TNF-a administration. that the expression of IL-1 genes is the direct effect of IL-2. An understanding of the role of secondary cytokines, such as However, we cannot rule out the possibility that the induction IL-1 and TNF-a, in the generation of lytic activity may enable of IL-1 gene expression by IL-2 is the result of the release of a us to determine whether adoptive immunotherapy can be ren preformed intracellular of IL-1 (21) or membrane-bound forms dered less toxic to the patient by controlling the production of of IL-1 (22) or TNF-a (23). these mediators. It is unlikely, however, that IL-1 alone is the sole secondary cytokine responsible for activating LAK precursors, since the ACKNOWLEDGMENTS addition of IL-1 to cultures containing IL-2 had no effect on We thank Dr. Ron Steis and the Biological Response Modifiers the kinetics or magnitude of lytic activity (data not shown). The Program clinical staff and JoAnn Reman and the leukophoresis unit lack of effect of IL-1 on lytic activity may be a result of the 4 M. C. Galli, unpublished observation. rapid accumulation of endogenous IL-1 in the culture medium 5E. J. Kovacs, H. A. Young, and L. Varesio, manuscript submitted for of cells treated with IL-2 or that the cell-associated (membrane- publication. 943

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1989 American Association for Cancer Research. CYTOKINE GENE EXPRESSION IN LAK CELLS staff for providing patient cells. Bonnie Ferguson prepared the LAK Spontaneous human lymphocyte mediated cytotoxicity against tumor target cells. Human monocytes, T-cells, and LGL were provided by Dr. John cells. J. Clin. Immunol., /: 51-63, 1981. 17. Chirgwin, J. M., Przybyla, A. E., McDonald, R. J., and Rattner, W. J. R. Ortaldo. We thank Sue-Ming Yang for her excellent photographic Isolation of biologically active ribonucleic acid from sources rich in ribonu- assistance. Dr. Joost J. Oppenheim and Dr. Francis W. Ruscetti clease. Biochemistry, IS: 5294-5299, 1979. 18. Glisin, V., Crvenjqakav, R., and Byus. C. Ribonucleic acid isolation by cesium reviewed the manuscript. chloride centrifugation. Biochemistry, 13: 2633-2637, 1974. 19. Kovacs, E. J., Oppenheim, J. J., and Young, H. A. 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Elizabeth J. Kovacs, Suzanne K. Beckner, Dan L. Longo, et al.

Cancer Res 1989;49:940-944.

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