Lysis of Chinese hamster embryo fibroblast mutants by human natural cytotoxic (NK) cells (tumorigenicity/anchorage dependence/serum requirement)

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1 Proc, Natl. Acad. Sc. USA Vol. 80, pp , December 1983 Genetics Lysis of Chinese hamster embryo fibroblast mutants by human natural cytotoxic (NK) cells (tumorigenicity/anchorage dependence/serum requirement) DEVENDRA P. DUBEY*t, DONALD E. STAUNTON*t, BABARA L. SMIT4H $, EDMOND J. YUNIS*t, AND RuTH SAGER0 II Divisions of *Immunogenetics and of *Cancer Genetics, Dana-Farber Cancer Institute, Boston, MA 02115; and Departments of tpathology and of IMicrobiology and Molecular Genetics, Harvard Medical School, Boston, MA Contributed by Ruth Sager, August 29, 1983 ABSTRACT The nontansformed, nontumorigenic CHEF/18 Chinese hamster embryo fibroblast line, as well as nontumorigenic CHEF/18 mutants that had become anchorage independent or acquired a reduced serum requirement for growth, and fully transformed, tumorgenic CHEF cell lines were analyzed for their sensitivity to killing in vitro by human natural killer (NK) cells. Nontumorigenic but transformed anchorage-independent and lowserum-requiring mutants remained insensitive to NK-mediated lysis like the parent CHEF/18 line. Only fully tumorigenic CHEF lines were found to be sensitive to NK-mediated lysis, although a few tumorigenic lines were resistant to NK lysis. These results indicate that NK sensitivity is not the result of any cellular changes associated with acquisition of an anchorage-independent or lowserum-requiring phenotype but is the result of some additional change(s) found only in fully tumorigenic CHEF cells. Our studies also show that, whatever the NK target structure is, it is evolutionarily conserved so that human NK cells are able to distinguish between Chinese hamster tumorigenic and nontumorigenic cells. The immune surveillance hypothesis suggests that tumor cells arising de novo are destroyed by cytotoxic cells. The natural killer (NK) cells, a heterogeneous subpopulation of lymphocytes thought to be of either T-cell (1) or promonocyte (2) lineage, have been shown to have potent lytic activity against tumor cells (3). NK cells also recognize and lyse certain nontumorigenic cells, such as normal embryonic thymocytes and some bone marrow stem cells (4, 5). Several reports suggest that a target antigen (NK-TA) recognized by NK cells is present on cells that are sensitive to NK-mediated lysis. More than one target antigen has been identified on susceptible targets (6). Some of these antigens appear to be evolutionarily conserved, since a variety of T-cell lymphoma tumor cells, from a number of mammalian species, are recognized and killed by xenogeneic NK cells (7). Xenogeneic NK cell activity has been reported in human-mouse (7), mouse-human and mouse-rat (8), and nonhuman primate cell interactions (9). Evidence has been presented that NK-TA may be glycoprotein (10) or glycolipid (11). Recent studies have suggested that NK target antigens are differentiation-type antigens present on embryonic cells and reappear during tumorigenesis (12-14). Tumorigenic cells cultured in vitro exhibit an array of characteristics that distinguish them from their normal counterparts. Although none of these in vitro transformation phenotypes has been found to correlate absolutely with the acquisition of the capacity for forming tumors in vivo, several phenotypes correlate well with tumorigenicity. These include, especially, anchorage independence and low serum requirement for growth The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. (15-18). Since the acquisition of tumorigenicity appears to be a multistep process (19, 20), it is of interest to identify the stage of transformation at which NK target determinants and sensitivity to lysis are expressed. The present study utilized the unique Chinese hamster embryo fibroblast line CHEF/18 (20, 21). CHEF/18 is a stable diploid, nontumorigenic, anchorage-dependent line with a high serum requirement for growth. CHEF/16, isolated from the same embryo preparation as CHEF/18, is a spontaneous tumorigenic anchorage-independent line that has a low serum requirement for growth. Mutant lines were selected from chemically mutagenized CHEF/18, for the ability to grow without anchorage, for the ability to clone in low (1% or 3%) concentrations of serum, or for tumor formation in athymic nude mice ([able 1). Nearly all anchorage-independent and low-serum-requiring (LS) mutants retained the nontumorigenic phenotype CHEF/18 as tested by tumor formation in nude mice (20). The availability of CHEF/18-derived anchorage-independent and LS mutants, and tumors derived after mutagen treatment of these mutants, allows us to address the question of when NK target-determinants appear during malignant transformation. Using the progressively more transformed hierarchy of (i) normal CHEF/18; (ii) partially transformed, nontumorigenic, anchorage-independent or LS mutants; and (iii) tumor cells derived from the mutants, one may identify the point at which sensitivity to NK lysis appears. Our experimental protocol utilized a xenogeneic system: target cells of Chinese hamster origin and human-derived effector cells. The premise on which this study is based is that human NK cells recognize and kill xenogeneic tumor cells by identifying evolutionarily conserved NK target antigens. We report here that neither acquisition of anchorage independence nor a low serum requirement is expressed concomitantly with increased sensitivity to NK lysis. Only those mutants that have acquired tumorigenic potential are more susceptible to lysis than the parent clone CHEF/18. MATERIALS AND METHODS Cell Lines. The cell lines used in this study were the Chinese hamster embryo fibroblast cell lines CHEF/16 and CHEF/18 and mutant lines derived from them. The origin and characteristics of these lines have been described in detail (20). Briefly, mutant cells were selected after treatment of CHEF/ Abbreviations: CHEF, Chinese hamster embryo fibroblasts; LS, low serum requiring; NK, natural killer; Tu', tumorigenic; Tu-, nontumongenic. I Present address: Department of Surgery, Brigham and Women's Hospital, Boston, MA II To whom reprint requests should be addressed. 7303

2 7304 Genetics: Dubey et al. 18 cells with the mutagens ethyl methanesulfonate, N-methyl- N'-nitro-N-nitrosoguanidine, or 4-nitroquinoline-1-oxide. Mutagenized cells were grown and selected for growth in low serum (1% or 3% fetal calf serum) or for growth in methylcellulose suspension medium as an indication of anchorage independence. Cell lines were maintained at 370C in a humidified 6.5% CO2 atmosphere in a minimal essential medium (KC Biological, Lenexa, KS) supplemented with penicillin (100 Aug/ml), streptomycin (100 Ag/ml), 2 mm glutamine (GIBCO), and 10% fetal calf serum (M.A. Bioproducts, Walkersville, MD). Tumorigenic mutants were selected by injection of 107 cells per site into nude mice (20). Phenotypes of various mutant cell lines and the origin of each are given in Table 1. Effector Cell Preparation. Human peripheral blood mononuclear cells were isolated from buffy coats obtained from plasmapheresed individuals by Ficoll/Hypaque density centrifugation. Cells were cryopreserved in 10% (vol/vol) dimethyl sulfoxide and 20% fetal calf serum. Before use, mononuclear cells were thawed and washed in Hanks' buffered salt saline solution. They were then plated in Falcon 250-ml culture flasks in RPMI 1640 medium (M.A. Bioproducts), supplemented with 10% fetal calf serum, 1 mm L-glutamine, and 50 tug of gentamycin per ml, for 60 min at 370C in a 6.5% CO2 atmosphere. Nonadherent cells were eluted, centrifuged, and resuspended at 5 x 106 per ml in a minimal essential medium with 10% heatinactivated fetal calf serum and L-glutamine. NK Assay. The NK cell assay was performed according to the procedure described previously (22). Subconfluent hamster cells, seeded 2-3 days prior to the day of the assay, were trypsinized, washed, and suspended in an a minimal essential medium supplemented with 10% fetal calf serum and 2 mm L-glutamine and kept at 40C for about 1 hr before 51Cr labeling. All cell lines Table 1. CHEF cell transformation phenotype and susceptibility to NK cell lysis Phenotype Anchor- Susceptibility Clone (parent line) age Serum Tumor to NK lysis* CHEF/18 AD HS Tu- - Anchorage mutants 18-m3 Al HS Tui - 18-m4 Al HS Tu - 18-m5 Al HS Tu Al HS Tu AI HS Tut Al HS Tu+ - LS mutants LS1-1 AD LS Tut - LS1-3 AD LS Tu- - LS3-10 AD LS Tu- - Tu+ cell lines CHEF/16 Al LS Tu Fl (CHEF/18) AD LS Tu m3 (21-2) Al HS Tu+ + T304 (21-2-m3) AI LS Tu+, TD + T21-4 (CHEF/18) AI LS Tu+, TD + m4-t1-a4 (18-m4) AI ND Tu+, TD LS1-1-T2/3 (LS1-1) ND ND TD + LS3-10-T3A/2 (LS3-10) ND ND TD + LS3-10-T6A/8 (LS3-10) ND ND TD - Al, anchorage-independent; AD, anchorage-dependent; LS, low serum; HS, high serum; Tu+, tumorigenic; Tu+, weakly tumorigenic; Tu, nontumorigenic; TD, tumor-derived; ND, not determined. * Lines lysed to a greater extent than CHEF/18 are scored as NK+. Proc. Natl. Acad. Sci. USA 80 (1983) were subjected to identical procedures. Target cells were pelleted, resuspended in 100 til of Na 5'CrO4 ( Ci/g; New England Nuclear; 1 Ci = 3.7 X 10 0 Bq), and incubated for 60 min at 37C. Labeled cells were then washed thrice and resuspended in assay media at 2 x 105 cells per ml. For effector-to-target ratios (E/T) of 25, 50, and 100, 1.25, 2.5, and 5.0 x 105 effector cells, respectively, were mixed with 5 x 103 labeled target cells in conical microtiter wells to a final volume of 200 al. All assays were performed in a minimal essential medium supplemented with 2 mm L-glutamine and 10% fetal calf serum. After the addition of target cells, plates were centrifuged at 200 x g for 10 min, incubated at 370C in 6.5% CO2 atmosphere for various times, and then centrifuged again at 40C. Cell mixtures were harvested and counted in a gamma counter. Total or maximal radioactivity incorporated and spontaneous release were determined from wells containing 5 x 103 of each labeled target cell. Monolayer NK assays were performed as follows: 105 cells were seeded 2 days prior to testings in Falcon flat-bottom microtest wells and incubated at 370C in a humidified 6.5% CO2 atmosphere. On the day of the assay, cells from one well were trypsinized and counted, of Na25CrO4 was added to each well, and the cells were incubated for 1 hr before washing five or six times with cold Hanks' solution with 5% fetal calf serum. Effector cells were added to each well in appropriate dilutions, and the final volume was adjusted to 200 AI. After overnight incubation, supernatants were harvested and released radioactivity was measured. Percent cytotoxicity in both assays was calculated according to the formula: Exp. (cpm) -SR (cpm) ~10 % specific cytotoxicity = Ep pm-sr(m)x 100, T (cpm) - SR (cpm) in which Exp. (cpm) represents the amount of radioactivity released, in cpm, from the targets in the presence of effector cells, SR (cpm) represents the radioactivity released from the targets incubated in a minimal essential medium alone, and T (cpm) represents the total radioactivity incorporated by an equivalent number of target cells. All experiments were performed in triplicate or quadruplicate. Wherever required, the cytotoxicity was expressed in terms of lytic value and was computed from the linear regression line obtained by the least-squares fit of cytotoxicity data for at least three E/T values. Data Analysis. Comparisons of sensitivity to NK-mediated lysis between mutant clones were performed by means of nonparametric ranking tests. Rank order tests were performed by using rank test and Spearman's tests (23). The point biserial correlation coefficient was used in calculation of correlation between percent cytotoxicity and tumorigenic potential. Statistical significance was determined by F test or Student's t test. RESULTS Lysis of CHEF Cells by Human NK Cells. The susceptibility of parental CHEF/18 cells and mutant cells to human NK cell-mediated cytotoxicity was measured in a 15- to 16-hr 51Cr-release assay using labeled cell lines CHEF/18, LS mutant LSL-1, and tumor-derived T304. These lines were tested simultaneously at E/T of 25, 50, and 100 with a 15- to 16-hr incubation. It may be seen (Fig. 1) that for all E/T the tumorderived T30-4 is most extensively lysed, CHEF/18 shows a low extent of lysis, and LSL-1 is not lysed to any significant extent. There is evidence that human NK cell activity resides primarily in the nylon wool- or plastic-nonadherent lymphocyte subpopulation that was used as effector cells in these experiments (24). Although no further attempt was made to charac-

3 Genetics: Dubey et al. Proc. Natl. Acad. Sci. USA 80 (1983) 7305 x40 UV U~~~~~~~ 10 O _ E/T FIG. 1. Lytic activity mediated by nonadherent lymphocytes directed against different CHEF/18 clones. Nonadherent lymphocytes from a normal donor were incubated with appropriately labeled target cells in triplicate and incubated for 16 hr. The cells used as targets were LS1-1 (o), CHEF/18 (e), and T30-4 (A). Percent spontaneous release values were 24.5,24.8, and 21.2 for targets LS1-1, CHEF/18, and T30-4, respectively. Results represent the mean percent cytotoxicity ± SEM. terize the subpopulation(s) involved in lysis of Chinese hamster cells, involvement of NK-like cells in the lysis of CHEF cells was confirmed by the use of a cytotoxic T-cell clone (B31-18) with NK-like activity (25). This clone, which was generated in mixed lymphocyte culture, has the phenotype T3+, T4-, T8-, and T10+. B31-18 cells, used at E/T = 1, gave cytotoxicity values of 32 and 11.5% when tested against the target cells T30-4 and the parent CHEF/18, respectively. To investigate the possibility that trypsin treatment of CHEF clones might differentially affect the expression of various antigens involved in the recognition and lysis of targets, one NKsensitive and two NK-resistant clones were compared by means of a monolayer cytotoxicity assay. The latter procedure requires no trypsin treatment and subjected the target cells to a minimum of chemical and mechanical manipulations. Using this method, nonlysis of resistant clones rules out the possibility that NK sensitivity is modified by trypsin treatment. In fact, at E/T = 100, with 20-hr incubations, cytotoxicity against the NKsensitive clone T21-4 was 48 ± 1.4%, whereas that against the resistant clones LSl-1 and 18m3 was essentially zero ( % and -0.8 ± 0.5%, respectively). This suggests that trypsinization does not significantly alter sensitivity to NK lysis. Similar results have been obtained by using other CHEF/18 mutant clones (data not shown). CHEF/18-derived cell lines demonstrating high (T30-4), low (LSl-1), or intermediate (parent cell CHEF/18) NK susceptibility were studied in order to examine the time course of xenogeneic lysis during 24-hr exposure to NK-effector cells (Fig. 2). Cells at E/T 25, 50, and 100 were plated simultaneously and harvested after 8, 12, 16, 20, and 24 hr of incubation. T30-4 exhibited a steady linear increase in specific cytotoxicity with time, reaching 51% at 24 hr. Cytolysis of the intermediate target cell type, CHEF 18, increased gradually and linearly for 20 hr. No significant lysis of LSl-1 was observed over 20 hr (4%) and cytotoxicity remained slight (10%) after 24 hr of incubation. Susceptibility of CHEF/18 Mutants to NK-Mediated Lysis. A total of 18 different anchorage-independent and LS mutants Co 10 _,,I I Duration of assay, hr FIG. 2. Representative experiment depicting the kinetics of 51Cr release of human NK-mediated lysis of CHEF/18 mutant LS1-1 (0), CHEF/18 (e), and T30-4 (A). Data reported in this experiment pertain to testing performed when nonadherent lymphocytes and targets (E/T = 100) were mixed at the same time in triplicate and harvested after 8, 12, 16, 20, and 24 hr of incubation. Percent spontaneous release values were 12.9, 17.2, and 17.8 at 8 hr; 26.6, 24.9, and 22.7 at 12 hr; 28.3, 24.8, and 21.2 at 16 hr; 38.7, 40.2, and 24.8 at 20 hr; and 39.0, 44.2, and 30.0 at 24 hr for target cells LS1-1, CHEF/18, and T30-4, respectively. Results represent percent cytotoxicity ± SEM. and tumors derived from them, the nontumorigenic parent line CHEF/18, and also the tumorigenic CHEF/16 line (Table 1) were tested as targets for the cytotoxic effect of nonadherent lymphocytes from several different human donors. Fig. 3 describes a typical experiment in which nonadherent lymphocytes from two unrelated donors (donors 83 and 85) were simultaneously tested against a panel of 10 targets. The tumor-derived T30-4 clone exhibited maximal sensitivity to lysis, cytotoxicity being 45 and 32% for effectors 83 and 85, respectively. The nonparametric method of ranking variates (cytotoxicity measurements) was applied to analysis of data obtained from different donors' cells as effector. Even when different donors' cells were tested on different days for cytotoxicity with a set of targets, the rank order of lysis of these mutant clones was the same. Table 2 gives the rank of cytotoxicity values obtained on different occasions, using different donors, and with testing against eight different mutant clones. The Spearman method (23) gave the coefficient of concordance (Xr)09, with a high level of significance (F test, P < 0.001). Thus, it may be concluded 4._ 40 No. 85 :.:.:.:..:.:.: Cn 40 _ 20 40V77 D T30- T CHEF/ m4-21- CHEF/ 4 4 Fl TL2/1 18 m3 m4 Tl-A FIG. 3. Nonadherent lymphocytes from two healthy donors (nos. 83 and 85) were tested simultaneously against 10 different targets in a 16-hr 51Cr release assay. Percent cytotoxicity values were determined at E/T = 100. Spontaneous release in all targets tested was less than 25%. The results are expressed as percent cytotoxicity ± SEM.

4 7306 Genetics: Dubey et al. Proc. Nati. Acad. Sci. USA 80 (1983) Table 2. Rank order of CHEF mutants based on cytotoxicity data Rank order of cytotoxicity of eight mutants tested concomitantly Sum Final Tumori- Clone Exp. 1 Exp. 2 Exp. 3 Exp. 4 of ranks ranking genicity T Tu+ T Tu m Tu+ CHEF/ Tu Tu Tu- LS Tu- LSl Tu- Coefficient of concordance among different experiments = 0.90; rank order coefficient = 0.88; and F statistics = 28.1 (P < 0.001). that although the magnitude of lysis varies, the pattern of cytotoxicity is reproducible when a panel of target cells is testedi.e., the degree of susceptibility to lysis of various clones retains the same rank order. The significance of association between levels of cytotoxicity and tumorigenicity, a dichotomous variable, was also determined. Cytotoxicity values and tumorigenic potentials were found to be correlated (P < 0.02). The pattern emerging from this and several other experiments is that (i) lines T30-4, T214, 205-Fl, and 21-2-TL2/1, all of which are found to be tumorigenic in nude mice, always appear to be more susceptible to NK-mediated lysis than does the CHEF/ 18 line. However, two tumorigenic lines, LS-3-10-T6A/8 and m4-t14a, were found repeatedly to be resistant to NK lysis; (ii) nontumorigenic anchorage-independent and LS mutants show significant resistance to NK cell-mediated lysis. Taking the parent cell line CHEF/18 as a model nontumorigenic cell line, all cell lines showing higher NK susceptibility to lysis than CHEF/18 were scored as having "NK-sensitive" (NKs)+ phenotype, while clones showing lower NK cytotoxicity were scored as having "NK resistant" (NKs)- phenotype. Nontumorigenic mutant lines, such as LS-1, LS3-10, 18m3, etc., were consistently more resistant to NK cell-mediated lysis than was CHEF/18. Thus, neither the anchorageindependent nor the LS phenotype confers sensitivity to NK cell lysis in the CHEF system. Only fully tumorigenic CHEF Table 3. Expression of tumorigenicity and NK sensitivity of CHEF clones derived sequentially Cytotoxicity % specific Tumori- Step Clone and derivation cytotoxicity* LUt genicity 1 CHEF/18-U 12.7 ± Tu- Ethyl methaneufonate mutagenesis ± Tuh I Spontaneous mutation m ± 1.2* 15.9 Tu+ Spontaneous tumor formation in nude mouse 4 T ± Tu+ * Specific cytotoxicity determined at E/T = 100. tthe lytic unit, LU, is defined as the number of cells required to lyse 25% of 104 target cells. Cytotoxicity is expressed as LU/107 lymphocytes. The lytic value was calculated from the linear least-squares fit of cytotoxicity data obtained at at least three E/T values. The correlation coefficient for each set of data was better than tcomparison between steps 1 and 3, P < 0.01 (Student's t test). Comparison between steps 1 and 4, P < (Student's t test). cell lines are highly susceptible to NK cell lysis, although some tumorigenic cell lines remain resistant to NK lysis. Progressive Acquisition of Tumorigenic Potential and Sensitivity to NK Lysis. Most of the tumor lines tested for NK sensitivity were derived from anchorage-independent or LS mutants of CHEF-18. It was, therefore, possible to look for the appearance of NK sensitivity over several progressively more transformed steps: (i) nontransformed, nontumorigenic CHEF/ 18; (ii) partially transformed, nontumorigenic anchorage-independent LS mutant; and (iii) tumorigenic lines arising from an anchorage-independent or LS mutant. Six such hierarchies were examined: (i) CHEF-18, LSl-l, and LSl-l-T2/3; (ii)chef/ 18, LS3-10, and LS3-10-T3A/2; (iii) CHEF/18, LS3-10, and LS3-10-T6A/8; (iv) CHEF/18, 21-2, 21-2-m3, and T30-4; (v) CHEF/18, 21-2, and 21-TL2/1; and (vi) CHEF/18, 18m4, and m4-t1-a4. In all hierarchies, susceptibility to NK cell lysis and tumorigenicity appeared simultaneously, although, as noted above, not all tumorigenic cell lines were susceptible to NK lysis (e.g., m4-tl-a4 and LS3-10-T6A/8). Hierarchy iv, CHEF/18, 21-2, 21-2-m3, and T30-4, was studied in detail (Table 3). The parent line CHEF/18, which is anchorage dependent for growth, was mutagenized with ethyl methanesulfonate and mutants were selected for anchorage-independent growth in methylcellulose medium. Clone 21-2 was anchorage dependent and very weakly tumorigenic in nude mice (tumors in 2/29). This clone was again selected in methylcellulose medium to obtain 21-2-m3, which was highly tumorigenic when injected into nude mice. A tumor-derived subclone of 21-2-m3, T30-4, was selected from a tumor arising in a nude mouse. When NK sensitivity of these clones was determined, an increasing sensitivity to NK lysis was observed during the acquisition of tumorigenic potential. Table 3 describes the NK lysis of CHEF/18, 21-2, 21-2-m3, and T30-4. The tumorigenic clones 21-2-m3 and T304, when tested simultaneously at a fixed E/T of 100, yielded lytic values of 22.9 ± 1.2% and %, respectively, whereas clones CHEF/18 and 21-2 yielded 12.7 ± 1.2% and 13.9 ± 3.0%. When cytotoxicity data are expressed in terms of lytic units, LU, the tumorigenic clones 21-2-m3 and T30-4 yielded values of 15.9 and 36.1 LU, whereas clones CHEF/18 and 21-2 yielded 1.4 and 5.5 LU, suggesting an increase in sensitivity of tumorigenic target cells by 10- and 24-fold. DISCUSSION We have examined the susceptibility to human NK cell lysis of CHEF/18, nontumorigenic anchorage-independent or LS CHEF/18 mutants, and CHEF tumor lines, including tumor lines derived from anchorage-independent or LS mutants. NK sensitivity appears only in tumorigenic cell lines; no nontumorigenic anchorage-independent or LS mutants showed an

5 Genetics: Dubey et al. increase in NK sensitivity over that of the parent CHEF/18 line. A similar result was obtained in the mouse system by Collins et al. (26). Since anchorage independence was genetically inseparable from tumorigenicity in this mouse system, enhanced NK sensitivity appeared to be correlated with anchorage independence as well as with tumorigenicity. It is not yet known what feature of tumorigenic cells is recognized by NK cells. Previous work has shown that NK susceptibility is dependent upon the stage of differentiation of embryonic fibroblasts. Undifferentiated embryonal carcinoma cells are more susceptible to NK-mediated lysis than are the more differentiated endodermal cells (13, 14). As shown by Hansson et al. (4, 5), NK cells recognize some antigens present on embryonal cells, since murine and human thymocytes and bone marrow cells are sensitive to NK-mediated lysis. The facts that human tumor cell lines K562 and HL-60 can be made to differentiate in the presence of inducers of differentiation and that cells so treated show loss of NK sensitivity concomitant with differentiation support the view that NK -target antigens are differentiation antigens similar to tumor antigens (27), which are known to disappear when cells undergo differentiation. NK target antigens have thus been termed "oncofetal." The studies reported here show that the NK target antigen is evolutionarily conserved such that human NK cells are able to distinguish between Chinese hamster tumorigenic and nontumorigenic cells. Neither acquisition of anchorage independence nor a lowered serum requirement generated NK target antigens, since neither anchorage-independent nor LS mutants were susceptible to NK lysis. Only fully tumorigenic CHEF cell lines were lysed by NK cells. Not all tumorigenic CHEF cell lines are susceptible to NK lysis, however. It is thus possible for a CHEF line to acquire tumor-forming ability without acquiring NK sensitivity. It is possible that the CHEF tumor lines that are resistant to NK cell lysis are in a more differentiated state than are those tumor lines sensitive to NK cell lysis. Alternatively, the NK target antigen may be related to some function not strictly required for tumorigenicity in the CHEF system. Lysis of different targets by an effjtctor population may occur through the action of large granular lymphocyte fractions of nonadherent mononuclear cells (25, 28). In this study, no attempt was made to characterize the nature of the NK cell population specifically involved in recognition of CHEF/18 mutants. However, when a human large granular lymphocyte clone (B31-18) with NK-like activity and T-cell surface markers (25) was used as an effector cell, the pattern of lytic activity was the same as that seen with our standard nonadherent mononuclear effector cell population: 3-fold greater lysis of T30-4 than of CHEF/18 was obtained (data not presented). The similarity in lytic patterns of CHEF/18 mutants with standard nonadherent lymphocytes and with the large granular lymphocyte (B31-18) clone suggests that the lytic mechanisms of these two effectors are similar. The possibility that the increase in NK sensitivities of the tumorigenic clones was due to a cell membrane defect, was ruled out by testing the sensitivity of T30-4 to clone B31-23, a noncytotoxic T-cell clone with T-helper phenotype, and a HLA-B7 specific cytotoxic T-cell clone (B31-9). The mutant T30-4 was resistant to lysis by these clones (data not shown). All CHEF cell lines lysed by NK cells in vitro were tumorigenic in the nude mouse. These results imply that nude mouse NK cell activity does not play an important role in inhibiting tumor formation by CHEF cells. Our findings are similar to those of Stanbridge and Ceredig (29), who concluded that nude mouse NK cell activity was not significant in determining the tumorigenicity of human HeLa-fibroblast hybrids in nude mice. Examination of the susceptibility to NK cell lysis of anchorage-independent and LS mutants has helped to confirm Proc Nati. Acad. Sci. USA 80 (1983) 7307 their transformed, but not tumorigenic, phenotype. The nontumorigenic and weakly tumorigenic, anchorage-independent, and LS mutants were not sensitive to NK cell lysis in vitro. This finding, along with the demonstration that several anchorageindependent mutants were nontumorigenic, even in x-irradiated nude mice (20), strongly suggests that the mutants' nontumorigenic phenotypes are not the result of immunologically based rejection by the nude mouse. We believe that an assay of NK cell sensitivity in vitro may be an important control for other cell lines found to be nontumorigenic -in the nude mouse, as recent evidence has suggested that NK cell activity may play a role in nude mouse resistance to certain tumors (29). If such cell lines are insenstiive to NK cell lysis in vitro, it is much more likely that the cells are truly nontumorigenic. If, on the other hand, the cell line proves to be NK sensitive, it would suggest that the nontumorigenic phenotype is actually the result of NKcell-mediated inhibition of tumor formation by the nude mouse. This work was supported by National Institutes of Health Grants CA20531 and CA06516 to E.J.Y. and CA24828 to R.S. 1. Herberman, R. B., Nunn, M. E. & Holden, H. T. (1978)J. Immunol. 121, Lohman-Mathes, M. L. & Domzig, W. (1980) in Natural Cell-Mediated Immunity Against Tumors, ed. Herberman, R. B. (Academic, New York), pp Herberman, R. B., Nunn, M. E. & Lavrin, D. H. (1975) Int. J. Cancer 16, Hansson, M., Karre, K., Kiessling, R., Roder, J. C., Andersson, B. & Hayry, P. (1979) J. Immunol. 123, Hansson, M., Kiessling, R. & Andersson, B. (1981) Eur. 1 Immunol. 11, Roder, J. C., Ahrlund-Richter, L. & Jondal, M. (1979) J. Exp. Med. 150, Hansson, M., Karre, K., Kiessling, R. & Klein, G. (1978) J Immunol. 121, Nunn, M. E. & Herbeihnan, R. BP (1979) J. Natl. Cancer Inst. 62, Clarke, E. A. & Sturge, J. C. (1981)J. Immunol. 126, Roder, J. C., Rosen, A., Fenyo, E. M. & Troy-Frederic, A. (1979) Proc. Nati. Acad. Sci. USA 76, Gronberg, A., Hansson, M., Kiessling, R., Andersson, B., Karre, K. & Roder, J. (1980) J. Natl. Cancer Inst. 64, Ahrlund-Richter, L., Masucci, G. & Klein, G. (1980) Somatic Cell Genet. 6, Stern, P., Gidlund, M., Orn, A. & Wigzell, H. (1980) Nature (London) 285, Gidlund, M., Orn, A., Pattenagale, P. K., Jansson, M., Wigzell, H. & Nilsson, K. (1981) Nature (London) 292, Colburn, N. H., Bruegge, W F. V., Bates, J. R., Gray, R. H., Rossen, J. D. & Shimada, T. (1978) Cancer Res. 38, Barrett, C. J., Crawford, B. D., Mixter, L. D., Schechtman, L. M., T'So, P. 0. P. & Pollack, R. (1979) Cancer Res. 39, Dulbecco, R. (1970) Nature (London) 227, Gospodarowicz, D. & Moran, J. (1976) Annu. Rev. Biochem. 45, Cairns, J. (1975) Nature (London) 255, Smith, B. L. & Sager, R. (1982) Cancer Res. 42, Sager, R. & Kovac, P. E. (1978) Somatic Cell Genet. 4, Dubey, D. P., Staunton, D., Azocar, J., Stux, S., Essex, M. & Yunis, E. J. (1982) Clin. Immunol. Immunopathol. 23, Walker, H. M. & Lev, J. (1953) Statistical Inference (Holt, Rinehart & Winston, New York), pp Jenson, P. M., Amos, D. B. & Koren, H. S. (1979)1. Immunol. 123, Sheehy, M. J., Quintieri, F. B., Leung, D. Y. M., Geha, R. S., Dubey, D. P., Limmer, C. E. & Yunis, E. J. (1983) 1. Immunol. 130, Collins, J. L., Patek, P. Q. & Cohn, M. (1980) J. Exp. Med. 153, Werkmeister, J. A., Helfand, S. L., Haliotis, T., Pross, H. F. & Roder, J. C. (1982)1. Immunol. 129, Landazuri, M. O., Lopez-Botet, M., Timonen, T., Ortaldo, J. R. & Herberman, R. B. (1981)1 Immunol. 127, Stanbridge, E. J. & Ceredig, R. (1981) Cancer Res. 41,