Int. J . Cancer: 22, 528-534 (1978)

STIMULATION OF HUMAN NK-CELL ACTIVITY BY CULTURED CELLS. 1. RESPONSE OF NORMALS P. GROHMANN, W. HOLMES, F. PORZSOLT,~ I. QUIRT, R. G. MILLERand R. A. PHILLIPS Ontario Cancer Institute, Division of Biological Research and Department of Medicine, 500 Sherbourne Street, Toronto, Ontario M4X IK9, Canada

Natural celI.mediated cytotoxicity, observed on co-cultivation of peripheral blood lymphocytes (PBL) with malignant melanoma tumor cells (M,), i s described. The activity was inhibited when PBL were cultured in autologous serum. Studies using competitive inhibition with cold targets and stirnulation of PBL wlth other tumor cell lines failed t o detect specificity.

Several investigators have detected tumor-specific cell-mediated immune responses in patients with malignant melanoma (Hellstrom et af., 1973; Heppner et al., 1973; Pavie-Fisher et al., 1975; Golub, 1977a, b). For example, Golub (1977a, 6 ) reported that incubation of peripheral blood leukocytes (PBL) from healthy individuals and from melanoma patients with autochthonous and allogeneic melanoma tumor cells induced a cytotoxic response, which reached a peak on the 5th day of culture. Unsensitized lymphocytes showed a low cytotoxic activity. Sensitization with allogeneic melanoma cells gave a stronger response than with autochthonous tumor cells, and this increased cytotoxic activity was observed even when autochthonous target cells were used. The two possible explanations proposed were : (1) melanoma cells may have two types of antigen, one specific to the individual tumor and the other shared by other lines of the same histological tumor; (2) a restriction in cytotoxic activity, by autochthonous-sensitized PBL to target-carrying melanoma-associated antigens and autochthonous antigens of the major histocompatibility locus, could occur. It has also been reported that some melanoma lines induce a stronger anti-tumor response than others (Pavie-Fisher et al., 1975; Golub, 1977a, b). In a preliminary attempt to repeat these observations, we found that cytotoxicity increased very rapidly in culture with both PBL and melanoma cells, peaking within 24 h of incubation. However, this reaction lacked specificity in the sense that cytotoxicity was induced in normal individuals as often as in patients with melanoma. Herberman e f al. (1977) observed that natural cell-mediated cytotoxicity in animals could be increased by inoculation of syngeneic and allogeneic tumor cells. This cytotoxic activity reached a peak at day 3 after inoculation and fell rapidly thereafter. The effector cells of this cytotoxic activity had the cell surface characteristics of the natural killer cell. Herberman suggested that such responses may play a role in resistance to virusinduced tumors.

The anti-melanoma responses observed by us have some characteristics of a natural cytotoxic response and may be important in resistance to solid tumor. The data presented below provide a partial characterization of the rapidly induced cytotoxic response observed with PBL from normal individuals. Other papers in this series will summarize the response of the melanoma patients, the properties of the effector cells, and the inhibitory effect of salicylate on the induced cytotoxic response. MATERIAL AND METHODS

Cultured tumor cells The line of malignant melanoma tumor cells, Mq, was obtained from Dr. S. K. Liao, Henderson General Hospital, Hamilton, Ontario. The cells were grown in tissue culture flasks (Falcon No. 3024) in a-MEM (K.C. Biologicals, Inc., Lenexa, Kansas) supplemented with 100 ,ug/ml of streptomycin and 100 IU/ml of penicillin plus 20% FCS (Flow Labs., Inc., lot No. 4057017) or AB serum as indicated. They were incubated a t 37" C in a humidified atmosphere with 5 % CO,. M4 grows as a monolayer. When the culture is confluent, large numbers of cells detach spontaneously. When the cultures are fed daily, these detached cells are 90 to 100% viable, as determined by dye exclusion. Other melanoma cell lines, SK Me1 12, SK Me1 26, and MeWo were obtained from Dr. J. Fogh, Sloan-Kettering Institute, New York. They were grown as an attached monolayer and needed to be trypsinized before use. After trypsinization, they were washed three times in phosphate-buffered saline (PBS) and incubated overnight in 15 ml polystyrene tubes (Falcon No. 2006) in a-MEM plus 20% FCS, before being used as stimulator or target cells. The retinoblastoma lines, Weri and Y-79, used to study specificity, were obtained from Dr. B. L. Gallie, Wellesley Hospital, Toronto, Ontario. They were grown in suspension in a mixture of RPMI 1640 (K.C. Biologicals, Inc., Lenexa, Kansas) supplemented with 10%FCS.

Received: July 24, 1978. Present address: Universitat Ulm, Department fur Innere Medizin, Steinhovelstr. 9, D-79 UlmjDonau, West Germany. Reprint requests should be addressed to: Dr. R. A. Phillips, Ontario Cancer Institute, Division of Biological Research, 500 Sherbourne Street, Toronto, Ontario, Canada M4X 1K9.

529

IN VITRO ANTI-MELANOMA RESPONSE

Colon carcinoma lines, SW 403 and SW 602, were obtained from Dr. A. Leibovitz, Scott and White Clinic, Temple, Texas. They were grown with a-MEM+20% FCS as an attached monolayer and trypsinized before use. Lymphoblastoid lines, K 562 and Daudi, were obtained from Dr. J. Minowada of Roswell Park Memorial Institute, Buffalo, N.Y., and were grown in suspension in a-MEM+20% FCS. P815, a mouse mastocytoma line, was grown in suspension in a-MEM+20% FCS. Preparation of stimulator and labelled targets The tumor cells were adjusted to 2 x 105/ml when used as a stimulator. The melanoma and colon carcinoma cell lines were irradiated with 1,500 rads, Weri and Y-79 with 3,000 rads and Daudi and K 562 with 10,OOO rads. When used as targets, lo6 tumor cells were suspended in 0.5 ml of Hanks' balanced salt solution without calcium and magnesium (Grand Island Biological Co., control No. C263612) and 0.1 ml of Na2[51Cr]04(6.5 mCi/ml) was added. The cells were incubated for 1 h at 37" C, washed three times in PBS and adjusted to 2x1O4 cells/ml in a-MEM+20% FCS. Sheep red blood cells were also labelled with 51Cr by the same procedure as described above and adjusted to 2 x lo5 cells/ml. Collection of peripheral blood leukocytes (PBL) Heparinized peripheral blood was collected from normal donors and diluted 1:3 with PBS. The diluted blood was layered onto Ficoll-Hypaque prepared as follows: Ficoll was dissolved in distilled water to give a 9 % (w/v) solution. Hypaque (Winthrop) commercially available as a 50% (w/v) solution was diluted with PBS to a 34% solution. The two were mixed in a ratio of 40:25 Ficoll: Hypaque. The optical density of the final mixture was 1.370. The gradient was prepared in a 50-ml siliconized glass centrifuge tube with a conical bottom. The tubes were centrifuged for 30 min at 400 g and 4" C. The cells were removed from the interface, washed three times in PBS and adjusted to 2x106/ml in a-MEM+20% of the desired serum. With this procedure 90% of the mononuclear cells were recovered from blood with a viability of 95 % by dye exclusion and less than 2 % contamination with neutrophils. Incubation of PBL and assay for cytotoxicity Nine-tenths ml of PBL (2 x lo6) and 0.1 ml of irradiated tumor cell (2x105) were placed in 5-ml plastic tubes (Falcon No. 2058). In some experiments 0.5 ml of 4 x lo6 cells/ml were mixed with 0.5 ml of M, and Weri supernate. The cultures were incubated for varying periods of time at 37" C in 5 % CO,. At the desired time, the cells were harvested and tested for cytotoxicity. At the time of the assay, triplicate 0.1-ml aliquots of the cultures (direct and 1:2, 1:4 or 1:8 dilutions) were placed in wells in a microtiter tray (Linbro Scientific Co., Inc.) with 0.1 ml of labelled target (2x104/ml). In some experiments, unlabelled targets were added to test specificity of the cytotoxic activity. The ratio of

unlabelled to labelled targets ranged from 1O:l to 40:l. In these experiments, the labelled targets were prepared at 4 x lo4 cells/ml and diluted with an equal volume of unlabelled cells at the desired concentration. After effector and target cells were mixed, the trays were centrifuged for 5 min at 170 g and then incubated for 4 h at 37" C in 5 % CO,. After incubation, 0.1 ml of supernatant from each well was removed and the radioactivity measured in an LKB gamma counter. The spontaneous release (S) was determined by incubation of labelled tumor cells in medium alone for 4 h at 37" C. The total releasable counts (TR) were determined by adding 0.1 ml of 1 % acetic acid to 0.1 ml of labelled M4. The specific 51Chromium release was calculated in the standard way, p = (E-S)/(TR-S) where p is the fractional specific 51Chromium release and E is the cpm in the experimental group. To convert specific Cr-release to a value directly related to the number of cytotoxic lymphocytes, the method described by Miller and Dunkley (1974) was used. According to their model, Nat = -In (1 -p) where N = number of cells tested, a is a constant proportional to the absolute number of effector cells in culture, and t is time of the Crrelease assay. All the results below are expressed as Nat. Nat and p are very similar when p is equal to or less than 0.20 (20 % release). When p is higher, the difference between the two values increases. For example, when p is 0.7, Nat is 1.2. RESULTS

Culture conditions When PBL were incubated with irradiated M, cells, a reproducible, strong cytotoxic response was observed which usually reached a peak on day 1 and sometimes on day 2 (Fig. 1). In some experiments, a high background cytotoxicity was seen on these days when PBL were cultured without M, but the net stimulation was always significant except when indicated. Titration of day 1 cytotoxic activity is shown in Figure 2. At later times, after day 3, cytotoxicity sometimes increased in cultures

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FIGURE 1 - Cytotoxic activity as a function of time in culture. - - -, PBL with M, stimulator cells; -, PBL without M, stimulator cells.

530

GROHMANN ET AL.

containing only PBL. This increase is dependent on certain batches of FCS and was not studied further. The day 1cytotoxicity induced by M, was observed in 16 of 18 normal individuals tested, in a total of 38 experiments. One individual was tested eight

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times and gave quite reproducible levels of cytotoxicity, except in one instance in which a higher level was seen. The coefficient of variation of the seven samples was 0.102. The two individuals who did not respond to M, were each tested twice with similar results. Both individuals are healthy and have no history of chronic illness. To measure accurately the rate of activation of PBL, a large number of cultures were established with PBL from three donors. At 3-h intervals, cultures were harvested and tested for cytotoxicity. The results of one of these experiments are summarized in Figure 3. Cytotoxicity begins to increase above background after 9 h of incubation and continues to increase until 18 h. If lymphocytes were incubated in autologous serum (AS), no increase in cytotoxicity was observed. Different amounts of AS were mixed with FCS in three experiments. The results of one of these are shown in Figure 4. When 10% FCS plus 10% AS or 15% FCS plus 5 % AS were tested, no anti-M, activity was seen. When 19% FCS plus 1 % AS was used, the level of cytotoxicity increased but the response was still lower than when no AS was added. To exclude the possibility that the cytotoxicity was directed against FCS components bound to M,, the cell line was grown for 1 week in human AB serum. Since activation of PBL requires FCS, effector cells were induced under standard conditions. After 18 h the cells were harvested, washed and assayed for cytotoxicity in human AB serum,

FIGURE 2 -Titration of PBL cytotoxic activity cultured with (- - -) and without (-) M4.

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FIGURE4 - Cytotoxic activity observed when PBL with (- - -) and without (-) M, are cultured with 20% FCS (top, left), 20 % AS (top, right), mixture of 10 % FCS and 10% AS (bottom, left) and 19% FCS and 1% AS (bottom, right).

531

IN VITRO ANTI-MELANOMA RESPONSE

using as targets the M, cultured in AB serum. The cytotoxicity was identical to that in a normal control using FCS throughout. Horse serum was used in two experiments and the response was identical to that of cells incubated in FCS. Stimulation of PBL with other melanoma lines Stimulation of PBL with SK Me1 12 and MeWo was studied in four experiments. When SK Me1 12 was used as stimulator and target, a pattern similar to that of M, was observed except that cytotoxicity was lower. No stimulation was seen with MeWo. Stimulation with other tumor cell lines Kinetics of PBL sensitized with K 562, Daudi and Weri showed two different patterns. When K 562 and Weri were used as stimulator and target, both unsensitized (PBL cultured without tumor cells) and sensitized lymphocytes were highly cytotoxic when assayed against the line used as stimulator. Weri showed lower levels of activity than K 562. One representative kinetic experiment obtained with K 562 is shown in Figure 5. When Daudi was used as stimulator and target, the PBL incubated with irradiated Daudi produced strong cytotoxicity on each of the 5 days tested with two peaks of activity. The first peak was on day 1 and the second on day 4 (Fig. 6).

280 260

Stimulation with tumor line supernate and filtrate A significant increase in cytotoxic activity was observed in three out of four experiments in which M4 supernate was used as stimulator. The same results with Weri supernate were observed in two experiments. No increase in cytotoxic activity was seen when this supernatant was filtered. See Table I. Specz3city at the level of the efector When PBL were stimulated with M4 and assayed against other tumor cells or vice versa, different results were obtained for each cell line used. When PBL were stimulated with M4 and assayed against either labelled M4 or Weri, either similar levels of cytotoxicity (four experiments) or somewhat higher levels for Weri (two experiments) were observed. Stimulation with Weri resulted in similar killing of both targets (Table IT). Stimulation of peripheral blood lymphocytes with M, and assay against K 562 was performed twice. It resulted in greater killing than when M, was used as a target. Stimulation with K 562 and test against M, resulted in similar (one experiment) or lower anti-M, cytotoxicity (one experiment) when compared to the activity seen with K 562. Similar results were seen when M4 and Daudi were used as stimulator and target (data in Table 111). In two experiments, PBL were stimulated with M, and tested against SK Me1 12, SK Me1 26 and MeWo (see Table IV). No kifling of Iabelied SRBC was detected in two experiments in which PBL were stimulated with M,. Specificity at the level of the target The ability to inhibit the anti-M, cell-mediated cytotoxicity with cold targets was studied using several cell lines. Unlabelled M, effectively inhibited the activity induced by M4 at least 50% when the ratio unlabelled/labelled was 20:l or more.

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532

GROHMANN ET AL. TABLE I COMPARISON OF CYTOTOXIC ACTIVITY INDUCED BY TUMOR CELLS AND THEIR SUPERNATES ________________~~

PBL PBL+M4 PBL+M, supernate PBL+M4 filtered '1

~~

Exp. I

Exp. 2

Exp. 3

Exp. 4

Target

0.37k0.02 1.09 f0.09 0.83+0.10 0.26 10.03

0.22h0.02 1.12h0.16 0.38 hO.01 0.26&0.02

0.15 f0.02 1.15 +0.12 0.48 3Z0.05 0.08 1 0

0.16h0.01 0.89h0.06 0.37f0.03 0.1OfO

M4 M4 M4 M,

0.31 50.05 1.21+0.21 1.43h0.32 0.8650.23

0.61 50.07 2.18k0.72 2.09f0.79 0.63k0.09

Weri Weri Weri Weri

PBL PBL Weri PBL+ Weri supernate * PBL Weri filtered

+ +

'

Anti-melanoma Nat values for PBL cultures. - * All PEL cultures contained 2 x lo6cells and stimulators were always at 2 x 10' cells/ml. Tests were performed 24 h after cultivation and values are in Nat. - * As in but with M, stimulator cells added. As in but with 50% M, supernate included. - As in a but supernate filtered before inclusion. - Anti-retinoblastoma Nat values for PBL cultures. - 7 As in 8 but with Weri stimulator cells added. - As in but with 50% Weri supernate included. - As in but supernate filtered before inclusion.

-

The mouse line, P815, and colon carcinoma lines, SW 403 and SW602, were tested twice and inhibited the cytotoxicity induced by M4 only to 40% or less when the ratio unlabelled/labelled was higher than 40:l (Fig. 7). When the unlabelled retinoblastoma line, Y-79, was added, significant inhibition of anti-M, cytotoxicity was observed. Comparison of the ratio of M4and Y-79 needed to inhibit 50% of the activity is shown in Table V. There is no significant difference between the amount of unlabelled MI and the amount of unlabelled Y-79 that caused 50% inhibition, as indicated by the Wilcoxon Rank Sum Test. DISCUSSION

That cultivation of PBL from normal individuals with tumor cells results in tumor killing has been 0 10:1 20:1 40:l reported by several workers (Pavie-Fisher et al., Ratio of cold:hot target 10680 1975; Golub, 1977a, b; Takasugi et al., 1973). In the experiments described here, incubation of PBL FIGURE 7 - Cold target competitive experiment. PBL were cultured for 24 h with M, and 2 x los labelled M4 with malignant melanoma cells induced a rapid targets were added to each culture, always with cold cytotoxic activity in 16 out of 18 normal individuals target multiplicities shown. 0, unlabelled M4 com- tested, if the PBL were cultured in FCS. This effect petitor; A, unlabelled Y-79 competitor; 0 , P815 com- was not detected when the leukocytes were incupetitor; 7 , SW 602 competitor; a, SW 403 competitor. bated in autologous serum. Thus there is probably 0

TABLE I1 COMPARISON OF CYTOTOXIC ACTIVITY WHEN STIMULATOR AND TARGET CELLS ARE THE SAME OR DIFFERENT

PBL PBL PBL+Md PBL M, PBL+ Weri PBL Weri

+ +

Exp. 1

Exp. 2

Exp. 3

Exp. 4

0.16h0.01 0.49h0.04 0.81 &0.06 0.75 *0.05 1.06 f0.16 0.50h0.06

0.15h0.02 0.43 10.05 0.71 A0.12 0.97k0.07 1.25 h0.17 0.83f0.08

0.15 %0.02 0.31 10.05 1.15 h0.12 0.85 50.17 1.21 10.21 1.36h0.30

0.16 h0.01 0.61 3Z0.07 0.89k0.06 1.51 h0.15 2.18 f0.72 1.233Z0.20

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Target

M4

Weri M4

Weri Weri Ma

Anti-melanoma Nat values for PEL cultures. - ' Anti-retinoblastoma Nat values for PEL cultures. a As in but with M. stimulator Anti-retinoblastoma Nat values for cultures of 2 x loJ PEL with 2 x 10' M, added stimulator cells cultured for 24 h. - 8 Aa cells added. in but with Weri stimulator cells added. ' Anti-melanoma Nat values for culture of 2 x lo6 PBL with 2 x 10' Weri added stimulator cells cultured for 24 h.

-

-

533

IN VITRO ANTI-MELANOMA RESPONSE TABLE 111 COMPARISON OF CYTOTOXIC ACTIVITY WHEN STIMULATOR AND TARGET CELLS ARE THE SAME OR DIFFERENT

PBL lPBL PBL PBL+M4' PBLtM, PBL+M4 ' PBL+K 562 PBL+K 562 PBL-kDaudi PBL-kDaudi

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l1

Exp. 1

Exp. 2

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1.08k0.17 2.53+0.32 0.22z0.05 1.744~0.28 3.57 %2.20 0.77*0.21 2.18f0.55 2.10;t0.49 1.17f0.14 2.16rt0.50

0.14rt0.01 1.14+0.23 0.2010.02 0.3750.02 I.80g0.77 0.63 &0.05 2.82& 1.7

M1 K 562 Daudi Ma K 562 Daudi K

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1.12&0.08 0.20iz0.03

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D

1 Anti-melanoma Nat values for PBL cultures. - a PBL cultures were always at 2 x lo6 cells/ml with 2 x LOs added stimulator. Tests were performed 24 h after Cultivation and the values are in Nat. a Anti-K 562 response for PBL cultures. - Anti-Daudi response for PBL cultures. ' As in but with M, stimulator ceIIs added. Anti-K 562 response for PBL cultures stimulated with M,. 7 Anti-Daudi response for PBL cultures stimulated with M,. As in but with K 562 stimulator cells added. - Anti-M, response for PBL cultures stimulated with K 562. - As in ' but with Daudi stimulator cells added. - l1 Anti-M, response for PEL cultures stimulated with Daudi.

-

-

-

TABLE IV ANTI-MELANOMA RESPONSE USING M, AS STIMULATOR AND M, OR OTHER MELANOMA CELL LINES AS TARGET Targets Cultures M,

PBL PBL+M, PBL PBL+M4

0.11 a 0.58 0.12 0.72

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0.42 0.42

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TABLE V COMPARISON OF THE MULTIPLICITY OF M, AND Y-19 REQUIRED TO PRODUCE 50% INHIBITION OF THE ANTI-M. RESPONSE Cold target Exp.

1 2

' Difference not

test.

M.

Y-I9

9 9 10 14 20 9 19 8

13 9 18 16 28 9 19 20

significant as determined by Wilcoxon rank sum

some inhibitory factor present in normal human sera, since we have shown that cytotoxicity was not directed against a FCS component bound to M,. We observed that different lines of malignant melanoma induced variable levels of cytotoxic activity as has been previously reported by other workers (Pavie-Fisher et al., 1975;Golub, 1977a,b). Increase of cytotoxicity was also observed in experiments when only M 4 or Weri supernate was added to the cultures, suggesting that some factor released by the tumor cells is able to induce stimulation. Specificity tests are very difficult to validate in allogeneic systems and only indirect methods are available. Using different stimulators t o study specificity at the level of the effector, we found that, once the PBL are activated by co-cultivation with irradiated tumor cells, they may kill tumor cells other than the stimulating line. Nevertheless, the stimulated cells were not able to lyse SRBC. Competitive inhibition with cold targets showed that the cytotoxic activity detected when PBL were co-cultivated with M, could be inhibited by unlabelled M, as well as by unlabelled Y-79;unlabelled P815 or colon carcinoma tumor cells produced little inhibition. Several investigators recently reported in vitro evidence for natural cell-mediated cytotoxicity when PBL from healthy donors are cultivated with tumor cells [e.g., as described by Jondal and Pross (1975) for bladder carcinoma, lung carcinoma and. lymphoblastoid cell lines; Rosenberg et al. (1974) for breast cancer and Hodgkin's disease; Takasugi et al. (1973) for cervix, breast, colon, endometrial and prostate cancer]. Peter et al. (1976)observed cytotoxic activity after 6 h of co-cultivation of normal donor PBL plus human malignant melanoma cell line. This activity was not smcific since killing of a tumor line other than the stimulating one was also observed. They suggested that a )ymphotoxin-]ike mediator bi involved in the activity. Analysis Of Cell Surface markers suggested that activity was not mediated by a B cell or a T cell (Pross and Jondal, 1975; Peter et at., 1975, 1976;Herberman et al., 1977). The activity described by us is rapidly induced in normal donors, without evidence of specificity. Analysis of the effector cell of this cytotoxic activity reveals that it has cell surface characteristics of a natural killer cell, as will be described in subsequent papers (in preparation). We believe that the experiments described herein could represent an in vitro human phenomenon similar to that described by Herberman in their in vivo mouse system, in which natural cell-mediated cytotoxicity can be increased by inoculation with syngeneic and allogeneic tumor cells.

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I

ACKNOWLEDGEMENTS

P.G. was supported by the Universidade Federal do Rio de Janeiro and by the National Cancer Institute of Canada. This study was supported by the Ontario Cancer Treatment and Research Foundation.

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GROHMANN ET AL.

STIMULATION DE L'ACTIVITfi DES CELLULES NK HUMAINES PAR DES CELLULES EN CULTURE. I. RBPONSE DES SUJETS NORMAUX La cytotoxicite cellulaire spontante observte pendant la co-culture de lymphocytes sanguins (PBL) et de cellules de mtlanome (M,) est dkrite. Cette activite est inhibte lorsque les lymphocytes sanguins sont cultives en presence de strum autologue. Des etudes sup la competition par des lymphocytes sanguins non stimults ou stimults avec des cellules tumorales autres que les M, n'ont pu demontrer la spCcificitt de cette cytotoxicitt cellulaire spontante.

REFERENCES

GOLUB,H. S., In vitro sensitization of human lymphoid cells to antigens on cultured melanoma cells. I. Culture conditions in augmented cell-mediated cytotoxicity. Cell. Immunol., 28, 371-378 (1977a). GOLUB,H. S., In vitro sensitization of human lymphoid cells to antigens on cultured melanoma cells. 11. Sensitization against melanoma-associated antigens. Cell. Immunol., 28, 379-389 (19776). HELLSTROM, I., WARNER, G. P., HELLSTROM, K. E., and SJOGREN, H. O., Sequential studies on cell-mediated tumor immunity and blocking serum activity in ten patients with malignant melanoma, Int. J . Cancer, 11, 280-292 (1973).

L., BYRNE,M., CUMMINGS, F. J., HEPPNER, G. H., STOLBACH, MCDONOUGH, E., and CALABRESI, P., Cell mechanism and serum blocking activity to tumor antigens in patients with malignant melanoma. fnt. J. Cancer, 11, 245-260 (1973). HERBERMAN, R. B., NUNN,M. E., HOLDEN,H. T., STAAL, S., and DJEN,J. Y., Augmentation of natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic target cells. Int. J. Cancer, 19, 555-564 (1977). JONDAL, M., and PROSS,H., Surface markers on human B and T lymphocytes. VI. Cytotoxicity against cell lines as a functional marker for lymphocyte subpopulations. Int. J . Cancer, IS, 596-605 (1975). MILLER,R. G., and DUNKLEY, M., Quantitative analysis

of the "Cr release cytotoxicity assay for cytotoxic lymphocytes. Cell. Immunol., 14, 284-302 (1974). J., KOURILSKY, F. M., PICARD,F., BANZET, P., PAVIE-FISHER, and PUISSANT, A., Cytotoxicity of lymphocytes from healthy subjects and from melanoma patients against cultured melanoma cells. Clin. exp. Immunol., 21, 430-441 (1975). PETER,H. H., EIFE,R. F., and KALDEN, J. R., Spontaneous cytotoxicity of normal human lymphocytes against a human melanoma cell line: a phenomenon due to a lymphotoxinlike mediator. J. Immunol., 116, 342-348 (1976). J., FRIDMAN, W. H., AUBERT, C., PETER, H. H., PAVIE-FISHER, F. M., Cell CESSINI,5. P., ROUBIN,R., and KOURILSKY, mediated cytotoxicity in v i m of human lymphocytes against a tissue culture melanoma cell line (igr3). J . Immunol., 115,539-548 (1975). PROSS,H. F., and JONDAL, M., Cytotoxic lymphocytes from normal donors. A functional marker of human non Tlymphocytes. Clin. exp. Immunol., 21, 226-235 (1975). ROSENBERG, E. B., McCoy, J. L., GREEN,S. S.,DONELLY, F. C., SLWARSKI, D. F., LEVINE,P. H., and HERBERMAN, R. B., Destruction of human lymphoid tissue culture cell lines by human peripheral lymphocytes in 0-release cellular cytotoxicity assays. J. nut. Cancer Inst., 52, 345-352 (1 974). TAKASUGI, M., MICKEY, M. R., and TERASAKI, P. I., Reactivity of lyrnphocytes from normal persons on cultured tumor cells. Cancer Res., 33, 2898-2902 (1973).

Stimulation of human NK-cell activity by cultured cells. I. Response of normals.

Int. J . Cancer: 22, 528-534 (1978) STIMULATION OF HUMAN NK-CELL ACTIVITY BY CULTURED CELLS. 1. RESPONSE OF NORMALS P. GROHMANN, W. HOLMES, F. PORZSO...
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