Cytotoxic Antibodies to Cultured Melanoma Cells in the Sera of Melanoma Patients

s. Ferrone




and M. A. Pellegrino 4 , 6

ABSTRACT-By means of the complement-dependent microcytotoxicity test, cytotoxic antibodies to melanoma cells in longterm culture were detected in 34 of 90 sera from melanoma patients. The incidence of cytotoxic antibodies in melanoma patients was significantly greater than in subjects free of malignant disease but not significantly greater than in patients with other types of cancer. The sera were cytolytic to melanoma cells only in conjunction with rabbit complement, and they reacted with the panel of melanoma cells in a distinct fashion. No association was found between presence of cytotoxic antibodies and the occurrence of metastasis.-J Natl Cancer Inst 58: 1201-1204, 1977.

Quiescence of activity and even spontaneous regression may follow the appearance of a melanoma (1). This sequence of events certainly could result from the interplay of the host's immune system with the tumor and its products. In examining this possibility, several investigators found antibodies to melanoma-associated antigens in sera from melanoma patients, mainly by the use of freshly explanted tumor and short-term cultured tumor cells (2--6). Because of the limited availability of sera from melanoma patients, the characterization of antibodies present in such sera is limited. Moreover, the need for cells freshly ex planted from patients further limits these investigations and makes comparison of results among various laboratories impractical. To surmount these obstacles, we decided to apply the microcytotoxicity test extensively utilized for HLA antigens (7) to detect complement-dependent cytotoxic antibodies to melanoma-associated antigens. Only 2 ILl of serum per patient is necessary for this procedure (7). To overcome the problems deriving from the use of cells freshly explanted from patients, we used cultured melanoma cells as targets. Our results indicate a statistically significant difference in the incidence of cytotoxic antibodies to melanoma cells between patients suffering from melanoma and subjects free of malignant disease. MATERIALS AND METHODS

Cultured melanoma cell lines. -The melanoma lines were established at the University of California, Los Angeles, Division of Oncology, Department of Surgery Laboratory by procedures previously described (8) and at the City and County Hospital of Denver, Colorado. The 5 cell lines, M21 (HLA-A2, -A3, -B5, -BI2), Colo 38 (HLA-A2), M51 (HLA-A3, -A9, -B5), Colo 53 (HLA-A2, -B 12), and M54 (HLA-Al, -A2) were grown in monolayers inside T-flasks containing medium RPMI-1640 and 10% FCS. The cells, harvested either by scraping or by trypsinization, were washed twice with Hanks' balanced salt solution and then adjusted to a concentration of 3 X 106 cells/rnl. Cultured human lymphoid cells.-The cultured human lymphoid cells Daudi (no detectable HLA antigens), RAJI (HLA-A3), 1301 (HLA not known), RPMI 1788 (HLA-A2, -AIO, -B7, -BI4), RPMI 4098 (HLA-A3, VOL. 58, No.5, MAY 1977 Downloaded from by University of Durham user on 07 March 2018

-A29), RPMI 6410 (HLA-A2, -B7, -BI2), RPMI 8866 (HLA-A2, -A3, -B7, -BI2), and WI-L2 (HLA-Al, -A2, -B5, -BI7) were used as controls. The properties of these cell lines have been described previously (9). Sera.-Sera from patients with melanomas, with other types of tumors, and with nonmalignant disease and sera from normal subjects were obtained from the Mayo Clinic (Rochester, Minn.). The normal subjects were blood bank donors and hospital workers. The sera were coded and sent to our laboratory at Scripps Clinic and Research Foundation. The code was broken after the results had been sent to Dr. R. B. Herberman ofNCI. Complement.-Human complement was from a pool of sera from 5 donors with no previous pregnancies or blood transfusions. Guinea pig complement was from a pool of sera from 12 randomly chosen guinea pigs. Two sources of rabbit complement were used: One (indicated as #3), obtained from 100 randomly chosen rabbits, had a titer of natural antibodies (10) to cultured human lymphoid cells lower than 1:1; the other one (#6), pooled from 50 randomly chosen rabbits, had a titer of natural antibodies to cultures human lymphoid cells of 1:4. Microcytotoxicity test.-The eosin exclusion test was used for human cultured lymphoid (11) and melanoma cells. In Meller-Coates plates under mineral oil, 1 ILl of cell suspension was incubated with 2 ILl of serum at room temperature for 30 minutes. Then 3 ILl of rabbit complement was added, and incubation was prolonged for 2 hours or for 18 hours when cultured human lymphoid cells and melanoma cells were the targets, respectively. At the end of the incubation, 2 ILl of a 5% solution of eosin was added, and the reaction was then stopped by the addition of 2 ILl of 18% formaldehyde solution. The reactions were read under a phase-contrast microscope, and the percentage of "killed" cells was determined. The reactions were classified as follows: Negative when the percentage of killed cells was lower than 50; positive when the percentage was between 50 and 80; and strongly positive when higher than 80.


ABBREVIATION USED: FCS=fetal calf serum. Received July 9, 1976; accepted October 15, 1976. Supported by Public Health Service grants A113154 from the National Institute of Allergy and Infectious Diseases and CA16069 and CA16071 from the National Cancer Institute (NCI). 3 Publication #1183 from Scripps Clinic and Research Foundation. 4 Department of Molecular Immunology, Scripps Clinic and Research Foundation, 476 Prospect St., La Jolla, Calif. 92037. 5 Recipient of an American Heart Association Established Investigatorship. 6 We thank Dr. R. B. Herberman, NCI, for arranging the shipment of the panel of sera from the Mayo Clinic to our laboratory. We appreciate the excellent technical assistance of Ms. S. Janis and Ms. L. Harris, the editorial assistance of Mrs. P. Minick, and the secretarial assistance of Ms. D. Walton. 1







Preliminary experiments were performed to select the optimal conditions for the microcytotoxicity test with melanoma cells. Incubation of cultured melanoma cells, sera, and complement had to continue for 18 hours to obtain significant killing of target cells. No significant differences in the results were observed when the reactants were incubated at either 37 or 23° C. Therefore, the test was routinely performed at room temperature. Human complement and guinea pig complement in conjunction with sera from melanoma patients did not lyse melanoma cells. Rabbit serum proved to be the most efficient source of complement; however, we had to select sera with a low content of natural antihuman cell antibodies to avoid a high background of target cell killing. The melanoma cell lines also varied in their sensitivities to lysis by rabbit antibodies and complement native to rabbit serum. Rabbit complement #6 was diluted 1:5 with rabbit complement #3, for the cell lines M21 and M51, and 1:2 for the cell line Colo 53; straight rabbit complement #6 was preferable when Colo 38 and M54 were the targets. In these sources of complement, the level of xenoantibodies to cultured melanoma cells was such that it caused =30% killing of target cells in control reactions without alloantibodies to cultured melanoma cells. To minimize the possibility of considering background reactions as positive, a reaction with sera to be tested was considered positive only if the killing of target cells was higher than 50%. To rule out the possibility that trypsinization destroyed melanoma-associated antigens, cells harvested by trypsinization and by scraping were compared in their susceptibility to lysis by sera from melanoma patients and sera from a control group. No significant difference was detected; therefore, the two procedures for harvesting were used interchangeably. Some variability in results was observed when melanoma cells were tested with a battery of sera on separate occasions: Therefore, every serum was tested with all the cell lines five times, and a reaction was considered positive only when more than 50% lysis occurred at least three times, with at least 1 of the melanoma cell lines; a serum considered as negative may have reacted once or twice with melanoma cells (table 1). To determine the specificity of the reactions against tumor cells, attempts were made initially to use fibroblasts derived from the donor of melanoma cells as targets in the cytotoxicity test. However, because of the difficulties in growing these fibroblasts, this procedure I.-Number of sera from melanoma patients and from the control group that gave false positive reactions (l or 2 positive reactions/5 tests) with melanoma cell lines


Melanoma cell lines Donors


Colo 38


Colo 53


1/5 2/5 1/5 2/5 1/5 2/5 1/5 2/5 1/5 2/5 Melanoma patients 20 17 Control group


2. -Reproducibility of the reactions (3, 4, or 5 concordant reactions/5 tests) obtained with the various melanoma lines


17 14

23 28

5 10

21 33

12 16

12 22

7 14

14 7


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3 3

Melanoma cell lines M21 Colo 38 M51 Colo 53 M54

Reaction reproducibility: 5/5



50 48 39 54 80

25 35 40 27 13

25 17 21 19 7

TABLE 3.-Representative patterns of reactivity of sera from melanoma patients, normal subjects, and patients with tumors other than melanoma with the panel of cultured melanoma cells Melanoma cell linea Type of tumor Melanoma

None (free of malignancy) Other than melanoma

Serum No. 3529 4305 4317 4341 8391 8942 4331 7207 8587 8670 949 3178 4647

M21 + + + +

Colo 38 + + + +

+ (+) (+) +

(+) (+) + +



M51 + + + + + + + + + (+) + + +

Colo 53




+ +

+ +


+ +

Parentheses indicate that 3 out of 5 tests gave concordant reactions. a

became impractical. Therefore, a panel of cultured human lymphoid cells was substituted for the fibroblasts in these tests. A positive reaction to melanoma cells was considered specific if no reaction to cultured human lymphoid cells could be detected. Sera reacting both with cultured melanoma cells and with cultured human lymphoid cells were considered negative. Of 90 sera from melanoma patients, 34 (37%) contained cytotoxic antibodies to the panel of melanoma cells; of 109, 23 (21%) sera from normal subjects and patients with nonmalignant diseases gave positive reactions, and 7 of 21 (33%) sera from patients suffering from tumors other than melanoma reacted with the melanoma lines. The incidence of cytotoxic antibodies to melanoma cells in the sera of melanoma patients differed significantly from that in the sera of the control group of normal subjects and patients with nonmalignant diseases (P < 0.02) but not from a control group of patients with tumors other than melanoma. The latter two did not differ significantly from each other. The reproducibility of the reactions obtained by each cell line with the panel of sera is indicated in table 2. The sera reacted with the panel of cultured melanoma cells in a distinct fashion, and representative reaction patterns are indicated in table 3. Some sera reacted with all 5 melanoma cell lines, yet others reacted only with some of them; this suggested that antibodies from some sera are directed to antigens common to all melanoma lines, whereas others are directed to antigens specific for an individual cell line. The results were also analyzed by VOL. 58, NO.5, MAY 1977



determination of the percentage of positive reactions obtained with each cell line alone and in various combinations. In analysis of single cell lines, only the M21 cells significantly discriminated between sera from melanoma patients and those from the control group (P < 0.002) (table 4). Similar conclusions could be reached for the combinations of cell lines M21-M51 and M21-M54 analyzed at one time. Some combinations almost reached the limit of significance (table 5), but the remaining groups did not distinguish between melanoma patients and the control group. On the other hand, no significant difference in the occurrence of cytotoxic antibodies to melanoma cells could be detected between patients with metastases and those without. DISCUSSION

In the present investigation, the complement-dependent microcytotoxicity test was adapted to detect cytotoxic antibodies to melanoma cells because it functions efficiently with minute amounts of serum and is suitable for assay of large numbers of samples at a time. Compared with the use of the test to study HLA antigens, the main modification needed to seek antibodies in serum of melanoma patients is the extended incubation time of the reaction mixture. Such an approach was utilized successfully also by Takasugi and Terasaki (12) to detect antibodies in sera from a large number of cancer patients, 9 of whom sufferad from melanomas. The fact that the prolonged incubation increases the test's sensitivity and is required to detect antibodies in sera from melanoma patients suggests that these antibodies are weak, as also indicated by the low titer and by the killing of only 50-80% of the target cells in the majority of cases. Another reason for the prolonged incubation time is that melanoma cells are more resistTABLE 4.-Reactivity of various melanoma cell lines with sera from melanoma patients and a control group Melanoma cell line M21 Colo 38 M51 Colo 53 M54

Melanoma patients

Control group


Percent positive


Percent positive

88 92 89 86 89

19.3 20.6 30.3 27.9 10.1

128 129 130 127 128

4.7 20.9 25.4 20.5 4.7

P value 0.001 0.2 0.521 0.274 0.198

TABLE 5.-Combinations of cell lines that can discriminate between sera from melanoma patients and from the control group Percentage of positive reactions

P value

Melanoma cell lines Melanoma patients

Control group

27 11 10 10 11 11 10

14 3 3

M21, M51 M21, M54 M21, Colo 38, M54 M21, Colo 38, Colo 53 M21, M51, M54 Colo 38, M51, M54 M21, Colo 38, M51, M54

3 4 4 3

Cytotoxic antibodies to cultured melanoma cells in the sera of melanoma patients.

Cytotoxic Antibodies to Cultured Melanoma Cells in the Sera of Melanoma Patients s. Ferrone 4 ,5 1,2,3 and M. A. Pellegrino 4 , 6 ABSTRACT-By me...
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