Humangenetik 28, 177--181 (1975) © by Springer-Verlag 1975

Original Investigations Gm(1) and Gin(2) Immunglobulin Allotypes in Patients with Malignant Melanoma W. Schultheis, H. H. Peter 1, and H. Deicher Abteilung fiir Klinische Immunologic und Transfusionsmedizin, Medizinische Klinik der Medizinischen ttochschule, ttannover Received February 24, 1975

Summary. Gm allotype markers were determined in sera from 71 melanoma patients and 400 control persons. There was no significant difference between both groups in Gm distribution. The results were compared to a recent report. Furthermore, in 25 melanoma patients the capacity of serum to interfere with cell-mediated cytotoxieity (CMC) of autologous lymphoeytes was determined and related to the Gm allotype. Introduction I n m a n y reports, malignant melanoma has been associated with genetic factors. I n general, a genetic predisposition exists for caucasians, particularly those with red hair and freckled skin, while among North American negroes melanoma is an extremely rare condition (Cipollaro, 1966; Brown et al., 1971). Furthermore, numerous cases of multiple primary and familial melanomas have been reported (Anderson et al., 1967, 1971 ; Korting et al., 1969; Bauman, 1971 ; Greenhalgh et al., 1971 ; Wallace et al., 1971) and malignant melanoma has repeatedly been observed in siblings (Andrews et al., 1968; Nagel et al. 1970; JSrgensen and Klostermann, 1968). JSrgensen and Lal (1972) were the first to suggest t h a t the genetic predisposition for melanoma m a y be linked with Gm allotype markers on h u m a n immunoglobulins. Since the Gm markers are located in the Fc p a r t of IgG1 the observation of JSrgensen and Lal, if confirmed, would have a certain theoretical importance in view of the recently discovered antibody-dependent cellular tyrotoxicity (ADCC) (Maclennan et al., 1970). Unlike target-cell damage mediated b y specifically sensitized cytotoxic T lymphocytes (Cerottini and Brunner, 1974) ADCC requires the interaction between the Fe p a r t of membrane bound IgG1 or IgG3 with a Fc receptor carrying, non-specific effector lymphocyte, provisionally called " K " cell (MacLennan, 1972; Perlman et al., 1972). B y functional criteria this cell type can be clearly distinguished from classical T and B cells. I n h u m a n t u m o r immunology there is increasing evidence for an important role of non-T cell mediated cytotoxicity (O'Toole et al., 1974; Peter et al., 1975a), which seems to be at least partially identical with " K " cell activity (Peter et al., 1975b). 1 Supported by DFG grant Pe 151/3. 13 Humangenetik, Bd. 28

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I n t h e present s t u d y we e x a m i n e d t h e G m allotype d i s t r i b u t i o n i n 71 m e l a n o m a patients, 7 of whom h a d a choroid m e l a n o m a , a n d i n 400 control persons from t h e area of H a n n o v e r . F u r t h e r m o r e , we i n v e s t i g a t e d i n 25 m e l a n o m a p a t i e n t s serumm e d i a t e d a c t i v a t i o n or i n h i b i t i o n of CMC of m e l a n o m a p a t i e n t s l y m p h o c y t e s against a n allogeneie 51Cr labeled m e l a n o m a cell line M1. The results of the 5~Cr release assays were correlated to t h e G m allotype i n the p a t i e n t s ' sera.

Material and Methods Sera and Hemagglutination Assay. Melanoma and control sera were collected in collaboration with the Departments of Blood Transfusion, Surgery, Dermatology and the Oneological Policlinic of the Medizinisehe Hochschule, Hannover. The sera were tested at 1:2 and 1:20 dilutions, either freshly or after storage at --85°C. Erythroeytes were taken from a Rh(D) positive donor. GIn(H-l) and GIn(H-2) anti-D antisera as well as appropriate anti-Gm(-kl) and anti-Gm(-{-2) antisera were obtained from Behring-Werke (Marburg, Germany). Standard hemagglutination inhibition tests were performed as recommended by Behring-Werke. All melanoma and control sera were coded and tested as unknowns. Furthermore, in most melanoma patients sera from different bleedings were tested. I n no case were discordant results obtained with ser& from the same patient. 51Cr Release Assays/or Determination o/Cell-Mediated Cytotoxicity (CMC) and AntibodyDependent Cellular Cytotoxicity (ADCC). Details of the methodology have been described elsewhere (Peter et al., 1975a). Briefly, target cells of the allogeneic human melanoma cell line M1 were labeled with Na2SlCrOa and 10a cells were incubated for 8 hrs together with 50 × 10~ purified peripheral blood lymphocytes in quadruplicate assays. The isotope released into the supernatant served as indicator for target-cell destruction by the lymphocytes. The results of tests in which lymphoeytes and target cells were incubated alone (CMC assays) were compared to simultaneously performed assays in which 1:6 diluted, heat-inactivated serum of the lymphocyte donor was added to the lymphocyte target-cell mixture (ADCC assays). Changes of more than 4% specific ~lCr release (for details see Peter et al., 1974a) in ADCC assays as compared to CIVICassays were considered as activation or inhibition and the results were correlated to the Gm allotype of the patient's serum.

Results Distribution o/Gm Allotypes in Melanoma and Control Sera [rom the Hannover Area. As can be seen i n T a b l e 1 t h e observed frequencies of G m ( + l - - 2 ) , Gin ( + I A - 2 ) , a n d G m ( - - 1 - - 2 ) i n our group of m e l a n o m a p a t i e n t s were n o t signifi c a n t l y different from the control group. There was also n o essential difference between our results a n d other reports concerning the Gm d i s t r i b u t i o n i n t h e W e s t

Table 1. Distribution of Gm allotype markers in melanoma patients and control persons from the area of Hannover

Melanoma patients Control persons ~2 (d] 1) a Not significant.

n

Gin(H-l-k2)

Gm(-kl--2)

Gm(q-l~2) Gin(q-l--2)

Gin(--1--2)

71 400

15.5~o 16.3~/o 0.000 a

28.1% 32.7% 0.367 a

43.6~o 49.0% 0.208 a

56.4% 51.0% 0.474 a

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Table 2. Changes of cell-mediated cytotoxicity of melanoma lymphocytes against an allogeneic melanoma target cell (M1) upon addition of 1:6 diluted serum of the lymphocyte donor. Relationship to the Gm allotype of the serum Effect of serum on CMC activity

Gm(-}-l+2)

Gm(~l--2)

Gin(--1--2)

Increasea Decrease a No change

1/3b 1/3 1/3

2/10 2/10 6/10

4/14 1/14 10/14

a Changes of more than 4% specific 51Cr release as compared to CMC assays without autologous serum. b Fraction of total number of persons tested.

German population. Consequently, our findings do not confirm the increased occurrence of Gm(~-2) in melanoma patients observed b y JSrgensen and Lal (1972) in the nearby area of GSttingen.

Gm Allotypes o] Melanoma Sera and their Relationship to Stimulatory and inhibitory E~ects on CMC Activity o/Autologous Lymphocytes. Based on JSrgensen's data of a strong representation of the Gm(-}-2) allotype among melanoma patients it was expected t h a t our Gm(~-1-}-2) melanoma sera would exert a predominantly inhibitory effect on CMC activity of their own lymphoeytes against an allogeneic melanoma target cell. Gm(-}-l--1) and G m ( - - 1 - - 2 ) sera, on the other hand, should have either no effect or a slightly stimulatory effect on lymphocytotoxicity. As is shown in Table 2, there is no evident relationship between the Gm allotype of a melanoma patient and the ability of his serum to stimulate or to inhibit CMC of his own lymphocytes. Discussion Based on the observation t h a t negroes, who have a low incidence of melanoma, lack the Gm(~-2) marker (Grubb, 1970; Natvig and Kunkel, 1973), the finding of JSrgensen and Lal (1972) of an increased representation of Gm(~-2) in Caucasian melanoma patients suggested a relationship between both phenomena. However, in our geographically comparable group of 71 melanoma patients the Gm distribution did not differ significantly from t h a t observed in 400 comparable control persons. Furthermore, our percentages Gm(~-l~-2), Gm(-]-l--2) and Gm(--1---2) were within the range of studies performed in Germany (Wendt et al., 1963; Deicher and Schupp, 1963). The rare condition of G m ( - - l ~ - 2 ) was not observed among our tested persons. The second p a r t of our experimentation was designed to test the ability of melanoma sera to interfere with CMC of autologous lymphocytes. I n this particular test system using an allogeneie melanoma cell line as target cell, it has been shown t h a t the effector cell is a non-T lymphocyte, most likely the so-called " K " cell (Peter et al., 1975b). The hypothesis to test was therefore whether the Gm allotype of individual melanoma sera would influence CMC of autologous lympho13"

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cytes or whether there was a r a n d o m distribution of stimulatory and inhibitory effects t h r o u g h o u t the three groups of Gm allotypes. The results of these experiments have to be interpreted with great caution since 1:6 diluted serum was added t o CMC tests and n o t isolated IgG1 fractions. U n d e r such conditions blocking and stimulating effects on CMC m a y be due to several factors other t h a n Gm allotypes, for instance free antibody, antibody-antigen complexes and circulating antigen. Therefore, only in case effector l y m p h o c y t e s from donors with different Gm allotypes would have reacted in a clearly diffrent fashion u p o n addition of their autologous serum to the target-effector cell mixtures, the results could have been used as suggestive evidence for a possible role of Gm markers in controlling cellular cytotoxicity. Our d a t a show, however, an entirely r a n d o m distribution of stimulatory and inhibitory effects on CMC within the groups of Gm(~-l-~2), G m ( + l - - 2 ) and G i n ( - - 1 - - 2 ) sera. Therefore, the Gm allotype does n o t seem to influence the activation of Fc receptor l y m p h o c y t e s in melanoma patients.

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Perlmann, P., Perlmann, H., Wigzell, H. : Lymphocyte mediated eytotoxicity in vitro. Induction and inhibition by humoral antibody and nature of effeetor cells. Transplant. Rev. 1], 91 (1972) Peter, H. H., Kalden, J. R., Seeland, P., Diehl, V., Eekert, G.: Humoral and cellular immune reactions "in vitro" against allogeneic and autologous human melanoma cells. Clin. exp. Immunol. 19 (1975a, in press) Peter, H. It., Pavie-Fiseher, J., Fridman, W. It., Aubert, Ch., Cesarini, J. P., Roubin, R., Kourilsky, F. M. : Cell-mediated eytotoxieity "in vitro" of human lymphocytes against a tissue culture melanoma cell line (IGR3). J. Immunol. (1975b, in press) Wallace, D. C., Exton, L. A., MeLeod, G. R. • Genetic factor in malignant melanoma. Cancer 27, 1262 (1971) Wendt, G. G., Deicher, H., v. Koppenfels, I. M., Puls, D. : Der Gammaglobulinfaktor Gin(a) und seine Anwendung in Vaterschaftsgntachten. Z. Morph. Anthrop. 54, 216 (1963) Prof. Dr. It. Deieher Abteilung fiir klinische Immunologie und Transfusionsmedizin Medizinische Klinik der Medizinischen Hochschule D-3000 ttannover, P.O. Box 180 Federal Republic of Germany