[CANCER RESEARCH 53, 3343-3348. July 15, 1993] Differential Expression of Cell Adhesion Molecules CD54/CDlla and CD58/CD2 by Human Melanoma Cells and Functional Role in Their Interaction with Cytotoxic Cells1 Maresa Altomonte, Annunciata Gloghini, Giulio Bertela, Aldo Gasparollo, Antonino Carbone, Soldano Ferrone, and Michele Maio2 Advanced Immunotherapeutics Unit and Division of Experimental Oncology 2 [M. A., A. Ca., M. M.], Pathology [A. GÃOE, A. C.], Surgical Oncology I [G. B.J, and Medical Oncology 1 ¡A.Ga.¡,C. R. O., Aviano, Italy 33081, and Department of Microbiology and Immunology. New York Medical College, Valhalla, New York 10595 [S. F.J ABSTRACT Immunohistochemical staining with monoclonal antibodies showed a differential distribution of intercellular adhesion molecule 1 (ICAM-1/ CD54) and lymphocyte function-associated antigen 3 (LFA-3/CD58) and their respective counterreceptors lymphocyte function-associated antigens 1 (LFA-1/CDlla) and 2 (LFÀ-2/CD2) on ten melanoma cell lines and in 46 surgically removed metastatic melanoma lesions. CDlla and CD2 were not detected on melanoma cells while CD54 and CD58 were coexpressed on the majority of the melanoma cell populations investigated. CD54 showed a higher degree of intra- and intertumor heterogeneity than CD58. •¿y-Interferon and/or tumor necrosis factor a upregulated the expression of CD54 by melanoma cells, but neither modulated that of CD58 nor induced that of CDlla and CD2. Anti-CD54 and anti-CD58 monoclonal antibodies partially inhibited the lysis of melanoma cells by allogeneic natural killer cells, lymphokine-activated killer cells and, to a greater extent, by autol- ogous tumor-infiltrating lymphocytes. Soluble CD54 (cCD54) purified from serum of patients with melanoma inhibited the lysis of melanoma cells FO-1 by natural killer cells in a dose-dependent fashion. These results suggest that membrane-bound CD54 and CD58 and cCD54 play a role in host-tumor interactions in patients with malignant melanoma and may account for the relationship between CD54 expression in primary lesions and the clinical course of disease. INTRODUCTION Several cell membrane proteins that mediate homotypic and het- erotypic cell-to-cell adhesive phenomena have been assigned to the large family of CAM3 (1). Among CAM, ICAM-1/CD54 with its counterreceptor LFA-1/CDlla and LFA-3/CD58 with its counter- receptor LFA-2/CD2 are of note for their role in cell-cell interactions required to generate an immune response (2). Furthermore, the het- erotypic interaction of CD54/CDlla and CD58/CD2 molecules strengthens the lysis of target cells by MHC antigen-restricted CTL (3, 4), and the two ligand-receptor interacting pairs can act synergis- tically in this phenomenon (5). In contrast, conflicting data have been reported on the role of these CAM in the NK and LAK cell-mediated lysis of various types of target cells (6-12). The potential involvement of CAM in the interaction of melanoma cells with the host's immune system and the previously described Received 12/30/92; accepted 5/6/93. 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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Associazione Italiana per la Ricerca sul Cancro and by USPHS Grant CA39559 awarded by the National Cancer Institute. 2 To whom requests for reprints should be addressed, at Advanced Immunotherapeutics Unit, C. R. O., Aviano, Italy 33081. 3 The abbreviations used are: CAM, cell adhesion molecules; APAAP, alkaline phosphatase-antialkaline phosphatase; CTL, cytotoxic T-lymphocytes; DTAF, di- chlorotriazynylaminofluorescein; ELISA, enzyme-linked immunosorbent assay; PCS, fetal calf serum; ICAM-1, intercellular adhesion molecule 1; IFN, interferon; IgG, immunoglobulin G; IIP, indirect immunofluorescence; IL, interleukin; LAK, lymphokine- activated killer; LFA, lymphocyte function-associated antigen; mAb, monoclonal anti body; MACAM, melanoma-associated cell adhesion molecules; MHC, major histocom- patibility complex; NK, natural killer; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; rIFN, recombinant interferon; rIL, recombinant interleukin; rTNF, recombinant tumor necrosis factor; TIL, tumor-infiltrating lymphocytes; TNF, tumor necrosis factor. association of the clinical course of the disease with CD54 expression in primary melanoma lesions (13-15) and with the levels of soluble CD54 (cCD54) in the serum of patients with malignant melanoma (16, 17) have stimulated interest in the characterization of the expres sion of CAM in melanoma cells and of their modulation by cytokines. Therefore, in the present study, we have investigated the expression of CD54 and CD58 and their counterreceptors CDlla and CD2 on a panel of melanoma cell lines and in a large series of surgically re moved metastatic melanoma lesions. In addition, we have analyzed the role of membrane-bound CD54 and CD58 in the lysis of mela noma cells by allogeneic and autologous cytotoxic cells. Lastly, we have evaluated the role of soluble CD54, purified from the serum of patients with melanoma, in the lysis of melanoma cells by NK cells. MATERIALS AND METHODS Cells. The human melanoma cell lines A375, Colo 38, FO-1, Me Wo and its highly metastatic variant MeM 50-10, SK-MEL-19, SK-MEL-33, SK-MEL- 93, 3S5, and 70-W were grown in RPMI Medium 1640 (Flow Laboratories, Inc., McLean, VA) supplemented with 10% heat-inactivated PCS (Flow) and 2 mm L-glutamine. PMBC were separated from heparinized blood of healthy volunteers by Ficoll-Hypaque (Pharmacia Fine Chemicals AB, Uppsala, Sweden) density gradient centrifugation (400 X g for 30 min) and used either as a source of NK cells or for LAK cell generation. LAK cells were generated by culturing PBMC in the continuous presence of IL-2 (1000 units/ml); half of the medium was changed every 48 h with an equal volume of fresh medium containing IL-2. Melanoma Lesions and Sera. Metastatic melanoma lesions were obtained from 43 patients with no history of chemotherapy or immunotherapy who had been admitted for surgery at the National Cancer Institute of Aviano, Italy. Tissues were processed within 30 min following surgical removal. Each spec imen was divided into three parts under sterile conditions. One third was fixed in Bouin's solution, embedded in paraffin, and processed for routine histopa- thology. One third was snap frozen in liquid nitrogen and stored at -80°C for immunohistochemical analysis. The remaining tissue was used to isolate TIL and melanoma cells as previously described and with minor modifications (18). For the generation of primary cultures of melanoma cells and for TIL isolation, cells (5 X 105/ml) obtained by mechanical mincing and enzymatic digestion of tumor specimens were seeded in T25 tissue culture flasks in RPMI Medium 1640 supplemented with 10% PCS, 10% AB-type human serum, 1% nonessential amino acids (Flow), and 2 min t-glutamine. Following a 24-h incubation at 37°Cin a 5% CO2-humidified atmosphere, culture supernatant was harvested and spun at 400 x g for 10 min. Nonadherent cells were recovered and used as effector cells in the cytotoxicity assay with autologous melanoma cells. Sera were collected from patients with melanoma and frozen at -20°C until use. Monoclonal Antibodies and Conventional Antisera. The anti-CD54 mAbs RR1/1 and CL203.4, the anti-CDlla mAb TS1/22, the anti-CD58 mAb TS2/9, the anti-CD2 mAb TS2/18, and the anti-HLA Class I mAb W6/32 were developed as described elsewhere (19-22). mAbs were purified from ascitic fluid by sequential precipitation with caprylic acid and ammonium sulfate (23). The purity of mAb preparations was monitored by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (24) under reducing and nonreducing con ditions. 3343 on June 26, 2015. © 1993 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from