Differential Expression of Cell Adhesion Molecules CD54/Cdlla and CD58/CD2 by Human Melanoma Cells and Functional Role in Their Interaction with Cytotoxic Cells1

[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. GÃŒ,A. C.], Surgical Oncology I [G. B.J, and Medical Oncology 1 Â¡A.Ga.Â¡,C. R. O., Aviano, Italy 33081, and Department of Microbiology and Immunology. New York Medical College, Valhalla, New York 10595 [S. F.J

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

DTAF F(ab')2 fragments of goat anti-mouse IgG antibodies, F(ab')2 frag ment-specific antibodies, and ChromePure mouse immunoglobulin were pur IBB2ue'Ã±M8> chased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). â€¢¿1;.â€¢8 , l .tâ€¢f888889â€¢ Reagents for APAAP were purchased from Dakopatts (Glosturp, Denmark). Human Recombinant Cytokines and Reagents. rIFN-a and ill-V-v were obtained from Hoffmann LaRoche, Inc. (Nutley. NJ). rIL-la was purchased from Genzyme Biochemicals, Ltd. (Maidstone, England). rIL-2 and rTNF-a were obtained from Cetus Corporation (Emeryville, CA). Human hemoglobin was purchased from Sigma Chemical Co. (St. Louis, MO). Serological Assays. IIP was performed as previously described (25). Sam ples were analyzed for cell surface fluorescence utilizing a FACScan flow cytometer (Becton Dickinson Immunocytometry System, Mountain View, CA) equipped with a Model 9I35C Hewlett-Packard computer (Hewlett-Packard Q- 8 8 8 Co., Palo Alto. CA). Fluorescence was collected by using a four-decade log arithmic amplifier. Viable cells (1 X IO4, volume gated) were collected in a list 1BBB3)'MU~ mode fashion for data analysis. The latter was performed with Consort C32 90 software (Becton-Dickinson). Results are expressed as the percent of positive 0' cells and mean values of fluorescence intensity on a logarithmic scale. A IBBu â€¢¿* sample was classified as positive when more than 5% of the cells were stained with relevant mAb. "88838er*â€¢¿â€¢¿â€¢¿ uÂ§ Â»Bâ€¢¿Â«. Immunohistochemical staining of cryostat tissue sections by the APAAP method was performed as previously described (26). IBMÂ£o The double determinant immunoassay to measure cCD54 was performed utilizing an ELISA kit from Bender MedSystems (Wien, Austria) according to 8 8 8s.s the manufacturer's instructions. Cytotoxicity Assays. All the assays were performed in triplicate in round- au. i1I bottomed 96-well microtiter plates (Costar, Cambridge, MA) as previously described (27). After labeling with 5lCr, target cells (1 x IO4) were incubated fofoâ€” Kl OÂ» for 4 h at 37Â°Cin a total volume of 200 /il of RPMI Medium 1640 containing 1s 1_l1 1 â€”¿1LUâ€”¿i s 0 LUIII LU 10% FCS, mAb (10 /xg/ml), and different numbers of effector cells to obtain 3i 100:1 effectortarget ratios in the NK- and 20:1 ratios in the LAK- and TIL- itt i i in mediated cytotoxicity assay. Plates were then centrifuged for 5 min at 30 x g S 1u 3u. 11Å“ Z Zâ€¢ SuÂ» Â«o^ ^M Mio r> and supernatant (100 (xl) was harvested from each well and counted in a Fig. 1. Expression of CAM on human melanoma cell lines. Melanoma cells (1 X IO5) gamma counter. The percentage of cytotoxicity was calculated utilizing the were Â«suspended in PBS-bovine serum albumin-0.01% sodium azide and sequentially formula: incubated with anti-CD54 mAb CL203.4 (â€¢),anti-CDlla mAb TS1/22 (O), anti-CD58 mAb TS2/9 (â€¢).and anti-CD2 mAb TS2/18 (D) and with DTAF-conjugated F(ab')2 MCr experimental release â€”¿spontaneousrelease fragments of goat anti-mouse IgG xenoantibodies. Then cells (1 X IO4, volume gated) maximum release â€”¿spontaneousrelease X 100 were analyzed by flow cytometry. Staining of melanoma cells with anti-CD54 mAb RR1/1 was similar to that obtained with anti-CD54 mAb CL203.4 (data not shown). The per centage of melanoma cells stained by isotype-matched mouse immunoglobulin and the Spontaneous release represents the radioactivity released by 1 X IO4 target mean values of fluorescence intensity were lower than 5% and 10, respectively, on all cell cells labeled with "Cr during a 4-h incubation; maximum release is the lines tested. radioactivity released by 1 X IO4 target cells lysed with 1% Nonidet-40 (Sigma). Only experiments with a spontaneous release lower than 15% were were expressed by all the melanoma cell lines, and the expression of used for data analysis. both molecules was heterogeneous in terms of percentage of stained Purification of cCD54. cCD54 was purified from the serum of clinical Stage IV (distant mÃ©tastases)melanoma patients as described elsewhere (28). cells and/or intensity of cell staining (Fig. 1). More than 90% of cells Briefly, 4 ml of patient's serum diluted 1:10 with PBS were passed through an from the melanoma cell lines A375, Colo 38, FO-1, SK-MEL-33, immunoaffmity column with anti-CD54 mAb CL203.4 coupled to cyanogen SK-MEL-93, and 70-W were stained by the anti-CD54 mAb CL203.4 bromide-activated Sepharose 4B (Pharmacia) to a final concentration of 5 and the anti-CD58 mAb TS2/9. The latter mAb stained more than 90% mg/ml of packed resin. All chromatography steps were at 4Â°C.The column was of cells from the melanoma cell lines MeWo and 3S5, while the washed, and bound CD54 was eluted with 50 HIMtriethanolamine-HCl:0.15 M former mAb stained about 50% of cells. The anti-CD54 mAb CL203.4 NaCl (pH 9.0) and neutralized immediately. The amount of cCD54 recovered stained 97% of cells from the melanoma cell line SK-MEL-19, but did was determined by the double determinant immunoassay described above. not stain melanoma cell MeM 50-10. The anti-CD58 mAb TS2/9 Statistical Analysis. Data were analyzed by the Student paired t test uti stained 14% of cells from the melanoma cell line SK-MEL-19 and lizing the StatWorks statistical package from Cricket Software, Inc. (Philadel 33% of cells from the melanoma cell line MeM 50-10. The mean phia. PA). Differences with P < 0.05 were considered statistically significant. fluorescence intensity values, which are an indirect expression of antigen density, were more heterogeneous for CD54 than for CD58 as RESULTS they ranged from 8 to 257 for the former antigen and from 9 to 92 for Expression of CAM on Melanoma Cell Lines. IIP staining with the latter one. the anti-CD54 mAb CL203.4, with the anti-CD58 mAb TS2/9, with No relationship was found between the percentage of cells stained the anti-CDlla mAb TS1/22, and with the anti-CD2 mAb TS2/18 for CD54 or CD58 and the mean fluorescence intensity values. Fur showed a differential expression of CAM on the melanoma cell lines thermore, no relationship was found between the expression of CD54 A375, Colo 38, FO-1, MeWo and its highly metastatic variant MeM and that of CD58 in terms of the percentage of stained cells and mean 50-10, SK-MEL-19, SK-MEL-33, SK-MEL-93, 3S5, and 70-W (Fig. fluorescence intensity. 1). CDlla and CD2 were not detected on the melanoma cell lines Expression of CAM in Metastatic Melanoma Lesions. Immu investigated. The percentage of stained cells and the mean fluores nohistochemical staining with anti-CD54 mAb CL203.4, with anti- cence intensity values obtained with mAb TS1/22 and mAb TS2/18 CD58 mAb TS2/9, with anti-CDl la mAb TS1/22, and with anti-CD2 were lower than 5 and 10%, respectively (Fig. 1). CD54 and/or CD58 mAb TS2/18 of 46 metastatic melanoma lesions surgically removed 3344

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1993 American Association for Cancer Research. CELL ADHESION MOLECULES IN MELANOMA-HOST INTERACTION from 43 patients showed a differential distribution of the four CAM leee (Table 1). Neither GDI la nor CD2 was detected in the melanoma lesions tested. In contrast, CD54 and CD58 were detected in 42 of the 46 tumor specimens tested (Table 1). CD54 showed a higher degree of CD54 intra- and intertumor heterogeneity than did CD58. The percentage of tea CD54-positive melanoma cells was lower than 25% in four lesions, = = =4 ranged between 2 and 50% in five and between 51 and 75% in nine, and was higher than 76% in the remaining 24 lesions. In contrast, the percentage of CD58-positive melanoma cells ranged between 51 and cosa ia 75% in two lesions and was higher than 76% in the remaining 40 12 24 48 72 lesions. Moreover, the expression of CD54 for percentage of mela noma cells stained and/or intensity of staining differed in autologous Time (hours) melanoma lesions removed from distinct anatomical sites from Pa Fig. 2. Time-dependent modulation by cytokines of CD54 and CD58 expressed by cultured Colo 38 melanoma cells. Colo 38 melanoma cells were incubated with 500 tients Mel 8, Mel 11, and Mel 31, while the expression of CD58 units/ml of IFN-a (â€¢).IFN--y(G). TNF-a ('.â€¢),theircombination (â€¢).IL-la (A), and 1L-2 displayed no detectable difference (Table 1). No relationship was (*) at 37Â°Cfor up to 72 h. Control cultures (Al were incubated under the same experi mental conditions, but without cytokines. At the indicated times cells were harvested, found between the percentage of melanoma cells expressing CD54 washed twice with Hanks' balanced salt solution, and frozen in RPMI Medium 1640 containing 40% PCS and 20% dimethyl sulfoxide. Then cells were defrosted, washed twice with Hanks' balanced salt solution, resuspended in PBS-bovine serum albumin- Table 1 Immunohistochemical analysis of CAM expression in metastalic 0.01% sodium azide and sequentially incubated with either anti-CD54 mAb CL203.4 melanoma lesions (dashed lines) or anti-CD58 mAb TS2/9 (solid lines) and with DTAF-conjugated F(ab'): Cryostat sections of surgically removed metastatic melanoma lesions were stained with fragments of goat anti-mouse IgG xenoantibodies. Then cells (1 X IO4 volume gated) anti-CD54 mAb CL203.4, anti-CDlla mAb TSI/22, anti-CD58 mAb TS2/9. and anti- were analyzed by flow cytometry. The mean values of fluorescence intensity obtained by CD2 mAb TS2/18 by APAAP. staining melanoma cells with isotype-matched mouse immunoglobulin were lower than 10 and did not change with cell culture. of PatientMel CD2Skinmetastasis Pigment CD54 CD Ila CD58 1Mel +-H-W*Lymph -" and CD58 and their level of expression; the percentage of melanoma 2 node + +-H- cells stained by anti-CD54 mAb CL203.4 and by anti-CD58 mAb Lymph node -/+ - Mel 3 TS2/9 was high in a number of metastatic melanoma lesions with a Mel4Mel Lvmph node -/+ ++++w 5Mel Skin++++Lymph -+++ weak intensity of staining (Table 1). Furthermore, the expression of 6 node - ++ - CAM by melanoma cells did not correlate with the anatomical site of Mel 7 Lymph node + +++w NTf Mel8aMel Skin+wLymph +++-+++ mÃ©tastasesand the level of pigmentation of melanoma cells. 8b node + Modulation of CAM Expression on Melanoma Cell Lines by Mel9Mel Skin++Lymph -/+ -tt+ Cytokines. To investigate the modulation and/or the induction of 10 node - +++ MelIlaMel Skin-Lymph - - -+++ CAM on melanoma cells by cytokines, the melanoma cell lines A375, lib node - +++ - FO-1, MeM 50-10, Me Wo. SK-MEL-19, and 3S5 were incubated with Mel12Mel SkinSkin -++W IFN-7 and TNF-a for 24 h, and the melanoma cell line Colo 38 was 13Mel ++-M-Skin--M-+Lymph 14Mel -/+ ++W++++++ incubated with IFN-a, IFN-y and/or TNF-a, IL-la, and IL-2 for up to 15 node -/+ +++ ++w 72 h. At the end of the incubation, cells were stained with anti-CD54 Mel 16 Lymph node ++++ ++++ mAb CL203.4, with anti-CD58 mAb TS2/9, with anti-CDlla mAb Mel 17 Lymph node - ++++ ++++ - Mel 18 Lymph node ++++ ++++ TSI/22, and with anti-CD2 mAb TS2/18 and analyzed by flow cy Mel 19 Lymph node - ++++ ++++ tometry. Neither CDlla nor CD2 expression was induced on mela Mel20Mel Muscle-Lymph - + noma cells (data not shown). IFN--y and TNF-a, both individually and 21 node + +++ NT Mel22Mel Skin++++WMucosa + to a greater extent in combination, enhanced the expression of CD54, -Skin + ++++ - ++++W 23Mel but did not affect that of CD58. Representative results obtained with 24Mel NTLymph -/+ ++++ ++++W 25 node -/+ ++++w ++++ Colo 38 melanoma cells are shown in Fig. 2. The increase of CD54 Mel 26 Lymph node + +-M-+W expression induced by the combination of the two cytokines was Mel27Mel Skinâ€”¿Skin + ++++ â€”¿ ++++ 28Mel ++++Lymph - +++ higher than that induced by the individual ones. The upregulation of 29Mel node - MM - ++++ - CD54 expression reached its maximum between 12 h and 24 h of 30 Lymph node - ++ - ++++ - incubation of melanoma cells with the cytokines and decreased there Mel 3laMel ++++GutSkin ++ after. IFN-a, IL-la, and IL-2 modulated neither CD54 nor CD58 31bMel â€”¿Lymph â€”¿/+ ++++ â€”¿ ++++ 32 node - ++++ ++++ expression by Colo 38 melanoma cells. However, both IFN-a and Mel 33 Lymph node + ++++ - ++++ - IL-lo induced a time-dependent upregulation of the expression of Mel 34 Lymph node -/+ +++ - ++++ - Mel35Mel Skin++++Lymph ++++W HLA Class I antigens by Colo 38 cells (data not shown). 36 node - ++++ ++++W Role of CD54 and CD58 in NK and LAK Cell-mediated Lysis of Mel37Mel ++++SkinSkin ++++ Melanoma Cells. FO-1, SK-MEL-19, and SK-MEL-33 melanoma 38Mel ++++Lymph - 39Mel node - ++++ MM cells were susceptible to NK and LAK cell-mediated lysis. The mean 40Mel MMLymphSkin -/+ + values of specific 5lCr release induced by NK cells from three dif node -n-++w M MW 41 ferent donors were 37 Â±15, 14 Â±2, and 29 Â±2% for FO-1, SK- Mel 42 Lymph node - MM ++++W Mel 43Site Lymph node - ++++ + ++++ - MEL-19, and SK-MEL-33 melanoma cells, respectively. The mean " Results were classified as - when no melanin was present in malignant cells and as values of specific 31Cr release induced by LAK cells from three +/- or + when less than or more than 50% of malignant cells contained melanin, different donors were 86 Â±2, 54 Â±7, and 76 Â±6% for FO-1, respectively. * Results were classified as - when less than 5% of malignant cells were stained: as SK-MEL-19, and SK-MEL-33 melanoma cells, respectively. + when 6 to 25% of malignant cells were stained; as ++ when 26 to 50% of malignant cells The anti-CD54 mAb CL203.4 had no detectable effect on the lysis were stained; as +++ when 51 to 75% of malignant cells were stained; and as ++++ when of the melanoma cell lines FO-1, SK-MEL-19, and SK-MEL-33 by 76 to 100% of malignant cells were stained. W, weak intensity of staining. '' NT, not tested. NK and LAK cells from three donors (Fig. 3). In contrast, the anti- 3345

Fig. 3. Inhibitory effect of mAb to CD54 and/or mRb FB-1 SK-MEL-I9 SK-MEl-33 CD58 on NK cell- and LAK cell-mediated cyto- Ml/I CL2I3.4TS2/9 toxicity of cultured melanoma cells. MCr-labeled melanoma cells (I X IO4) were incubated in 96- well round-bottomed plates with NK (top) cells (I X IO6) or LAK (bottom) cells (2 X IO5) in the presence of anti-CD54 mAbs RR1/1 and CL203.4, anti-CD58 mAb TS2/9. or the combination of anti- CD54 mAb RR1/I and anti-CD58 mAb TS2/9 (fi nal concentration, 10 ^tg/ml). Control cultures were incubated under the same experimental conditions but without mAb. At the end of a 4-h incubation at 37Â°C,plates were spun for 5 min at 30 X g; the LRK supernatant ( 100 ^il) was harvested from each well, and released radioactivity was counted in a gamma counter. Columns, mean percentage of specific MCr release inhibition as compared to that of control cultures obtained in three experiments; bars. SD. *, P < 0.05 versus mAb RR1/1 Cr release inhibition (%)

CD54 mAb RR1/1 and the anti-CD58 mAb TS2/9 inhibited the lysis Rl/l CL2H.4 TS2/9 of the three melanoma cell lines by NK cells to an almost similar extent. Furthermore, the extent of inhibition of NK cell-mediated lysis of melanoma cells FO-1 and SK-MEL-19 induced by the combination of the two mAbs was significantly (P < 0.05) higher than that induced by the anti-CD54 mAb RR1/1. The inhibitory effect of the two mAbs on both NK and LAK cell-mediated lysis varied among the three melanoma cell lines. The inhibition of NK cell-mediated lysis with the anti-CD54 mAb RR1/1 and the anti-CD58 mAb TS2/9 ranged be tween a minimum of 15 and 14% with the cell line SK-MEL-19 and a maximum of 52 and 57% with the cell line SK-MEL-33. LAK cell-mediated lysis of melanoma cells was inhibited by the anti-CD54 mAb RR1/1; lysis of SK-MEL-19 cells was inhibited to a greater Cr release inhibition (%) extent than that of FO-1 and SK-MEL-33 cells. The anti-CD58 mAb TS2/9 inhibited LAK cell-mediated lysis of melanoma cells to a lower Fig. 5. Inhibitory effect of mAb to CD54 and/or CD58 on TIL-mediated cytotoxicity of autologous melanoma cells. "Cr-labeled melanoma cells (1 X IO4) were incubated in extent than the anti-CD54 mAb RR1/1, and the combination of the 96-well round-bottomed plates with TIL (2 X IO5) in the presence of anti-CD54 mAbs two mAbs had an effect similar to that of the anti-CD54 mAb RR1/1 RR1/1 and CL203.4, anti-CD58 mAb TS2/9, or the combination of anti-CD54 mAb used alone. The inhibition of LAK cell-mediated lysis by mAb RR1/1 RR1/1 and anti-CD58 mAb TS2/9 (final concentration. 10 fig/ml). Control cultures were incubated under the same experimental conditions, but without mAb. At the end of a 4-h and TS2/9 ranged between a minimum of 14 and 2% with the cell line incubation at 37Â°C,plates were spun at 30 X g; the supernatant (100 (Â¿1)washarvested FO-1 and a maximum of 46 and 19% with the cell lines SK-MEL-19 from each well, and released radioactivity was counted in a gamma counter. Data are expressed as the percentage of specific "Cr release inhibition as compared to that of and SK-MEL-33 (Fig. 3). control cultures. Modulation of NK Cell-mediated Lysis of Melanoma Cells by Soluble CD54. To corroborate the role of CD54 in the lysis of mel anoma cells by NK cells, the effect of cCD54 in the lysis of FO-1 tal conditions did not affect the lysis of melanoma cells by NK cells melanoma cells by NK cells from three donors was investigated. (data not shown). cCD54 inhibited the lysis of FO-1 melanoma cells in a dose-dependent Role of CD54 and CD58 in TIL-mediated Lysis of Autologous fashion (Fig. 4). Human hemoglobin used under the same experimen- Melanoma Cells. Melanoma cells Mel PA, Mel NN, Mel BM, and Mel SM were susceptible to autologous TIL-mediated lysis; the values of specific 51Cr release were 33, 41, 13, and 13%, respectively. The anti-CD54 mAb CL203.4 had no detectable effect on the lysis of autologous melanoma cells by TIL (Fig. 5). In contrast, the anti- CD54 mAb RR1/1 and the anti-CD58 mAb TS2/9 inhibited the lysis of melanoma cells by autologous TIL; the extent of the inhibition was

C 38 higher with the former than with the latter mAb. The inhibition of TIL-mediated lysis induced by the combination of the two mAbs was â€¢¿ M m similar to that induced by the anti-CD54 mAb RR1/1 used alone (Fig. 5). The inhibitory effect of mAb RR1/1 and TS2/9 ranged from a Â£ 18 minimum of 51 and 20%, respectively, with Mel BM melanoma cells to a maximum of 92 and 85%, respectively, with Mel PA and Mel SM 3.12 2s.ee 12.SB 6.25 melanoma cells (Fig. 5). (ng/ml) Fig. 4. Dose-dependent inhibition by cCD54 of NK cell-mediated lysis of FO-1 mel anoma cells. 51Cr-labeled melanoma cells (1 X IO6) were incubated in 96-well round- DISCUSSION bottomed plates with NK cells ( 1 X 10~6) in the continuous presence of scalar doses of cCD54. Control cultures were incubated under the same experimental conditions, but The present study has shown a differential distribution of CAM on without soluble CD54. At the end of a 4-h incubation at 37Â°C.plates were spun at 30 X melanoma cell lines and in metastatic melanoma lesions. CDlla and g; the supernatant ( 100 Â¿il)was harvested from each well, and released radioactivity was counted in a gamma counter. Columns, mean percentage of specific 5lCr-release inhibition CD2 were not detected on melanoma cells, while CD54 and/or CD58 as compared to that of control cultures; bars, SD. were detected on all the cell lines and melanoma lesions tested. 3346

However, CD54 and CD58 display distinct characteristics in their inhibited the lysis of M26 melanoma cells by LAK cells (34). These expression by melanoma cells: CD54 is more heterogeneous than observations, although preliminary, suggest that soluble CD54 can be CD58 in its distribution within and among metastatic melanoma le considered a molecule that melanoma cells use in vivo to escape sions than CD58. Furthermore, CD54 is susceptible to modulation by immune cell surveillance by cytotoxic cells. cytokines, while CD58 is not. Lastly, CD54 is shed by melanoma The data reported in this paper demonstrate that CD54 and CD58 cells, and the shedding is enhanced by cytokines (29), while CD58 may play a critical role in melanoma-host interaction(s), although was not detected in the spent medium of cultured melanoma cells.4 other MACAM may be relevant in this phenomenon. However, the The distributions of the CAM tested on malignant cells of the mel- shedding of MACAM in a functional form should be taken into anocytic lineage imply that interactions of melanoma cells with im appropriate consideration for the evaluation of their biological signif mune cells are mediated by CD Ila and CD2 expressed on the latter icance and for their potential role in influencing the clinical course of cells and by CD54 and CD58 expressed on the former cells. In disease in melanoma patients. addition, the expression of CD54 and/or CD58 on melanoma cells is of potential relevance in vivo, since effector cells may use either one of the two molecules for their contact with neoplastic cells. ACKNOWLEDGMENTS The functional involvement of CD54 and CD58 in the lysis of The authors thank Dr. T. A. Springer for the gift of mAb RR1/1 and melanoma cells by cytotoxic cells is demonstrated by the ability of the acknowledge the excellent technical and secretarial assistance of Francesca anti-CD54 mAb RR1/1 and of the anti-CD58 mAb TS2/9 to inhibit the Colizzi and Paola Cattaneo. lysis of melanoma cells by allogeneic NK and LAK cells and by autologous TIL. The combination of the two mAbs had a synergistic REFERENCES effect on NK cell lysis but not on LAK cell and TIL lysis of melanoma Albelda. S. M., and Buck. C. A. Integrins and other cell adhesion molecules. FASEB cells, suggesting that the two pathways might be differentially in J.. 4: 2868-2880. 1990. volved in the lysis of melanoma cells by different effector cells. Springer, T. A. Adhesion receptors of the immune system. Nature (Lond.). 346: Furthermore, it is noteworthy that the anti-CD54 mAb RR1/1, the 425-134, 1990. Shaw. S., Ginther Luce, G. E., QuiÃ±ones. R., Gress, R. E., Springer. T. 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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1993 American Association for Cancer Research. Differential Expression of Cell Adhesion Molecules CD54/CD11a and CD58/CD2 by Human Melanoma Cells and Functional Role in Their Interaction with Cytotoxic Cells

Maresa Altomonte, Annunziata Gloghini, Giulio Bertola, et al.

Cancer Res 1993;53:3343-3348.

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