IMMUNOSUPPRESSIVE EFFECTS OF MELANOMA-DERIVED HEAVY-CHAIN FERRITIN ARE DEPENDENT ON STIMULATION OF IL-10 PRODUCTION Christian P. GRAY 1 , Agustin V. FRANCO 1 , P. AROSIO 2 and Peter HERSEY 1 * 1 Department of Oncology and Immunology, Newcastle Mater Hospital, Newcastle, New South Wales, Australia 2 Protein Engineering Unit, Department of Biological and Technological Research, San Raffaele, Milan, Italy Cultured melanoma cells release soluble factors that influ- ence immune responses. Screening of a cDNA library with anti-sera from a melanoma patient identified an immunore- active plaque, which encoded heavy-chain ferritin (H-fer- ritin). Previous studies have drawn attention to the immu- nosuppressive effects of this molecule and prompted further studies on its biochemical and functional properties in human melanoma. These studies demonstrated, firstly, that H-fer- ritin appeared to be secreted by melanoma cells, as shown by immunoprecipitation of a 21.5 kDa band from supernatants. It was also detected in extracts of melanoma cells by West- ern blotting as 43 and 64 kDa dimers and trimers of the 21.5 kDa fraction. Secondly, flow-cytometric analysis of H- and light-chain ferritin (L-ferritin) expression on melanoma showed a wide variation in L-ferritin expression and conse- quently of the ratio of H- to L-ferritin expression. Suppres- sion of mitogenic responses of lymphocytes to anti-CD3 showed a correlation with the ratio of H- to L-ferritin in the supernatants and was specific for H-ferritin, as shown by inhibition studies with a monoclonal antibody (MAb) against H-ferritin. Similar results were obtained with H- and L-fer- ritin from other sources. Suppression of mitogenic responses of lymphocytes to anti-CD3 by H-ferritin was inhibited using a MAb against IL-10, which suggested that the immunosup- pressive effect of H-ferritin was mediated by IL-10. Assays of cytokine production from anti-CD3–stimulated lymphocytes showed that H-ferritin markedly increased production of IL-10 and IFN- and had only slight effects on IL-2 and IL-4 production. Our results suggest that melanoma cells may be a major source of H-ferritin and that production of the latter may account for some of the immunosuppressive effects of melanoma. © 2001 Wiley-Liss, Inc. Key words: heavy-chain ferritin; human melanoma; immunosuppres- sion; IL-10; light-chain ferritin; SEREX The mechanisms by which melanoma avoids destruction by the immune system are of much interest. One possibility is that mel- anoma cells release soluble factors, which inhibit immune re- sponses in the local tumour environment. Such factors have been found in the supernatants from sarcoma, lung carcinoma, mela- noma, head-and-neck carcinoma and colon cancer; they inhibit mitogen and other strong lymphocyte responses. 1–3 The nature of the factors responsible for the immunosuppressive effects in many instances are unknown, but in others, a wide variety of factors have been implicated, such as PGE 2 , 4 TGF-2, 5,6 IL-10, 7,8 -MSH, 7 IL-6, 9 hydrogen peroxide 10 and Fas ligand. 11 Immunoscreening of a cDNA library from the MM200 mela- noma line with sera from a melanoma patient identified several plaques that were shown by cDNA sequence analysis to be heavy- chain ferritin (H-ferritin). Previous studies have shown that H- ferritin may suppress proliferation of T cells, 12–14 E-rosette forma- tion by T cells 15 and colony formation by normal human macrophages. 16 Ferritin is a major tissue iron-binding protein 17 and, in its native form, is approximately 500 kDa. It is composed of 24 subunits consisting of acid/heavy (H) and basic/light (L) chains. 18,19 The genes encoding H- and L-ferritin are found in different chromo- somes and are transcriptionally independent. 20 The 24-subunit polymer may form isoferritins, which are either more acidic (H- rich) or more basic (L-rich), depending on the relative proportions of H and L chains. Liver and spleen ferritins are basic because they are made up mainly of light chains, with very few heavy chains. In contrast, heart, kidney and placental ferritins are highly acidic because they are composed of mostly heavy chains. 21 Interest- ingly, ferritin in cancer cells consists mainly of heavy chains. 22,23 Others have suggested that there is another species of ferritin in cancer cells called super-heavy chain or P43. 24 In the present studies, we sought to characterize the nature of ferritin in mela- noma cells and to investigate its possible effects on immune responses. MATERIAL AND METHODS Cell lines Mel-JG was isolated from a fresh surgical biopsy of s.c. metas- tasis from a patient attending the Sydney Melanoma Unit (Sydney, Australia) and established in the laboratory. The derivation of MM200, Me1007, Me4405 and Me10538 is described else- where. 25 All melanoma cell lines were positive for tyrosinase and MART-1 mRNA by RT-PCR, described elsewhere. 26 MRC-5 lung fibroblasts were obtained from BioWhittaker (Walkersville, MD). All melanoma cell lines and MRC-5 were cultured in DMEM containing 5% FCS (Commonwealth Serum Laboratories, Mel- bourne, Australia). Human umbilical vein endothelial cells (HUVECs) were kindly supplied by Dr. A.D. Hibberd and Mr. D. Clark (Transplantation Unit, John Hunter Hospital, Newcastle, Australia); they were isolated from umbilical veins of placenta by digestion in collagenase, as described elsewhere, 27 and grown in M199 medium (Life Technologies, Gaithersburg, MD) supple- mented with 100 mg/l L-glutamine (Life Technologies), 20% FCS, 135 mg/l heparin (Sigma, St. Louis, MO) and 16.7 g/l endothelial cell growth supplement (Sigma). Over 95% of the cells expressed CD31 and von Willebrand factor using flow cytometry. Cell lines were tested routinely for Mycoplasma by the Mycoplasma Detec- tion Set PCR method (Takara Shuzo, Tokyo, Japan). Antibodies and recombinant proteins The monoclonal antibody (MAb) rH02, specific for human H-ferritin, was kindly provided by Dr P. Arosio and is described elsewhere. 28 Dr T.L. Nagabhushan (Schering-Plough, Bloomfield, NJ) kindly provided the MAb JES3-9D7, specific for human IL-10 and described elsewhere. 29 The 10-F10 MAb against H-ferritin was purchased from Fitzgerald (Concord, MA). A MAb against L-ferritin, 1A-4E2, was purchased from Bioclone Sydney, Austra- lia). The IgG1 isotype MAb against TNP was purchased from Pharmingen, (San Diego, CA). The rat IgG isotype control was Grant sponsor: Hunter Melanoma Foundation and Melanoma and Skin Cancer Institute, New South Wales, Australia. *Correspondence to: Oncology and Immunology Unit, Room 443, David Maddison Clinical Sciences Building, Cnr. King and Watt Streets, Newcastle, NSW 2300, Australia. Fax: +61-2-492-36184. E-mail: Peter.Hersey@newcastle.edu.au Received 25 August 2000; Revised 13 November 2000; Accepted 19 January 2001 Published online 2 April 2001 Int. J. Cancer: 92, 843– 850 (2001) © 2001 Wiley-Liss, Inc. Publication of the International Union Against Cancer