Association of Increased Levels of Heavy-Chain Ferritin with Increased CD4  CD25  Regulatory T-Cell Levels in Patients with Melanoma 1 Christian P. Gray, Paolo Arosio, and Peter Hersey 2 Deparment of Oncology and Immunology, Newcastle Mater Hospital, Newcastle, New South Wales, 2300 Australia [C. P. G., P. H.], and Materno Infantile Tecnologie Biomediche, Universita ` di Brescia, Brescia, Italy [P. A.] ABSTRACT We have shown previously that melanoma cells in cul- ture release heavy-chain ferritin (H-Ferritin) into superna- tants and that this is responsible for the suppression of responses of peripheral blood lymphocytes stimulated by anti-CD3. These effects were mediated by activation of reg- ulatory T cells to produce interleukin (IL)-10. In the present study, we examined whether a similar relation might exist between levels of H-Ferritin and activation of regulatory T cells in patients with melanoma. Ferritin levels were evalu- ated by ELISA and regulatory T-cell numbers were assessed by three-color flow cytometry to identify CD4  CD25  CD69  T cells. CD69 positive cells were excluded to avoid inclusion of normal activated CD4, CD25 expressing T cells. Measurements of H- and light-chain (L)-Ferritin by ELISA revealed that H- but not L-Ferritin was elevated in the circulation of melanoma patients. In addition, these studies revealed a marked increase in the number of CD4  CD25  CD69  T cells in such patients, compared with age-matched controls. The ratio of H-Ferritin:L-Ferritin correlated with the levels of regulatory T cells consistent with a causal relation between unbound H-Ferritin levels and the activa- tion of regulatory T cells. H-Ferritin or regulatory T cells did not, however, correlate with the stage of the melanoma. These results provide evidence for the importance of H- Ferritin in the induction of regulatory T cells in patients with melanoma and provide additional insight into the sup- pression of immune responses in such patients. INTRODUCTION Ferritin is a major tissue iron-storage protein (1) and, in its native form, has a molecular weight of M r 500,000. It is composed of 24 subunits consisting of acid/H 3 and basic/L chains (2, 3). The 24-subunit polymer may form iso-ferritins, 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 L chains and very few H chains. In contrast, heart, kidney, and placental ferritins are highly acidic because they are com- posed of mostly H chains (4). The ferritin found in cancer cells was found to consist mainly of H chains (5, 6). Ferritin is localized predominantly intracellular; however, small amounts are present in plasma (7), urine (8), cerebrospinal fluid (9), ascitic fluid (10), milk (11), and all body fluids thus far tested (12), which suggests that it is actively secreted by cells. A number of functions have been attributed to the extra- cellular ferritins: regulation of myelopoiesis (13), regulation of lymphocyte migration (14), and as an immunosuppressive agent (15). The immunological and structural properties of extracel- lular ferritin vary in different fluids (12), which probably relates to its origin and function. A large body of evidence indicates that the level of plasma ferritin parallels the concentration of storage iron within the body, regardless of the cell type in which it is stored (16). This relationship of plasma ferritin to body iron stores, however, is altered in inflammatory states and liver disease, conditions in which ferritin is disproportionately ele- vated (17). Elevated levels have also been reported in the plasma of patients with different malignancies; however, the source of the ferritin is unknown (18 –20). We have previously shown that there was marked variability found in the ratios of H-Ferritin: L-Ferritin released from melanoma cultures. It was shown that some melanoma cells released predominantly H-Ferritin, whereas others contained more equal proportions of H- and L-Ferritin (21). In addition, we have previously shown that H-Ferritin from melanoma cells may suppress immune responses (21). This was attributable to changes in antigen presenting cells, which re- sulted in the preferential activation of regulatory T cells that produce interleukin 10 (22). The regulatory T cell implicated in this suppression appeared to be a particular subpopulation of CD4 + T cells, which constitutively express the CD25 compo- nent of the interleukin 2 receptor and the CTLA-4 T cell activation receptor for CD80 (B7.1) and CD86 (B7.2; Refs. Received 10/08/02; revised 1/23/03; accepted 1/23/03. 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 Supported by a grant from Hunter Melanoma Foundation and Cure Cancer Australia Foundation, New South Wales, Australia. 2 To whom requests for reprints should be addressed, at Oncology and Immunology Unit, Room 443, David Maddison Clinical Sciences Build- ing, Corner King and Watt Streets, Newcastle, NSW 2300 Australia. Phone: 61-2-49236828; Fax: 61-2-49236184; E-mail: Peter.Hersey@ newcastle.edu.au. 3 The abbreviations used are: H, heavy (chain); L, light (chain); H- Ferritin, heavy-chain ferritin; L-Ferritin, light-chain ferritin; MAb, monoclonal antibody; AJCC, American Joint Commission of Cancer; PBL, peripheral blood lymphocytes. 2551 Vol. 9, 2551–2559, July 2003 Clinical Cancer Research Cancer Research. on November 24, 2021. © 2003 American Association for clincancerres.aacrjournals.org Downloaded from