Concurrent Induction of Antitumor Immunity and Autoimmune Thyroiditis in CD4 + CD25 + Regulatory T Cell–Depleted Mice Wei-Zen Wei, 1 Jennifer B. Jacob, 1,2 John F. Zielinski, 1 Jeffrey C. Flynn, 2 K. David Shim, 1 Ghazwan Alsharabi, 3 Alvaro A. Giraldo, 3 and Yi-chi M. Kong 2 1 Karmanos Cancer Institute and 2 Department of Immunology and Microbiology, School of Medicine, Wayne State University, and 3 Division of Immunopathology, St. John Hospital and Medical Center, Detroit, Michigan Abstract When CD4 + CD25 + regulatory T cells are depleted or inacti- vated for the purpose of enhancing antitumor immunity, the risk of autoimmune disease may be significantly elevated because these regulatory T cells control both antitumor immunity and autoimmunity. To evaluate the relative benefit and risk of modulating CD4 + CD25 + regulatory T cells, we established a new test system to measure simultaneously the immune reactivity to a tumor-associated antigen, neu, and an unrelated self-antigen, thyroglobulin. BALB/c mice were inoculated with TUBO cells expressing an activated rat neu and treated with anti-CD25 monoclonal antibody to deplete CD25 + cells. The tumors grew, then regressed, and neu-specific antibodies and IFN-;–secreting T cells were induced. The same mice were also exposed to mouse thyroglobulin by chronic i.v. injections. These mice produced thyroglobulin- specific antibody and IFN-;–secreting T cells with inflamma- tory infiltration in the thyroids of some mice. The immune responses to neu or thyroglobulin were greater in mice undergoing TUBO tumor rejection and thyroglobulin injection than in those experiencing either alone. To the best of our knowledge, this is the first experimental system to assess the concurrent induction and possible synergy of immune reactivity to defined tumor and self-antigens following reduction of regulatory T cells. These results illustrate the importance of monitoring immune reactivity to self-antigens during cancer immunotherapy that involves immunomodu- lating agents, and the pressing need for novel strategies to induce antitumor immunity while minimizing autoimmunity. (Cancer Res 2005; 65(18): 8471-8) Introduction CD4 + CD25 + regulatory T (Treg)–like cells have been described in patients with different types of cancers (1–3). We and others have shown that removal of CD4 + CD25 + cells from tumor-bearing mice resulted in the regression of certain mouse tumors (4, 5), suggesting that Treg may negatively regulate antitumor immunity and depletion of Treg may be a powerful way to control tumor growth. In addition to CD4 and CD25, Treg express CTLA-4 (6), a glucocorticoid-induced tumor necrosis factor receptor family member (TNFRSF18; ref. 7), CD80 (8), CD62L, membrane-bound transforming growth factor h (9), as well as the transcription factor scurfin, encoded by foxp3 (10). They do not proliferate when stimulated in vitro via CD3. Treg suppressive activity is triggered through the T-cell receptor by specific antigen and can inhibit T-cell activation in an antigen-specific (11) or nonspecific (12, 13) manner through a contact-dependent mechanism. In this study, rat neu is used as the model tumor-associated antigen. Overexpression of erbB-2 or Her-2/neu in a number of common cancers, such as breast, ovarian, colorectal, prostate, and pancreatic adenocarcinoma (14–17), is correlated with a more aggressive course of disease (18, 19), rendering Her-2 an important target of cancer therapy. The therapeutic effect of anti–Her-2 monoclonal antibody (mAb), Herceptin, in stage IV breast cancer patients further distinguishes this molecule as an exceptional target of immunotherapy and vaccination. Because of self-tolerance, it is difficult to elicit strong immune responses to Her-2, as we showed in Her-2 transgenic mice (20), and Treg depletion may be a plausible strategy to amplify anti–Her-2/neu immunity. Depletion of CD4 + CD25 + cells combined with CTLA-4 blockade has been shown to enhance the efficacy of B16 melanoma cell vaccine with an increase in autoimmune skin depigmentation, demonstrating the concurrent induction of antitumor immunity and autoimmunity directed at common antigens (21). Autoimmu- nity induced through modulation of regulatory T cells is, however, not restricted to such common antigens. Autoimmune thyroiditis and a spectrum of other autoimmune diseases have been observed in cancer patients receiving melanoma gp100 or Her-2 peptide vaccines with immunomodulating agents (22, 23). In this study, we examined the induction of autoimmunity in the thyroid which does not share common antigens with Her-2. We have shown that depletion of Treg in CBA/J mice increased their susceptibility to experimental autoimmune thyroiditis (24), the murine model of Hashimoto’s thyroiditis. Hashimoto’s thyroid- itis, the leading cause of hypothyroidism, is characterized by mononuclear cell infiltration and destruction of the thyroid, elevation of thyroid-stimulating hormone, and decrease of thyroid hormones (T3 and T4). The production of autoantibodies (25) and T-cell proliferation to thyroid antigens (26) are indicators of autoreactivity. Susceptibility to thyroiditis is strongly influenced by the haplotype of class II MHC. For example, human HLA- DRB1*0301 (DR3) transgene (27) and murine H2 k (CBA/J) confer susceptibility to autoimmune thyroiditis, whereas murine H2 d (BALB/c) is associated with resistance (28). In genetically susceptible mice, experimental autoimmune thyroiditis is induced by injection of mouse thyroglobulin (mTg), usually in the presence of a strong adjuvant (e.g., complete Freund’s Adjuvant or lipopolysaccharide; ref. 29), or by repeated injections of mTg for 4 weeks (30). Like Hashimoto’s thyroiditis, experimental Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Wei-Zen Wei, Karmanos Cancer Institute, Wayne State University, 110 East Warren Avenue, Detroit, MI 48201. Phone: 313-833-0715 Ext 2360; Fax: 313-831-7518; E-mail: weiw@karmanos.org. I2005 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-0934 www.aacrjournals.org 8471 Cancer Res 2005; 65: (18). 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