Unmasking immunosurveillance against a syngeneic colon cancer by elimination of CD4  NKT regulatory cells and IL-13 Jong Myun Park 1 , Masaki Terabe 1 , Leon T. van den Broeke 1 , Debra D. Donaldson 2 and Jay A. Berzofsky 1 * 1 Vaccine Branch, Center for Cancer Research, National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, MD, USA 2 Wyeth Research, Cambridge, MA, USA We have previously observed a novel role of natural killer T (NKT) cells in negative regulation of antitumor immune responses against an immunogenic regressor tumor expressing a transfected viral antigen. Here, we investigated whether hidden spontaneous antitumor immunosurveillance, in the absence of a vaccine, could be revealed by disruption of this negative regulatory pathway involving CD4  NKT cells and interleukin-13 (IL-13), in a murine pulmonary metastasis model of a nontransfected, nonregressor, syngeneic tumor, the CT26 colon carcinoma. Lung metastases of CT26 were decreased in CD4  T cell– depleted BALB/c mice, suggesting that CD4  T cells were involved in negative regulation of antitumor responses. CD1-knock out (CD1-KO) mice, which have conventional CD4  T cells and CD4  CD25  regulatory T cells but lack CD1-restricted CD4  NKT cells, were significantly resistant to lung metastasis of CT26. The metastases were not further decreased in CD4  T cell– depleted CD1-KO mice, imply- ing that CD4  NKT cells might be the primary negative regulator of antitumor immune responses in BALB/c mice. CD8  T cells were found to act as effectors in antitumor immune responses, since the inhibition of lung metastases observed in naı ¨ve CD1-KO or CD4  T cell– depleted mice was abrogated by depletion of CD8  T cells. Lung metastases were significantly decreased by treatment of mice with an IL-13 inhibitor, but not by deficiency or inhibition of IL-4. Thus, even for a nonregressor tumor, immuno- surveillance exists but is negatively regulated via CD4  NKT cells possibly mediated by IL-13, and can be unmasked by removal of these negative regulatory components. Published 2004 Wiley-Liss, Inc. † Key words: lung metastasis; CT26; NKT cells; IL-13 inhibitor; im- munosurveillance The success of immunotherapy against cancer depends very much on how efficiently tumor antigens are presented to antigen- specific T cells and how effectively those T cells are activated to eradicate tumor cells. However, many tumor antigens targeted for immunotherapy by active immunization are self-antigens ex- pressed not only in tumor cells but also in normal tissues. Thus, immune responses to tumor antigens are often limited by immu- nologic tolerance. 1,2 Most self-reactive T cells are deleted in the thymus and those leaking to the periphery are severely suppressed and fail to respond to antigens sufficiently to kill tumor cells. 3,4 Therefore, in order to increase the immunogenicity of tumor an- tigens and T cell responses against tumor cells, the suppressive mechanisms of peripheral tolerance must be overcome. Diverse approaches to accomplish this have been reported, such as expres- sion of cytokines like granulocyte macrophage-colony stimulatory factor (GM-CSF), or costimulatory molecules, in tumor cells, 5–7 elimination of CD4 + CD25 + T regulatory cells 8 –11 and blockade of CTLA-4 12 or a combination of these. 13 A number of negative regulatory cells have been found to play a role in both autoimmune disease and cancer. 14 –16 One of the most widely studied T cells associated with immune suppression or tolerance is the immunoregulatory CD4 + T cell constitutively expressing CD25 (the CD4 + CD25 + regulatory T cell). CD4 + CD25 + regulatory cells are considered to inhibit T cell activation irrespective of antigenic specificity. 14,17 Effective T cell immune responses against tumor were enhanced in mice by de- pletion of this T cell subpopulation in vivo. 8 –11 Another important but less widely studied regulatory cell is the natural killer T (NKT) cell. NKT cells comprise a novel T cell subset expressing NK cell markers, are CD4 + CD8 – or CD4, CD8 double negative, and have a skewed  T cell receptor (TCR) repertoire restricted by the nonclassic MHC class I-like CD1d molecule, which presents mostly glycolipid antigens rather than peptides. 18,19 While their detailed immunological functions remain unknown, NKT cells have been recently identified as one of the negative regulatory T cells, besides their roles as effector cells. 20 Regulatory CD4 + NKT cells produce large amounts of interleu- kin-4 (IL-4), which drives the differentiation and growth of T helper 2 (Th2) cells. 21,22 Thus, CD1d-restricted NKT cells could control physiological Th2-driven immune responses and indirectly regulate Th1-mediated immune responses. 23 Defects in develop- ment and function of NKT cells are related to the occurrence of experimental autoimmune diseases where Th1 immune responses dominate, 24 but the enhancement of NKT cell activity by the glycolipid, -galactosylceramide, protects NOD mice from auto- immunity. 25–27 NKT cell production of IL-4 and IL-13 has also been implicated in allergen-induced airway hypersensitivity. 28 In addition to their role in the regulation of autoimmunity, CD4 + NKT cells have been implicated in influencing immune responses against tumors by suppression of T cell activity to maintain self-tolerance. 18,29,30 Considering the immune regulatory properties of NKT cells, it would be possible to enhance the effectiveness of active immunotherapy against tumors if we could manipulate NKT cell function in vivo. 31 There are few reports studying negative influences of CD4 + NKT cells on the immune responses against tumors. 29,32,33 Studies by our laboratory identi- fied CD4 + NKT cell–mediated suppression as one of the mecha- nisms to downregulate tumor immunosurveillance and defined IL-13 as a mediator of this pathway. 29 However, these studies involved an immunogenic regressor tumor expressing a transfected foreign viral tumor antigen. In this study, we tested the generality of this regulatory pathway in tumor immunosurveillance by using the widely studied nonregressor CT26 syngeneic colon carcinoma in BALB/c mice. We show that lung metastases were significantly decreased in BALB/c mice depleted of CD4 + T cells and also in CD1-knockout (CD1-KO) mice, which lack CD1-restricted NKT cells. By CD8 + T cell depletion in CD1-KO mice, cytotoxic T lymphocyte (CTL) activity against CT26 was found to be neces- sary for the inhibition of CT26 lung metastasis. Furthermore, IL-13 was implicated as a mediator of this NKT-cell suppression of immunosurveillance. Abbreviations: FACS, fluorescence activated cell sorter; FITC, floures- cein isothiocyanate; IFN, interferon; IgG, immunoglobulin G; i.v., intra- venous; MAb, monoclonal antibody; MHC, major histocompatibility com- plex; PBMC, peripheral blood mononuclear cells; PE, phycoerytherin; RBC, red blood cell; s.c., subcutaneous; NKT, natural killer T; IL, inter- leukin; GM-CSF, granulocyte macrophage-colony stimulatory factor; TCR, T cell receptor; KO, knock-out; CTL, cytotoxic T lymphocyte; Th, T helper. *Correspondence to: Building 10, Room 6B-04, Vaccine Branch, Cen- ter for Cancer Research, National Cancer Institute, NIH, Bethesda, Mary- land 20892, USA. Fax: (301) 480-0681. E-mail: berzofsk@helix.nih.gov Received 13 April 2004; Accepted after revision 25 August 2004 DOI 10.1002/ijc.20669 Published online 2 November 2004 in Wiley InterScience (www. interscience.wiley.com). Int. J. Cancer: 114, 80 – 87 (2005) Published 2004 Wiley-Liss, Inc. † This article is a US Government work and, as such, is in the public domain in the United States of America. Publication of the International Union Against Cancer