A variety of tumor-derived antigens have been defined by IgG antibodies in tumor bearers' sera with serological identification of antigens by recombinant expression cloning (SEREX), a serological expression cloning method. The majority of these antigens show no structural abnormality and seem to be wild-type autoantigens. Coimmunization with DNA encoding these autoantigens and tumor-specific cytotoxic T lymphocytes epitopes heightened CD8⁺ T cell responses and increased resistance to tumor challenge in a CD4⁺ T cell-dependent manner. In contrast, immunization with these SEREX-defined autoantigens alone leads to heightened susceptibility to tumor challenge. This suppressive effect of immunization is mediated by CD4⁺ CD25⁺ T cells. In mice immunized with one of the SEREX-defined autoantigens, Dna J-like 2, the number of α-GalCer/CD1d tetramer⁺ CD3⁺ T cells [representing natural killer T (NKT) cells] was reduced in the pulmonary compartment, whereas no evident change in the number of other T cell subsets was observed. Experiments with Jα281^–/– mice lacking most NKT cells indicate that NKT cells are primarily responsible for metastasis suppression and that their activity is inhibited by immunization with Dna J-like 2. We propose that SEREX identifies a pool of autoantigens that maintains and regulates immunological homeostasis via CD4⁺ CD25⁺ regulatory T cells.