While studying Ag-pulsed syngeneic dendritic cell (DC) immunization, we discovered that surprisingly, unpulsed DCs induced protection against tumor lung metastases resulting from i.v. injection of a syngeneic BALB/c colon carcinoma CT26 or a syngeneic C57BL/6 lung carcinoma LL/2. Splenocytes or immature splenic DCs did not protect. The protection was mediated by NK cells, in that it was abrogated by treatment with anti-asialo-GM1 but not anti-CD8, and was induced by CD1−/− DCs unable to stimulate NKT cells, but did not occur in beige mice lacking NK cells. Protection correlated with increased NK activity, and increased infiltration of NK but not CD8+ cells in lungs of tumor-bearing mice. Protection depended on the presence of costimulatory molecules CD80, CD86, and CD40 on the DCs, but surprisingly did not require DCs that could make IL-12 or IL-15. Unexpectedly, protection sensitive to anti-asialo-GM1 and increased NK activity were still present 14 mo after DC injection. As NK cells lack memory, we found by depletion that CD4+ not CD8+ T cells were required for induction of the NK antitumor response. The role of DCs and CD4+ T cells provides a novel mechanism for NK cell induction and innate immunity against cancer that may have potential in preventing clinical metastases.

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. The success of immunotherapy against cancer depends very much on how efficiently tumor antigens are presented to antigenspecific 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 expressed not only in tumor cells but also in normal tissues. Thus, immune responses to tumor antigens are often limited by immunologic 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 antigens 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 expression of cytokines like granulocyte macrophage-colony stimulatory factor (GM-CSF), or costimulatory molecules, in tumor cells, 5-7 elimination of CD4 ϩ CD25 ϩ 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 ϩ T cell constitutively expressing CD25 (the CD4 ϩ CD25 ϩ regulatory T cell). CD4 ϩ CD25 ϩ 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 depletion 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 ϩ 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 CD4ϩ NKT cells produce large amounts of interleukin-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 indire...

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