Recent studies revealed that two novel interleukin (IL)-12-related cytokines, IL-23 and IL-27, have potent antitumor activities. However, the antitumor effects were mainly evaluated in relatively highly immunogenic tumors and have not been fully evaluated against nonimmunogenic or poorly immunogenic tumors. In this study, we investigated the antitumor efficacies of IL-23 and IL-27 on poorly immunogenic B16F10 melanoma and found that the antitumor responses mediated by IL-23 and IL-27 were clearly different. In syngeneic mice, mouse single-chain (sc) IL-23-transfected B16F10 (B16/IL-23) tumors exhibited almost the same growth curve as B16F10 parental tumor about until day 20 after tumor injection and then showed growth inhibition or even regression. In contrast, scIL-27-transfected B16F10 (B16/ IL-27) tumors exhibited significant retardation of tumor growth from the early stage. In vivo depletion assay revealed that the antitumor effect of B16/IL-23 was mainly mediated by CD8 + T cells and IFN-; whereas that of B16/IL-27 mainly involved natural killer cells and was independent of IFN-;. We also found that antitumor effects of B16/IL-23 and B16/IL-27 were synergistically enhanced by treatment with IL-18 and IL-12, respectively. Furthermore, B16/IL-23-vaccinated mice developed protective immunity against parental B16F10 tumors but B16/IL-27-vaccinated mice did not. When combined with prior in vivo depletion of CD25 + T cells, 80% of B16/ IL-23-vaccinated mice completely rejected subsequent tumor challenge. Finally, we showed that the systemic administration of neither IL-23 nor IL-27 induced such intense toxicity as IL-12. Our data support that IL-23 and IL-27 might play a role in future cytokine-based immunotherapy against poorly immunogenic tumors. (Cancer Res 2006; 66(12): 6395-404)

IL-27 is a member of the IL-6/IL-12 family and activates both STAT1 and STAT3 through its receptor, which consists of WSX-1 and gp130. We previously demonstrated that IL-27 has potent antitumor activities, which are mediated through CD8+ T cells, NK cells, or its own antiangiogenic activity. In this study, we demonstrate that IL-27 also possesses a direct antiproliferative activity on melanoma. Although WSX-1 expression was hardly detected in parental mouse melanoma B16F10 cells, IL-27 activated STAT1 and STAT3 and up-regulated MHC class I in B16F10 transfectants expressing wild-type WSX-1. In contrast, IL-27 failed to activate STAT1 and up-regulate MHC class I in those expressing mutant WSX-1, in which the putative STAT1-binding Tyr-609 of the cytoplasmic region was replaced by Phe. IL-27 inhibited the tumor growth of transfectants expressing wild-type WSX-1 in a dose-dependent manner. IL-27 augmented the expression of IFN regulatory factor (IRF)-1 and IRF-8, which possess tumor suppressor activities, in B16F10 transfectants expressing wild-type WSX-1. Down-regulation of IRF-1 but not IRF-8 with small interfering RNA partially blocked the IL-27-induced growth inhibition. A small, but significant, direct antiproliferative effect of IL-27 was also observed in vivo. Moreover, several human melanoma cells were revealed to express both IL-27 receptor subunits, and activation of STAT1 and STAT3 and growth inhibition by IL-27 were detected. These results suggest that IL-27 has an antiproliferative activity on melanomas through WSX-1/STAT1 signaling. Thus, IL-27 may be an attractive candidate as an antitumor agent applicable to cancer immunotherapy.

CD4(+)CD25(+) T cells are an important population that plays a crucial role in the maintenance of peripheral self-tolerance. Recently, it was shown that the elimination of these cells by in vivo administration of anti-CD25 monoclonal antibody (mAb) caused the regression of highly immunogenic tumors in syngeneic mice. In this study, we examined whether B16F10 melanoma cells regressed with the elimination of CD25(+) regulatory T cells. We found the melanoma cells were not affected at all by in vivo anti-CD25 mAb administration alone but tumor rejection resulted in all mice when the administration was combined with IL-12 gene transfer to tumor cells. In vivo, depletion of natural killer (NK) cells or CD8(+) T cells cancelled the tumor rejection. NK-cell depletion allowed IL-12-transfected B16F10 melanoma (B16/IL-12) to grow from an early stage and resulted in a more rapid tumor growth of B16/IL-12 than that in mice without administration of anti-CD25 mAb. On the other hand, CD8(+) T-cell depletion did not affect the tumor growth in the early phase but allowed B16/IL-12 to grow in rather a late phase and resulted in almost the same degree of tumor growth as in mice without administration of anti-CD25 mAb. In a previous study, we showed that the elimination of CD4(+) T cells enhanced the antitumor effect of B16/IL-12 and induced vitiligo-like coat color alteration. Therefore, we also examined the frequency of the change to a vitiligo-like coat color in mice showing tumor rejection caused by CD25(+) T-cell elimination to compare with the mechanism enhancing the antitumor effects by cell elimination. The elimination of CD25(+) T cells did not induce vitiligo-like coat color changes, though that of CD4(+) T cells induced the change in 60% of mice. Furthermore, we confirmed that elimination of CD25(+) T cells did not affect the T-helper (Th) 1/Th2 cytokine profile, while that of CD4(+)T cells abrogated the Th2 cytokines (IL-4 and IL-10) and resulted in a Th1-dominant cytokine profile in the tumor-draining lymph nodes (TDLNs) of B16/IL-12-bearing mice. These results indicate that in vivo depletion of CD25(+) regulatory T cells is a potent useful adjuvant in immunotherapy of B16F10 melanoma, when combined with IL-12 gene transfer and that the enhancement of the antitumor effect by CD25(+) T-cell depletion is mediated through CD8(+) T cells and may differ from the enhancing mechanism caused by CD4(+) T-cell depletion.

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Alopecia areata (AA) is generally regarded as an organ-specific autoimmune disease. Although it has been hypothesized that the autoimmunity is mediated by T cells and that hair follicle melanocyte is one of the targets, definitive evidence is lacking. We here demonstrate that AA-like lesions can be induced in mice by inducing CD8(+) T-cell-mediated immunity to hair follicle melanocytes. We found that hair loss was induced in mice-bearing interleukin-12-producing B16 melanoma cells by the depletion of CD4(+) T cells, accompanied by vitiligo-like coat color change. The alopecic lesions varied in size from pachy to extensive. In many instances, hair loss developed and was followed by the regrowth of white hairs. Histological analysis revealed that mononuclear cells infiltrated in and around the bulb region of hair follicles. Furthermore, immunohistochemical examination clearly showed the intra-follicular infiltration of CD8(+) T cells. Neither the vitiligo-like coat color nor AA-like lesions were induced when CD8(+) T cells were codepleted. These observations indicate that the induction of CD8(+) T-cell-mediated immunity against hair follicle melanocytes causes alopecia. It is thought that there are many types of AA with different mechanisms, targets etc. Although hair follicle melanocytes have long been thought to be one of the targets of AA, evidence to support the hypothesis is sparse. Therefore, we believe that our observation is significant to support the hypothesis.