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Interleukin (IL)-27 is an IL-12 family cytokine playing a pivotal role in the induction of Th1 immune responses, although its action on natural killer (NK) cells has not been fully elucidated. Here, we show that IL-27 is capable of inducing phosphorylation of signal transducers and activators of transcription 1 and 3, as well as expression of T-bet and granzyme B in murine DX-5+ NK cells. IL-27 also enhances cytotoxic activity of NK cells both in vitro and in vivo, while the in vitro viability of NK cells is also improved by this cytokine. Therapeutic administration of the IL-27 gene drastically suppressed the growth of NK-unsusceptible SCCVII tumors that had been preestablished in syngenic mice, resulting in significant prolongation of the survival of the animals. This can likely be ascribed to the antibody-dependent cellular cytotoxicity machinery because IL-27 successfully induced tumor-specific IgG in the sera of the tumor-bearing mice, and supplementation of the sera enabled IL-27–activated NK cells to kill SCCVII cells in an Fcγ receptor III–dependent manner. These findings strongly suggest that IL-27 may offer a powerful immunotherapeutic tool to eradicate head and neck squamous cell carcinoma and other poorly immunogenic neoplasms through activating NK cells and inducing tumor-specific immunoglobulin that may cooperatively elicit antibody-dependent cellular cytotoxicity activity. [Cancer Res 2009;69(6):2523–30]

Although much promising data that interleukin (IL)-12 could be a powerful therapeutic agent against cancer were reported in animal models, its excessive toxicity has become a problem for its clinical application. IL-27 is a novel IL-12 family member that plays a role in the early regulation of T helper cell 1 initiation, including induction of T-bet and IL-12 receptor β2 expression. In the present study, we have evaluated the antitumor activity of IL-27 against a murine tumor model of colon carcinoma C26. C26 cells, which were transduced with the single-chain IL-27 cDNA and became secreting IL-27 (C26-IL-27), exhibited minimal tumor growth in vivo, and all of the mice inoculated with these cells survived healthily with complete tumor remission. Inoculation of mice with C26-IL-27 induced enhanced IFN-γ production and cytotoxic T-lymphocyte activity against C26 tumor in spleen cells. Recovered mice from the inoculation showed a tumor-specific protective immunity to the following challenge with parental C26 tumor. The antitumor activity of IL-27 was almost diminished in nude mice, and depletion of CD8+ T cells and neutralization of IFN-γ in immunocompetent mice reduced greatly the antitumor activity. Moreover, the antitumor activity was abolished in T-bet-deficient mice, whereas it was observed unexpectedly in mice deficient of signal transducer and activator of transcription (STAT) 4. These results suggest that IL-27 has potent abilities to induce tumor-specific antitumor activity and protective immunity and that the antitumor activity is mediated mainly through CD8+ T cells, IFN-γ, and T-bet but not through STAT4.

Antigen specific immune response by both MHC class I-restricted CD8+ T cells and MHC class II-restricted CD4+ T cells is a key event in cancer immunotherapy. In DC vaccine therapy, the short epitope peptides (9 or 10-mer) are mainly used to load, but these short peptides are presented on MHC class I molecule and can stimulate only CD8+ T cells. Using whole protein as antigen might be better to induce both antigen specific CD8+ and CD4+ T cells. The PepTivator® is a peptide pool: consisting mainly of 15-mer sequences with 11 amino acids overlap and covering the complete sequence of target protein. In this study, we used PepTivator® derived from ovalbumin protein (PepTivator® OVA) instead of whole protein as antigen, and evaluated the inducing activity of antigen specific CD8+ and CD4+ T cell responses in mouse model. In order to examine the antigen presenting ability of PepTivator® OVA-pulsed DCs, CD8+ T cells (106 cells/mL) isolated from OT-I transgenic mouse (OT-I CD8T) or CD4+ T cells (106 cells/mL) isolated from OT-II transgenic mouse (OT-II CD4T) were cultured with PepTivator® OVA-pulsed DCs (5×104 cells/mL) for 4 days. As positive control, OT-I epitope peptide (OVA257-264aa) and OT-II epitope peptide (OVA323-339aa) were used. DCs were prepared from bone marrow cells of C57BL/6 mice after cultivation with medium containing GM-CSF, IL-4 and LPS for 10 days. In preparation of peptide-pulsed DCs, DCs were treated with each peptide at 2 µg/mL for 4 hours and washed with medium to deplete free peptide before use. During the cultivation of OT-I CD8T or OT-II CD4T cells with each peptide-pulsed DCs, the responsive T cell proliferation was measured daily by flow cytometric analysis with absolute count beads and CFSE dye dilution. In addition, cytokines (IL-2 and IFN-gamma) in culture supernatant were measured by ELISA. As a result, the proliferation of OT-I CD8T cells cocultured with PepTivator® OVA-pulsed DCs seemed to be similar to that in coculture with OT-I epitope peptide-pulsed DCs. In the case of proliferation assay using OT-II CD4T cells, PepTivator® OVA-pulsed DCs showed similar T cell expansion in comparison with OT-II epitope peptide-pulsed DCs. As for cytokines secretion, no difference was observed in concentration of IL-2 and IFN-gamma in both cases using OT-I CD8T and OT-II CD4T cells when PepTivator® OVA was compared with OT-I or OT-II epitope peptide, respectively. These results showed that PepTivator® OVA pulsed-DCs could induce both antigen specific CD8+ and CD4+ T cell responses in mouse model. It suggests that PepTivator® OVA is at least superior to short epitope peptide in inducing antigen specific T cell response. In addition, it might be big advantage that PepTivator® derived from tumor antigen could be useful in many candidate patients without their HLA restriction. It is expected that therapeutic benefit of PepTivator® pulsed-DCs will be shown in clinical. Citation Format: Kenji Miki, Koji Nagaoka, Hermann Bohnenkamp, Takayuki Yoshimoto, Takashi Kamigaki, Ryuji Maekawa. Induction of both OVA specific CD4+ and CD8+ T cells by using PepTivator® OVA-pulsed DCs in mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2815. doi:10.1158/1538-7445.AM2014-2815

For successful dendritic cell (DC) therapy of cancer, it is important to choose the optimal method to load DCs with antigens to induce higher anti-tumor immunity, particularly tumor-specific CTL responses. The co-incubation (Co) method is conventionally used to load DCs with tumor antigens in DC therapy. Although DCs could uptake tumor antigens by endocytosis in this method, they are thought to be processed in lysosome and presented mainly on MHC class II, but not efficiently on MHC class I. Electroporation (Ep) is one of the promising antigen-loading method because it is reported that DCs could uptake much larger amount of FITC-dextran by Ep in comparison with Co method. In this study, we performed side-by-side comparisons of these two antigen loading methods using murine bone marrow derived DCs. First, we compared antigen presentation using purified ovalbumin (OVA) and OVA-specific OT-I CD8 and OT-II CD4 T cells. To this end, DCs were loaded with OVA by either Co or Ep, and subsequently cultured with T cells. Two or three days later, T cell expansion and cytokine production were evaluated. As expected, OVA-Co-DCs induced moderate expansion and cytokine production of OT-II T cells, but had little effect on OT-I T cells. In contrast, OVA-Ep-DCs potently stimulated both OT-I and OT-II T cells to expand and produce cytokines, such as IL-2 and IFN-γ. Using monoclonal antibody specific for MHC class I/OVA peptide complex, we confirmed efficient presentation of the OVA peptide on MHC class I by OVA-Ep-DCs. Furthermore, when we used the lysate of EG7 tumor cell line (OVA gene-transfected) as antigen instead of purified OVA, Ep-DCs induced expansion of both OT-I and OT-II T cells efficiently compared with Co-DCs. In the case of Alexa488-OVA, unexpectedly, similar amounts of uptake were seen in Ep-DCs and Co-DCs by FCM analysis. By using confocal microscopy, however, there was a critical difference of intracellular localization of Alexa488-OVA. Although Alexa488-OVA was localized in intracellular vesicles of both DCs, the cytosolic localization was observed only in Ep-DCs, suggesting that antigen loaded by Ep entered cytosol of DCs and would be processed by proteasome and presented on MHC class I efficiently. Finally, to determine the ability of these DCs to induce anti-tumor immunity in vivo, mice were subcutaneously immunized twice at 7 days intervals with 3-5 x 105 cells of either OVA-Ep-DCs or OVA-Co-DCs and then challenged with 3 x 106 of EG7 cells at 7 days after the 2nd immunization. When OVA-Co-DCs were used for immunization, tumor formations were partially prevented. On the contrary, mice immunized with OVA-Ep-DCs were almost completely protected from tumor formations. These results demonstrate that DCs loaded with tumor antigens by Ep have greater antigen presentation capacity, especially through MHC class I pathway, and could induce stronger anti-tumor immunity in vivo than those loaded by Co. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1562. doi:1538-7445.AM2012-1562