Cancer immunosurveillance failure is largely attributed to the insufficient activation of tumor-specific class I major histocompatibility complex (MHC) molecule (MHC-I)-restricted CD8+ cytotoxic T lymphocytes (CTLs). DEC-205+ dendritic cells (DCs), having the ability to cross-present, can present captured tumor antigens on MHC-I alongside costimulatory molecules, inducing the priming and activation of tumor-specific CD8+ CTLs. It has been suggested that reduced levels of costimulatory molecules on DCs may be a cause of impaired CTL induction and that some tumors may induce the downregulation of costimulatory molecules on tolerogenic DCs. To examine such possibilities, we established two distinct types of murine hepatoma cell lines, named Hepa1-6-1 and Hepa1-6-2 (derived from Hepa1-6 cells), and confirmed that they display similar antigenicities, as well as identical surface expression of MHC-I. We found that Hepa1-6-1 had the ability to grow continuously after subcutaneous implantation into syngeneic C57BL/6 mice and did not prime CD8+ CTLs. In contrast, Hepa1-6-2 cells, which display reduced levels of adhesion molecules, such as Intercellular Adhesion Molecule 1 (ICAM-1), failed to grow in vivo and efficiently primed CTLs. Moreover, Hepa1-6-1-derived factors, such as transforming growth factor (TGF)-β1, vascular endothelial growth factor (VEGF) and α-fetoprotein (AFP), converted CD11chigh MHC-IIhigh DEC-205+ DC subsets into tolerogenic cells, displaying downregulated costimulatory molecules and having impaired cross-presenting capacities. These immunosuppressive tolerogenic DCs appeared to inhibit the induction of tumor-specific CD8+ CTLs and suppress their cytotoxic functions within the tumor. Together, the findings presented here provide a new method of cancer immunotherapy using the selective suppression, depletion or alteration of immunosuppressive tolerogenic DCs within tumors.

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Priming of CTLs at mucosal sites, where various tumors are originated, seems critical for controlling tumors. In the present study, the effect of the oral administration of OVA plus adjuvant cholera toxin (CT) on the induction of Ag-specific mucosal CTLs as well as their effect on tumor regression was investigated. Although OVA-specific TCRs expressing lymphocytes requiring in vitro restimulation to gain specific cytotoxicity could be detected by OVA peptide-bearing tetramers in both freshly isolated intraepithelial lymphocytes and spleen cells when OVA was orally administered CT, those showing direct cytotoxic activity without requiring in vitro restimulation were dominantly observed in intraepithelial lymphocytes. The magnitude of such direct cytotoxicity at mucosal sites was drastically enhanced after the second oral administration of OVA with intact whole CT but not with its subcomponent, an A subunit (CTA) or a B subunit (CTB). When OVA plus CT were orally administrated to C57BL/6 mice bearing OVA-expressing syngeneic tumor cells, E.G7-OVA, in either gastric tissue or the dermis, tumor growth was significantly suppressed after the second oral treatment; however, s.c. or i.p. injection of OVA plus CT did not show any remarkable suppression. Those mucosal OVA-specific CTLs having direct cytotoxicity expressed CD8αβ but not CD8αα, suggesting that they originated from thymus-educated cells. Moreover, the infiltration of such OVA-specific CD8+ CTLs was observed in suppressed tumor tissues. These results indicate that the growth of ongoing tumor cells can be suppressed by activated CD8αβ CTLs with tumor-specific cytotoxicity via an orally administered tumor Ag with a suitable mucosal adjuvant.

Two major distinct subsets of dendritic cells (DCs) are arranged to regulate our immune responses in vivo; 33D1+ and DEC-205+ DCs. Using anti-33D1-specific monoclonal antibody, 33D1+ DCs were successfully depleted from C57BL/6 mice. When 33D1+ DC-depleted mice were stimulated with LPS, serum IL-12, but not IL-10 secretion that may be mediated by the remaining DEC-205+ DCs was markedly enhanced, which may induce Th1 dominancy upon TLR signaling. The 33D1+ DC-depleted mice, implanted with syngeneic Hepa1-6 hepatoma or B16-F10 melanoma cells into the dermis, showed apparent inhibition of already established tumor growth in vivo when they were subcutaneously (sc) injected once or twice with LPS after tumor implantation. Moreover, the development of lung metastasis of B16-F10 melanoma cells injected intravenously was also suppressed when 33D1+ DC-deleted mice were stimulated twice with LPS in a similar manner, in which the actual cell number of NK1.1+CD3− NK cells in lung tissues was markedly increased. Furthermore, intraperitoneal (ip) administration of a very small amount of melphalan (l-phenylalanine mustard; l-PAM) (0.25 mg/kg) in LPS-stimulated 33D1+ DC-deleted mice helped to induce H-2Kb-restricted epitope-specific CD8+ cytotoxic T lymphocytes (CTLs) among tumor-infiltrating lymphocytes against already established syngeneic E.G7-OVA lymphoma. These findings indicate the importance and effectiveness of selective targeting of a specific subset of DCs, such as DEC-205+ DCs alone or with a very small amount of anticancer drugs to activate both CD8+ CTLs and NK effectors without externally added tumor antigen stimulation in vivo and provide a new direction for tumor immunotherapy.

Cholera toxin (CT) is a potent mucosal adjuvant and oral administration of ovalbumin (OVA) antigens plus CT induces OVA-specific CD8+ cytotoxic T lymphocytes (CTLs) and IgA production in intestinal mucosa. However, the mechanisms of induction of these immune responses remain unknown. Intestinal OVA-specific CD8+ CTLs were not induced by oral administration of the CT active (CTA) or CT binding (CTB) subunit as an adjuvant and CD11c+ DCs were involved in cross-priming of intestinal CTLs. CD8+CD103+CD11c+CD11b−DCs and DCIR2+CD103+CD11c+CD11b+ DCs were distributed in the intestinal lamina propria and mesenteric lymph nodes, both DC subsets expressed DEC-205, and the expression of co-stimulatory molecules such as CD80 and CD86 was enhanced in both DC subsets after oral administration of intact CT but not the CTA or CTB subunit. Intestinal DCs activated by the oral administration of OVA plus CT cross-presented OVA antigens and DCs that captured OVA antigen through DEC-205, but not DCIR2, could cross-present antigen. We found that oral administration of intact CT, but not the CTA or CTB subunit, enhanced cell death, cytoplasmic expression of high-mobility group box 1 protein (HMGB1) in epithelial cell adhesion molecule (EpCAM)+CD45− intestinal epithelial cells (IECs), and HMGB1 levels in fecal extracts. HMGB1 dose-dependently enhanced the expression of CD80 and CD86 on DCs in vitro, and intravenous or oral administration of glycyrrhizin, an HMGB1 inhibitor, significantly suppressed activation of mucosal DCs and induction of intestinal OVA-specific CTLs and IgA by oral CT administration. These results showed that oral administration of intact CT triggers epithelial cell death in the gut and the release of HMGB1 from damaged IECs, and that the released HMGB1 may mediate activation of mucosal DCs and induction of CTLs and IgA in the intestine.