The Mycobacterium bovis bacillus Calmette-Guérin cell-wall skeleton (BCG-CWS) activates Toll-like receptor (TLR) 2 and TLR4, but unlike the typical TLR4 agonist bacterial lipopolysaccharide barely induces type 1 IFN. BCG-CWS has been used for adjuvant immunotherapy for patients with cancer. We investigated the adjuvant potential of BCG-CWS for induction of CTLs subsequent to TLR-mediated dendritic cell (DC) maturation, using a syngeneic mouse tumor model (B16 melanoma in C57BL/ 6). We evaluated the retardation of tumor growth and cytotoxic response in wild-type and MyD88؊/؊ mice immunized with tumor debris and/or BCG-CWS. Delays in tumor growth and cytotoxic response were induced by immunization with a mixture of BCG-CWS emulsion and the tumor. BCG-CWS was capable of activating DCs ex vivo by the criteria of CD80/CD86 up-regulation and cytokine (interleukin-12, tumor necrosis factor-␣) induction. Efficient tumor suppression and ex vivo cytokine induction did not occur in MyD88-deficient mice and cells, suggesting that the MyD88 adapter is crucial for induction of tumor cytotoxicity. Because TLR4 is involved in both MyD88-dependent and -independent pathways and the latter affects DC maturation, our findings indicate that both pathways cooperate to induce CTL-based tumor immunity.

Monocyte-derived dendritic cells (mDCs) and NK cells are reciprocally activate via cytokines and cell-cell contact. Although seven human NKG2D ligands (NKG2DLs), UL16-binding proteins (ULBP) 1, 2, 3 and 4, retinoic acid early transcript 1G (RAET1G) and MHC class I-related chains A and B, have been reported, the differential distribution and roles of these ligands in the maturation of human mDCs have not been elucidated. In the present study, we produced polyclonal antibodies (pAbs) directed against human ULBP1, 2 and 3. All these ULBPs were detected on human mDCs when probed by the pAbs, although their expression profiles were different. We next investigated what kinds of Toll-like receptor agonists and RNA viruses [influenza virus, human respiratory syncytial virus (RSV), measles virus and hepatitis C virus (HCV)] induced the expression of NKG2DLs on mDCs. ULBP1 was up-regulated on mDCs in response to LPS or infection with RSV. The expression of ULBP2 was induced by LPS and poly I:C, indicating that the TIR-containing adapter molecule-1 (TIR domain-containing adaptor-inducing IFN) pathway is associated with ULBP2 induction. Although infection with HCV did not cause up-regulation of NKG2DLs, other RNA virus infections and poly I:C promoted expression of ULBP2 and RAET1G in an IFN-alpha/beta-independent manner. Finally, the over-expression of ULBP1 and 2 on mDCs facilitated NK cell proliferation and IFN-gamma production through a mDC-NK cell interaction in the presence of IL-2. Hence, the results reflect the important role of NKG2DLs on human mDCs in mDC-mediated NK cell activation.

Oral administration of hot-water extract of Spirulina, cyanobacterium Spirulina platensis, leads to augmentation of NK cytotoxicity in humans. Here, we applied to syngeneic tumor-implant mice (C57BL/6 versus B16 melanoma) Spirulina to elucidate the mechanism of raising antitumor NK activation. A B16D8 subcell line barely expressed MHC class I but about 50% expressed Rae-1, a ligand for NK activation receptor NKG2D. The Rae-1-positive population of implant B16 melanoma was effectively eliminated in the tumor mass progressed in mice. This antitumor activity was induced in parallel with IFN-g and abolished in mice by treatment with asialoGM-1 but not CD8b Ab, suggesting the effector is NK cell. NK cell activation occurred in the spleen of wild-type mice medicated with Spirulina. This Spirulina-mediated enhanced NK activation was abrogated in MyD88 -/-mice but not in TICAM-1 -/-mice. The NK activating properties of Spirulina depending on MyD88 were confirmed with in vitro bone marrow-derived dendritic cells expressing TLR2/4. In D16D8 tumor challenge studies, the antitumor effect of Spirulina was abolished in MyD88 -/-mice. Hence, orally administered Spirulina enhances tumoricidal NK activation through the MyD88 pathway. Spirulina exerted a synergistic antitumor activity with BCG-cell wall skeleton, which is known to activate the MyD88 pathway via TLR2/4 with no NK enhancing activity. Spirulina and BCG-cell wall skeleton synergistically augmented IFN-g production and antitumor potential in the B16D8 versus C57BL/6 system. We infer from these results that NK activation by Spirulina has some advantage in combinational use with BCG-cell wall skeleton for developing adjuvant-based antitumor immunotherapy. (Cancer Sci 2009; 100: 1494-1501)