Artificially synthesized helper/killer-hybrid epitope long peptide (H/K-HELP): preparation and immunological analysis of vaccine efficacy.

To elucidate the immunologic mechanisms of artificially synthesized helper/killer-hybrid epitope long peptide (H/K-HELP), which indicated a great vaccine efficacy in human cancers, we prepared ovalbumin (OVA)-H/K-HELP by conjugating killer and helper epitopes of OVA-model tumor antigen via a glycine-linker. Vaccination of C57BL/6 mice with OVA-H/K-HELP (30 amino acids) but not with short peptides mixture of class I-binding peptide (8 amino-acids) and class II-binding peptide (17 amino-acids) combined with adjuvant CpG-ODN (cytosine-phosphorothioate-guanine oligodeoxynucleotides), induced higher numbers of OVA-tetramer-positive CTL with concomitant activation of IFN-γ-producing CD4(+) Th1 cells. However, replacement of glycine-linker of OVA-H/K-HELP with other peptide-linker caused a significant decrease of vaccine efficacy of OVA-H/K-HELP. In combination with adjuvant CpG-ODN, OVA-H/KHELP exhibited greater vaccine efficacy compared with short peptides vaccine, in both preventive and therapeutic vaccine models against OVA-expressing EG-7 tumor. The elevated vaccine efficacy of OVAH/K-HELP might be derived from the following mechanisms: (i) selective presentation by only professional dendritic cells (DC) in vaccinated draining lymph node (dLN); (ii) a long-term sustained antigen presentation exerted by DC to stimulate both CTL and Th1 cells; (iii) formation of three cells interaction among DC, Th and CTL. In comparative study, H/K-HELP indicated stronger therapeutic vaccine efficacy compared with that of extended class I synthetic long peptide, indicating that both the length of peptide and the presence of Th epitope peptide were crucial aspects for preparing artificially synthesized H/K-HELP vaccine.

The key role of IL-6-arginase cascade for inducing dendritic cell-dependent CD4(+) T cell dysfunction in tumor-bearing mice.

Evaluation of immune dysfunction during the tumor-bearing state is a critical issue in combating cancer. In this study, we initially found that IL-6, one of the cachectic factors, suppressed CD4(+) T cell-mediated immunity through downregulation of MHC class II by enhanced arginase activity of dendritic cells (DC) in tumor-bearing mice. We demonstrated that administration of Ab against IL-6R (anti-IL-6R mAb) greatly enhanced T cell responses and inhibited the growth of tumor in vivo. We also found that IL-6 upregulated the expression of arginase-1 and arginase activity of DC in vitro. Tumor-infiltrating CD11c(+) DC exhibited upregulated mRNA expression of arginase-1 but reduced expression of MHC class II in parallel with the increase in serum IL-6 levels at the late stage in tumor-bearing hosts. However, the administration of anti-IL-6R mAb into tumor-bearing mice inhibited both the downmodulation of MHC class II and the upregulation of arginase-1 mRNA levels in DC. Furthermore, we noted that N(ω)-hydroxy-L-arginine or L-arginine, an arginase-1 inhibitor, blocked the reduction in MHC class II levels on CD11c(+) DC during the tumor-bearing state. Finally, we demonstrated that the administration of N(ω)-hydroxy-L-arginine at the peritumor site significantly enhanced CD4(+) T cell responses and inhibited tumor growth. Thus, IL-6-mediated arginase activation and the subsequent reduction in MHC class II expression on DC appeared to be critical mechanisms for inducing dysfunction of the immune system in the tumor-bearing state. Blockade of the IL-6-arginase cascade is a promising tool to overcome the dysfunction of antitumor immunity in tumor-bearing hosts.

Extracts of Larix Leptolepis effectively augments the generation of tumor antigen-specific cytotoxic T lymphocytes via activation of dendritic cells in TLR-2 and TLR-4-dependent manner.

Type-1 immunity plays a crucial role in host defense against various tumors and infectious diseases. Here, we first demonstrated that extract of Larix Leptolepis (ELL), one of the most popular timbers at Hokkaido area in Japan, strongly activated Type-1 immunity. ELL induced production of Type-1 cytokines such as IL-12 and TNF-α from bone marrow-derived dendritic cells (BMDCs) in TLR2- and TLR4-dependent manner and remarkably up-regulated the expression of MHC and co-stimulatory molecules. In addition, antigen-specific CTLs were significantly augmented by the combined administration of ELL, antigen and BMDCs. Finally, we revealed that combination therapy using ELL, antigen and BMDCs significantly inhibited the growth of established tumor in mouse model. Thus, these findings suggested that ELL would be a novel adjuvant for inducing an activation of Type-1-dependent immunity including activation of BMDCs and induction of tumor-specific CTLs, which is applicable to the therapy of cancer and infectious diseases.

Anti-IL-6 receptor mAb eliminates myeloid-derived suppressor cells and inhibits tumor growth by enhancing T-cell responses.

CD11b(+) Gr-1(+) immature myeloid cells (ImCs), which are abnormally increased in tumor-bearing mice, were classified into three different subsets according to their phenotypic and morphological characteristics: Gr-1(low) F4/80(+) macrophages (MΦ-ImCs), Gr-1(mid) stab neutrophils (Neut(stab)-ImCs), and Gr-1(high) segmented neutrophils (Neut(seg)-ImCs). In the spleen, only MΦ-ImCs but not Neut(stab)-ImCs and Neut(seg)-ImCs exhibited a significant immunosuppressive activity in MLR. In contrast, tumor-infiltrating leukocytes (TILs) contained only two ImC subsets, MΦ-ImCs and Neut(seg)-ImC, both of which exhibited stronger inhibitory activity against T cells compared with spleen-MΦ-ImCs. Thus, we concluded that tumor-infiltrating MΦ-ImCs and Neut(seg)-ImCs were fully differentiated myeloid-derived suppressor cells (MDSCs) with stronger T-cell inhibitory activity. Indeed, spleen MΦ-ImCs were converted into stronger MΦ-MDSCs by tumor-derived factor (TDF). Moreover, both spleen Neut(stab)-ImCs and Neut(seg)-ImCs differentiated into Neut(seg)-MDSCs with suppressive activity after culture with TDF. We first demonstrated that administration of anti-IL-6R mAb could downregulate the accumulation of MΦ-MDSCs and Neut(seg)-MDSCs in tumor-bearing mice. The elimination of those MDSCs caused subsequent enhancement of antitumor T-cell responses, including IFN-γ-production. The therapeutic effect of anti-IL-6R mAb was further enhanced by combination with gemcitabine (GEM). Thus, we propose that anti-IL-6R mAb could become a novel tool for the downmodulation of MDSCs to enhance antitumor T-cell responses in tumor-bearing hosts.

Toll-like receptor-dependent IL-12 production by dendritic cells is required for activation of natural killer cell-mediated Type-1 immunity induced by Chrysanthemum coronarium L.

Type-1 immunity has an essential role for our host defenses against cancer and outer pathogens such as bacteria and virus. We demonstrated here that the edible plant extract of Chrysanthemum coronarium L. (C. coronarium) remarkably activates Type-1 immunity in a Toll-like receptor (TLR)2-, TLR4-, and TLR9-dependent manner. In the present experiments, the extract of C. coronarium significantly induces interferon (IFN)-γ production by mouse spleen cells. In addition, the IFN-γ production by spleen cells was completely blocked by the addition of anti-Interleukin (IL)-12 monoclonal antibodies. We confirmed that NK1.1(+) natural killer (NK) cells, NKT cells, and CD11c(+) dendritic cells (DC) were immediately activated after the stimulation with the extract of C. coronarium and the IFN-γ production was abolished in NK1.1(+) cell-depleted spleen cells. The stimulation with the extract of C. coronarium caused DC maturation involving with up-regulations of surface expression levels of MHC class I, MHC class II, CD40, and CD86 as well as induction of IL-12 production. The IFN-γ production induced by the extract was significantly reduced in the spleen cells depleted CD11c(+) cells. Furthermore, the IFN-γ production after the stimulation was strongly reduced in TLR4- and partially in TLR2- and TLR9-deficient spleen cells. Thus, we demonstrated the cellular mechanism for the activation of Type-1 immunity via NK cells, NKT cells, and DC by the extract of C. coronarium. These findings strongly suggest that C. coronarium would be a promising immuno-improving adjuvant, which might be useful for prevention of infectious, cancer, and allergic diseases through the activation of Type-1 immunity.