Hirotake Tsukamoto

Proliferation Potential-Related Protein, an Ideal Esophageal Cancer Antigen for Immunotherapy, Identified Using Complementary DNA Microarray Analysis

Purpose: To establish effective antitumor immunotherapy for esophageal cancer, we tried to identify an useful target antigen of esophageal cancer. Experimental Design: We did cDNA microarray analysis to find a novel candidate antigen, proliferation potential-related protein (PP-RP). We examined cytotoxicity against tumor cells in vitro and in vivo of CTLs specific to PP-RP established from esophageal cancer patients. Results: In 26 esophageal cancer tissues, an average of relative ratio of the expression of the PP-RP mRNA in cancer cells versus adjacent normal esophageal tissues was 396.2. Immunohistochemical analysis revealed that, in 20 of the 22 esophageal cancer tissues, PP-RP protein was strongly expressed only in the cancer cells and not so in normal esophageal epithelial cells. PP-RP protein contains 10 epitopes recognized by HLA-A24–restricted CTLs. These CTLs, generated from HLA-A24–positive esophageal cancer patients, had cytotoxic activity against cancer cell lines positive for both PP-RP and HLA-A24. Furthermore, adoptive transfer of the PP-RP–specific CTL line inhibited the growth of a human esophageal cancer cell line engrafted in nude mice. Conclusions: The expression of PP-RP in esophageal cancer cells was significantly higher than in normal cells, and the CTLs recognizing PP-RP killed tumor cells in vitro and also showed tumor rejection effects in a xenograft model. Therefore, PP-RP may prove to be an ideal tumor antigen useful for diagnosis and immunotherapy for patients with esophageal cancer. cDNA microarray analysis is a useful method to identify ideal tumor-associated antigens.

Identification of promiscuous KIF20A long peptides bearing both CD4+ and CD8+ T-cell epitopes: KIF20A-specific CD4+ T-cell immunity in patients with malignant tumor

Purpose: To identify long peptides (LP) derived from a novel tumor-associated antigen (TAA), kinesin family member 20A (KIF20A), which induce tumor-specific T-helper type 1 (TH1) cells and CTLs. Experimental Design: We combined information from a recently developed computer algorithm predicting HLA class II–binding peptides with KIF20A-derived CTL-epitope sequences presented by HLA-A2 (A*02:01) or HLA-A24 (A*24:02) to select candidate promiscuous TH1-cell epitopes containing CTL epitopes. Peripheral blood mononuclear cells (PBMC) derived from healthy donors or patients with head-and-neck malignant tumor (HNMT) were used to study the immunogenicity of KIF20A-LPs, and the in vitro cross-priming potential of KIF20A-LPs bearing CTL epitopes. We used HLA-A24 transgenic mice to address whether vaccination with KIF20A-LP induces efficient cross-priming of CTLs in vivo. The TH1-cell response to KIF20A-LPs in HNMT patients receiving immunotherapy with TAA-derived CTL-epitope peptides was analyzed using IFN-γ enzyme-linked immunospot assays. Results: We identified promiscuous KIF20A-LPs bearing naturally processed epitopes recognized by CD4+ T cells and CTLs. KIF20A-specific CTLs were induced by vaccination with a KIF20A-LP in vivo. KIF20A expression was detected in 55% of HNMT by immunohistochemistry, and significant frequencies of KIF20A-specific TH1 cell responses were detected after short-term in vitro stimulation of PBMCs with KIF20A-LPs in 50% of HNMT patients, but not in healthy donors. Furthermore, these responses were associated with KIF20A expression in HNMT tissues. Conclusions: These are the first results showing the presence of KIF20A-specific TH1 cell responses in HNMT patients and underline the possible utility of KIF20A-LPs for propagation of TH1 cells and CTLs. Clin Cancer Res; 19(16); 4508–20. ©2013 AACR.

Synthetic small interfering RNA targeting heat shock protein 105 induces apoptosis of various cancer cells both in vitro and in vivo

We previously reported that heat shock protein 105 (HSP105), identified by serological analysis of a recombinant cDNA expression library (SEREX) using serum from a pancreatic cancer patient, was overexpressed in various human tumors and in the testis of adult men by immunohistochemical analysis. In the present study, to elucidate the biological function of the HSP105 protein in cancer cells, we first established NIH3T3 cells overexpressing murine HSP105 (NIH3T3‐HSP105). The NIH3T3‐HSP105 cells acquired resistance to apoptosis induced by heat shock or doxorubicin. The small interfering RNA (siRNA)‐mediated suppression of HSP105 protein expression induced apoptosis in human cancer cells but not in fibroblasts. By a combination of siRNA introduction and doxorubicin or heat shock treatment, apoptosis was induced synergistically in a human colon cancer cell line, HCT116. In vivo, siRNA inoculation into the human gastric cancer cell line KATO‐3 established in the flank of an NOD SCID mouse suppressed the tumor growth. This siRNA‐induced apoptosis was mediated through caspases, but not the p53 tumor suppressor protein, even though the HSP105 protein was bound to wild‐type p53 protein in HCT116 cells. These findings suggest that the constitutive overexpression of HSP105 in cancer cells is involved in malignant transformation by protecting tumor cells from apoptosis. HSP105 may thus be a novel target molecule for cancer therapy and a treatment regimen using synthetic siRNA to suppress the expression of HSP105 protein may provide a new strategy for cancer therapy. (Cancer Sci 2006; 97: 623–632)

Systematic Analysis of the Combinatorial Nature of Epitopes Recognized by TCR Leads to Identification of Mimicry Epitopes for Glutamic Acid Decarboxylase 65-Specific TCRs

Accumulating evidence indicates that recognition by TCRs is far more degenerate than formerly presumed. Cross-recognition of microbial Ags by autoreactive T cells is implicated in the development of autoimmunity, and elucidating the recognition nature of TCRs has great significance for revelation of the disease process. A major drawback of currently used means, including positional scanning synthetic combinatorial peptide libraries, to analyze diversity of epitopes recognized by certain TCRs is that the systematic detection of cross-recognized epitopes considering the combinatorial effect of amino acids within the epitope is difficult. We devised a novel method to resolve this issue and used it to analyze cross-recognition profiles of two glutamic acid decarboxylase 65-autoreactive CD4+ T cell clones, established from type I diabetes patients. We generated a DNA-based randomized epitope library based on the original glutamic acid decarboxylase epitope using class II-associated invariant chain peptide-substituted invariant chains. The epitope library was composed of seven sublibraries, in which three successive residues within the epitope were randomized simultaneously. Analysis of agonistic epitopes indicates that recognition by both TCRs was significantly affected by combinations of amino acids in the antigenic peptide, although the degree of combinatorial effect differed between the two TCRs. Protein database searching based on the TCR recognition profile proved successful in identifying several microbial and self-protein-derived mimicry epitopes. Some of the identified mimicry epitopes were actually produced from recombinant microbial proteins by APCs to stimulate T cell clones. Our data demonstrate the importance of the combinatorial nature of amino acid residues of epitopes in molecular mimicry.

Myeloid-Derived Suppressor Cells Attenuate TH1 Development through IL-6 Production to Promote Tumor Progression

IL-6+/+ MDSC dampened the induction of TH1 cells and CD4+ T-cell cognate help for CD8+ T cells, and temporal blockade of IL-6 activity at the T-cell priming phase restored TH1 cell differentiation. Tsukamoto and colleagues identify Gr-1+ MDSC as a source of IL-6 in tumor-bearing mice and show that IL-6+/+ MDSC-sensitized CD4+ T cells were less potent in mounting antitumor immune responses. In the aggregate, these results suggest that MDSC-derived IL-6 contributes to the dysfunction of host antitumor responses. Collaborative action between tumor cells and host-derived suppressor cells leads to peripheral tolerance of T cells to tumor antigens. Here, we showed that in tumor-bearing mice, generation of tumor antigen-specific effector T-helper cells (TH1) was significantly attenuated, and impaired TH1 differentiation was restored by the temporal blockade of interleukin (IL)-6 activity at the T-cell priming phase. Furthermore, we found that Gr-1+ myeloid-derived suppressor cells (MDSC) served as a source of IL-6 in tumor-bearing mice. Adoptive transfer of effector CD4+ T cells revealed that MDSC-sensitized effector CD4+ T cells were less potent in mounting antitumor immune responses, although effector T cells generated together with Gr-1+ cells from tumor-free mice eradicated established tumors. CD8+ T cells, IFN-γ, and MHC-class II expression in host mice were indispensable for the antitumor activity initiated by effector CD4+ T cells. Despite comparable suppressive activity of IL-6+/+ and IL-6−/− MDSC on primary T-cell activation, transfer of IL-6+/+ MDSC, but not IL-6−/− MDSC, dampened the efficient induction of effector TH1 cells and counteracted CD4+ T cell–mediated antitumor immunity including cognate help for CD8+ T cells in vivo. These findings suggest that, apart from the inhibitory effects on primary T-cell activation, MDSC promote tumor progression by attenuating functional differentiation of tumor-specific CD4+ T cells into effector TH1 cells through IL-6 production to promote tumor progression. This novel mode of MDSC-induced tolerance of effector CD4+ T cells should be considered as the basis for the rational design of effective T cell–mediated antitumor therapies. Cancer Immunol Res; 1(1); 64–76. ©2013 AACR.

Corosolic acid impairs tumor development and lung metastasis by inhibiting the immunosuppressive activity of myeloid-derived suppressor cells

SCOPE Recent studies demonstrated that myeloid cells are associated with systemic immunosuppression in tumor-bearing hosts. In particular, myeloid cells positive for Gr-1 and CD11b in tumor-bearing mice are called myeloid-derived suppressor cells (MDSC) because of their suppression of T-cell activation. In this study, we investigated the antitumor effects of corosolic acid (CA) in murine sarcoma model. METHODS AND RESULTS The results from the in vivo study showed that CA administration did not suppress the tumor proliferation index, but significantly impaired subcutaneous tumor development and lung metastasis. Furthermore, CA administration inhibited signal transducer and activator of transcription-3 (Stat3) activation and increased in the number of infiltrating lymphocytes in tumor tissues. Ex vivo analysis demonstrated that a significant immunosuppressive effect of MDSC in tumor-bearing mice was abrogated and the mRNA expressions of cyclooxygenase-2 and CCL2 in MDSC were significantly decreased by CA administration. Furthermore, CA enhanced the antitumor effects of adriamycin and cisplatin in in vitro. CONCLUSION Since Stat3 is associated with tumor progression not only in osteosarcoma, but also in other malignant tumors, our findings indicate that CA might be widely useful in anticancer therapy by targeting the immunosuppressive activity of MDSC and through its synergistic effects with anticancer agents.

IL-6-mediated environmental conditioning of defective Th1 differentiation dampens antitumour immune responses in old age

Decline in immune function and inflammation concomitantly develop with ageing. Here we focus on the impact of this inflammatory environment on T cells, and demonstrate that in contrast to successful tumour elimination in young mice, replenishment of tumour-specific CD4+ T cells fails to induce tumour regression in aged hosts. The impaired antitumour effect of CD4+ T cells with their defective Th1 differentiation in an aged environment is restored by interleukin (IL)-6 blockade or IL-6 deficiency. IL-6 blockade also restores the impaired ability of CD4+ T cells to promote CD8+ T-cell-dependent tumour elimination in aged mice, which requires IFN-γ. Furthermore, IL-6-stimulated production of IL-4/IL-21 through c-Maf induction is responsible for impaired Th1 differentiation. IL-6 also contributes to IL-10 production from CD4+ T cells in aged mice, causing attenuated responses of CD8+ T cells. These findings suggest that IL-6 serves as an extrinsic factor counteracting CD4+ T-cell-mediated immunity against tumour in old age.

Identification of CDCA1-derived long peptides bearing both CD4+ and CD8+ T-cell epitopes: CDCA1-specific CD4+ T-cell immunity in cancer patients.

We recently identified a novel cancer‐testis antigen, cell division cycle associated 1 (CDCA1) using genome‐wide cDNA microarray analysis, and CDCA1‐derived cytotoxic T lymphocyte (CTL)‐epitopes. In this study, we attempted to identify CDCA1‐derived long peptides (LPs) that induce both CD4+ helper T (Th) cells and CTLs. We combined information from a recently developed computer algorithm predicting HLA class II‐binding peptides with CDCA1‐derived CTL‐epitope sequences presented by HLA‐A2 (A*02:01) or HLA‐A24 (A*24:02) to select candidate CDCA1‐LPs encompassing both Th cell epitopes and CTL‐epitopes. We studied the immunogenicity of CDCA1‐LPs and the cross‐priming potential of LPs bearing CTL‐epitopes in both human in vitro and HLA‐class I transgenic mice in vivo. Then we analyzed the Th cell response to CDCA1 in head‐and‐neck cancer (HNC) patients before and after vaccination with a CDCA1‐derived CTL‐epitope peptide using IFN‐γ enzyme‐linked immunospot assays. We identified two CDCA1‐LPs, CDCA139–64‐LP and CDCA155–78‐LP, which encompass naturally processed epitopes recognized by Th cells and CTLs. CDCA1‐specific CTLs were induced through cross‐presentation of CDCA1‐LPs in vitro and in vivo. In addition, CDCA1‐specific Th cells enhanced induction of CDCA1‐specific CTLs. Furthermore, significant frequencies of CDCA1‐specific Th cell responses were detected after short‐term in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with CDCA1‐LPs in HNC patients (CDCA139–64‐LP, 74%; CDCA155–78‐LP, 68%), but not in healthy donors. These are the first results demonstrating the presence of CDCA1‐specific Th cell responses in HNC patients and underline the possible utility of CDCA1‐LPs for propagation of both CDCA1‐specific Th cells and CTLs.

Identification of glypican-3-derived long peptides activating both CD8+ and CD4+ T cells; prolonged overall survival in cancer patients with Th cell response

In a recent phase I clinical trial, a vaccine consisting of glypican-3 (GPC3)-derived CTL epitopes was found to be safe and induced measurable immune and clinical responses in patients with hepatocellular carcinoma (HCC). The aim of this study was to identify GPC3-derived long peptides (GPC3-LPs) carrying promiscuous HLA class II-restricted T helper (Th) cell epitopes. Using a computer algorithm, we predicted GPC3-LPs that can bind to promiscuous HLA class II molecules. Their antigenicity for induction of specific CD4+ T cells in healthy donors or patients with HCC, before and after vaccination with GPC3-SPs, was proven by IFNγ enzyme-linked immunospot assays. Natural processing of these epitopes was confirmed by the immune response of helper T cells to dendritic cells (DCs) loaded with GPC3 proteins. Cross-presentation capacity was assessed in vitro using human DCs and LPs encapsulated in liposomes and in vivo in HLA-A2 transgenic mice (Tgm). All five LPs could induce Th1 cells and were presented by several frequently occurring HLA class II molecules in vitro. Four of them were likely to be naturally processed. One of the LPs encapsulated in liposomes was well cross-presented in vitro; it cross-primed CTLs in HLA-A2 Tgm. LP-specific and HLA class II-restricted CD4+ T-cell responses were observed in 14 of 20 HCC patients vaccinated with GPC3-SPs. Repeated vaccinations enhanced GPC3-LP-specific responses in 8 of 13 patients with HCC. Moreover, the presence of the specific Th cell was correlated with prolonged overall survival (OS). GPC3-LPs can be useful for cancer immunotherapy.

TCR ligand avidity determines the mode of B-Raf/Raf-1/ERK activation leading to the activation of human CD4+ T cell clone.

The interactions between peptide/MHC complexes and their cognate TCR are essential for various T cell responses. However, the relationship between the avidity of TCR ligand and the subsequent intracellular signaling through the TCR is still unclear. To investigate the effects of TCR ligand avidity on TCR‐mediated signaling, we established L cells expressing HLA‐DR4 molecules covalently linked with agonistic peptide (high‐affinity ligand) or altered peptide ligand (APL; low‐affinity ligand) at various densities as APC for a cognate human CD4+ T cell clone. Using this system, we demonstrated that the T cell clone stimulated with APL/HLA‐DR4 complexes presented at an excessive density provoked the up‐regulation of CD69, IL‐2 production and proliferation, but no detectable phosphorylation of ZAP‐70/LAT/SLP‐76. Furthermore, in contrast to the high‐affinity stimulation, the low‐affinity stimulation evoked delayed and sustained activation of the B‐Raf/extracellular signal‐regulated kinase (ERK) pathway without Raf‐1 activation. The strength and duration of B‐Raf/ERK activations closely correlated with the density of the TCR ligand. A knockdown approach confirmed that B‐Raf activation was indispensable for the APL‐induced T cell responses. These observations suggest that the differences in TCR‐peptide/MHC interactions reflect the strength and duration of B‐Raf/Raf‐1/ERK activation in the human CD4+ T cells.