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Although cancer immunotherapy using immune checkpoint inhibitors (ICIs) has been recognized as one of the major treatment modalities for malignant diseases, the clinical outcome is not uniform in all cancer patients. Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of immature myeloid cells that possess various strong immunosuppressive activities involving multiple immunocompetent cells that are significantly accumulated in patients who did not respond well to cancer immunotherapies. We reviewed the perspective of MDSCs with emerging evidence in this review. Many studies on MDSCs were performed in malignant diseases. Substantial studies on the participation of MDSCs on non-malignant diseases such as chronic infection and autoimmune diseases, and physiological roles in obesity, aging, pregnancy and neonates have yet to be reported. With the growing understanding of the roles of MDSCs, variable therapeutic strategies and agents targeting MDSCs are being investigated, some of which have been used in clinical trials. More studies are required in order to develop more effective strategies against MDSCs.

PDF file - 203K, Surface expression of calreticulin on TE2 after 30Gy-irradiation (10Gy irradiation on day 1, day 2, and day 3) or chemotherapy (5FU + CDDP + Docetaxel on day 1)

WMV file - 3260K, Live cell confocal photography of mock- (left panel) and NKG2D-DAP10-CD3ξ-transduced NK cells (right panel) co-cultured U2 OS osteosarcoma cells. NK cells were labeled with PKH26 (Sigma) before culture and appear red. Microscopy was performed with a Nikon TE2000E2 microscope equipped with a Nikon C1Si confocal using 488nm and 561nm DPSS lasers for excitation. Temperature was maintained at 37oC and CO2 at 5% using an environmental control chamber. Images were acquired with a Nikon 40x 1.3 NA DIC objective every 30s for 2hr using Nikon EZC1 software. Imaris (Bitplane Scientific Software) was used to analyze speed and displacement of NK cells.

Although it has been shown that chemoradiotherapy may induce immunogenic cell death, which could trigger T-cell immunity mediated by high-mobility group box 1 protein (HMGB1) and calreticulin, there is still limited information to support this theory directly in a clinical setting. In the present study, we evaluated antigen-specific T-cell responses against six cancer-testis antigens in peripheral blood lymphocytes from patients with esophageal squamous cell carcinoma (ESCC) receiving chemoradiation. Expression of HMGB1 and calreticulin within tumor microenvironment was also analyzed in resected samples with and without chemoradiotherapy in relation to patients survival. Tumor antigen–specific T-cell responses were confirmed in six (38%) of 16 patients with ESCC after chemoradiotherapy coexisting with elevated serum HMGB1. In addition, HMGB1 within tumor microenvironment was significantly upregulated in patients with ESCC with preoperative chemoradiotherapy, but not in those without chemoradiotherapy, and the degree of HMGB1 positively correlated with patient survival (n = 88). Both irradiation and chemotherapeutic drugs induced upregulation of HMGB1 and calreticulin in nine ESCC cell lines. Furthermore, HMGB1 was able to induce maturation of dendritic cells. Together, our findings indicate that chemoradiation induces tumor antigen–specific T-cell responses, and HMGB1 production is related to clinical outcome after chemoradiation. Cancer Res; 72(16); 3967–76. ©2012 AACR.

Natural killer (NK) cells rely on surface receptors to distinguish healthy cells from cancer cells. We designed a receptor termed NKG2D-DAP10-CD3ζ that is composed of the NK cell activating molecule NKG2D plus 2 key signaling molecules, DAP10 and CD3ζ, and evaluated its capacity to promote cancer cell killing. Retroviral transduction of NKG2D-DAP10-CD3ζ markedly increased NKG2D surface expression in NK cells, which became consistently more cytotoxic than mock-transduced cells against leukemia and solid tumor cell lines. In contrast, there was no increase in cytotoxicity against nontransformed blood and mesenchymal cells. NKG2D blockade abrogated gains in cytotoxicity to cancer cells. Receptor stimulation triggered signal transduction, secretion of IFN-γ, GM-CSF, IL-13, MIP-1α, MIP-1β, CCL5, and TNF-α, and massive release of cytotoxic granules, which persisted after 48 hours of continuous stimulation. NKG2D-DAP10-CD3ζ–expressing NK cells had considerable antitumor activity in a mouse model of osteosarcoma, whereas activated NK cells were ineffective. Thus, the cytotoxic potential of NK cells against a wide spectrum of tumor subtypes could be markedly enhanced by expression of NKG2D-DAP10-CD3ζ receptors. The development of an electroporation method that permits rapid expression of the receptor in a large number of human NK cells facilitates clinical translation of this NK-based strategy for a generalized cellular therapy that may be useful to treat a wide range of cancers. Cancer Res; 73(6); 1777–86. ©2012 AACR.

PDF file - 58K, Maturation phenotypes of HMGB1-treated DCs by flow cytometry

PDF file - 50K, Correlation of serum HMGB1 level with grade of antigen-specific T cell responses (Spot-forming cells) by ELISPOT assay against each antigen in patients receiving chemoradiotherapy

Background Several good results of clinical trial of nivolumab or involving nivolumab in advanced esophageal squamous cell carcinoma were reported. However, the response rate was still poor. A rare phenomenon called the “abscopal effect” refers to the regression of not only the irradiated tumor but also non-irradiated distant tumors after local radiotherapy. The mechanism is not completely clear, but it is thought that the activation of anti-tumor immunity induced by radiotherapy is the main factor. Case A 66-year-old man with recurred and nivolumab resistant esophageal squamous cell carcinoma in left-side cervical and abdominal para-aortal lymph node metastasis was treated with a total of 40 Gy (10 fractions) of radiotherapy to the left-side cervical lymph node metastasis which caused neck pain as a palliative treatment. Nivolumab was resumed the day after completion of radiotherapy. At 3 months after radiotherapy showed that the irradiated lesion in the left neck had regressed to a scar-like appearance. Notably, the abdominal para-aortal lymph nodes outside the irradiation area, which had previously tended to progress, had also shrunk (abscopal effect). The T cell receptor and B cell receptor (TCR/BCR) repertoire analysis before and after radiotherapy revealed that radiotherapy caused the changes in the TCR/BCR repertoire. Conclusion Changes in the TCR/BCR receptor repertoire repertoires were assumed to be a part of the mechanism of the abscopal effect. The findings in this patient suggest that combination of immune checkpoint inhibitors and radiotherapy can be a promising treatment approach, even for patients with immune checkpoint inhibitors resistant cancer.

The patient was a 66-year-old male who had undergone an operation for lung cancer and solitary brain metastases. Follow- up PET-CT after 1 year detected FDG accumulation in the stomach. We performed esophagogastroscopy and found an approximately 20 mm-sized Type 2 tumor on the greater curvature of the upper stomach. A pathological diagnosis of lung adenocarcinoma metastasis in the stomach was made. Laparoscopic surgery was performed on the metastatic lesion to prevent bleeding and perforation, and resection was achieved with minimal invasion. The current development of chemotherapy, including immunotherapy, has contributed to the improved prognosis of cancer patients, including those with lung metastasis in the stomach. Considering these backgrounds, preventive surgical resection under laparoscopy may be an effective approach for improving prognosis and preventing acute life-threatening adverse events. We report this case along with a literature review.

4027 Background: Although basic, translational and clinical research suggest a possibility of synergistic effect of radiation-induced immunogenic cell death with immune checkpoint inhibitors, the effectiveness of the combination therapy is not fully established. Therefore, we conducted a single-arm, phase 1/2 trial (ClinicalTrials.gov, NCT03453164) in gastric cancer (GC) patients treated with a combination of nivolumab and oligo-fractionated irradiation (22.5 Gy/5 fractions/5 days). Methods: Eligible patients (n = 40) had un-resectable advanced or recurrent GC which progressed after primary and secondary chemotherapy with more than one lesion assessable in diagnostic imaging (one lesion must be ≥2cm). PBMCs from enrolled patients underwent high-dimensional flow cytometry-based, multiplexed MHC multimer analysis using a total of 46 tumor-associated antigens (TAA) and 10 virus epitopes and next-generation sequencing-based repertoire analysis of TCR β-chain. Results: The disease control rate (DCR) for the non-irradiated target as abscopal effect as the primary endpoint was 22.5%, and the DCR for the irradiated lesion was 40.0%. The median survival time was 230 days (157-330 days, 95%CI) and probability of 1-year survival rate was 28.6%. Although most TAA-specific T cells could be tracked longitudinally pre- and post-treatment, some several novel TAA-specific CD8 T cells were detected de novo after irradiation, indicating that potential irradiation-driven antigen spreading. Moreover, irradiation was associated with phenotypical changes of TAA-specific CD8 T cells towards higher expressions of KLRG1, HLA-DR, TIGIT and CD160 and lower expression of CD27 and CD127. Furthermore, the T cell clonality evaluated by the inverted Pielou’s evenness indicated that longer survival patients had more diverse TCR beta repertoire during treatment in comparison to shorter survivors. Also, we confirmed several new sequence-reads after radiation and nivolumab treatment in the top 30 most frequent clonotypes. Conclusions: Taken together, our results suggest that irradiation may induce, through immunogenic cell death, an immune-modulating effect with potential antigen spreading and a more diverse TCR repertoire, ultimately resulting in better survival during combination therapy of radiation with nivolumab. Clinical trial information: NCT03453164.

Background We conducted a phase I/Ib, open-label, single-arm trial to assess the safety, tolerability and optimal scheduling regimen of OTSGC-A24 cancer vaccine in patients with advanced gastric cancer. Methods Patients with advanced gastric cancer with HLA-A*24:02 haplotype were included in this study. OTSGC-A24 was administered at 1 mg in 3-weekly (3w), 2-weekly (2w), and weekly (1w) cohorts to evaluate the safety, immunological response and schedule. Based on the highest specific cytotoxic T lymphocyte (CTL) induction rate at 4 weeks, using the ELISPOT test, cohorts were expanded to define the optimal dosing schedule for OTSGC-A24. Results In this study, 24 advanced gastric cancer patients with HLA-A*24:02 haplotype were enrolled and treated in 3 cohorts (3w cohort: 3; 2w cohort: 11 and 1w cohort: 10 patients). The most common adverse events were decreased appetite (29%), diarrhea (21%), myalgia (25%). The most common treatment-related adverse event was injection site erythema (25%). No dose-limiting toxicities were observed in any cohort and OTSGC-A24 was well tolerated. Positive CTL responses after vaccination were observed in 15 patients (75%) at 4 weeks: 3w cohort (33%), 2w cohort (88%), 1w cohort (78%). At 12 weeks, 18 patients had responded (90%); 3w cohort (100%), 2w cohort (100%), 1w cohort (78%). The best radiological was stable disease (40%). Median progression free survival was 1.7 months (95% CI: 1.4 to 3.5) and median overall survival was 5.7 months (95% CI 3.8 to 8.6). Conclusions OTSGC-A24 combined peptide cancer vaccine was well tolerated. Significant responses in CTL were observed and the recommended phase 2 dose is 1 mg OTSGC-A24 sub-cutaneous, every 2 weeks. Although no radiological response was observed, a respectable overall survival was achieved, consistent with other immunotherapy agents being investigated in gastric cancer. Trial registration ClinicalTrials.gov Identifier: NCT01227772, Date registered: 21 Oct 2010.

Despite multidisciplinary treatment for patients with advanced gastric cancer, their prognosis remains poor. Therefore, the development of novel therapeutic strategies is urgently needed, and immunotherapy utilizing anti-programmed death 1/-programmed death ligand-1 mAb is an attractive approach. However, as there is limited information on how programmed death ligand-1 is upregulated on tumor cells within the tumor microenvironment, we examined the mechanism of programmed death ligand-1 regulation with a particular focus on interferon gamma in an in vitro setting and in clinical samples. Our in vitro findings showed that interferon gamma upregulated programmed death ligand-1 expression on solid tumor cells through the JAK-signal transducer and activator of transcription pathway, and impaired the cytotoxicity of tumor antigen-specific CTL against tumor cells. Following treatment of cells with anti-programmed death ligand-1 mAb after interferon gamma-pre-treatment, the reduced anti-tumor CTL activity by interferon gamma reached a higher level than the non-treatment control targets. In contrast, programmed death ligand-1 expression on tumor cells also significantly correlated with epithelial-mesenchymal transition phenotype in a panel of solid tumor cells. In clinical gastric cancer samples, tumor membrane programmed death ligand-1 expression significantly positively correlated with the presence of CD8-positive T cells in the stroma and interferon gamma expression in the tumor. The results suggest that gastric cancer patients with high CD8-positive T-cell infiltration may be more responsive to anti-programmed death 1/-programmed death ligand-1 mAb therapy.

Natural killer (NK) cells can react with tumor cells through the balance of inhibitory and stimulatory signals between NK cell surface receptors and their ligands, such as MHC class I chain-related A (MICA), MHC class I chain-related B (MICB), and several UL16-binding proteins (ULBPs). In the present study, we evaluated the relationship between NKG2D ligand expression and matrix metalloproteinase (MMP) activity in in vitro culture systems of a panel of gastric cancer cell lines (n = 10) and clinical samples (n = 102). First, the surface expression of NK group 2 member D (NKG2D) ligands (MICA, MICB, ULBP-2, and ULBP-3) on tumor cells was markedly downregulated on in vitro culture, in parallel to the upregulation of MMPs analyzed by gelatin zymography and gene expression microarray, whereas the transcript levels of NKG2D ligands remained unchanged on in vitro culture. Second, MMP-specific inhibitors could restore the downregulated expression of NKG2D ligands and functionally improve susceptibilities to NK cells in vitro. Third, the production of soluble NKG2D ligands was increased on in vitro culture and was inhibited by MMP-specific inhibitors. Finally, there was a significant inverse correlation between MMP-9 expression and NKG2D ligand expression as analyzed by immunohistochemistry in clinical tumor samples. The present study is a comprehensive study demonstrating that upregulation of MMP activity can induce a downregulation of expression of NKG2D ligands in gastric cancer cells, leading to lower-level susceptibility to NK cells.

X-ray radiotherapy activates tumor antigen-specific T-cell responses, and increases in the serum levels of high mobility group box 1 (HMGB1) induced by X-ray irradiation play a pivotal role in activating anti-tumor immunity. Here, we examined whether carbon-ion beams, as well as X-rays, can induce HMGB1 release from human cancer cell lines. The study examined five human cancer cell lines: TE2, KYSE70, A549, NCI-H460 and WiDr. The proportion of cells surviving X- or carbon-ion beam irradiation was assessed in a clonogenic assay. The D10, the dose at which 10% of cells survive, was calculated using a linear-quadratic model. HMGB1 levels in the culture supernatants were assessed by an ELISA. The D10 dose for X-rays in TE2, KYSE70, A549, NCI-H460 and WiDr cells was 2.1, 6.7, 8.0, 4.8 and 7.1 Gy, respectively, whereas that for carbon-ion beams was 0.9, 2.5, 2.7, 1.8 and 3.5 Gy, respectively. X-rays and carbon-ion beams significantly increased HMGB1 levels in the culture supernatants of A549, NCI-H460 and WiDr cells at 72 h post-irradiation with a D10 dose. Furthermore, irradiation with X-rays or carbon-ion beams significantly increased HMGB1 levels in the culture supernatants of all five cell lines at 96 h post-irradiation. There was no significant difference in the amount of HMGB1 induced by X-rays and carbon-ion beams at any time-point (except at 96 h for NCI-H460 cells); thus we conclude that comparable levels of HMGB1 were detected after irradiation with iso-survival doses of X-rays and carbon-ion beams.

The response rate of anti-PD-1/anti-PD-L1antibody alone is about 20 to 30%and the development of biomarker for them is important to know their indication. Based on previous reports and our research results, we suggested that basic candidates of biomarker for anti-PD-1/anti-PD-L1antibody are the expression of PD-L1and HLA class I on cancer cells and the invasion of CD8 positive T cells in tumor microenvironment. Furthermore, in addition to these conditions, regulatory T cells and immune cells expressing PD-L1in tumor microenvironment, and microsatellite instability of cancer cells will be considered in the future.

It has been reported that chemo-radiotherapy can induce immunogenic tumor cell death (ICD), which triggers T-cell immunity mainly mediated by high-mobility group box 1 protein (HMGB1) and calreticulin. However, there is still limited information to support this theory relating to chemotherapy alone. In the present study, the expression of HMGB1 and calreticulin was evaluated by immunohistochemistry in pre-treatment biopsy specimens and surgically resected specimens, which were obtained from patients with breast cancer (n=52) and esophageal squamous cell carcinoma (ESCC) (n=8) who had been treated with neoadjuvant chemotherapy (NAC). We also analyzed HMGB1 and calreticulin expression in breast cancer cell lines treated with chemotherapeutic drugs. As a result, both HMGB1 and calreticulin expression levels were significantly upregulated after NAC in both breast cancer and ESCC tissues. However, no significant correlation was observed between HMGB1 expression and pathological response after NAC or between HMGB1 expression and patient survival. Furthermore, although overall survival in the high infiltration group of CD8-positive T cells was significantly superior to that in the low infiltration group in breast cancer patients, there were no correlations between the number of CD8-positive T cells and HMGB1 or calreticulin expression levels. In addition, chemotherapeutic drugs induced upregulation of HMGB1 and calreticulin in all tested cell lines. Our findings indicate that chemotherapy alone can significantly induce ICD regardless of the degree of pathological response after chemotherapy.

Programmed cell death 1(PD-1)/PD-ligand 1(PD-L1)immune checkpoint blockade has emerged as a promising therapeutic strategy in various types of cancer. In a recent phase II clinical trial, treatment with the anti-PD-1 agent, pembrolizumab, resulted in considerable clinical benefit in patients with mismatch repair(MMR)-deficient colorectal cancer(CRC). Upregulation of PD-1on T-cells and PD-L1 on tumor cells induces inhibitory signals to suppress T-cell activation, leading to an immune-suppressive microenvironment particularly in MMR-deficient tumors. However, the regulation of PD-L1 expression on CRC cells is poorly understood. We hypothesized that certain microRNAs(miRNAs)are involved in the immunosuppressive microenvironment by directly targeting PD-L1. We identified candidate miRNAs by RNA-sequence analyses for mRNA and miRNA expression obtained from the TCGA colon adenocarcinoma database combined with miRNA target prediction programs. We found that forced miRNA expression could decrease PD-L1 expression on cancer cell lines. Our findings may facilitate an understanding of the role of miRNAs in PD-L1 regulation and also suggest potential miRNAs to serve as biomarkers and therapeutic targets for cancer immunotherapy.

Therapeutic cancer vaccine enhances a specific immune response against tumor cells in vivo, resulting in exertion of antitumor effects. On the other hand, immune checkpoint inhibitors promote the induction of tumor-specific T cells and also enhance the cytotoxic abilityof these T cells in tumor microenvironment. There is a possibilitythat immune checkpoint inhibitors enhance tumor immune responses induced bytherapeutic cancer vaccine, and it is expected that additive or synergistic effects will be obtained bythe combination of them. Moreover, according to previous reports, we should use an immune checkpoint inhibitor to enhance the cytotoxic ability of tumor-specific T cells as the combination for therapeutic cancer vaccine. Furthermore, the combination of a specific antibodyagainst newlyidentified co-inhibitoryreceptors (Lag-3, Tim-3, TIGIT, etc)and a therapeutic cancer vaccine is also one of newlyexpected treatments in the future.

Immunotherapy has become a promising treatment strategy for cancer. Immune checkpoint blockade with anti-CTLA4 mAb and anti-PD-1 mAb has demonstrated clear evidence of objective responses including improved overall survival and tumor shrinkage, driving renewed enthusiasm for cancer immunotherapy in multiple cancer types including esophageal squamous cell carcinoma (ESCC). There are several clinical trials using anti-PD1 mAb for ESCC in early phases and the results are currently promising. In this review, recent advances in cancer immunotherapy for ESCC are discussed with particular focus on immune checkpoint inhibitors and cancer vaccine.

To develop more effective therapies for patients with advanced gastric cancer, we examined the potential of ex vivo expanded natural killer (NK) cells. We assessed the expression of ligands for NK Group 2 Member D (NKG2D, an important NK activation molecule) in primary tumors from 102 patients with gastric cancer by immunohistochemistry and determined their prognostic value. We then examined the in vitro and in vivo cytotoxicity of NK cells from healthy donors and patients with gastric cancer. The cytotoxicity of resting and of interleukin (IL)-2-activated NK cells was compared to that of NK cells expanded for 7 days by coculture with the K562-mb15-4.1BBL cell line. As a result, the expression of NKG2D ligands in primary tumors was correlated with favorable presenting features and outcomes, suggesting that gastric cancer may be sensitive to NK cell cytotoxicity. Although resting NK cells showed minimal cytotoxicity against gastric cancer cells, K562-mb15-4.1BBL-expanded NK cells were highly cytotoxic and significantly more powerful than IL-2-activated NK cells. Cytotoxicity was correlated with NKG2D ligand expression and could be modulated by mitogen-activated protein kinase and AKT-PI3 kinase inhibitors. The cytotoxicity of expanded NK cells against HER2-positive gastric cancer cells could be increased by Herceptin and further augmented by Lapatinib. Finally, expanded NK cells exhibited strong antitumor activity in immunodeficient mice engrafted with a gastric cancer cell line. In conclusion, gastric cancer tumors express NKG2D ligands and are highly susceptible to killing by NK cells stimulated by K562-mb15-4.1BBL. These results provide a strong rationale for clinical testing of these NK cells in patients and suggest their use to augment the effects of antibody therapy.

Background Immune checkpoint inhibitors have been reported to be ineffective in patients with high levels of MDSC (myeloid-derived suppressor cells) and MDSC has been proven to be stimulated by inflammation involving VEGF and IL-17. We have reported that MDSC, IL-17 and VEGF are closely associated with immunosuppression and poor prognosis. Methods Stimulation index (SI) obtained by PHA-blastogenic response of lymphocyte was used as a marker of cell-mediated immunity and was tested in 658 patients with unresectable diseases including 260 esophageal, 110 gastric and 288 colorectal cancers. These patients received chemotherapy and SI were analyzed in correlation with nutritional status, treatment outcome (RECIST) and prognosis. These patients were divided into 2 groups with a median level of SI and, chemotherapy outcome and overall survival were compared between these groups (Study 1). To characterize the mechanisms of immunosuppression, PBMC (peripheral blood cells) were separated from the patients and the production of IL-17 (ELISA) by PBMC. Circulating levels of MDSC (CD14-CD11b+CD33+/flow cytometry), serum levels of VEGF (ELISA) were measured and analyzed in 43 patients with gastric cancer and 64 with colorectal cancer (Study 2). Results (Study 1) The overall survival was significantly worse in patients with lower SI than in those with higher SI in each disease, and the responses (RECIST) to chemotherapy were significantly better in patients with higher SI than those with lower SI. SI were significantly inversely correlated with NLR (neutrophil/lymphocyte ratio: inflammatory parameter) and nutritional factors including prealbumin, retinol-binding protein and transferrin. (Study 2) SI were significantly and inversely correlated with the levels of MDSC, the production of IL-17, and the levels of VEGF. The levels of MDSC, VEGF, IL-17 were significantly correlated with each other and inversely with nutritional parameters. Conclusion The key mechanisms of immunosuppression in these patients are systemic inflammation involving MDSC, VEGF and IL-17. Immune checkpoint inhibitors might be more effective with a decreased MDSC by controlling VEGF and IL-17. Legal entity responsible for the study Masahiko Shibata. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

HLA class I and PD-L1expressed on cancer cells play a pivotal role in the CTL recognition mechanism against cancer cells in the tumor microenvironment. It is well known that IFN-g upregulates PD-L1as well as HLA class I expression in cancer cells, and it is suggested that TILs, including CTL, produce IFN-g in the tumor microenvironment. Therefore, there is a possibility that IFN-g produced by activated TILs upregulate both HLA class I and PD-L1expression in cancer cells in the tumor microenvironment. We propose that the anti-tumor effect of CTL could be enhanced if the inhibition of CTL recognition mechanism against cancer cells via the PD-1/PD-L1pathway is canceled by anti-PD-1or anti-PD-L1antibody.

Novel, tumor-specific drugs are urgently needed for a breakthrough in cancer therapy. Herein, we generated a first-in-class humanized antibody (PRL3-zumab) against PRL-3, an intracellular tumor-associated phosphatase upregulated in multiple human cancers, for unconventional cancer immunotherapies. We focused on gastric cancer (GC), wherein elevated PRL-3 mRNA levels significantly correlated with shortened overall survival of GC patients. PRL-3 protein was overexpressed in 85% of fresh-frozen clinical gastric tumor samples examined but not in patient-matched normal gastric tissues. Using human GC cell lines, we demonstrated that PRL3-zumab specifically blocked PRL-3+, but not PRL-3–, orthotopic gastric tumors. In this setting, PRL3-zumab had better therapeutic efficacy as a monotherapy, rather than simultaneous combination with 5-fluorouracil or 5-fluorouracil alone. PRL3-zumab could also prevent PRL-3+ tumor recurrence. Mechanistically, we found that intracellular PRL-3 antigens could be externalized to become “extracellular oncotargets” that serve as bait for PRL3-zumab binding to potentially bridge and recruit immunocytes into tumor microenvironments for killing effects on cancer cells. In summary, our results document a comprehensive cancer therapeutic approach to specific antibody-targeted therapy against the PRL-3 oncotarget as a case study for developing antibodies against other intracellular targets in drug discovery.

Adoptive transfer of immune cells, such as T lymphocytes and NK cells, has potential to control cancer growth. However, this can be counteracted by immune escape mechanisms within the tumor microenvironment, including those mediated by reactive oxygen species (ROS). Here, we determined the levels of anti-oxidant molecules in NK cells and their capacity to overcome ROS-induced immune suppression. We investigated the effect of H2O2 on resting NK cells, IL-2-activated NK cells and NK cells expanded by coculture with the K562 leukemia cell line genetically modified to express membrane-bound IL-15 and 4-1BB ligand (K562-mb15-41BBL). Expression of anti-oxidant and anti-apoptotic genes was evaluated by expression array, and protein levels of anti-oxidant molecules by Western blot. Activated NK cells, IL-2-activated NK cells and NK cells expanded by K562-mb15-41BBL were significantly more resistant to H2O2-induced cell death than resting NK. Thioredoxin-1 (TXN1) and peroxiredoxin-1 (PRDX1) were also up-regulated in activated NK cells. Moreover, H2O2-induced cell death after IL-2 activation was significantly induced in the presence of an anti-TXN1-neutralising antibody. Collectively, these data document that activated NK cells can resist to H2O2-induced cell death by up-regulation of TXN1.

HER2 is a promising target for immunotherapeutic interventions with T cell-based approaches since it is amplified and overexpressed in 20-30% of breast cancers. However, several previous studies including ours showed that HER2-overexpressing tumors may escape cytotoxic T lymphocyte-mediated lysis by downregulating MHC Class I and components of the antigen-processing machinery. The aims of the present study were to analyze the relationship between HER2 and MHC Class I expression and to elucidate the mechanisms underlying MHC Class I downregulation in breast cancer. We explored expression of HER2, MHC Class I, PTEN, Ki67, estrogen and progesterone expression in 70 breast cancer patients by immunohistochemistry (IHC) and analyzed their correlation. We also explored the components of the signal transduction pathway that are involved in the regulation of MHC Class I expression using small-interfering RNAs targeting HER2 as well as an inhibitor of HER2 signaling. HER2 expression in breast cancers correlated inversely with MHC Class I expression analyzed by IHC. HER2 depletion by small-interfering RNAs resulted in MHC Class I upregulation. Moreover, MHC Class I expression on breast cancer cell lines was upregulated by PD98059, an inhibitor of mitogen-associated protein kinases, in a dose-dependent manner. Thus, agents that target the MAPK signaling pathway may increase MHC Class I expression in breast cancer cells.

Although an increase in regulatory T cells (Tregs) is observed in tumor microenvironments, the underlying mechanism is not fully clarified. Since it was suggested that Tregs showed a lower sensitivity toward oxidative stress in comparison with conventional T cells, in the present study, we investigated the H(2)O(2) production and apoptosis of Tregs in gastric and esophageal cancer tissues, employing flow cytometric analysis using fresh samples (n = 93) and immunohistochemical analysis (n = 203).The increased tumor-infiltrating Tregs coexisted with elevated H(2)O(2) production according to disease progression. The grade of apoptosis in Tregs was less pronounced than that in conventional T cells, and there was a positive correlation between H(2)O(2) production and the grade of apoptosis in conventional T cells, while there was no correlation between H(2)O(2) production and the grade of apoptosis in Tregs. Moreover, Tregs were less sensitive to H(2)O(2)-induced apoptosis compared with conventional T cells in vitro.We have demonstrated that the increased prevalence of tumor-infiltrating Tregs closely related to their lower sensitivity to H(2)O(2)-induced apoptosis.

The extracellular matrix protein biglycan (Bgn) is a leucine-rich proteoglycan that is involved in the matrix assembly, cellular migration and adhesion, cell growth, and apoptosis. Although a distinct expression of Bgn was found in a number of human tumors, the role of this protein in the initiation and/or maintenance of neoplastic transformation has not been studied in detail. Using an in vitro model of oncogenic transformation, a down-regulation of Bgn expression as well as an altered secretion of different Bgn isoforms was found both in murine and human HER-2/neu oncogene-transformed cells when compared with HER-2/neu− cells. This was associated with a reduced growth, wound closure, and migration capacity. Vice versa, silencing of Bgn in HER-2/neu− fibroblasts increased the growth rate and migration capacity of these cells. Bgn expression was neither modulated in HER-2/neu+ cells by transforming growth factor-β1 nor by inhibition of the phosphoinositol 3-kinase and MAP kinase pathways. In contrast, inhibition of the protein kinase C (PKC) pathway led to the reconstitution of Bgn expression. In particular, the PKC target protein cAMP response element binding protein (CREB) is a major regulator of Bgn expression as the silencing of CREB by RNA interference was accompanied by ∼5000-fold increase in Bgn-mRNA expression in HER-2/neu+ cells. Thus, Bgn inhibits the major properties of HER-2/neu-transformed cells, which is inversely modulated by the PKC signaling cascade. Background: Oncogenic transformation has been shown to down-regulate components of the extracellular matrix. Results: HER-2/neu-mediated oncogenic transformation causes silencing of biglycan gene expression. Reconstitution of biglycan expression led to an impaired proliferation and migration of oncogenic transformed cells. Conclusion: HER-2/neu-mediated silencing of Bgn expression may promote tumor cell proliferation and migration. Significance: Biglycan represents a putative therapeutic target for the treatment of HER-2/neu+ tumor cells.

Human NK cells can be divided into two subsets, CD56dimCD16(+)NK and CD56brightCD16(−)NK cells, based on their expression of CD56 and CD16. In the present study, we analyzed the relationship between CD56dim/CD56bright NK cells and H2O2 in tumor-infiltrating NK cells in patients with gastric (n = 50) and esophageal (n = 35) cancer. The ratio of CD56dim NK cells infiltrating tumors gradually decreased according to disease progression. H2O2 was abundantly produced within tumor microenvironments, and there was an inverse correlation between CD56dim NK cell infiltration and H2O2 production. CD56dim NK cells are more sensitive to apoptosis induced by physiological levels of H2O2 than CD56bright NK cells. Furthermore, the exposure of NK cells to H2O2 resulted in the impairment of ADCC activity. In conclusion, H2O2 produced within tumor microenvironments inversely correlated with the infiltration of CD56dim NK cells, possibly due to their preferentially induced cell death. These observations may explain one of the mechanisms behind NK cell dysfunction frequently observed in tumor microenvironments.

Although Th17 cells reportedly play critical roles in the development of autoimmunity and allergic reactions, information on Th17 cells in cancer-bearing hosts is still limited. In the present study, we investigated the distribution of Th17 cells in relation to regulatory T cells (Treg) in the tumor-infiltrating lymphocytes (TILs), regional lymph node lymphocytes, and peripheral blood lymphocytes of gastric cancer patients. Interleukin (IL)-17-producing CD4(+) cells as Th17 cells and CD4(+)CD25(+)FoxP3(+) cells as Treg were evaluated by flow cytometry and expressed as a percentage of the total CD4(+) cells, in addition to performing a Th1/Th2 balance assay. Moreover, immunohistochemical staining for IL-17 and FoxP3 were performed. In TILs from patients with early disease (n = 27), the frequency of Th17 cells was significantly higher than that in the normal gastric mucosa (23.7 ± 8.9 vs 4.5 ± 3.1%). In TILs from patients with advanced disease (n = 28), the frequency of Th17 cells was also significantly higher, but lower compared to early disease, than that in the normal gastric mucosa (15.1 ± 6.2 vs 4.0 ± 2.0%). This observation for Th17 cell-distribution was also confirmed by immunohistochemistry. When the ratio of Th17/Treg in TILs was evaluated in individual cases, it was more markedly increased in early than in advanced disease. In conclusion, the accumulation of Th17 cells as well as Treg in the tumor microenvironment of gastric cancer occurred in early disease and then the infiltration of Th17 cells gradually decreased according to the disease progression, in contrast to increased Treg.

Background: As HER2 is expressed in 30% of oesophageal squamous cell carcinomas (ESCCs), T-cell-based immunotherapy and monoclonal antibodies targeted against HER2 are attractive, novel approaches for ESCCs. However, it was shown that there is an inverse correlation between HER2 and MHC class I expression on tumours. Thus, the correlation between HER2 and MHC class I expressions on ESCC was evaluated. Methods: Expressions of MHC class I and HER2 in ESCC tissues (n=80) and cell lines were assessed by immunohistochemistry, fluorescence in situ hybridisation (FISH), and flow cytometry. We investigated whether HER2 downregulation with small interfering RNA (siRNA) in ESCC cell lines could upregulate the expression of MHC class I and the antigen presentation machinery components, and could increase their sensitivity for tumour antigen-specific CTLs. Results: There was an inverse correlation between HER2 and MHC class I expressions in both tumour tissues and cell lines. Downregulation of HER2 with siRNA resulted in the upregulation of MHC class I expression, leading to increased CTL recognition by tumour antigen-specific CTLs. Conclusion: HER2-overexpressing ESCC tumour cells showed a reduced sensitivity for CTLs through the downregulation of MHC class I.

85 Background: Programmed cell death 1 (PD-1) / PD-ligand 1 (PD-L1) immune checkpoint blockade has emerged as a promising therapeutic strategy in various types of cancer. Here we report that certain microRNAs (miRNAs) are involved in immunosuppressive microenvironment by directly targeting PD-L1 in mismatch repair deficient (dMMR) colorectal cancer (CRC). Methods: We identified candidate miRNAs by using RNA-sequence analyses for mRNA and miRNA expression obtained from The Cancer Genome Atlas (TCGA) Colon Adenocarcinoma combined with miRNA target prediction programs. HCT116 and SW837 CRC cell lines were transfected with miRNA mimics and inhibitors, and PD-L1 expression was examined by qRT-PCR, western blotting and flow cytometry. The CRC cell lines were co-cultured with activated T cells and then cytotoxicity for T cells were evaluated. Results: Using miRNA expression profiles of 260 tumors obtained through TCGA Colon Adenocarcinoma, 47 miRNA probes were found to be inversely correlated with PD-L1 expression. Among them, 19 mature miRNAs were down-regulated in dMMR tumors. Furthermore, eight in silico miRNA-target prediction programs were utilized to identify candidate miRNAs that target the 3’UTR of PD-L1 mRNA. We found that forced miRNA expression decreased PD-L1 expression in protein (total and surface) and mRNA levels, and they also dramatically decreased IFN-induced cell surface PD-L1 expression by 32% in CRC cell lines. Furthermore, we found that forced miRNA expression decreased PD-L1 associated apoptotic cell death in co-cultured activated T cells. Conclusions: Our findings suggest that PD-L1 expression is at least in part regulated by miRNAs and may also suggest potential miRNAs to serve as biomarkers and therapeutic targets for cancer immunotherapy.

Background: We previously reported that Trastuzumab- and Cetuximab-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) in cancer patients was impaired in comparison with that in healthy donors because of NK-cell dysfunction. In this study, we evaluated whether IL-21 could improve the impairment of ADCC in patients with oesophageal squamous cell carcinoma (ESCC), as IL-21 was reported to have the ability to activate NK cells. Methods: We examined Trastuzumab- and Cetuximab-mediated ADCC of peripheral blood mononuclear cells (PBMCs) or of enriched NK cells derived from ESCC patients (n=20) and healthy donors (n=16) in the presence of IL-21. We further analysed ADCC-related molecules (perforin, granzyme-B, and CD247) on NK cells in response to IL-21. Results: Trastuzumab- and Cetuximab-mediated ADCC of PBMCs or of enriched NK cells was enhanced by the addition of IL-21 in a dose-dependent manner and the levels of ADCC enhanced by IL-21 in patients were high enough in comparison with those in healthy donors, paralleling the upregulation of CD247 on NK cells. Conclusion: IL-21 could efficiently restore impaired ADCC in ESCC patients with the upregulation of CD247 molecules.

Patients with diseases characterized by chronic inflammation, caused by infection or cancer, have T cells and NK cells with impaired function. The underlying molecular mechanisms are diverse, but one of the major mediators in this immune suppression is oxidative stress caused by activated monocytes, granulocytes, or myeloid-derived suppressor cells. Reactive oxygen species can seriously hamper the efficacy of active immunotherapy and adoptive transfer of T and NK cells into patients. In this study, we have evaluated whether enhanced expression of the antioxidant enzyme catalase in human T cells can protect them against reactive oxygen species. Human CD4+ and CD8+ T cells retrovirally transduced with the catalase gene had increased intracellular expression and activity of catalase. Catalase transduction made CD4+ T cells less sensitive to H2O2-induced loss-of-function, measured by their cytokine production and ability to expand in vitro following anti-CD3 stimulation. It also enhanced the resistance to oxidative stress-induced cell death after coculture with activated granulocytes, exposure to the oxidized lipid 4-hydroxynonenal, or H2O2. Expression of catalase by CMV-specific CD8+ T cells saved cells from cell death and improved their capacity to recognize CMV peptide-loaded target cells when exposed to H2O2. These findings indicate that catalase-transduced T cells potentially are more efficacious for the immunotherapy of patients with advanced cancer or chronic viral infections.

Background and Methods:It has been shown that T-cell dysfunction, including apoptosis of peripheral blood T cells, commonly occurs in patients receiving chemotherapy. In order to evaluate whether concomitant administration of the oral biological response modifier protein-bound polysaccharide K (PSK) could induce anti-apoptotic effects in patients treated with the anti-cancer drug, S-1, peripheral blood T cells were analyzed for induction of apoptosis, caspase-3 activities and expression of proapoptotic protein Bax and anti-apoptotic protein Bcl-2 in patients with curatively resected stage III gastric cancer, who were randomly assigned to postoperative adjuvant therapy with S-1 alone (n = 10) or S-1 combined with PSK (n = 10). Results: T-cell apoptosis 5 weeks after adjuvant therapy was significantly higher in the S-1 group (24.1 ± 5.0%) than in the S-1 + PSK group (19.1 ± 3.9%). S-1 induced T-cell apoptosis and concomitantly elevated caspase-3 activities and Bax expression in peripheral blood T cells. In addition, PSK partially prevented the T-cell apoptosis induced by S-1. Conclusion:PSK could partially prevent the T-cell apoptosis induced by S-1.

To evaluate the possibility of treatment with antiepidermal growth factor receptor (EGFR) mAb, Cetuximab against esophageal squamous cell carcinoma (SCC), we performed detail analysis of the antibody-dependent cellular cytotoxicity (ADCC) mediated by Cetuximab against esophageal SCC. Esophageal SCC cell lines with various levels of EGFR (n = 8) were evaluated for their Cetuximab-mediated ADCC by (51)Cr-release assay. As a result, Cetuximab was able to induce ADCC against EGFR-expressing esophageal SCC and the activities reflected the degree of EGFR expression on the esophageal SCC. The activities of Cetuximab-mediated ADCC by patients' PBMC were impaired in comparison with those by healthy donors' PBMC. Moreover, the inhibition of transforming growth factor (TGF)-beta could enhance Cetuximab-mediated ADCC against TGF-beta-producing SCC. In conclusion, Cetuximab was able to induce ADCC against EGFR-expressing esophageal SCC. Some modalities aiming at enhancing the Cetuximab-mediated ADCC may be necessary for successful Cetuximab treatment of patients with esophageal SCC.

Dendritic cells (DCs) are the most potent antigen-presenting cells and play a central role in the host-antitumor immunity. Since it has been reported that vascular endothelial growth factor (VEGF) inhibits the functional maturation of immature-DCs and impairs DC differentiation, it is important to elucidate the mechanisms of VEGF-induced DC-dysfunction. To investigate the effects of VEGF against human mature DCs, we investigated how VEGF affects mature DCs with regards to phenotype, induction of apoptosis, IL-12(p70) production and the antigen-presenting function evaluated by allogeneic mixed leukocyte reaction (allo-MLR). We generated monocyte-derived DCs matured with lipopolysaccharide, OK-432 or pro-inflammatory cytokine cocktails. As a result, VEGF treatment did not alter the mature DCs with regard to phenotype, IL-12(p70) production and induction of apoptosis. As a novel and important finding, VEGF inhibited the ability of mature DCs to stimulate allogeneic T cells. Furthermore, this VEGF-induced DC dysfunction was mainly mediated by VEGF receptor-2 (VEGF R2). These observations were confirmed by the findings that the VEGF-induced DC dysfunction was recovered by anti-human VEGF neutralizing mAb or anti-human VEGF R2 blocking mAb, and that placenta growth factor (PlGF), VEGF R1-specific ligand, did not have any effect against mature DCs. Some modalities aiming at reversing mature-DC dysfunction induced by VEGF will be needed in order to induce the effective antitumor immunity.

Purpose: In order to broaden the possibility for anti-MAGE-3 immune targeting, it is important to identify HLA-A24-restricted epitopes derived from MAGE-3, since HLA-A24 is one of the most common alleles in Japanese and Asian people. In the present study, we defined a new MAGE-3 derived, HLA-A24-binding peptide presented as a CTL epitope on gastrointestinal cancer cells. Materials and Methods: A panel of MAGE-3-derived peptides (9mer and 10mer) with the HLA-A24-binding motif was selected, and identification of MAGE-3-derived, HLA-A24-restricted CTL epitopes was performed by a reverse immunology approach. To induce MAGE-3-peptide specific CTLs, PBMCs were repeatedly stimulated with monocyte-derived, mature DCs pulsed with the peptides. Subsequent peptide-induced T cells were tested for their specificities by ELISPOT, tetramer and cytotoxic assay. CTL clones were then obtained from the CTL line by limiting dilution. Results: The peptide-inducing CTLs revealed that MAGE-3(113)-peptide was reacted as a CTL epitope in a HLA-A24-restricted fashion, confirmed by ELISPOT and cytotoxic assays. In addition, the MAGE-3(113)-specific CTL clones, confirmed by tetramer assay, showed that the MAGE-3(113) epitope is naturally processed and presented as the CTL epitope on MAGE-3-expressing gastrointestinal cancer cells by evaluating the cold target inhibition assays. Conclusion: The newly identified MAGE-3(113)-peptide epitope is naturally processed and presented as the CTL epitope on MAGE-3-expressing gastrointestinal cancer cells, indicating that anti-MAGE-3 immune targeting with the MAGE-3(113) peptide is a promising approach for treatment.

Recently, we reported that human leukocyte antigen (HLA) class I expression is predominantly regulated by the mitogen-activated protein kinase (MAPK) pathway as one of the oncogenic regulations of HLA class I expression. In the present study, we examined mechanisms of how HLA class I and PD-L1 are regulated by MAPK inhibitors and interferon-γ (IFN-γ). Furthermore, we evaluated the expression of major signal transduction molecules by Western blot and anti-tumor CTL activity by a cytotoxic assay when HLA class I and PD-L1 were modulated by MAPK inhibitors and/or IFN-γ. As a result, we confirmed, as a more general phenomenon, that the inhibition of MAPK could upregulate HLA class I expression in a panel of human solid tumors (n = 26). Of note, we showed that MAPK inhibitors act on the upregulation of HLA class I expression through a different pathway from IFN-γ; there was an additive effect in the upregulation of HLA class I when treated with the combination of MAPK inhibitors and IFN-γ, and there was no overlapping activation of JAK2/STAT1 and Erk1/2 molecules when treated with either IFN-γ or MAPK inhibitors. Furthermore, we showed that IFN-γ–treatment impaired the tumor-specific CTL activity due to the upregulation of PD-L1 in spite of the upregulation of HLA class I, while MAPK inhibitors can augment the tumor-specific CTL activity due to the upregulated HLA class I without PD-L1 alterations. In conclusion, in addition to the original anti-proliferative activity, MAPK inhibitors may work toward the enhancement of T-cell-mediated anti-tumor immunity through the upregulation of HLA class I without the upregulation of PD-L1.

Downregulation of HLA class I expression may contribute to a poor prognosis in cancer patients. There is limited information about epigenetic and oncogenic regulation of HLA class I, and multiple mechanisms may be involved. In the current study, we examined the relationship between the HER2-signaling pathway (MAPK and PI3K-Akt) and the expression of HLA class I and Ag-processing machinery (APM) components. A panel of gastric and esophageal cancer cell lines was treated with wortmannin as an Akt-signal inhibitor; the MAPK signal inhibitor PD98059; lapatinib, which inhibits both the epidermal growth factor receptor and HER2 tyrosine kinase; or siRNA for MAPK. The levels of HER2-signaling molecules, APM components, and HLA class I were evaluated by Western blot, quantitative PCR, and flow cytometry. Resected gastric tumor tissues (n = 102) were analyzed for p-Erk and HLA class I expression by immunohistochemistry. As a result, inhibition of the MAPK pathway induced upregulation of HLA-A02 and HLA-A24 expression in parallel with an increase in APM components and enhanced target sensitivity to tumor Ag–specific CTL lysis. HLA-A expression was predominantly regulated by the MAPK pathway, but it was also influenced, in part, by the Akt pathway. There was a strong inverse correlation between p-Erk expression and HLA class I expression in clinical tumor samples. In conclusion, HLA-A expression is predominantly regulated by the MAPK pathway in gastric and esophageal cancer.

Chemoradiotherapy can induce immunogenic cell death, triggering danger signals such as high-mobility group box 1 protein, and resulting in T-cell immunity. This concept can potentially be harnessed for clinical therapy to enhance tumor-specific immunity. There is however limited information to translate this theory directly in a clinical setting. In this review, we will discuss and summarize molecular and cellular mechanisms underlying immunogenic tumor cell death induced by chemoradiotherapy, with emphasis on a clinical translation.