BACKGROUND: Gastric cancer is a major oncological challenge, ranking highly among causes of cancer-related mortality worldwide. This study was initiated to address the variability in patient responses to combination chemotherapy, highlighting the need for personalized treatment strategies based on genomic data. METHODS: We analyzed whole-genome and RNA sequences from biopsy specimens of 65 advanced gastric cancer patients before their chemotherapy treatment. Using machine learning techniques, we developed a model with 123 omics features, such as immune signatures and copy number variations, to predict their chemotherapy outcomes. RESULTS: The model demonstrated a prediction accuracy of 70-80% in forecasting chemotherapy responses in both test and validation cohorts. Notably, tumor-associated neutrophils emerged as significant predictors of treatment efficacy. Further single-cell analyses from cancer tissues revealed different neutrophil subgroups with potential antitumor activities suggesting their usefulness as biomarkers for treatment decisions. CONCLUSIONS: This study confirms the utility of machine learning in advancing personalized medicine for gastric cancer by identifying tumor-associated neutrophils and their subgroups as key indicators of chemotherapy response. These findings could lead to more tailored and effective treatment plans for patients.

BACKGROUND: The iPocc trial, a randomized, global phase 3 study that compared intraperitoneal (IP) and intravenous (IV) carboplatin with dose-dense paclitaxel chemotherapy in epithelial ovarian cancer (EOC) patients, demonstrated improved progression-free survival in patients who received IP chemotherapy. The present study aimed to investigate the role of preexisting tumor immunity in the clinical outcomes of patients receiving IP chemotherapy. METHODS: This study involved analyzing patient data from the iPocc trial, selectively of those whose tumor specimens were preserved at the time of primary surgery. A total of 116 cases ((IP; n = 59), (IV; n = 57)) were subjected to microarray analysis. Single-sample gene set enrichment analyses were performed to evaluate the tumor immune microenvironment. RESULTS: Patients with enhanced tumor infiltration of T cells, natural killer (NK) cells, and cytotoxic lymphocytes in the IP group had a longer overall survival (OS) than those in the IV group, but not in the group with low infiltration. IP therapy improved the OS of patients with high expression of immune-related genes such as CD8A and FOXP3. In patients' subdivided into "immune Hot" and "immune Cold" groups based on hierarchical clustering analysis using four parameters representing "Innate immunity," "T cells," "IFNG response" and "Inhibitory molecules," IP therapy significantly improved prognosis in the "immune Hot" group, but not in the "immune Cold" group compared to that of IV therapy. CONCLUSIONS: IP chemotherapy enhances the survival rates of patients with EOC with an immune-Hot phenotype in the tumor microenvironment prior to treatment. (Japan Registry of Clinical Trials number, jRCTs031180141.).

Protein complexes are crucial structures that control many biological processes. Harnessing these structures could be valuable for therapeutic therapy. However, their instability and short lifespans need to be addressed for effective use. Here, we propose an innovative approach based on a functional polymeric cloak that coordinately anchors different domains of protein complexes and assembles them into a stabilized nanoformulation. As the polymer-protein association in the cloak is pH sensitive, the nanoformulation also allows targeting the release of the protein complexes to the acidic microenvironment of tumors for aiding their therapeutic performance. Building on this strategy, we developed an IL-15 nanosuperagonist (Nano-SA) by encapsulating the interleukin-15 (IL-15)/IL-15 Receptor α (IL-15Rα) complex (IL-15cx) for fostering synergistic transpresentation in tumors. Upon intravenous administration, Nano-SA stably circulated in the bloodstream, safeguarding the integrity of IL-15cx until reaching the tumor site, where it selectively released the active complex. Thus, Nano-SA significantly amplified the antitumor immune signals while diminishing systemic off-target effects. In murine colon cancer models, Nano-SA achieved potent immunotherapeutic effects, eradicating tumors without adverse side effects. These findings highlight the transformative potential of nanotechnology for advancing protein complex-based therapies.

11550 Background: Improvements in surgical techniques and the introduction of perioperative chemotherapy have led to the improved 5-year survival rate of 91% for bone and tissue sarcoma in Japan (JCOG1306 trial). However, for invasive soft tissue sarcomas, the local control rate remains approximately 80% with a high recurrence rate. Undifferentiated pleomorphic sarcoma (UPS) and myxofibrosarcoma (MFS) frequently exhibit strong local invasiveness, yet the mechanisms underlying their invasiveness remain unclear. Thus, in this study, we aimed to explore the tumor microenvironment and genetic profiles associated with the invasiveness of UPS and MFS by using bulk RNA-sequencing data. Methods: We focused on 47 cases with UPS or MFS, whose RNA-sequencing data were obtained from surgical specimens at the Cancer Institute Hospital of JFCR between April 2017 and April 2023. The entire cohort was divided into two groups based on preoperative MRIs. Tumors showing a tail-like sign or irregular margins were categorized as invasive (Group I: 32 cases, average age 71±13 years, UPS 22 cases, MFS 10 cases), while tumors with clearly defined margins were categorized as non-invasive (Group NI: 15 cases, average age 68±13 years, UPS 10 cases, MFS 5 cases). We calculated immune cell fractions using CIBERSORTx and analyzed the differentially expressed genes (DEGs) and altered pathways using iDEP estimated from RNA-sequencing data. The results were analyzed for correlation with clinical outcomes. Results: The 5-year overall survival rate was significantly lower in Group I (74%) than in Group NI (100%, p=0.04), while the 5-year recurrence-free survival rate did not differ significantly between the groups (p=0.4). Group I showed significantly lower naive B cell count (p=0.04), and cases with higher naive B cell count had a significantly higher 5-year overall survival rate than those with lower count (93% vs. 69%, p=0.04). A total of 1260 DEGs were identified between Group I and NI, including 241 immune-related genes, and 11 genes were associated with overall survival. Multivariate analysis of these 11 immune-related genes and overall survival revealed the significant association of one gene, “Gene X” (p=0.01). In Group I, pathways related to fat metabolism were downregulated, while those related to sugar metabolism and rheumatoid arthritis were upregulated. Conclusions: This study revealed that soft tissue sarcoma invasiveness is correlated with overall survival. Specific tumor-infiltrating immune cells and immune-related genes may be regulated by the associated metabolic pathways. The pathways including “Gene X”, which reported with its antibodies, may contribute to the regulation of invasiveness of invasive soft tissue sarcoma and the improvement in survival rate. Thus, “Gene X” may be considered a potential novel therapeutic target for invasive soft tissue sarcoma.

BACKGROUND: Cancer immunotherapy aims to unleash the immune system's potential against cancer cells, providing sustained relief for tumors responsive to immune checkpoint inhibitors (ICIs). While promising in gastric cancer (GC) trials, the efficacy of ICIs diminishes in the context of peritoneal dissemination. Our objective is to identify strategies to enhance the impact of ICI treatment specifically for cases involving peritoneal dissemination in GC. METHODS: The therapeutic efficacy of anti-PD1, CTLA4 treatment alone, or in combination was assessed using the YTN16 peritoneal dissemination tumor model. Peritoneum and peritoneal exudate cells were collected for subsequent analysis. Immunohistochemical staining, flow cytometry, and bulk RNA-sequence analyses were conducted to evaluate the tumor microenvironment (TME). A Janus kinase inhibitor (JAKi) was introduced based on the pathway analysis results. RESULTS: Anti-PD1 and anti-CTLA4 combination treatment (dual ICI treatment) demonstrated therapeutic efficacy in certain mice, primarily mediated by CD8 + T cells. However, in mice resistant to dual ICI treatment, even with CD8 + T cell infiltration, most of the T cells exhibited an exhaustion phenotype. Notably, resistant tumors displayed abnormal activation of the Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) pathway compared to the untreated group, with observed infiltration of macrophages, neutrophils, and Tregs in the TME. The concurrent administration of JAKi rescued CD8 + T cells function and reshaped the immunosuppressive TME, resulting in enhanced efficacy of the dual ICI treatment. CONCLUSION: Dual ICI treatment exerts its anti-tumor effects by increasing tumor-specific CD8 + T cell infiltration, and the addition of JAKi further improves ICI resistance by reshaping the immunosuppressive TME.

BACKGROUND: Glioblastoma (GBM) is a highly lethal brain tumor. The effectiveness of temozolomide (TMZ) treatment in GBM is linked to the methylation status of O6-methyl-guanine DNA methyltransferase (MGMT) promoter. Patients with unmethylated MGMT promoter have limited treatment options available. Consequently, there is a pressing need for alternative therapeutic strategies for such patients. METHODS: Data, including transcriptomic and clinical information, as well as information on MGMT promoter methylation status in primary GBM, were obtained from The Cancer Genome Atlas (TCGA) (n=121) and Chinese Glioma Genome Atlas (CGGA) (n=83) datasets. Samples were categorized into high and low MGMT expression groups, MGMT-high (MGMT-H) and MGMT-low (MGMT-L) tumors. A comprehensive transcriptome analysis was conducted to explore the tumor-immune microenvironment. Furthermore, we integrated transcriptome data from 13 GBM patients operated at our institution with findings from tumor-infiltrating lymphocyte (TIL) cultures, specifically investigating their response to autologous tumors. RESULTS: Gene signatures associated with various immune cells, including CD8 T cells, helper T cells, B cells, and macrophages, were noted in MGMT-H tumors. Pathway analysis confirmed the enrichment of immune cell-related pathways. Additionally, biological processes involved in the activation of monocytes and lymphocytes were observed in MGMT-H tumors. Furthermore, TIL culture experiments showed a greater presence of tumor-reactive T cells in MGMT-H tumors compared to MGMT-L tumors. These findings suggest that MGMT-H tumors has a potential for enhanced immune response against tumors mediated by CD8 T cells. CONCLUSION: Our study provides novel insights into the immune cell composition of MGMT-H tumors, which is characterized by the infiltration of type 1 helper T cells and activated B cells, and also the presence of tumor-reactive T cells evidenced by TIL culture. These findings contribute to a better understanding of the immune response in MGMT-H tumors, emphasizing their potential for immunotherapy. Further studies are warranted to investigate on the mechanisms of MGMT expression and antitumor immunity.

Most of esophageal squamous cell carcinoma (ESCC) develop in smoking males in Japan, but the genomic etiology and immunological characteristics of rare non-smoking female ECSS remain unclear. To elucidate the genomic and immunological features of ESCC in non-smoking females, we analyzed whole-genome or transcriptome sequencing data from 94 ESCCs, including 20 rare non-smoking female cases. In addition, 31,611 immune cells were extracted from four ESCC tissues and subject to single-cell RNA-seq. We compared their immuno-genomic and microbiome profiles between non-smoking female and smoking ESCCs. Non-smoking females showed much better prognosis. Whole-genome sequencing analysis showed no significant differences in driver genes or copy number alterations depending on smoking status. The mutational signatures specifically observed in non-smoking females ESCC could be attributed to aging. Immune profiling from RNA-seq revealed that ESCC in non-smoking females had high tumor microenvironment signatures and a high abundance of eosinophils with a favorable prognosis. Single-cell RNA-sequencing of intratumor immune cells revealed gender differences of eosinophils and their activation in female cases. ESCCs in non-smoking females have age-related mutational signatures and gender-specific tumor immune environment with eosinophils, which is likely to contribute to their favorable prognosis.

We investigated the tumor immune response in gastric cancer patients receiving third-line nivolumab monotherapy to identify immune-related biomarkers for better patient selection. Nineteen patients (10 males, median age 67 years) who received nivolumab as a third- or later-line therapy were enrolled. We analyzed the tumor immune response in durable clinical benefit (DCB) and non-DCB patients. Pre-treatment and early-on-treatment tumor transcriptomes were examined, and gene expression profiles, immunograms, and T cell receptor (TCR) repertoire were analyzed. DCB was observed in 15.8% of patients, with comparable secondary endpoints (ORR; objective response rate, OS; overall survival, PFS; progression-free survival) to previous trials. The immunograms of individual subjects displayed no significant changes before or early in the treatment, except for the regulatory T cell (Treg) score. Moreover, there were no consistent alterations observed among cases experiencing DCB. The intratumoral immune response was suppressed by previous treatments in most third- or later-line nivolumab recipients. TCR repertoire analysis revealed newly emerged clonotypes in early-on-treatment tumors, but clonal replacement did not impact efficacy. High T cell/Treg ratios and a low UV-radiation-response gene signature were linked to DCB and treatment response. This study emphasizes the tumor immune response's importance in nivolumab efficacy for gastric cancer. High T cell/Treg ratios and specific gene expression signatures show promise as potential biomarkers for treatment response. The tumor-infiltrating immune response was compromised by prior treatments in third-line therapy, implying that, to enhance immunotherapeutic outcomes, commencing treatment at an earlier stage might be preferable. Larger cohort validation is crucial to optimize immune-checkpoint inhibitors in gastric cancer treatment.

多くの癌腫に対して免疫チェックポイント阻害剤(immune checkpoint inhibitor:ICI)による治療が効果を上げているが,ICIは腫瘍組織に対してだけではなく正常組織にも影響を与えてしまうことがあり,これは免疫関連有害事象(immune-related adverse events:irAE)と呼ばれる.現在臨床応用されているICIは,T細胞受容体シグナル伝達の抑制に関わるCTLA-4やPD-1をブロックすることで,免疫システムの活性化をまねき抗腫瘍効果を促進させると同時にirAEの原因となる.irAEで認める自己免疫反応のメカニズムは,破壊された癌細胞から腫瘍抗原だけでなく自己抗原もT細胞に提示されてしまうepitope spreadingによる自己反応性T細胞の活性化,B細胞の活性化による自己抗体の産生増加,自然免疫の賦活化,予期せぬ組織での標的分子の発現,腸内細菌等環境因子との関連など,非常に幅広い.irAEのメカニズム解明は,適切な患者に適切な治療を行い,ICIの効果を増強してリスクを減らすためには重要であると考えられる.(著者抄録)

Cytotoxic T lymphocytes (CTLs) control tumors via lysis of antigen-presenting targets or through secretion of cytokines such as interferon-γ (IFNG), which inhibit tumor cell proliferation. Improved understanding of CTL interactions within solid tumors will aid the development of immunotherapeutic strategies against cancer. In this study, we take a systems biology approach to compare the importance of cytolytic versus IFNG-mediated cytostatic effects in a murine melanoma model (B16F10) and to dissect the contribution of immune checkpoints HAVCR2, LAG3, and PDCD1/CD274 to CTL exhaustion. We integrated multimodal data to inform an ordinary differential equation (ODE) model of CTL activities inside the tumor. Our model predicted that CTL cytotoxicity played only a minor role in tumor control relative to the cytostatic effects of IFNG. Furthermore, our analysis revealed that within B16F10 melanomas HAVCR2 and LAG3 better characterize the development of a dysfunctional CTL phenotype than does the PDCD1/CD274 axis.

Treatment of immunologically cold tumors is a major challenge for immune checkpoint inhibitors (ICIs). Interleukin 12 (IL-12) can invigorate ICIs against cold tumors by establishing a robust antitumor immunity. However, its toxicity and systemic induction of counteracting immunosuppressive signals have hindered translation. Here, IL-12 activity is spatiotemporally controlled for safely boosting efficacy without the stimulation of interfering immune responses by generating a nanocytokine that remains inactive at physiological pH, but unleashes its full activity at acidic tumor pH. The IL-12-based nanocytokine (Nano-IL-12) accumulate and release IL-12 in tumor tissues, eliciting localized antitumoral inflammation, while preventing systemic immune response, counteractive immune reactions, and adverse toxicities even after repeated intravenous administration. The Nano-IL-12-mediated spatiotemporal control of inflammation prompt superior anticancer efficacy, and synergize with ICIs to profoundly inflame the tumor microenvironment and completely eradicate ICI-resistant primary and metastatic tumors. The strategy could be a promising approach toward safer and more effective immunotherapies.

The cutting edge of cancer immunotherapy extends to ecto-5'-nucleotidase (CD73), a cell membrane enzyme that targets the metabolism of extracellular adenosine. We herein focused on the expression of CD73 to clarify the state of CD73 positivity in cancer immunity and tumor microenvironment, thereby revealing a new survival predictor for patients with bladder cancer (BCa). We used clinical tissue microarrays of human BCa and simultaneously performed the fluorescent staining of cell type-specific markers (CD3, CD8, Foxp3, programmed cell death protein 1, and programmed death-ligand 1 [PD-L1]) and CD73 together with DAPI for nuclear staining. In total, 156 participants were included. Multiplexed cellular imaging revealed a unique interaction between CD73 expression and CD8+ cytotoxic T cells (CTLs) and Foxp3+ regulatory T (Treg) cells in human BCa, showing the high infiltration of CD8+CD73+ CTLs and Foxp3+CD73+ Treg cells in tumors to be associated with tumorigenesis and poor prognosis in BCa. Interestingly, from a biomarker perspective, the high infiltration of CD73+ Treg cells in tumors was identified as an independent risk factor for overall survival in addition to clinicopathologic features. Regarding the relationship between immune checkpoint molecules and CD73 expression, both CD73+ CTLs and CD73+ Treg cells tended to coexpress programmed cell death protein 1 as tumor invasiveness and nuclear grade increased. Additionally, they may occupy a spatial niche located distantly from PD-L1+ cells in tumors to interfere less with the cancerous effects of PD-L1+ cells. In conclusion, the present results on the status of CD73 in cancer immunity suggest that CD73 expression on specific T-cell types has a negative immunoregulatory function. These findings may provide further insights into the immunobiological landscape of BCa, which may be translationally linked to improvements in future immunotherapy practice.

INTRODUCTION: Regulatory T cells (Tregs) have attracted attention as a novel therapeutic target to augment the clinical efficacy of immunotherapy. We conducted phase Ia and Ib trials to examine the safety and efficacy of the anti-CCR4 antibody, KW-0761 (mogamulizumab), which may eliminate effector Tregs (eTregs). We herein overviewed the results of these trials, presented cases with a durable clinical response, and investigated factors associated with the clinical effects of KW-0761. METHODS: Forty-nine patients with CCR4-negative solid cancers were enrolled in the phase Ia and Ib trials on KW-0761. An integral analysis of safety, clinical responses, prognosis, blood laboratory data, and cancer testis antigen-specific immune responses was performed. RESULTS: Grade 3-4 treatment-related adverse events were reported in 21 (42.9%) out of 49 patients, all of which were manageable. A partial response and stable disease were observed in 1 and 9 patients, respectively. A durable clinical response was noted in 2 esophageal and 2 lung cancer patients. eTreg depletion in peripheral blood was confirmed in most patients, and eTreg depletion was sustained during the KW-0761 treatment. High lymphocyte levels at baseline and 2 weeks after the initiation of KW-0761 were associated with a favorable clinical outcome. CONCLUSIONS: A durable clinical response was noted in some patients, and high lymphocyte levels before treatment initiation may be a biomarker for the efficacy of KW-0761. The synergistic effect of KW-0761 for depleting Tregs and other immunotherapies is expected in the future.

Chronic kidney disease (CKD) affects kidney cancer patients' mortality. However, the underlying mechanism remains unknown. M2-like macrophages have pro-tumor functions, also exist in injured kidney, and promote kidney fibrosis. Thus, it is suspected that M2-like macrophages in injured kidney induce the pro-tumor microenvironment leading to kidney cancer progression. We found that M2-like macrophages present in the injured kidney promoted kidney cancer progression and induced resistance to anti-PD1 antibody through its pro-tumor function and inhibition of CD8+ T cell infiltration. RNA-seq revealed Slc7a11 was upregulated in M2-like macrophages. Inhibition of Slc7a11 with sulfasalazine inhibited the pro-tumor function of M2-like macrophages and synergized with anti-PD1 antibody. Moreover, SLC7A11-positive macrophages were associated with poor prognosis among kidney cancer patients. Collectively, this study dissects the characteristic microenvironment in the injured kidney that contributed to kidney cancer progression and anti-PD1 antibody resistance. This insight offers promising combination therapy with anti-PD1 antibody and macrophage targeted therapy.

Only a small fraction of tumor-infiltrating lymphocytes can specifically recognize and attack cancer cells in PD-1/PD-L1 blockade therapy. Here, we investigate approaches to expand the neoantigen-specific CD8+ T cells to overcome the difficulties in treating PD-1/PD-L1 blockade-resistant tumors. Mutation-associated neoepitopes of murine non-small cell lung cancer ASB-XIV were estimated by Whole-exome and RNA sequencing and predicted by MHC-I binding affinity (FPKM>1) in silico. Using ASB-XIV-specific CD8+ T cells, we screened a panel of 257 neoepitope peptides derived from ASB-XIV missense and indel mutations. Mutated Phf3 peptide (mPhf3) was successfully identified as an immunogenic neoepitope. Prophylactic mPhf3-DC vaccination inhibited ASB-XIV tumor growth through CD8+ T cell-mediated antitumor immunity. Combining the mPhf3-DC vaccine and anti-PD-1 treatment elicited robust antitumor activity through the induction of mPhf3-specific CD8+ T cells in the tumor microenvironment. Furthermore, the adoptive transfer of mPhf3-specific CD8+ T cells eradicated ASB-XIV tumors. Likewise, the combination of mutated Cdt1 peptide (mCdt1)-DC vaccine and anti-PD-1 treatment or adoptive transfer of mCdt1-specific CD8+ T cells also led to significant regression of PD-1 blockade-resistant murine gastric YTN16 tumors. In conclusion, a novel immunogenic neoepitope of ASB-XIV was identified for immunotherapy targeting neoantigens. Identification of immunogenic neoantigens can extend the therapeutic strategies by increasing the frequency of neoantigen-specific T cells, even for PD-1/PD-L1 blockade-resistant tumors. This article is protected by copyright. All rights reserved.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers and is primarily treated with platinum-based neoadjuvant chemotherapy (NAC). Some ESCCs respond well to NAC. However, biomarkers to predict NAC sensitivity and their response mechanism in ESCC remain unclear. We perform whole-genome sequencing and RNA sequencing analysis of 141 ESCC biopsy specimens before NAC treatment to generate a machine-learning-based diagnostic model to predict NAC reactivity in ESCC and analyzed the association between immunogenomic features and NAC response. Neutrophil infiltration may play an important role in ESCC response to NAC. We also demonstrate that specific copy-number alterations and copy-number signatures in the ESCC genome are significantly associated with NAC response. The interactions between the tumor genome and immune features of ESCC are likely to be a good indicator of therapeutic capability and a therapeutic target for ESCC, and machine learning prediction for NAC response is useful.

Immune checkpoint inhibitors have been approved as second-line therapy for patients with advanced urothelial carcinoma (UC). However, which patients will obtain clinical benefit remains to be determined. To identify predictive biomarkers for the pembrolizumab (PEM) response early during treatment, the present study investigated 31 patients with chemotherapy-resistant recurrent or metastatic UC who received 200 mg PEM intravenously every 3 weeks. Blood was taken just before the first dose and again before the second dose, and the peripheral blood mononuclear cells of all 31 pairs of blood samples were immune phenotyped by flow cytometry. Data were assessed by principal component analysis (PCA), correlation analysis and Cox proportional hazards modeling in order to comprehensively determine the effects of PEM on peripheral mononuclear immune cells. Absolute counts of CD45RA+CD27-CCR7- terminally differentiated CD8+ T cells and KLRG1+CD57+ senescent CD8+ T cells were significantly increased after PEM administration (P=0.042 and P=0.043, respectively). Senescent and exhausted CD4+ and CD8+ T cell dynamics were strongly associated with each other. By contrast, counts of monocytic myeloid-derived suppressor cells (mMDSCs) were not associated with other immune cell phenotypes. The results of PCA and non-hierarchical clustering of patients suggested that excessive T cell senescence and differentiation early during treatment were not necessarily associated with a survival benefit. However, decreased mMDSC counts after PEM were associated with improved overall survival. In conclusion, early on-treatment peripheral T cell status was associated with response to PEM; however, it was not associated with clinical benefit. By contrast, decreased peripheral mMDSC counts did predict improved overall survival.

Understanding the tumor microenvironment (TME) and anti-tumor immune responses in gastric cancer are required for precision immune-oncology. Taking advantage of next-generation sequencing technology, the feasibility and reliability of transcriptome-based TME analysis were investigated. TME of 30 surgically resected gastric cancer tissues was analyzed by RNA-Seq, immunohistochemistry (IHC) and flow cytometry (FCM). RNA-Seq of bulk gastric cancer tissues was computationally analyzed to evaluate TME. Computationally analyzed immune cell composition was validated by comparison with cell densities established by IHC and FCM from the same tumor tissue. Immune cell infiltration and cellular function were also validated with IHC and FCM. Cell proliferation and cell death in the tumor as assessed by RNA-Seq and IHC were compared. Computational tools and gene set analysis for quantifying CD8+ T cells, regulatory T cells and B cells, T cell infiltration and functional status, and cell proliferation and cell death status yielded an excellent correlation with IHC and FCM data. Using these validated transcriptome-based analyses, the immunological status of gastric cancer could be classified into immune-rich and immune-poor subtypes. Transcriptome-based TME analysis is feasible and is valuable for further understanding the immunological status of gastric cancer.

Abstract Microbes and their byproducts have been reported to regulate host health and immune functions. Here we demonstrated that microbial exopolysaccharide produced by Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 (EPS-R1) induced CCR6+ CD8+ T cells of mice and humans. In mice, ingestion of EPS-R1 augmented antitumor effects of anti–CTLA-4 or anti–PD-1 monoclonal antibody against CCL20-expressing tumors, in which infiltrating CCR6+ CD8+ T cells were increased and produced IFNγ accompanied by a substantial immune response gene expression signature maintaining T-cell functions. Of note, the antitumor adjuvant effect of EPS-R1 was also observed in germ-free mice. Furthermore, the induction of CCR6 expression was mediated through the phosphorylated structure in EPS-R1 and a lysophosphatidic acid receptor on CD8+ T cells. Overall, we find that dietary EPS-R1 consumption induces CCR6+ CD8+ T cells in Peyer's patches, favoring a tumor microenvironment that augments the therapeutic effect of immune-checkpoint blockade depending on CCL20 production by tumors. Significance: Gut microbiota- and probiotic-derived metabolites are attractive agents to augment the efficacy of immunotherapies. Here we demonstrated that dietary consumption of Lactobacillus-derived exopolysaccharide induced CCR6+ CD8+ T cells in Peyer's patches and improved the tumor microenvironment to augment the therapeutic effects of immune-checkpoint blockade against CCL20-producing tumors. See related commentary by Di Luccia and Colonna, p. 1189. This article is highlighted in the In This Issue feature, p. 1171

BACKGROUND: Regulatory T cells (Tregs) play an important role in the antitumor immune response in epithelial ovarian cancer (EOC). To understand the immune-inhibitory networks of EOC, we addressed the association between Tregs and immune checkpoint expression on T cells in the tumor microenvironment of EOC. METHODS: A total of 41 patients with stage IIIC and IV EOC were included in the analysis. We harvested cells from malignant ascites and investigated them using multi-color flow cytometry. We categorized the Tregs into 3 groups: effector-type Tregs, naïve Tregs and non-Tregs, based on the expression patterns of CD45RA and Foxp3 in CD4+ T cells. Furthermore, the relationships between the expression of various immune checkpoint molecules, such as PD-1, on CD8+ T cells and each of the Treg subtypes was also evaluated. RESULTS: The median frequency of naïve Tregs, effector-type Tregs and non-Tregs were 0.2% (0-0.8), 2.0% (0-11.4) and 1.5% (0.1-6.3) in CD4+ T cells of malignant ascites from EOC patients, respectively. A high frequency of effector-type Tregs was associated with high-grade serous carcinoma compared with the other histotypes. Patients with higher proportions of effector-type Tregs showed a trend towards increased progression-free survival. We also demonstrated a correlation between a higher proportion of effector-type Tregs and increased PD-1 expression on CD8+ T cells. In addition, C-C chemokine receptor 4 expression was also observed in effector-type Tregs. CONCLUSION: These data suggest that multiple immune-inhibitory networks exist in malignant ascites from EOC patients, suggesting an approach towards combinational immunotherapies for advanced EOC patients.

PURPOSE: The goal of this study is to clarify the underlying mechanisms of metastasis suppression by carbon-ion radiotherapy combined with immature dendritic cell immunotherapy (CiDC), which was shown previously to suppress pulmonary metastasis in an NR-S1-bearing C3H/He mouse model. METHODS AND MATERIALS: Mouse carcinoma cell lines (LLC, LM8, Colon-26, and Colon-26MGS) were grafted into the right hind paw of syngeneic mice (C57BL/6J, C3H/He, and BALB/c). Seven days later, the tumors on the mice were locally irradiated with carbon ions (290 MeV/n, 6 cm spread-out Bragg peak, 1 or 2 Gy). At 1.5 days after irradiation, bone marrow-derived immature dendritic cells (iDCs) were administrated intravenously into a subset of the mice. The number of lung metastases was evaluated within 3 weeks after irradiation. In vitro-cultured cancer cells were irradiated with carbon ions (290 MeV/n, mono-energy, LET approximately 70-80 keV/µm), and then cocultured with iDCs for 3 days to determine the DC maturation. RESULTS: CiDC effectively repressed distant lung metastases in cancer cell (LLC and LM8)-bearing C57BL/6J and C3H/He mouse models. However, Colon-26- and Colon-26MGS-bearing BALB/c models did not show enhancement of metastasis suppression by combination treatment. This result was evaluated further by comparing LM8-bearing C3H/He and LLC-bearing C57BL/6J models with a Colon-26-bearing BALB/c model. In vitro coculture assays demonstrated that all irradiated cell lines were able to activate C3H/He- or C57BL/6J-derived iDCs into mature DCs, but not BALB/c-derived iDCs. CONCLUSIONS: The genetic background of the host could have a strong effect on the potency of combination therapy. Future animal and clinical testing should evaluate host genetic factors when evaluating treatment efficacy.

Cancer histological images contain rich biological and clinical information, but quantitative representation can be problematic and has prevented the direct comparison and accumulation of large-scale datasets. Here, we show successful universal encoding of cancer histology by deep texture representations (DTRs) produced by a bilinear convolutional neural network. DTR-based, unsupervised histological profiling, which captures the morphological diversity, is applied to cancer biopsies and reveals relationships between histologic characteristics and the response to immune checkpoint inhibitors (ICIs). Content-based image retrieval based on DTRs enables the quick retrieval of histologically similar images using The Cancer Genome Atlas (TCGA) dataset. Furthermore, via comprehensive comparisons with driver and clinically actionable gene mutations, we successfully predict 309 combinations of genomic features and cancer types from hematoxylin-and-eosin-stained images. With its mounting capabilities on accessible devices, such as smartphones, universal encoding for cancer histology has a strong impact on global equalization for cancer diagnosis and therapies.

With the advent of immune checkpoint inhibitors, the importance of immunotherapy in cancer treatment has been widely recognized. However, it is also true that immune checkpoint inhibitors alone have limited therapeutic effects. Therefore, in addition to the combination among immune checkpoint inhibitors such as the combination therapy of anti-CTLA-4 antibody and anti-PD-1 antibody, the combinations with chemotherapy, radiotherapy, and molecular target drug are extensively being developed. In order to link the antitumor effect of the immune response equipped in the body to cancer treatment, it is necessary to understand and overcome the immunosuppressive environment that inhibits it. This article outlines the understanding of tumor immunity by the immune genome profiling of cancer tissues.

BACKGROUND: The aims of this study were (1) to clarify the impact of tertiary lymphoid structure (TLS) status on the outcome and immunogenomic profile of human clear cell renal cell carcinoma (ccRCC) and (2) to determine phenotypic differences in TLSs between different types of genitourinary cancer, that is, urinary ccRCC and bladder cancer. METHODS: We performed a quantitative immunohistological analysis of ccRCC tissue microarrays and conducted integrated genome mutation analysis by next-generation sequencing and methylation array analysis. Since the tumor immune microenvironment of ccRCC often differs from that of other cancer types, we analyzed the phenotypic differences in TLSs between ccRCC and in-house bladder cancer specimens. RESULTS: Varying distribution patterns of TLSs were observed throughout ccRCC tumors, revealing that the presence of TLSs was related to poor prognosis. An analysis of genomic alterations based on TLS status in ccRCC revealed that alterations in the PI3K-mTOR pathway were highly prevalent in TLS-positive tumors. DNA methylation profiling also revealed distinct differences in methylation signatures among ccRCC samples with different TLS statuses. However, the TLS characteristics of ccRCC and bladder cancer markedly differed: TLSs had the exact opposite prognostic impact on bladder cancer as on ccRCC. The maturity and spatial distribution of TLSs were significantly different between the two cancer types; TLSs were more mature with follicle-like germinal center organization and likely to be observed inside the tumor in bladder cancer. Labeling for CD8, FOXP3, PD-1, and PD-L1 showed marked differences in the diversity of the immune microenvironment surrounding TLSs. The proportions of CD8-, FOXP3-, and PD-L1-positive cells were significantly higher in TLSs in bladder cancer than in TLSs in ccRCC; rather the proportion of PD-1-positive cells was significantly higher in TLSs in ccRCC than in TLSs in bladder cancer. CONCLUSION: The immunobiology of ccRCC is unique, and various cancerous phenomena conflict with that seen in other cancer types; therefore, comparing the TLS characteristics between ccRCC and bladder cancer may help reveal differences in the prognostic impact, maturity and spatial distribution of TLSs and in the immune environment surrounding TLSs between the two cancers.

Elimination of cancerous cells by the immune system is an important mechanism of protection from cancer, however, its effectiveness can be reduced owing to development of resistance and evasion. To understand the systemic immune response in advanced untreated primary colorectal cancer, we analyze immune subtypes and immune evasion via neoantigen-related mechanisms. We identify a distinctive cancer subtype characterized by immune evasion and very poor overall survival. This subtype has less clonal highly expressed neoantigens and high chromosomal instability, resulting in adaptive immune resistance mediated by the immune checkpoint molecules and neoantigen presentation disorders. We also observe that neoantigen depletion caused by immunoediting and high clonal neoantigen load are correlated with a good overall survival. Our results indicate that the status of the tumor microenvironment and neoantigen composition are promising new prognostic biomarkers with potential relevance for treatment plan decisions in advanced CRC.

Serum autoantibody to cancer/testis antigens (CTAs) is a critical biomarker that reflects the antitumor immune response. Quantitative and multiplexed anti-CTA detection arrays can assess the immune status in tumors and monitor therapy-induced antitumor immune reactions. Most full-length recombinant CTA proteins tend to aggregate. Cysteine residue-specific S-cationization techniques facilitate the preparation of water-soluble and full-length CTAs. Combined with Luminex technology, we designed a multiple S-cationized antigen-immobilized bead array (MUSCAT) assay system to evaluate multiple serum antibodies to CTAs. Reducible S-alkyl-disulfide-cationized antigens in cytosolic conditions were employed to develop rabbit polyclonal antibodies as positive controls. These control antibodies sensitively detected immobilized antigens on beads and endogenous antigens in human lung cancer-derived cell lines. Rabbit polyclonal antibodies successfully confirmed the dynamic ranges and quantitative MUSCAT assay results. An immune monitoring study was conducted using the serum samples on an adenovirus-mediated REIC/Dkk-3 gene therapy clinical trial that showed a successful clinical response in metastatic castration-resistant prostate cancer. Autoantibody responses were closely related to clinical outcomes. Notably, upregulation of anti-CTA responses was monitored before tumor regression. Thus, quantitative monitoring of anti-CTA antibody biomarkers can be used to evaluate the cancer-immunity cycle. A quality-certified serum autoantibody monitoring system is a powerful tool for developing and evaluating cancer immunotherapy.

To develop combination immunotherapies for gastric cancers, immunologically well-characterized preclinical models are crucial. Here, we leveraged two transplantable murine gastric cancer cell lines, YTN2 and YTN16, derived from the same parental line but differing in their susceptibility to immune rejection. We established their differential sensitivity to immune checkpoint inhibitors (ICI) and identified neoantigens. Although anti-CTLA-4 mAbs eradicated YTN16 tumors in 4 of 5 mice, anti-PD-1 and anti-PD-L1 mAbs failed to eradicate YTN16 tumors. Using whole-exome and RNA sequencing, we identified two and three neoantigens in YTN2 and YTN16, respectively. MHC class I ligandome analysis detected the expression of only one of these neoantigens, mutated Cdt1, but the exact length of MHC binding peptide was determined. Dendritic cell vaccine loaded with neoepitope peptides and adoptive transfer of neoantigen-specific CD8+ T cells successfully inhibited the YTN16 tumor growth. Targeting mutated Cdt1 had better efficacy for controlling the tumor. Therefore, mutated Cdt1 was the dominant neoantigen in these tumor cells. More mCdt1 peptides were bound to MHC class I and presented on YTN2 surface than YTN16. This might be one of the reasons why YTN2 was rejected while YTN16 grew in immune-competent mice.

Regulatory T (Treg) cells are important negative regulators of immune homeostasis, but in cancers they tone down the anti-tumor immune response. They are distinguished by high expression levels of the chemokine receptor CCR4, hence their targeting by the anti-CCR4 monoclonal antibody mogamulizumab holds therapeutic promise. Here we show that despite a significant reduction in peripheral effector Treg cells, clinical responses are minimal in a cohort of patients with advanced CCR4-negative solid cancer in a phase Ib study (NCT01929486). Comprehensive immune-monitoring reveals that the abundance of CCR4-expressing central memory CD8+ T cells that are known to play roles in the antitumor immune response is reduced. In long survivors, characterised by lower CCR4 expression in their central memory CD8+ T cells possessed and/or NK cells with an exhausted phenotype, cell numbers are eventually maintained. Our study thus shows that mogamulizumab doses that are currently administered to patients in clinical studies may not differentiate between targeting effector Treg cells and central memory CD8+ T cells, and dosage refinement might be necessary to avoid depletion of effector components during immune therapy.

An in-depth understanding of the tumor microenvironment (TME) is required for the development of improved combination immunotherapies for gastric cancer. Recently, we classified these cancers into four main types defined by their immunological attributes, namely Hot 1, Hot 2, Intermediate and Cold. Of these, the T cell-inflamed "Hot" tumors were further divided into Hot 1 and Hot 2 with different clinical outcomes. Thus, overall survival and progression-free survival of patients with Hot 1 tumors were shorter than with Hot 2. In the present study, we re-evaluated RNA-Seq data of 6 Hot 1 and 6 Hot 2 gastric cancers to elucidate the underlying reason for the poor prognosis and T cell dysfunction in the former. In addition, 56 Hot 1 and 27 Hot 2 tumors in The Cancer Genome Atlas (TCGA) were analyzed. We report that single sample Gene Set Enrichment Analysis (ssGSEA) and differential gene expression analysis identified differences between Hot 1 and Hot 2 tumors involved in metabolism and cell adhesion pathways. Therefore, it is suggested that strategies to modulate active metabolism in Hot 1 tumors should be integrated into the treatment of these gastric cancers.

PD-1 blockade exerts antitumor effects by reinvigorating tumor antigen–specific CD8+ T cells. Whereas neoantigens arising from gene alterations in cancer cells comprise critical tumor antigens in antitumor immunity, a subset of non–small cell lung cancers (NSCLCs) harboring substantial tumor mutation burden (TMB) lack CD8+ T cells in the tumor microenvironment (TME), which results in resistance to PD-1 blockade therapy. To overcome this resistance, clarifying the mechanism(s) impairing antitumor immunity in highly mutated NSCLCs is an urgent issue. Here, we showed that activation of the WNT/β-catenin signaling pathway contributed to the development of a noninflamed TME in tumors with high TMB. NSCLCs that lacked immune cell infiltration into the TME despite high TMB preferentially up-regulated the WNT/β-catenin pathway. Immunologic assays revealed that those patients harbored neoantigen-specific CD8+ T cells in the peripheral blood but not in the TME, suggesting impaired T cell infiltration into the TME due to the activation of WNT/β-catenin signaling. In our animal models, the accumulation of gene mutations in cancer cells increased CD8+ T cell infiltration into the TME, thus slowing tumor growth. However, further accumulation of gene mutations blunted antitumor immunity by excluding CD8+ T cells from tumors in a WNT/β-catenin signaling-dependent manner. Combined treatment with PD-1 blockade and WNT/β-catenin signaling inhibitors induced better antitumor immunity than either treatment alone. Thus, we propose a mechanism-oriented combination therapy whereby immune checkpoint inhibitors can be combined with drugs that target cell-intrinsic oncogenic signaling pathways involved in tumor immune escape.

An important factor associated with primary resistance to immune-checkpoint therapies (ICT) is a "cold" tumor microenvironment (TME), characterized by the absence of T cell infiltration and a non-inflammatory milieu. Whole-exome and RNA sequencing to predict neoantigen expression was performed on the LLC1 cell line which forms "cold" tumors in mice. Dendritic cell (DC)-based vaccination strategies were developed using candidate neoantigen long peptides (LPs). A total of 2536 missense mutations were identified in LLC1 and of 132 candidate neoantigen short peptides, 25 were found to induce CD8+ T cell responses. However, they failed to inhibit LLC1 growth when incorporated into a cancer vaccine. In contrast, DCs pulsed with LPs induced CD4+ and CD8+ T cell responses and one of them, designated L82, delayed LLC1 growth in vivo. By RNA-Seq, CD38 was highly expressed by LLC1 tumor cells and, therefore, anti-CD38 antibody treatment was combined with L82-pulsed DC vaccination. This combination effectively suppressed tumor growth via a mechanism relying on decreased regulatory T cells in the tumor. This study demonstrated that an appropriate vaccination strategy combining neoantigen peptide-pulsed DC with anti-CD38 antibody can render an ICT-resistant "cold" tumor susceptible to immune rejection via a mechanism involving neutralization of regulatory T cells.

BACKGROUND: Cabozantinib is an oral tyrosine kinase inhibitor in renal cell carcinoma (RCC), whose targets include oncogenic AXL and unique ligand GAS6. Critical gaps in basic knowledge need to be addressed to devise an exclusive biomarker and candidate when targeting the AXL/GAS6 axis. METHODS: To clarify the effects of the AXL/GAS6 axis on RCC, we herein performed a large-scale immunogenomic analysis and single-cell counts including various metastatic organs and histological subtypes of RCC. We further applied genome-wide mutation analyses and methylation arrays. RESULTS: Varying patterns of AXL and GAS6 expression were observed throughout primary RCC tumours and metastases. Scoring individual AXL/GAS6 levels in the tumour centre and invasive margin, namely, the AXL/GAS6 score, showed a good ability to predict the prognosis of clear cell RCC. Metastasis- and histological subtype-specific differences in the AXL/GAS6 score existed since lung metastasis and the papillary subtype were weakly related to the AXL/GAS6 axis. Cell-by-cell immunohistological assessments clarified an immunosuppressive environment in tumours with high AXL/GAS6 scores. Genomic alterations in the PI3K-mTOR pathway and DNA methylation profiling revealed distinct differences with the AXL/GAS6 score in ccRCC. CONCLUSION: The AXL/GAS6 scoring system could predict the outcome of prognosis and work as a robust biomarker for the immunogenomic state in RCC.

Tregs infiltrate tumors and inhibit antitumor immunity. KW-0761 (Mogamulizumab) is a humanized anti-CCR4 monoclonal antibody that could eliminate activated Tregs with high immunosuppressive activity that express CCR4. In this phase Ib trial, KW-0761 was used as a cancer immunotherapeutic reagent to deplete Tregs in patients with advanced or recurrent solid CCR4-negative tumors. Thirty-nine patients with solid cancer were treated with KW-0761 at a dose of 0.1 or 1.0 mg/kg. The safety, clinical responses, and effects of Treg depletion were analyzed. Any grade and grade 3-4 treatment-related adverse events (AEs) were observed in 36 (92%) and 14 (36%) out of 39 patients, respectively. All treatment-related AEs were manageable. One and 5 patients achieved a partial response and stable disease, respectively, during treatment and were long survivors. The efficient depletion of Treg in peripheral blood was confirmed in both cohorts. Therefore, the administration of KW-0761 was safe, resulting in the depletion of Tregs in peripheral blood and potential immune responses in patients with solid cancer. The combined use of KW-0761 to deplete Tregs and other immunotherapies is a promising approach to augment immune responses.

Transient expression of human intracellular proteins in human embryonic kidney (HEK) 293 cells is a reliable system for obtaining soluble proteins with biologically active conformations. Contrary to conventional concepts, we found that recombinantly expressed intracellular cancer-testis antigens (CTAs) showed frequent aggregation in HEK293 cells. Although experimental subcellular localization of recombinant CTAs displayed proper cytosolic or nuclear localization, some proteins showed aggregated particles in the cell. This aggregative property was not observed in recombinant housekeeping proteins. No significant correlation was found between the aggregative and biophysical properties, such as hydrophobicity, contents of intrinsically disordered regions and expression levels, of CTAs. These results can be explained in terms of structural instability of CTAs, which are specifically expressed in the testis and aberrantly expressed in cancer cells and function as a hub in the protein-protein network using intrinsically disordered regions. Hence, we speculate that recombinantly expressed CTAs failed to form this protein complex. Thus, unfolded CTAs formed aggregated particles in the cell.

Despite the high sensitivity of renal cell carcinoma (RCC) to immunotherapy, RCC has been recognized as an unusual disease in which CD8+ T-cell infiltration into the tumor beds is related to a poor prognosis. To approach the inner landscape of immunobiology of RCC, we performed multiplexed seven-color immunohistochemistry (CD8, CD39, PD-1, Foxp3, PD-L1, and pan-cytokeratin AE1/AE3 with DAPI), which revealed the automated single-cell counts and calculations of individual cell-to-cell distances. In total, 186 subjects were included, in which CD39 was used as a marker for distinguishing tumor-specific (CD39+) and bystander (CD39-) T-cells. Our clear cell RCC cohort also revealed a poor prognosis if the tumor showed increased CD8+ T-cell infiltration. Intratumoral CD8+CD39+ T-cells as well as their exhausted CD8+CD39+PD-1+ T-cells in the central tumor areas enabled the subgrouping of patients according to malignancy. Analysis using specimens post-antiangiogenic treatment revealed a dramatic increase in proliferative Treg fraction Foxp3+PD-1+ cells, suggesting a potential mechanism of hyperprogressive disease after uses of anti-PD-1 antibody. Our cell-by-cell study platform provided spatial information on tumors, where bystander CD8+CD39- T-cells were dominant in the invasive margin areas. We uncovered a potential interaction between CD8+CD39+PD-1+ T-cells and Foxp3+PD-1+ Treg cells due to cell-to-cell proximity, forming a spatial niche more specialized in immunosuppression under PD-1 blockade. A paradigm shift to the immunosuppressive environment was more obvious in metastatic lesions; rather the infiltration of Foxp3+ and Foxp3+PD-1+ Treg cells was more pronounced. With this multiplexed single-cell pathology technique, we revealed further insight into the immunobiological standing of RCC.

A cutting edge therapy for future immuno-oncology is targeting a new series of inhibitory receptors (IRs): LAG-3, TIM-3, and TIGIT. Both immunogenomic analyses and diagnostic platforms to distinguish candidates and predict good responders to these IR-related agents are vital in clinical pathology. By applying an automated single-cell count for immunolabelled LAG-3, TIM-3, and TIGIT, we reveal that individual IR levels with exclusive domination in each tumour can serve as valid biomarkers for profiling human renal cell carcinoma (RCC). We uncover the immunogenomic landscape associated with individual IR levels in human RCC tumours with metastases in various organs and histological subtypes. We then externally validate our results and devise a workflow with optimal biomarker cut-offs for discriminating the LAG-3, TIM-3, and TIGIT tumour profiles. The discrimination of LAG-3, TIM-3, and TIGIT profiles in tumours may have a broad impact on investigations of immunotherapy responses after targeting a new series of IRs.

BACKGROUND: Metformin (Met) is the first-line treatment for type 2 diabetes mellitus and plays an effective role in treating various diseases, such as cardiovascular disease, neurodegenerative disease, cancer, and aging. However, the underlying mechanism of Met-dependent antitumor immunity remains to be elucidated. METHODS: MitoTEMPO, a scavenger of mitochondrial superoxide, abolished the antitumor effect of Met, but not antiprogrammed cell death (PD-1) antibody (Ab) treatment. Consequently, we studied the mechanism of the Met-induced antitumor effect. Expressions of glucose transporter (Glut)-1, mitochondrial reactive oxygen species (mtROS), interferon (IFN)-γ, Ki67, autophagy markers, activation markers for NF-E2-related factor 2 (Nrf2), and mammalian target of rapamaycin complex 1 (mTORC1) in CD8+ tumor-infiltrating T lymphocytes (CD8TILs) were examined by flow cytometry analysis. In addition, conditional knockout mice for Nrf2 and p62 were used to detect these markers, together with the monitoring of in vivo tumor growth. RNA sequencing was performed for CD8TILs and tumor cells. Melanoma cells containing an IFN-γ receptor (IFNγR) cytoplasmic domain deletion mutant was overexpressed and used for characterization of the metabolic profile of those tumor cells using a Seahorse Flux Analyzer. RESULTS: Met administration elevates mtROS and cell surface Glut-1, resulting in the production of IFN-γ in CD8TILs. mtROS activates Nrf2 in a glycolysis-dependent manner, inducing activation of autophagy, glutaminolysis, mTORC1, and p62/SQSTM1. mTORC1-dependent phosphorylation of p62 at serine 351 (p-p62(S351)) is also involved in activation of Nrf2. Conditional deletion of Nrf2 in CD8TILs abrogates mTORC1 activation and antitumor immunity by Met. In synergy with the effect of anti-PD-1 Ab, Met boosts CD8TIL proliferation and IFN-γ secretion, resulting in decreased glycolysis and oxidative phosphorylation in tumor cells. Consequently, Glut-1 is elevated in CD8TILs, together with the expansion of activated dendritic cells. Moreover, tumor cells lacking in IFNγR signaling abolish IFN-γ production and proliferation of CD8TILs. CONCLUSIONS: We found that Met stimulates production of mtROS, which triggers Glut-1 elevation and Nrf2 activation in CD8TILs. Nrf2 activates mTORC1, whereas mTORC1 activates Nrf2 in a p-p62(S351)-dependent manner, thus creating a feedback loop that ensures CD8TILs' proliferation. In combination with anti-PD-1 Ab, Met stimulates robust proliferation of CD8TILs and IFN-γ secretion, resulting in an IFN-γ-dependent reprogramming of the tumor microenvironment.

Introduction: Although immune checkpoint inhibitors (ICIs) have been considered as promising agents for the treatment of advanced hepatocellular carcinoma (HCC), previous clinical trials revealed that the response to anti-programmed cell death protein 1 (anti-PD-1) monotherapy was as low as 20%. Identifying subgroups that respond well to ICIs is clinically important. Here, we studied the prognostic factors for anti-PD-1 antibody treatment based on the molecular and immunological features of HCC. Methods: Patients who were administered anti-PD1 antibody for advanced HCC at Kindai University Hospital were included. Clinicopathological backgrounds and antitumor responses were examined in 34 cases where tumor tissues before treatment were available. Transcriptome analysis was performed using 40 HCC samples obtained from surgical resection, and immune status was compared between 20 HCCs with activating mutations in β-catenin and those without the mutations using transcriptome-based immunogram. Results: Univariate analysis showed that the disease control rate was significantly better in patients with α-fetoprotein < 400 ng/mL, negative for β-catenin/glutamate synthetase (GS) staining, high combined positive score (CPS) of programmed death-ligand 1 (PD-L1), and increased infiltration of CD8+ cells in tumor tissues. Among them, negative staining of β-catenin/GS, CPS of PD-L1 ≥ 1, and high degree of CD8+ tumor-infiltrating lymphocytes (TILs) were significantly associated with longer survival in both progression-free survival (PFS) and overall survival (OS). The combination of these factors well stratified the survival of the patients on anti-PD-1 antibody in both PFS and OS (p < 0.0001 and p = 0.0048 for PFS and OS, respectively). In addition, the immunogram revealed that tumor-carrying mutations in β-catenin showed downregulation of immune-related genes, especially in those related to priming and activation by dendritic cells, interferon-γresponse, inhibitory molecules, and regulatory T cells. Discussion/Conclusion: The combined score including Wnt/β-catenin activation, CPS of PD-L1, and degree of CD8+ TILs in HCC is informative for predicting the response to ICI in HCC cases. Constitutive activation of β-catenin can induce an immune cold phenotype with downregulation of immune-related genes, and immunohistochemistry-based evaluation is beneficial for identifying the subgroup that shows a good response to ICI.

がん免疫治療は、がんに対する免疫応答を増強し治療に活用する治療法である。生体には、過剰な免疫応答による正常組織の破壊を避けるためのプログラムが備わっている。従来のがんワクチン治療や養子免疫細胞療法(ACT)で効果が得られなかったことが示すとおり、抗腫瘍リンパ球を誘導したり増殖させたりすることで治療効果を得ることは容易ではなかった。遺伝子改変技術により改良を加えることによって、T細胞の抗原特異性(腫瘍特異性)、エフェクター機能、代謝をリプログラミングしたキメラ抗原受容体T(CAR-T)細胞を用いた治療は、生体内でも強い抗腫瘍効果を発揮し、がん患者に治癒をもたらすことが可能な治療である。腫瘍浸潤リンパ球(TIL)療法で養われたT細胞の培養増殖技術、T細胞の機能と分化の制御に重要な分子の同定などに、ウイルスベクターを用いたT細胞への遺伝子導入技術の確立が加わり、現在のCAR-T細胞療法の開発に至った歴史を振り返ってみたい。(著者抄録)

BACKGROUND AIMS: After therapy with platinum, 5-fluorouracil and taxane, no further recommended therapy is available for recurrent or metastatic esophageal cancer (r/mEC). Here the authors report two phase 1 trials of adoptive γδT-cell therapy, one for treatment-refractory r/mEC (γδT-monotherapy-P1, UMIN000001419) and the other for r/mEC with no prior systemic therapy (DCF-γδT-P1, UMIN000008097). METHODS: For γδT-monotherapy-P1, patients received four weekly and four biweekly injections of autologous γδT cells. For DCF-γδT-P1, patients received docetaxel, cisplatin and 5-fluorouracil (DCF) chemotherapy consisting of docetaxel (60 mg/m2) and cisplatin (60 mg/m2) on day 1 and continuous injection of 5-fluorouracil (600 mg/m2/day) on days 1-5 of each 28-day cycle; additionally, they received autologous γδT-cell injections on day 15 and day 22 of each cycle. RESULTS: Twenty-six patients were enrolled for γδT-monotherapy-P1. No severe adverse events were associated with γδT-cell therapy. Median overall survival was 5.7 months (95% confidence interval [CI], 4.3-10.0), and median progression-free survival was 2.4 months (95% CI, 1.7-2.8). Eighteen patients received DCF-γδT-P1. All treatment-related adverse events were associated with DCF chemotherapy, not γδT injection. Median overall survival was 13.4 months (95% CI, 6.7-not reached), and median progression-free survival was 4.0 months (95% CI, 2.5-5.7). The response rate and disease control rate were 39% and 78%, respectively. CONCLUSIONS: The use of γδT-cell immunotherapy with or without chemotherapy was safe and feasible for r/mEC patients. Although the authors failed to demonstrate any clinical benefit of γδT-monotherapy-P1, survival benefits were observed in the DCF-γδT-P1 trial.

Background/Aim: A mixture of anticancer agents and iodized poppy seed oil (IPSO) has been widely used for intra-arterial chemotherapy of hepatocellular carcinoma. However, the anticancer agents can easily separate from IPSO, so the therapeutic potential is limited. We developed epirubicinentrapped water-in-oil-in-water emulsion (WOW-Epi) using a double-membrane emulsification technique. Materials and Methods: We delivered WOW-Epi through a hepatic arterial injection to VX2 hepatic tumor rabbit model (1.2 mg/kg). Results: VX2 tumor growth was selectively suppressed in the WOW-Epi-treated group compared with the control treated groups. The accumulation of WOW in nearby cancer cells was confirmed via electron-microscopy. Endocytosis seemed to be the mechanism underlying the uptake of WOW. Conclusion: WOW-Epi led to tumour growth suppression in vivo. WOW does not cause toxicity to arterial vessels. WOW-Epi will be hopefully used for repeated intra-arterial chemotherapy to HCC patients in the near future.

Because of the complexity of cancer-immune system interactions, combinations of biomarkers will be required for predicting individual patient responses to treatment and for monitoring combination strategies to overcome treatment resistance. To this end, the "immunogram" has been proposed as a comprehensive framework to capture all relevant immunological variables. Here, we developed a method to convert transcriptomic data into immunogram scores (IGS). This immunogram includes 10 molecular profiles, consisting of innate immunity, priming and activation, T cell response, interferon γ (IFNG) response, inhibitory molecules, regulatory T cells, myeloid-derived suppressor cells (MDSCs), recognition of tumor cells, proliferation, and glycolysis. Using genes related to these 10 parameters, we applied single-sample gene set enrichment analysis (ssGSEA) to 9417 bulk RNA-Seq data from 9362 cancer patients with 29 different solid cancers in The Cancer Genome Atlas (TCGA). Enrichment scores were z-score normalized (Z) for each cancer type or the entire TCGA cohort. The IGS was defined by the formula IGS = 3 + 1.5 × Z so that patients would be well distributed over a range of scores from 1 to 5. The immunograms constructed in this way for all individual patients in the entire TCGA cohort can be accessed at "The RNA-Seq based Cancer Immunogram Web" (https://yamashige33.shinyapps.io/immunogram/).

Serum cytokine and chemokine networks may reflect the complex systemic immunological interactions in cancer patients. Studying groups of cytokines and their networks may help to understand their clinical biology. A total of 178 cases of ovarian cancer were analyzed in this study, including 73 high-grade serous (HGSC), 66 clear cell (CCC) and 39 endometrioid carcinomas. Suspension cytokine arrays were performed with the patients' sera taken before the primary surgery. Associations between each cytokine and clinicopathological factors were analyzed in all patients using multivariate linear regression models, and cluster analyses were performed for each histotype. In the multivariate analyses, twelve of 27 cytokines were correlated with histotypes. Cluster analyses in each histotype revealed 2 cytokine signatures S1 and S2 in HGSC, and similarly C1 and C2 in CCC. Twenty-two of 27 cytokines were commonly clustered in HGSC and CCC. Signature S1 and C1 included IL-2,6,8,15, chemokines and angiogenic factors, whereas signature S2 and C2 included IL-4,5,9,10,13, TNF-α and G-CSF. Four subgroups based on a high or low level for each signature were identified, and this cluster-based classification demonstrated significantly different progression-free and overall survivals for CCC patients (P = 0.00097 and P = 0.017).

BACKGROUND: Although immune checkpoint blockade is effective for several malignancies, a substantial number of patients remain refractory to treatment. The future of immunotherapy will be a personalized approach adapted to each patient's cancer-immune interactions in the tumor microenvironment (TME) to prevent suppression of antitumor immune responses. To demonstrate the feasibility of this kind of approach, we developed combination therapy for a preclinical model guided by deep immunophenotyping of the TME. METHODS: Gastric cancer cell lines YTN2 and YTN16 were subcutaneously inoculated into C57BL/6 mice. YTN2 spontaneously regresses, while YTN16 grows progressively. Bulk RNA-Seq, single-cell RNA-Seq (scRNA-Seq) and flow cytometry were performed to investigate the immunological differences in the TME of these tumors. RESULTS: Bulk RNA-Seq demonstrated that YTN16 tumor cells produced CCL20 and that CD8+ T cell responses were impaired in these tumors relative to YTN2. We have developed a vertical flow array chip (VFAC) for targeted scRNA-Seq to identify unique subtypes of T cells by employing a panel of genes reflecting T cell phenotypes and functions. CD8+ T cell dysfunction (cytotoxicity, proliferation and the recruitment of interleukin-17 (IL-17)-producing cells into YTN16 tumors) was identified by targeted scRNA-Seq. The presence of IL-17-producing T cells in YTN16 tumors was confirmed by flow cytometry, which also revealed neutrophil infiltration. IL-17 blockade suppressed YTN16 tumor growth, while tumors were rejected by the combination of anti-IL-17 and anti-PD-1 (Programmed cell death protein 1) mAb treatment. Reduced neutrophil activation and enhanced expansion of neoantigen-specific CD8+ T cells were observed in tumors of the mice receiving the combination therapy. CONCLUSIONS: Deep phenotyping of YTN16 tumors identified a sequence of events on the axis CCL20->IL-17-producing cells->IL-17-neutrophil-angiogenesis->suppression of neoantigen-specific CD8+ T cells which was responsible for the lack of tumor rejection. IL-17 blockade together with anti-PD-1 mAb therapy eradicated these YTN16 tumors. Thus, the deep immunological phenotyping can guide immunotherapy for the tailored treatment of each individual patient's tumor.

BACKGROUND: Not all non-small cell lung cancer (NSCLC) patients possess drug-targetable driver mutations, and response rates to immune checkpoint blockade therapies also remain unsatisfactory. Therefore, more effective treatments are still needed. Here, we report the results of a phase 2 clinical trial of adoptive cell therapy using zoledronate-expanded autologous Vγ9Vδ2 T-cells for treatment-refractory NSCLC. METHODS: NSCLC patients who had undergone at least two regimens of standard chemotherapy for unresectable disease or had had at least one treatment including chemotherapy or radiation for recurrent disease after surgery were enrolled in this open-label, single-arm, multicenter, phase 2 study. After preliminary testing of Vγ9Vδ2 T-cell proliferation, autologous peripheral blood mononuclear cells were cultured with zoledronate and IL-2 to expand the Vγ9Vδ2 T-cells. Cultured cells (>1×109) were intravenously administered every 2 weeks for six injections. The primary endpoint of this study was progression-free survival (PFS), and secondary endpoints included overall survival (OS), best objective response rate (ORR), disease control rate (DCR), safety and immunomonitoring. Clinical efficacy was defined as median PFS significantly >4 months. RESULTS: Twenty-five patients (20 adenocarcinoma, 4 squamous cell carcinoma and 1 large cell carcinoma) were enrolled. Autologous Vγ9Vδ2 T-cell therapy was administered to all 25 patients, of which 16 completed the foreseen course of 6 injections of cultured cells. Median PFS was 95.0 days (95% CI 73.0 to 132.0 days); median OS was 418.0 days (179.0-479.0 days), and best overall responses were 1 partial response, 16 stable disease (SD) and 8 progressive disease. ORR and DCR were 4.0% (0.1%-20.4%) and 68.0% (46.5%-85.1%), respectively. Severe adverse events developed in nine patients, mostly associated with disease progression. In one patient, pneumonitis and inflammatory responses resulted from Vγ9Vδ2 T-cell infusions, together with the disappearance of a massive tumor. CONCLUSIONS: Although autologous Vγ9Vδ2 T-cell therapy was well tolerated and may have an acceptable DCR, this trial did not meet its primary efficacy endpoint. TRIAL REGISTRATION NUMBER: UMIN000006128.

BACKGROUND: Programmed death-ligand 1 (PD-L1) expression on immune cells (ICs) is a predictive marker for PD-L1 checkpoint blockade in patients with triple-negative breast cancer (TNBC). However, the level of PD-L1 expression and the percentage of cells that are PD-L1+ are continuous variables not dichotomous variables for tumor-infiltrating lymphocytes (TILs) and other cells. METHODS: Multiplexed immunohistochemistry was applied to 31 archived surgical specimens from untreated TNBC patients. TIL levels were visually scored, and CD8+ T cells and PD-L1+ ICs were quantified using an automated multispectral imaging system. PD-L1 expression was assessed within a multiplexed context (CD8 combined spectral composite). RESULTS: The mean value of stromal TILs (i.e., the percentage of the stromal area with a dese mononuclear infiltrate) was 20%. The frequency of patients with PD-L1-positive tumor cells (TC) and ICs was 38.7% and 32.2%, respectively, with a significant association between them. TIL levels were correlated with CD8+ T cell infiltration in the stroma (Spearman r = 0.795, p < 0.0001). PD-L1 expression on IC was significantly associated with TIL levels (Spearman r = 0.790, p < 0.001) and infiltration of CD8+ T cells (Spearman r = 0.683, p < 0.0001). CONCLUSIONS: The level of PD-L1 on IC was correlated with the level of PD-L1 on TC as well as TIL levels and infiltration of CD8+ T cells. These results suggest that high PD-L1 on IC may reflect T cell-inflamed tumors with the amount of TILs present, including the CD8+ T cells required for anti-tumor responses.

BACKGROUND: There is increasing evidence for the benefit of poly ADP ribose polymerase (PARP) inhibitors in a subset of high-grade serous ovarian carcinoma (HGSC) patients, especially those with homologous recombination (HR)-deficient tumors. However, new treatment strategies, such as immune checkpoint inhibition, are required for patients with HR-proficient tumors. METHODS: A total of 80 cases of HGSC were analyzed in this study. Whole exome and RNA sequencing was performed for these tumors. Methylation arrays were also carried out to examine BRCA1 and RAD51C promoter methylation status. Mutations, neoantigen load, antigen presentation machinery, and local immune profile were investigated, and the relationships of these factors with clinical outcome were also analyzed. RESULTS: As expected, the numbers of predicted neoAgs were lower in HR-proficient (n=46) than HR-deficient tumors (n=34). However, 40% of the patients with HR-proficient tumors still had higher than median numbers of neoAgs and better survival than patients with lower numbers of neoAgs. Incorporation of human leukocyte antigen (HLA)-class I expression status into the survival analysis revealed that patients with both high neoAg numbers and high HLA-class I expression (neoAghiHLAhi) had the best progression-free survival (PFS) in HR-proficient HGSC (p=0.0087). Gene set enrichment analysis demonstrated that the genes for effector memory CD8 T cells, TH1 T cells, the interferon-γ response, and other immune-related genes, were enriched in these patients. Interestingly, this subset of patients also had better PFS (p=0.0015) and a more T-cell-inflamed tumor phenotype than patients with the same phenotype (neoAghiHLAhi) in HR-deficient HGSC. CONCLUSIONS: Our results suggest that immune checkpoint inhibitors might be an alternative to explore in HR-proficient cases which currently do not benefit from PARP inhibition.

The establishment of cancer cell lines, which have different metastatic abilities compared with the parental cell, is considered as an effective approach to investigate mechanisms of metastasis. A highly metastatic potential mouse colon cancer cell subline, Colon-26MGS, was derived from the parental cell line Colon-26 by in vivo selection using continuous subcutaneous implanting to immunocompetent mice. To clarify the mechanisms involved in the enhancement of metastasis, morphological characteristics, cell proliferation, and gene expression profiles were compared between Colon-26MGS and the parental cell. Colon-26MGS showed over 10 times higher metastatic ability compared with the parental cell, but there were no differences in morphological characteristics and in vitro proliferation rates. In addition, the Colon-26MGS-bearing mice exhibited no marked change of splenocyte population and lung pre-metastatic niche with tumor-free mice, but there were significant differences compared to Colon-26-bearing mice. RNA-seq analyses indicated that immune costimulatory molecules were significantly up-regulated in Colon-26MGS. These results suggest that Colon-26MGS showed not only higher metastatic activity, but also less induction property of host immune response compared to parental Colon-26. Colon-26MGS has proven to be a novel useful tool for studying multiple mechanisms involving metastasis enhancement.

Treatment with molecular targeted agents together with immune checkpoint inhibitors will most likely improve the efficacy of current cancer immunotherapy. Because molecular targeted agents not only directly affect cancer cells, but also influence immune cells and modulate the tumor microenvironment, a better understanding of the overall immunological effects of these drugs will contribute to the rational design of combination therapies. Therefore, this study performed extensive immune monitoring of patients' peripheral blood mononuclear cells (PBMCs) to investigate the immunological effects of the molecular targeted agents sunitinib, everolimus and temsirolimus, which have been widely used for the treatment of renal cell carcinoma (RCC). Immunophenotyping and functional analysis of PBMCs revealed that these molecular targeted agents exerted different immunological effects on patients with RCC. Sunitinib decreased the percentage of early‑stage myeloid‑derived suppressor cells (eMDSCs) and increased natural killer cells, but did not affect the phenotypes and effector functions of CD4+ or CD8+ T cells. Everolimus decreased effector regulatory T cells, but also decreased IL‑2‑producing CD4+ T cells and increased dysfunctional CD8+ T cells. Conversely, temsirolimus decreased programmed cell death protein 1+CD8+ T cells and eMDSCs, but increased interferon‑γ and tumor necrosis factor‑α double producers at the same time as decreasing dysfunctional CD8+ T cells, albeit not significantly. In conclusion, although everolimus and temsirolimus are mTOR inhibitors, their effects on overall T‑cell functions are very different. Therefore, although it may increase the risk of immune‑related toxicity, temsirolimus is expected to offer the best outcome when combined with other immunomodulators for the development of cancer immunotherapy.

BACKGROUND: The tumor microenvironment can be classified into immunologically active "inflamed" tumors and inactive "non-inflamed" tumors based on the infiltration of cytotoxic immune cells. Previous studies on liver cancer have reported a superior prognosis for inflamed tumors compared to non-inflamed tumors. However, liver cancer is highly heterogeneous immunologically and genetically, and a finer classification of the liver cancer microenvironment may improve our understanding of its immunological diversity and response to immune therapy. METHODS: We characterized the immune gene signatures of 234 primary liver cancers, mainly virus-related, from a Japanese population using RNA-Seq of tumors and matched non-tumorous hepatitis livers. We then compared them with the somatic alterations detected using the whole-genome sequencing. FINDINGS: Liver cancers expressed lower levels of immune marker genes than non-tumorous hepatitis livers, indicating immunosuppression in the tumor microenvironment. Several immunosuppression mechanisms functioned actively and mutually exclusively, resulting in four immune subclasses of liver cancer: tumor-associated macrophage (TAM), CTNNB1, cytolytic activity (CYT), and regulatory T cell (Treg). The CYT and Treg subclasses represented inflamed tumors, while the TAM and CTNNB1 subclasses represented non-inflamed tumors. The TAM subclass, which comprised 31% of liver cancers, showed a poor survival, expressed elevated levels of extracellular matrix genes, and was associated with somatic mutations of chromatin regulator ARID2. The results of cell line experiments suggested a functional link between ARID2 and chemokine production by liver cancer cells. INTERPRETATION: Primary liver cancer was classified into four subclasses based on mutually exclusive mechanisms for immunosuppression. This classification indicate the importance of immunosuppression mechanisms, such as TAM and Treg, as therapeutic targets for liver cancer. FUNDING: The Japan Agency for Medical Research and Development (AMED).

Objectives: A better understanding of antitumor immunity will help predict the prognosis of gastric cancer patients and tailor the appropriate therapies in each patient. Therefore, we propose a novel immunological classification of gastric cancer. Methods: We performed whole-exome sequencing (WES), RNA-Seq and flow cytometry in 29 gastric cancer patients who received surgery. The TCGA data set of 323 gastric cancer patients and RNA-Seq data of 45 patients who received pembrolizumab (Kim et al. Nat Med 2018; 24: 1449-1458) were also analysed. Results: Immunogram analysis of cancer-immunity interaction of gastric cancer revealed immune signatures of four main types, designated Hot1, Hot2, Intermediate and Cold. Immunologically hot tumors displayed a dysfunctional T-cell signature, while cold tumors had an exclusion signature. Ex vivo tumor-infiltrating lymphocyte analysis documented T-cell dysfunction with the expression of checkpoint molecules and impaired cytokine production. The T-cell function was more profoundly damaged in Hot1 than Hot2 tumors. Patients in Hot2 subtypes had better survival in our cohort and TCGA cohort. Although these immunological subtypes overlapped to some degree with the molecular subtypes in the TCGA, intratumoral immune responses cannot be predicted solely based on histological or molecular subtyping of gastric cancer. Molecular and immunological classifications complement each other to predict the responses to anti-PD-1 therapy and have the potential to be a biomarker for the treatment of gastric cancer. Conclusion: The immunological classification of gastric cancer resulted in four subtypes. Hot tumors were further divided into two subtypes, between which the functional status of T cells was different.

INTRODUCTION: Programmed cell death-1 (PD-1) inhibitors effectively treat NSCLC and prolong survival. Robust biomarkers for predicting clinical benefits of good response and long survival with anti-PD-1 therapy have yet to be identified; therefore, predictive biomarkers are needed to select patients with benefits. METHODS: We conducted a prospective study to explore whether serum antibody against NY-ESO-1 and/or XAGE1 cancer-testis antigens predicted primarily good clinical response and secondarily long survival with anti-PD-1 therapy for NSCLC. The serum antibody was detected by enzyme-linked immunosorbent assay, and tumor immune microenvironment and mutation burden were analyzed by immunohistochemistry and next-generation sequencing. RESULTS: In the discovery cohort (n = 13), six antibody-positive NSCLC cases responded to anti-PD-1 therapy (two complete and four partial responses), whereas seven antibody-negative NSCLC cases did not. Antibody positivity was associated with good response and survival, regardless of tumor programmed death ligand 1 (PD-L1) expression, mutation burden, and CD8+ T-cell infiltration. In the validation cohort (n = 75), 17 antibody-positive NSCLC cases responded well to anti-PD-1 therapy as compared with 58 negative NSCLC cases (objective response rate 65% versus 19%, p = 0.0006) and showed significantly prolonged progression-free survival and overall survival. Antibody titers highly correlated with tumor reduction rates. In the multivariate analysis, response biomarkers were tumor programmed death ligand 1 expression and antibody positivity, and only antibody positivity was a significantly better predictive biomarker of progression-free survival (hazard ratio = 0.4, p = 0.01) and overall survival (hazard ratio = 0.2, p = 0.004). CONCLUSIONS: Our results suggest that NY-ESO-1 and/or XAGE1 serum antibodies are useful biomarkers for predicting clinical benefits in anti-PD-1 therapy for NSCLC and probably for other cancers.

Immune-based therapies have shown limited efficacy in glioma thus far. This might be at least in part due to insufficient numbers of neoantigens, thought to be targets of immune attack. In addition, we hypothesized that dynamic genetic and epigenetic tumor evolution in gliomas might also affect the mutation/neoantigen landscape and contribute to treatment resistance through immune evasion. Here, we investigated changes in the neoantigen landscape and immunologic features during glioma progression using exome and RNA-seq of paired primary and recurrent tumor samples obtained from 25 WHO grade II-IV glioma patients (glioblastoma, IDH-wild-type, n = 8; grade II-III astrocytoma, IDH-mutant, n = 9; and grade II-III oligodendroglioma, IDH-mutant, 1p/19q-codeleted, n = 8). The number of missense mutations, predicted neoantigens, or expressed neoantigens was not significantly different between primary and recurrent tumors. However, we found that in individual patients the ratio of expressed neoantigens to predicted neoantigens, designated the "neoantigen expression ratio," decreased significantly at recurrence (P = 0.003). This phenomenon was particularly pronounced for "high-affinity," "clonal," and "passenger gene-derived" neoantigens. Gene expression and IHC analyses suggested that the decreased neoantigen expression ratio was associated with intact antigen presentation machinery, increased tumor-infiltrating immune cells, and ongoing immune responses. Our findings imply that decreased expression of highly immunogenic neoantigens, possibly due to persistent immune selection pressure, might be one of the immune evasion mechanisms along with tumor clonal evolution in some gliomas.

PURPOSE: Irreversible electroporation (IRE) differs from thermal radiofrequency (RF) ablation, especially in terms of the reparative process in the ablation zone induced. To elucidate this, the systemic immune responses after 2 mechanistically different types of ablation (IRE and RF ablation) were evaluated in patients with hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Twenty-one patients with HCC who underwent either RF ablation (n = 11) or IRE (n = 10) were studied. Peripheral blood samples were collected from all patients at 4 timepoints: before ablation, within 1 hour after ablation, 1 day after ablation, and 4 days after ablation. The phenotypes and functions of immune cells in peripheral blood and serum levels of cytokines and chemokines were monitored and analyzed using the mixed-effects model. Follow-up radiological images were also obtained to assess temporal changes in the ablation zone. RESULTS: The most significant difference was seen in the levels of macrophage migration inhibitory factor (MIF) in the IRE group compared to the RF ablation group (P = .0011): the serum levels of MIF in the IRE group significantly increased immediately after IRE and then rapidly decreased to the pre-ablation range 1 day after IRE, but, in contrast, no consistent trend was observed in the RF ablation group. The axial diameter (P = .0009) and area (P = .0192) of the ablation zone of IRE were significantly smaller than those of RF ablation 1 year after ablation. CONCLUSIONS: IRE was found to be associated with a significant early increase in MIF levels, which may facilitate the early reparative process and result in significant shrinkage of the ablation zone.

Development of chronic lung allograft dysfunction involves various alloimmune-independent insults including those mediated by Toll-like receptor (TLR) signaling, which is known to activate alloimmune responses. We hypothesized that TLR signaling may also contribute to the activation of fibroblasts and promoting allograft airway fibrosis. Mouse orthotopic tracheal transplants were conducted between major histocompatibility complex (MHC)-mismatched Balb/c donor and wild-type C3H or C3H-derived TLR4 mutant recipients (nonfunctional TLR4). Immunohistochemistry on day 21 showed significantly smaller alpha-smooth muscle actin (α-SMA)-positive areas in TLR4 mutant recipients than wild-type recipients (P =.01). No difference was found for CD3+ T-cell infiltration. Proliferation of alloreactive T cells derived from the recipient spleen showed no difference between TLR4 mutant and wild-type recipients in a mixed lymphocyte reaction. The effect of TLR4 signaling was examined in primary pulmonary fibroblast cultures both with lipopolysaccharide (LPS) and transforming growth factor (TGF)-β1. Stimulation with LPS significantly increased expression of α-SMA mRNA in wild-type fibroblasts cultured with TGF-β1 compared with the control without LPS (P =.001). Taken together, these findings suggest disruption of TLR signaling leads to reduced activation of fibroblasts without affecting T-cell infiltration and proliferation in this model. TLR4-mediated activation of fibroblasts may be a potentially important mechanism of allograft remodeling.

Background: Clinical trials of anti-CCR4 antibody for solid cancers with or without other immune-modulating agents including immune checkpoint blockade therapy are currently underway. However, little is known about the roles of CCR4+ lymphocytes and their prognostic impact in lung cancer. We hypothesized that high CCR4 expression in the tumor microenvironment would be associated with a poor prognosis and would act as a biomarker in lung adenocarcinoma. Methods: First, the prognostic impact of CCR4 gene expression was explored using pooled data from public transcriptomic databases with online survival analysis software. Second, tissue microarrays (TMAs) were constructed from resected lung adenocarcinoma specimens from tumors up to 3 cm in size. The density of CCR4+ lymphocytes infiltrating the tumor was then assessed by immunohistochemistry and related to survival. Confounding factors were controlled for by multivariate analysis using the Cox proportional hazards model. Results: Higher than median expression of the CCR4 gene was identified as an independent poor prognostic factor for overall survival (OS) by multivariate analysis of 720 lung adenocarcinoma patients in the public databases [HR =1.55 (95% CI: 1.03-2.35), P=0.037]. Consistent with this, high CCR4+ tumor-infiltrating lymphocyte (TIL) density was found to be an independent poor prognostic factor for both OS [HR =2.24 (1.01-5.34), P=0.049] and recurrence-free survival (RFS) [HR =2.20 (1.16-4.39), P=0.017] in the patients from whom TMA were obtained (n=180). Age, male gender, predominantly non-lepidic histological subtype, nodal involvement, and low CD8+ TIL density were also independent poor prognostic factors. However, FOXP3 gene expression and Foxp3+ lymphocyte infiltration did not possess any prognostic value in either study. Conclusions: High CCR4 expression in the tumor microenvironment may be a poor prognostic factor in lung adenocarcinoma. Patients with high CCR4+ lymphocyte infiltration may have a poor prognosis and thus be suitable candidates for clinical trials of anti-CCR4 antibody treatment.

Previously no mouse gastric cancer cell lines have been available for transplantation into C57BL/6 mice. However, a gastric cancer model in immunocompetent mice would be useful for analyzing putative therapies. N-Methyl-N-nitrosourea (MNU) was given in drinking water to C57BL/6 mice and p53 heterozygous knockout mice. Only 1 tumor from a p53 knockout mouse could be cultured and the cells s.c. transplanted into a C57BL/6 mouse. We cultured this s.c. tumor, and subcloned it. mRNA expression in the most aggressive YTN16 subline was compared to the less aggressive YTN2 subline by microarray analysis, and fibroblast growth factor receptor 4 (FGFR4) in YTN16 cells was knocked out with a CRISPR/Cas9 system and inhibited by an FGFR4 selective inhibitor, BLU9931. These transplanted cell lines formed s.c. tumors in C57BL/6 mice. Four cell lines (YTN2, YTN3, YTN5, YTN16) were subcloned and established. Their in vitro growth rates were similar. However, s.c. tumor establishment rates, metastatic rates, and peritoneal dissemination rates of YTN2 and YTN3 were lower than for YTN5 and YTN16. YTN16 established 8/8 s.c. tumors, 7/8 with lung metastases, 3/8 with lymph node metastases and 5/5 with peritoneal dissemination. FGFR4 expression by YTN16 was 121-fold higher than YTN2. FGFR4-deleted YTN16 cells failed to form s.c. tumors and showed lower rates of peritoneal dissemination. BLU9931 significantly inhibited the growth of peritoneal dissemination of YTN16. These studies present the first transplantable mouse gastric cancer lines. Our results further indicate that FGFR4 is an important growth signal receptor in gastric cancer cells with high FGFR4 expression.

Expression of immune checkpoint molecules, including programmed cell death protein-1 (PD-1), has been reported on T cells in various types of cancer. However, the expression status of these molecules in the tumor microenvironment of epithelial ovarian cancer (EOC) has not yet been studied. A total of 54 cases of malignant ascites from patients with EOC were analyzed in the present study. The expression of PD-1, lymphocyte-activation gene-3 (LAG-3), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) and B and T lymphocyte attenuator (BTLA) on cluster of differentiation (CD)4+ and CD8+ T cells in malignant EOC ascites were investigated using multicolor flow cytometric analysis. The expression of PD-L1 in tumor cells, PD-L2 in HLA-DR-positive cells and galectin-9 in ascitic fluid was also analyzed. In addition, cytokine profiling of ascitic fluid was performed to understand the immune microenvironment of EOC. PD-1, LAG-3 TIM-3, and BTLA were expressed on 65.8, 10.6, 4.3 and 37.6% of CD4+ T cells, and on 57.7, 5.0, 4.9 and 15.7% of CD8+ T cells, respectively. Programmed cell death protein-1 (PD-1), LAG-3 and BTLA were more frequently expressed on CD4+ compared with CD8+ T cells. The co-expression of immune checkpoints was further investigated and results indicated that 39 (72.2%) and 37 patients (68.5%) expressed multiple immune checkpoints on CD4+ T cells and CD8+ T cells, respectively. In addition, lower levels of TNF-α and interleukin-6 in ascitic fluid were significantly associated with multiple immune checkpoint expression on CD8+ T cells. The present findings indicated that multiple immune checkpoint molecules were expressed on T cells in the EOC tumor microenvironment and the results may suggest the significance of simultaneous blockade of immune checkpoints to control EOC.

To facilitate the optimization of cancer immunotherapy lacking immune-related adverse events, we performed TCR repertoire analysis of tumor-infiltrating CD8+ T-cells in B16 melanoma-bearing mice receiving anti-PD-1, anti-CTLA-4, anti-4-1BB, anti-CD4 or a combination of anti-PD-1 and 4-1BB antibodies. Although CD8+ T-cells in the tumor were activated and expanded to a greater or lesser extent by these therapies, tumor growth suppression was achieved only by anti-PD-1, anti-PD-1/4-1BB combined, or by anti-CD4 treatment, but not by anti-CTLA-4 or anti-4-1BB monotherapy. Increased CD8+ T cell effector function and TCR diversity with enrichment of certain TCR clonotypes in the tumor was associated with anti-tumor effects. In contrast, polyclonal activation of T-cells in the periphery was associated with tissue damage. Thus, optimal combination therapy increases TCR diversity with extended activation of selective CD8+ T-cells specifically in the tumor but not in the periphery. Incorporation of the concept of evenness for the TCR diversity is proposed.

To establish prognostic biomarkers and to identify potential novel therapeutic targets, we performed integrative immunomonitoring of blood and tumor in patients with resectable pancreatic cancer. Flow cytometry (FC) was employed for phenotyping immune cells, multiplex bead assays for plasma cytokine and chemokine determination, and RNA-Seq for the analysis of gene expression in the tumor. Nineteen pancreatic cancer patients were stratified into those with longer or shorter than median recurrence-free survival after surgery (median, 426 days). There were no significant differences between the two groups for clinical parameters including age, sex, surgical procedure, stage, or postoperative adjuvant therapy. However, we found that the percentages of NK cells as assessed by FC in peripheral blood mononuclear cells were higher in patients with late recurrence (P = .037). RNA-Seq data indicated no differences in the amount of immune cells or stromal cells between the two groups, although NK cells in the tumor did tend to be higher in patients with late recurrence (P = .058). Type I and II IFN signatures were enriched in late-recurring tumors (FDR q-value <0.001), while genes related to KRAS signaling and the epithelial mesenchymal transition (EMT) were enriched in early recurrence. We conclude that tumor-intrinsic properties of metastasis and recurrence influence prognosis, whereas NK cells that might contribute to prevent metastasis are associated with longer recurrence-free survival. Therefore, enhancement of NK cell activity and inhibition of the EMT and KRAS signaling might represent appropriate therapeutic targets following surgical resection of pancreatic cancer.

The success of immune checkpoint blockade has unequivocally demonstrated that anti-tumor immunity plays a pivotal role in cancer therapy. Because endogenous tumor-specific T-cell responsiveness is essential for the success of checkpoint blockade, combination therapy with cancer vaccination may facilitate tumor rejection. To select the best vaccine strategy to combine with checkpoint blockade, we compared dendritic cell-based vaccines (DC-V) with peptide vaccines for induction of anti-tumor immunity that could overcome tumor-induced immunosuppression. Using B16 melanoma and B16-specific TCR-transgenic T-cells (pmel-1), we found that DC-V efficiently primed and expanded pmel-1 cells with an active effector and central memory phenotype that were not exhausted. Vaccine-primed cells were metabolically distinct from naïve cells. DC-V-primed pmel-1 cells contained the population that shifted metabolic pathways away from glycolysis to mitochondrial oxidative phosphorylation. They displayed better effector function and proliferated more than those induced by peptide vaccination. DC-V inhibited tumor growth in prophylactic and therapeutic settings. Only DC-V but not peptide vaccine showed augmented anti-tumor activity when combined with anti-PD-1 therapy. Thus, DC-V combined with PD-1 checkpoint blockade mediates optimal anti-cancer activity in this model.

Depletion of CD4+ cells using an anti-CD4 monoclonal antibody (anti-CD4 mAb) induces the expansion of tumor-reactive CD8+ T cells and strong antitumor effects in several murine tumor models. However, it is not known whether the anti-CD4 mAb treatment activates a particular or a broad spectrum of tumor-reactive CD8+ T cell clones. To investigate the changes in the TCR repertoire induced by the anti-CD4 mAb treatment, we performed unbiased high-throughput TCR sequencing in a B16F10 mouse subcutaneous melanoma model. By Inter-Organ Clone Tracking analysis, we demonstrated that anti-CD4 mAb treatment increased the diversity and combined frequency of CD8+ T cell clones that overlapped among the tumor, draining lymph node (dLN), and peripheral blood repertoires. Interestingly, the anti-CD4 mAb treatment-induced expansion of overlapping clones occurred mainly in the dLN rather than in the tumor. Overall, the Inter-Organ Clone Tracking analysis revealed that anti-CD4 mAb treatment enhances the mobilization of a wide variety of tumor-reactive CD8+ T cell clones into the Cancer-Immunity Cycle and thus induces a robust antitumor immune response in mice.

Carbon-ion radiotherapy (CIRT) is an advanced radiotherapy and has achieved good local control, even in tumors that are resistant to conventional photon beam radiotherapy (PBRT). However, distant metastasis control is an important issue. Recently, the combination of radiotherapy and immunotherapy has attracted the attention. In immunotherapy, dendritic cells (DCs) play a pivotal role in the anti-tumor immune system. However, the mechanisms underlying the combination therapy of DCs and radiotherapy have been unclear. In the present study, we evaluated anti-metastatic effects of this combination therapy, focused on the irradiation type and the route of DC administration, using a mouse model. C3H/He mice bearing NR-S1 cells were treated with CIRT or PBRT, using biologically equivalent doses. Subsequently, DCs were administered intratumorally (IT) or intravenously (IV). IV and IT DC administrations combined with CIRT to the local tumor, but not alone, significantly suppressed pulmonary metastasis, whereas the combination of DCs with PBRT suppressed metastasis at a relatively higher dose. Additionally, the anti-metastatic effect was greater in IV DC administration compared with in IT DC administration in both CIRT and PBRT. The expression levels of CD40 and IL-12 in DCs were significantly increased after co-culturing with CIRT-treated NR-S1 cells. In addition, IV administration of those co-cultured DCs significantly suppressed pulmonary metastasis. Furthermore, ecto-calreticulin levels from CIRT-treated NR-S1 cells significantly increased compared with those of a PBRT-treated tumor. Taken together, these results suggest that local CIRT combined with IV DCs augments an immunogenicity of the tumor cells by ecto-calreticulin expression and the maturation of DCs to stimulate anti-tumor immunity to decrease lung metastases.

INTRODUCTION: The interaction of immune cells and cancer cells shapes the immunosuppressive tumor microenvironment. For successful cancer immunotherapy, comprehensive knowledge of antitumor immunity as a dynamic spatiotemporal process is required for each individual patient. To this end, we developed an immunogram for the cancer-immunity cycle by using next-generation sequencing. METHODS: Whole exome sequencing and RNA sequencing were performed in 20 patients with NSCLC (12 with adenocarcinoma, seven with squamous cell carcinoma, and one with large cell neuroendocrine carcinoma). Mutated neoantigens and cancer germline antigens expressed in the tumor were assessed for predicted binding to patients' human leukocyte antigen molecules. The expression of genes related to cancer immunity was assessed and normalized to construct a radar chart composed of eight axes reflecting seven steps in the cancer-immunity cycle. RESULTS: Three immunogram patterns were observed in patients with lung cancer: T-cell-rich, T-cell-poor, and intermediate. The T-cell-rich pattern was characterized by gene signatures of abundant T cells, regulatory T cells, myeloid-derived suppressor cells, checkpoint molecules, and immune-inhibitory molecules in the tumor, suggesting the presence of antitumor immunity dampened by an immunosuppressive microenvironment. The T-cell-poor phenotype reflected lack of antitumor immunity, inadequate dendritic cell activation, and insufficient antigen presentation in the tumor. Immunograms for both the patients with adenocarcinoma and the patients with nonadenocarcinoma tumors included both T-cell-rich and T-cell-poor phenotypes, suggesting that histologic type does not necessarily reflect the cancer immunity status of the tumor. CONCLUSIONS: The patient-specific landscape of the tumor microenvironment can be appreciated by using immunograms as integrated biomarkers, which may thus become a valuable resource for optimal personalized immunotherapy.

Background aims. The outcome for pancreatic cancer after surgery remains highly unsatisfactory, and development of more effective therapies is urgently needed. Therefore, we conducted a phase I clinical study of a novel combination of gemcitabine (GEM) and autologous gamma delta T-cell therapy for patients with curatively resected pancreatic cancer (University Hospital Medical Information Clinical Trials Registry identifier 000000931). Methods. From July 2008 to December 2012, 56 consenting patients were recruited. After preliminary testing of gamma delta T-cell proliferative capacity, 28 patients were eligible to receive combined GEM plus gamma delta T-cell therapy. Results. During treatment, most of the adverse events observed were due to GEM, including myelosuppression and gastrointestinal disorders. No severe adverse events were obviously related to the gamma delta T-cell therapy. To evaluate clinical efficacy, patients receiving combined therapy (Group A, n = 28) were compared with those receiving GEM alone (Group B, n = 20). No significant differences were observed between the two groups in recurrence-free survival or overall survival. However, we found that, relative to progressing patients, more gamma delta T-cells were detectable in the blood of recurrence-free patients after only two injections (P < .0388) and more so five injections (P < .0175). Patients with >15% peripheral gamma delta T-cells after two injections and >20% after five injections had a chance of a more favorable clinical outcome. Accumulation of gamma delta T cells was positively related to the quality of the infused products, with those having >80% gamma delta T cells being optimal. Discussion. High quality of the gamma delta T-cell product is crucial to achieve a high percentage of gamma delta T cells in the blood and to achieve better clinical outcome.

The importance of neoantigens for cancer immunity is now well-acknowledged. However, there are diverse strategies for predicting and prioritizing candidate neoantigens, and thus reported neoantigen loads vary a great deal. To clarify this issue, we compared the numbers of neoantigen candidates predicted by four currently utilized strategies. Whole-exome sequencing and RNA sequencing (RNA-Seq) of four non-small-cell lung cancer patients was carried out. We identified 361 somatic missense mutations from which 224 candidate neoantigens were predicted using MHC class I binding affinity prediction software (strategy I). Of these, 207 exceeded the set threshold of gene expression (fragments per kilobase of transcript per million fragments mapped 1), resulting in 124 candidate neoantigens (strategy II). To verify mutant mRNA expression, sequencing of amplicons from tumor cDNA including each mutation was undertaken; 204 of the 207 mutations were successfully sequenced, yielding 121 mutant mRNA sequences, resulting in 75 candidate neoantigens (strategy III). Sequence information was extracted from RNA-Seq to confirm the presence of mutated mRNA. Variant allele frequencies 0.04 in RNA-Seq were found for 117 of the 207 mutations and regarded as expressed in the tumor, and finally, 72 candidate neoantigens were predicted (strategy IV). Without additional amplicon sequencing of cDNA, strategy IV was comparable to strategy III. We therefore propose strategy IV as a practical and appropriate strategy to predict candidate neoantigens fully utilizing currently available information. It is of note that different neoantigen loads were deduced from the same tumors depending on the strategies applied.

Neoantigens derived from tumor-specific somatic mutations are excellent targets for anti-tumor immune responses. In ovarian clear cell carcinoma (OCCC), checkpoint blockade yields durable responses in a subset of patients. To approach the question of why only some patients respond, we first investigated neoantigen loads and immune signatures using exome sequencing and expression array data for 74 OCCC patients treated conventionally. Neither the number of missense mutations nor total predicted neoantigens assessed in the tumor correlated with clinical outcomes. However, the number of neoantigens per missense mutation ("neoAg frequency") did correlate with clinical outcomes. Cox multivariate regression analysis demonstrated that low neoAg frequencies correlated with increased progression-free survival (PFS) and was an independent predictive factor for PFS in OCCC (p = 0.032), especially at stage I-II (p D 0.0045). Immunity-associated genes including those related to effector memory CD8 T cells were dominantly expressed in tumors with low neoAg frequencies in stage I-II patients, suggesting CD8 T cell-mediated elimination of immunogenic sub-clones expressing neoantigens (immunoediting) had occurred. In contrast, we observed decreased HLA-A, -B, and -C expression (p = 0.036, p = 0.026, and p = 0.030, respectively) as well as increased ratios of CTLA-4, PD-1, Tim-3, and LAG3 to CD8A expression (p = 0.0064, p = 0.017, p = 0.033 and p = 0.0136, respectively) in stage I-II tumors with high neoAg frequencies. Constrained anti-tumor immunity may thus result in limited immunoediting, and poor prognosis. Our results show that neoAg frequency in OCCC is an independent prognostic factor for clinical outcome and may become a potential candidate biomarker for immunomodulatory agentbased treatments.

[This corrects the article DOI: 10.1371/journal.ppat.1003490.].

There are currently three major approaches to T cell-based cancer immunotherapy, namely, active vaccination, adoptive cell transfer therapy and immune checkpoint blockade. Recently, this latter approach has demonstrated remarkable clinical benefits, putting cancer immunotherapy under the spotlight. Better understanding of the dynamics of anti-tumor immune responses (the "Cancer-Immunity Cycle") is crucial for the further development of this form of treatment. Tumors employ multiple strategies to escape from anti-tumor immunity, some of which result from the selection of cancer cells with immunosuppressive activity by the process of cancer immunoediting. Apart from this selective process, anti-tumor immune responses can also be inhibited in multiple different ways which vary from patient to patient. This implies that cancer immunotherapy must be personalized to (1) identify the rate-limiting steps in any given patient, (2) identify and combine strategies to overcome these hurdles, and (3) proceed with the next round of the "Cancer-Immunity Cycle". Cancer cells have genetic alterations which can provide the immune system with targets by which to recognize and eradicate the tumor. Mutated proteins expressed exclusively in cancer cells and recognizable by the immune system are known as neoantigens. The development of next-generation sequencing technology has made it possible to determine the genetic landscape of human cancer and facilitated the utilization of genomic information to identify such candidate neoantigens in individual cancers. Future immunotherapies will need to be personalized in terms of the identification of both patient-specific immunosuppressive mechanisms and target neoantigens.

Neoantigens derived from tumor-specific somatic mutations are excellent targets for anti-tumor immune responses. In ovarian clear cell carcinoma (OCCC), checkpoint blockade yields durable responses in a subset of patients. To approach the question of why only some patients respond, we first investigated neoantigen loads and immune signatures using exome sequencing and expression array data for 74 OCCC patients treated conventionally. Neither the number of missense mutations nor total predicted neoantigens assessed in the tumor correlated with clinical outcomes. However, the number of neoantigens per missense mutation ("neoAg frequency") did correlate with clinical outcomes. Cox multivariate regression analysis demonstrated that low neoAg frequencies correlated with increased progression-free survival (PFS) and was an independent predictive factor for PFS in OCCC (p = 0.032), especially at stage I-II (p = 0.0045). Immunity-associated genes including those related to effector memory CD8 T cells were dominantly expressed in tumors with low neoAg frequencies in stage I-II patients, suggesting CD8 T cell-mediated elimination of immunogenic sub-clones expressing neoantigens (immunoediting) had occurred. In contrast, we observed decreased HLA-A, -B, and -C expression (p = 0.036, p = 0.026, and p = 0.030, respectively) as well as increased ratios of CTLA-4, PD-1, Tim-3, and LAG3 to CD8A expression (p = 0.0064, p = 0.017, p = 0.033 and p = 0.0136, respectively) in stage I-II tumors with high neoAg frequencies. Constrained anti-tumor immunity may thus result in limited immunoediting, and poor prognosis. Our results show that neoAg frequency in OCCC is an independent prognostic factor for clinical outcome and may become a potential candidate biomarker for immunomodulatory agent-based treatments.

[This corrects the article DOI: 10.1371/journal.ppat.1003490.].

In addition to the majority of T cells which carry the alpha beta T cell receptor (TCR) for antigen, a distinct subset of about 1-5% of human peripheral blood T cells expressing the gamma delta TCR contributes to immune responses to infection, tissue damage and cancer. T cells with the V delta 2+ TCR, usually paired with V gamma 9, constitute the majority of these gamma delta T cells. Analogous to alpha beta T cells, they can be sorted into naive (CD27(+)CD45RA(+)), central memory (CD27(+)CD45RA(-)), effector memory (CD27(-)CD45RA(-)), and terminally-differentiated effector memory (CD27(-)CD45RA(+)) phenotypes. Here, we found that CD27(-)CD45RA(+) gamma delta T cells can be further divided into two populations based on the level of expression of CD45RA: CD27(-)CD45RA(int) and CD27(-)CD45RA(hi). Those with the CD27(-)CD45RA(hi) phenotype lack extensive proliferative capacity, while those with the CD27(-)CD45RA(int) phenotype can be easily expanded by culture with zoledronate and IL-2. These CD27-CD45RA(hi) potentially exhausted gamma delta T cells were found predominantly in cancer patients but also in healthy subjects. We conclude that gamma delta T cells can be divided into at least 5 subsets enabling discrimination of gamma delta T cells with poor proliferative capacity. It was one of our goals to predict the feasibility of gd T cell expansion to sufficient amounts for adoptive immunotherapy without the necessity for conducting small-scale culture tests. Fulfilling the >1.5% criterion for gamma delta T cells with phenotypes other than CD27(-)CD45RA(hi), may help avoid small-scale culture testing and shorten the preparation period for adoptive gamma delta T cells by 10 days, which may be beneficial for patients with advanced cancer. (C) 2016 Elsevier Inc. All rights reserved.

Tumors commonly harbor multiple genetic alterations, some of which initiate tumorigenesis. Among these, some tumor-specific somatic mutations resulting in mutated protein have the potential to induce antitumor immune responses. To examine the relevance of the latter to immune responses in the tumor and to patient outcomes, we used datasets of whole-exome and RNA sequencing from 97 clear cell renal cell carcinoma (ccRCC) patients to identify neoepitopes predicted to be presented by each patient's autologous HLA molecules. We found that the number of nonsilent or missense mutations did not correlate with patient prognosis. However, combining the number of HLA-restricted neoepitopes with the cell surface expression of HLA or beta(2)-microglobulin (beta M-2) revealed that an A-neohi/HLA-Ahi or ABC-neohi/beta(2)Mhi phenotype correlated with better clinical outcomes. Higher expression of immune-related genes from CD8 T cells and their effector molecules [CD8A, perforin (PRF1) and granzyme A (GZMA)], however, did not correlate with prognosis. This may have been due to the observed correlation of these genes with the expression of other genes that were associated with immunosuppression in the tumor microenvironment (CTLA-4, PD-1, LAG-3, PD-L1, PDL2, IDO1, and IL10). This suggested that abundant neoepitopes associated with greater antitumor effector immune responses were counterbalanced by a strongly immunosuppressive microenvironment. Therefore, immunosuppressive molecules should be considered high-priority targets for modulating immune responses in patients with ccRCC. Blockade of these molecular pathways could be combined with immunotherapies targeting neoantigens to achieve synergistic antitumor activity. (C) 2016 AACR.

The melanoma-associated antigen A (MAGE-A) family comprises cancer-testis antigens that represent promising prognostic biomarkers and immunotherapy targets in several cancer types. The aim of this study was to investigate the significance of MAGE-A expression in upper urinary tract urothelial carcinoma in relation to clinicopathological features, lymphocytic infiltration, and clinical outcome. We immunohistochemically examined the expression of MAGE-A in 171 patients with upper urinary tract urothelial carcinoma. High (>= 50% positive) and low MAGE-A expression levels were observed in 33 (19%) and 49 (29%) cases, respectively. MAGE-A was negative in 89 cases (52%). MAGE-A expression was positively correlated with high histologic grade; concomitant carcinoma in situ; higher Ki-67 proliferation index; and infiltration of CD3-, CD8-, and CD45RO-positive T lymphocytes, but not with CD20-positive B lymphocytes. High MAGE-A expression was significantly associated with shorter metastasis-free survival after nephroureterectomy (log-rank P = .019; multivariate hazard ratio, 1.98; 95% confidence interval, 1.00-3.92). MAGE-A expression in metastatic lymph nodes was highly correlated with its expression in primary lesions. MAGE-A expression was retained in chemotherapy resistant metachronous metastatic lesions of urothelial carcinoma. MAGE-A may be a promising prognostic biomarker and potential immunotherapeutic target for patients with upper urinary tract urothelial carcinoma. (C) 2015 Elsevier Inc. All rights reserved.

Introduction: Two strategies for selecting neoantigens as targets for non-small cell lung cancer vaccines were compared: (1) an "off-the-shelf" approach starting with shared mutations extracted from global databases and (2) a personalized pipeline using whole-exome sequencing data on each patient's tumor. Methods: The Catalogue of Somatic Mutations in Cancer database was used to create a list of shared missense mutations occurring in more than 1% of patients. These mutations were then assessed for predicted binding affinity to HLA alleles of 15 lung cancer patients, and potential neoantigens (pNeoAgs) for each patient were selected on this basis. In the personalized approach, pNeoAgs were selected from missense mutations detected by whole-exome sequencing of the patient's own samples. Results: The list of shared mutations included 22 missense mutations for adenocarcinoma and 18 for squamous cell carcinoma (SCC), resulting in a median of 10 off-the-shelf pNeoAgs for each adenocarcinoma (range 5-13) and 9 (range 5-12) for each SCC. In contrast, a median of 59 missense mutations were identified by whole-exome sequencing (range 33-899) in adenocarcinoma and 164.5 (range 26-232) in SCC. This resulted in a median of 46 pNeoAgs (range 13-659) for adenocarcinoma and 95.5 (range 10-145) for SCC in the personalized set. We found that only one or two off-the-shelf pNeoAgs were included in the set of personalized pNeoAgs and then in only three patients, with no overlap seen in the remaining 12 patients. Conclusions: Use of an off-the-shelf pipeline is feasible but may not be satisfactory for most patients with non-small cell lung cancer. We recommend identifying personal mutations by comprehensive genome sequencing for developing neoantigen-targeted cancer immunotherapies.

γδ T cell-based cancer immunotherapy is attracting attention for the treatment of various malignancies because these cells secrete Th1-type cytokines, exert potent cytotoxicity against a variety of cancer cells irrespective of MHC class I expression, and bridge innate and adaptive immunity. They comprise 1-5 % of peripheral blood mononuclear cell (PBMC) the majority of them express the Vγ9Vδ2 T cell receptor that recognizes phosphoantigens. There are two strategies to develop γδ T cell-based cancer immunotherapy. One is in vivo direct activation/ expansion of γδ T cells in cancer patients the other is adoptive transfer of ex vivo- expanded γδ T cells. Both strategies have been tested in several clinical trials. We have established a large-scale in vitro expansion method for Vγ9Vδ2 T cells using zoledronate and interleukin-2. We found that Vγ9Vδ2 T cells from patients with advanced cancer underwent extensive proliferation under these conditions. Such cultured Vγ9Vδ2 T cells retained cytokine secretion capacity and mediated cytotoxicity against a variety of cancer cell lines. Recently, we conducted phase I clinical studies to evaluate safety and potential antitumor effects of intravenous injection of Vγ9Vδ2 T cells in patients with non-small cell lung cancer (NSCLC) and intraperitoneal injection of Vγ9Vδ2 T cells for the treatment of gastric cancer with malignant ascites. γδ T cells produced IFN-γ immediately upon recognition of cancer cells in ascites. Measuring IFN-γ in patients' sera might be a good prognostic marker in lung cancer patients receiving γδ T cells. γδ T cell therapy trials are being conducted at present with safety and response results already reported for selected cases. Despite the limited number of patients in the phase I studies, the clinical response is thus far promising and warrants further study.

Humoral immune responses against tumor-associated antigens (TAAs) or cancer/testis antigens (CTAs) aberrantly expressed in tumor cells are frequently observed in cancer patients. Recent clinical studies have elucidated that anticancer immune responses with increased levels of anti-TAA/CTA antibodies improve cancer survival rates. Thus, these antibody levels are promising biomarkers for diagnosing the efficiency of cancer immunotherapy. Full-length antigens are favored for detecting anti-TAA/CTA antibodies because candidate antigen proteins contain multiple epitopes throughout their structures. In this study, we developed a methodology to prepare purified water-soluble and full-length antigens by using cysteine sulfhydryl group cationization (S-cationization) chemistry. S-Cationized antigens can be prepared from bacterial inclusion bodies, and they exhibit improved protein solubility but preserved antigenicity. Anti-TAA/CTA antibodies detected in cancer patients appeared to recognize linear epitopes, as well as conformational epitopes, and because the frequency of cysteine side-residues on the epitope-paratope interface was low, any adverse effects of S-cationization were virtually negligible for antibody binding. Furthermore, S-cationized antigen-immobilized Luminex beads could be successfully used in highly sensitive quantitative-multiplexed assays. Indeed, patients with a more broadly induced serum anti-TAA/CTA antibody level showed improved progression-free survival after immunotherapy. The comprehensive anti-TAA/CTA assay system, which uses S-cationized full-length and water-soluble recombinant antigens, may be a useful diagnostic tool for assessing the efficiency of cancer immunotherapy.

Purpose: FoxP3(+) Tregs inhibit immune responses against tumors. KW-0761 is a humanized anti-human CCR4 monoclonal antibody (mAb) that has antibody-dependent cellular cytotoxicity activity. Depletion of CCR4-expressing FoxP3(+) CD4 Tregs by KW-0761 infusion was investigated in solid cancer patients. Experimental Design: We conducted a phase Ia clinical trial of KW-0761 infusion in 7 lung and 3 esophageal cancer patients. Toxicity, clinical efficacy, changes in lymphocyte subpopulations, including Tregs, and induction of immune responses were analyzed. Results: The results showed that KW-0761 infusion in a dose range between0.1mg/kg and 1.0mg/kgwas safe andwell tolerated. Nodose-limiting toxicitywas observed. Four of 10 patients showed stable disease during treatment and were long survivors. The monitoring of FoxP3(+) Tregs in the peripheral blood mononuclear cells during treatment indicated efficient depletion of those cells, even at the lowest dose of 0.1 mg/kg used. The reduction in Th 1 CD4 T cells and CD8 T cells was limited, whereas a significant reduction was observed with Th 2 and Th 17 CD4 T cells. Immune responses to cancer/testis (CT) antigens and an autoantibody response to thyroid peroxidase were observed in some patients. Conclusions: The findings showed Tregs depletion and the possible occurrence of an immune response following KW-0761 infusion. Combined use of KW-0761 to deplete FoxP3(+) Tregs with other immunotherapies, such as cancer vaccines or checkpoint inhibitors, is a promising approach to augment immune responses. (C) 2015 AACR.

Adoptive immunotherapy with cytotoxic T lymphocytes (CTLs) can result in robust and durable antitumor responses. Tumor-infiltrating CTLs produce IFNγ and mediate antitumor activity, but they simultaneously induce counter-regulatory immunosuppressive mechanisms in the tumor by recruiting monocytic myeloid-derived suppressor cells (MDSCs) that limit their proliferation and effector function. Using a murine model of adoptive immunotherapy for B16 melanoma, we developed a strategy to augment CTL activity by downregulating immunosuppression by MDSCs. Intravenous injection of transgenic pmel-1 CTLs into tumor-bearing mice, resulted in their infiltration into the tumor, but this was accompanied by the accumulation of large numbers of monocytic MDSCs (M-MDSCs). These cells hampered CTL function and reduced their numbers in the tumor. We determined that one mechanism responsible for this immunosuppression was the production of nitric oxide (NO) by MDSCs in the tumor. Therefore, mice were given the NO scavenger carboxy-PTIO (C-PTIO) on the day after CTL transfer. This led to the restoration of impaired proliferative capacity and function of the CTLs, resulting in sustained suppression of tumor growth. Thus, we conclude that CTL therapy can be improved by counter-acting immunosuppression. Targeting NO, one mediator of the immunosuppressive activity of M-MDSCs, may be an appropriate strategy to restore impaired CTL function and improve the efficacy of immunotherapy.

Comprehensive immunological evaluation is crucial for monitoring patients undergoing antigen-specific cancer immunotherapy. The identification and quantification of T cell responses is most important for the further development of such therapies. Using well-characterized clinical samples from a high responder patient (TK-f01) in an NY-ESO-1f peptide vaccine study, we performed high-throughput T cell receptor beta-chain (TCRB) gene next generation sequencing (NGS) to monitor the frequency of NY-ESO-1-specific CD8(+) T cells. We compared these results with those of conventional immunological assays, such as IFN-gamma capture, tetramer binding and limiting dilution clonality assays. We sequenced human TCRB complementarity-determining region 3 (CDR3) rearrangements of two NY-ESO-1f specific CD8(+) T cell clones, 6-8L and 2F6, as well as PBMCs over the course of peptide vaccination. Clone 6-8L possessed the TCRB CDR3 gene TCRBV11-03*01 and BJ02-01*01 with amino acid sequence CASSLRGNEQFF, whereas 2F6 possessed TCRBV05-08*01 and BJ02-04*01 (CASSLVGTNIQYF). Using these two sequences as models, we evaluated the frequency of NY-ESO-1-specific CD8(+) T cells in PBMCs ex vivo. The 6-8L CDR3 sequence was the second most frequent in PBMC and was present at high frequency (0.7133%) even prior to vaccination, and sustained over the course of vaccination. Despite a marked expansion of NY-ESO-1-specific CD8(+) T cells detected from the first through 6th vaccination by tetramer staining and IFN-gamma capture assays, as evaluated by CDR3 sequencing the frequency did not increase with increasing rounds of peptide vaccination. By clonal analysis using 12 day in vitro stimulation, the frequency of B*52:01-restricted NY-ESO-1f peptide-specific CD8(+) T cells in PBMCs was estimated as only 0.0023%, far below the 0.7133% by NGS sequencing. Thus, assays requiring in vitro stimulation might be underestimating the frequency of clones with lower proliferation potential. High-throughput TCRB sequencing using NGS can potentially better estimate the actual frequency of antigen-specific T cells and thus provide more accurate patient monitoring.

Depletion of CD4(+) cells in tumor-bearing mice has strong antitumor effects. However, the mechanisms underlying these effects and the therapeutic benefits of CD4(+) cell depletion relative to other immunotherapies have not been fully evaluated. Here, we investigated the antitumor effects of an anti-CD4-depleting mAb as a monotherapy or in combination with immune checkpoint mAbs. In B16F10, Colon 26, or Lewis lung carcinoma subcutaneous tumor models, administration of the anti-CD4 mAb alone had strong antitumor effects that were superior to those elicited by CD25(+) Treg depletion or other immune checkpoint mAbs, and which were completely reversed by CD8(+) cell depletion. CD4(+) cell depletion led to the proliferation of tumor-specific CD8(+) T cells in the draining lymph node and increased infiltration of PD-1(+)CD8(-) T cells into the tumor, with a shift toward type I immunity within the tumor. Combination treatment with the anti-CD4 mAb and immune checkpoint mAbs, particularly anti-PD-1 or anti-PD-L1 mAbs, synergistically suppressed tumor growth and greatly prolonged survival. To our knowledge, this work represents the first report of robust synergy between anti-CD4 and anti-PD-1 or anti-PD-L1 mAb therapies. (C)2015 AACR.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population with the ability to suppress immune responses and are currently classified into three distinct MDSC subsets: monocytic, granulocytic and non-monocytic, and non-granulocytic MDSCs. Although NK cells provide an important first-line defense against newly transformed cancer cells, it is unknown whether NK cells can regulate MDSC populations in the context of cancer. In this study, we initially found that the frequency of MDSCs in non-Hodgkin lymphoma (NHL) patients was increased and inversely correlated with that of NK cells, but not that of T cells. To investigate the regulation of MDSC subsets by NK cells, we used an EL4 murine lymphoma model and found the non-monocytic and non-granulocytic MDSC subset, i.e.,Gr1(+)CCD11b(+)Ly6G(med)Ly6C(med) MDSC, is increased after NK cell depletion. The MDSC population that expresses MHC class II, CD80, CD124, and CCR2 is regulated mainly by CD27(+)CCD11b(+)NK cells. In addition, this MDSC subset produces some immunosuppressive cytokines, including IL-10 but not nitric oxide (NO) or arginase. We also examined two subsets of MDSCs (CD14(+)HLA-DR- and CD14(-) HLA-DR- MDSC) in NHL patients and found that higher IL-10-producing CD14(+)HLA-DR-MDSC subset can be seen in lymphoma patients with reduced NK cell frequency in peripheral blood. Our analyses of MDSCs in this study may enable a better understanding of how MDSCs manipulate the tumor microenvironment and are regulated by NK cells in patients with lymphoma.

Adoptive immunotherapy with cytotoxic T lymphocytes (CTLs) can result in robust and durable antitumor responses. Tumor-infiltrating CTLs produce IFN gamma and mediate antitumor activity, but they simultaneously induce counter-regulatory immunosuppressive mechanisms in the tumor by recruiting monocytic myeloid-derived suppressor cells (MDSCs) that limit their proliferation and effector function. Using a murine model of adoptive immunotherapy for B16 melanoma, we developed a strategy to augment CTL activity by downregulating immunosuppression by MDSCs. Intravenous injection of transgenic pmel-1 CTLs into tumor-bearing mice, resulted in their infiltration into the tumor, but this was accompanied by the accumulation of large numbers of monocytic MDSCs (M-MDSCs). These cells hampered CTL function and reduced their numbers in the tumor. We determined that one mechanism responsible for this immunosuppression was the production of nitric oxide (NO) by MDSCs in the tumor. Therefore, mice were given the NO scavenger carboxy-PTIO (C-PTIO) on the day after CTL transfer. This led to the restoration of impaired proliferative capacity and function of the CTLs, resulting in sustained suppression of tumor growth. Thus, we conclude that CTL therapy can be improved by counter-acting immunosuppression. Targeting NO, one mediator of the immunosuppressive activity of M-MDSCs, may be an appropriate strategy to restore impaired CTL function and improve the efficacy of immunotherapy.

To understand global effector mechanisms of CTL therapy, we performed microarray gene expression analysis in a murine model using pmel-1 T-cell receptor (TCR) transgenic T cells as effectors and B16 melanoma cells as targets. In addition to upregulation of genes related to antigen presentation and the MHC class I pathway, and cytotoxic effector molecules, cell-cycle-promoting genes were downregulated in the tumor on days 3 and 5 after CTL transfer. To investigate the impact of CTL therapy on the cell cycle of tumor cells in situ, we generated B16 cells expressing a fluorescent ubiquitination-based cell-cycle indicator (B16-fucci) and performed CTL therapy in mice bearing B16-fucci tumors. Three days after CTL transfer, we observed diffuse infiltration of CTLs into the tumor with a large number of tumor cells arrested at the G(1) phase of the cell cycle, and the presence of spotty apoptotic or necrotic areas. Thus, tumor growth suppression was largely dependent on G(1) cell-cycle arrest rather than killing by CTLs. Neutralizing antibody to IFN gamma prevented both tumor growth inhibition and G(1) arrest. The mechanism of G(1) arrest involved the downregulation of S-phase kinase-associated protein 2 (Skp2) and the accumulation of its target cyclin-dependent kinase inhibitor p27 in the B16-fucci tumor cells. Because tumor-infiltrating CTLs are far fewer in number than the tumor cells, we propose that CTLs predominantly regulate tumor growth via IFN gamma-mediated profound cytostatic effects rather than via cytotoxicity. This dominance of G(1) arrest over other mechanisms may be widespread but not universal because IFN gamma sensitivity varied among tumors. (C) 2014 AACR.

Cytotoxic T lymphocytes (CTLs) play a central role in antitumor immunity. We utilized B16 melanoma cells expressing the fluorescent ubiquitination-based cell cycle indicator B16-fucci implanted in host mice and adoptively transferred with pmel-1-TCR transgenic T cells to demonstrate that tumor growth reduction is largely dependent on interferon g-mediated cell cycle arrest rather than the cytotoxic killing of tumor cells by CTLs.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population with the ability to suppress immune responses and are currently classified into three distinct MDSC subsets: monocytic, granulocytic and non-monocytic, and non-granulocytic MDSCs. Although NK cells provide an important first-line defense against newly transformed cancer cells, it is unknown whether NK cells can regulate MDSC populations in the context of cancer. In this study, we initially found that the frequency of MDSCs in non-Hodgkin lymphoma (NHL) patients was increased and inversely correlated with that of NK cells, but not that of T cells. To investigate the regulation of MDSC subsets by NK cells, we used an EL4 murine lymphoma model and found the non-monocytic and non-granulocytic MDSC subset, i.e., Gr1+CD11b+Ly6GmedLy6+med MDSC, is increased after NK cell depletion. The MDSC population that expresses MHC class II, CD80, CD124, and CCR2 is regulated mainly by CD27+CD11b+NK cells. In addition, this MDSC subset produces some immunosuppressive cytokines, including IL-10 but not nitric oxide (NO) or arginase. We also examined two subsets of MDSCs (CD14+HLA-DR- and CD14+ HLA-DR- MDSC) in NHL patients and found that higher IL-10-producing CD14+HLA-DR-MDSC ubset can be seen in lymphoma patients with reduced NK cell frequency in peripheral blood. Our analyses of MDSCs in this study may enable a better understanding of how MDSCs manipulate the tumor microenvironment and are regulated by NK cells in patients with lymphoma.

This prospective study was conducted to identify predictive markers for the response of metastatic renal cell carcinoma (RCC) to tyrosine kinase inhibitors (TKIs). Patients with histologically proven RCC with at least one measurable metastatic lesion were enrolled in this study. Blood samples were collected prior to treatment and the plasma levels of 27 cytokines were measured. Tumor response was assessed 8-12 weeks after the initiation of TKI treatment. A total of 13 patients (11 men and 2 women) with a median age of 63 years received sunitinib (8 cases), sorafenib (1 case), or axitinib (4 cases). Partial response (PR) was achieved in 5 patients (38%), stable disease (SD) in 4 (30%) and progressive disease (PD) was noted in 4 (30%). The plasma granulocyte macrophage colony-stimulating factor (GM-CSF) level in PR cases was significantly higher compared to that in SD or PD cases (P=0.012). Therefore, GM-CSF may be a predictive biomarker of the response of RCC to TKI treatment, suggesting that TKIs may exert clinical effects not only through suppression of the vascular endothelial growth factor, but also through immune system modulation.

Preventing protein aggregation is a major goal of biotechnology. Since protein aggregates are mainly comprised of unfolded proteins, protecting against denaturation is likely to assist solubility in an aqueous medium. Contrary to this concept, we found denatured total cellular protein mixture from mammalian cell kept high solubility in pure water when the mixture was nucleic acids free. The lysates were prepared from total cellular protein pellet extracted by using guanidinium thiocyanate-phenol-chloroform mixture of TRIzol, denatured and reduced total protein mixtures remained soluble after extensive dialysis against pure water. The total cell protein lysates contained fully disordered proteins that readily formed large aggregates upon contact with nucleic acids or salts. These findings suggested that the highly flexible mixtures of disordered proteins, which have fully ionized side chains, are protected against aggregation. Interestingly, this unusual solubility is characteristic of protein mixtures from higher eukaryotes, whereas most prokaryotic protein mixtures were aggregated under identical conditions. This unusual solubility of unfolded protein mixtures could have implications for the study of intrinsically disordered proteins in a variety of cells.

Background: Sunitinib, a tyrosine kinase inhibitor currently in use for the treatment of metastatic renal cell carcinoma (mRCC), has been reported to modulate immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) in addition to exerting anti-angiogenic effects. We conducted a clinical trial of dendritic cell (DC)-based immunotherapy together with sunitinib in mRCC patients in an effort to enhance immunotherapeutic efficacy by inhibiting immunosuppressive cells.Methods: Patients aged ≥20 years with advanced or recurrent mRCC who underwent nephrectomy were eligible for this study. Autologous tumor samples were obtained by surgery under aseptic conditions and used for preparing autologous tumor lysate. About 4 weeks after surgery, leukapheresis was performed to isolate peripheral blood mononuclear cells (PBMCs). DCs were generated from adherent PBMCs in the presence of recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) (500 IU/ml) and recombinant human IL-4 (500 IU/ml). Autologous tumor lysate was loaded into mature DC by electroporation. Eight patients were enrolled in the study and received sunitinib at a dose of 50 mg p.o. daily for 28 days followed by 14 days of rest. Tumor lysate-loaded DCs were administered subcutaneously every two weeks, with concomitant sunitinib.Results: No severe adverse events related to vaccination were observed. Sunitinib decreased the frequencies of MDSCs in peripheral blood of 5 patients and of Tregs in 3. Tumor lysate-reactive CD4 or CD8 T cell responses were observed in 5 patients, 4 of whom showed decreased frequencies of Tregs and/or MDSCs. The remaining 3 patients who failed to develop tumor-reactive T cell responses had high levels of IL-8 in their sera and did not show consistent reductions in MDSCs and Tregs.Conclusions: DC-based immunotherapy combined with sunitinib is safe and feasible for patients with mRCC.Trial registration: UMIN000002136.

Evidence suggests that CD204-positive (CD204(+)) tumor-infiltrating macrophages are associated with aggressive behavior of various cancers; however, the clinical, pathological, and prognostic associations of tumor-infiltrating CD204(+) macrophages in urothelial cancer have not been reported. A tissue microarray was constructed from the centers and peripheries of 171 upper urinary tract cancers treated with nephroureterectomy. CD204 immunohistochemistry was performed. The density of CD204(+) cells was calculated using image analysis software, and survival analyses were performed using the Kaplan-Meier method and multivariate Cox proportional hazards regression models. High CD204(+) cell density at the centers and peripheries of tumors was significantly associated with several adverse prognostic factors, including sessile architecture, histological high-grade, presence of lymphovascular invasion, concomitant carcinoma in situ, higher tumor stage, and lymph node metastasis. High CD204(+) cell density was significantly associated with shorter metastasis-free and cancer-specific survival (log-rank p < 0.001) and shorter metastasis-free survival in multivariate analysis. A high density of tumor-infiltrating CD204(+) macrophages was associated with aggressive behavior of upper urinary tract cancer. Our results suggest that a specific immune microenvironment may be associated with the biological behavior of urothelial cancer and that CD204 may serve as a novel prognostic biomarker for these tumors.

Complex interactions among multiple cell types contribute to the immunosuppressive milieu of the tumor microenvironment. Using a murine model of adoptive T-cell immunotherapy (ACT) for B16 melanoma, we investigated the impact of tumor infiltrating cells on this complex regulatory network in the tumor. Transgenic pmel-1-specific cytotoxic T lymphocytes (CTLs) were injected intravenously into tumor-bearing mice and could be detected in the tumor as early as on day 1, peaking on day 3. They produced IFN-, exerted anti-tumor activity and inhibited tumor growth. However, CTL infiltration into the tumor was accompanied by the accumulation of large numbers of cells, the majority of which were CD11b(+)Gr1(+) myeloid-derived suppressor cells (MDSCs). Notably, CD11b(+)Gr1(int)Ly6G(-)Ly6C(+) monocytic MDSCs outnumbered the CTLs by day 5. They produced nitric oxide, arginase I and reactive oxygen species, and inhibited the proliferation of antigen-specific CD8(+) T cells. The anti-tumor activity of the adoptively-transferred CTLs and the accumulation of MDSCs both depended on IFN- production on recognition of tumor antigens by the former. In CCR2(-/-) mice, monocytic MDSCs did not accumulate in the tumor, and inhibition of tumor growth by ACT was improved. Thus, ACT triggered counter-regulatory immunosuppressive mechanism via recruitment of MDSCs. Our results suggest that strategies to regulate the treatment-induced recruitment of these MDSCs would improve the efficacy of immunotherapy.

Malignant ascites caused by peritoneal dissemination of gastric cancer is chemotherapy-resistant and associated with poor prognosis. We conducted a pilot study to evaluate the safety of weekly intraperitoneal injections of in vitro expanded Vc gamma Vd delta T cells together with zoledronate for the treatment of such malignant ascites. Patient peripheral blood mononuclear cells were stimulated with zoledronate (5 mu mol/L) and interleukin-2 (1000 IU/mL). After 14 days culture, Vc gamma Vd delta T-cells were harvested and administered intraperitoneally in four weekly infusions. The day before T-cell injection, patients received zoledronate (1 mg) to sensitize their tumor cells to Vc gamma Vd delta T-cell recognition. Seven patients were enrolled in this study. The number of Vc gamma Vd delta T-cells in each injection ranged from 0.6 to 69.8 x 10(8) (median 59.0 x 10(8)). There were no severe adverse events related to the therapy. Intraperitoneal injection of Vc gamma Vd delta T cells allows them access to the tumor cells in the peritoneal cavity. The number of tumor cells in the ascites was significantly reduced even after the first round of therapy and remained substantially lower over the course of treatment. IFN-gamma was detected in the ascites on treatment. Computed tomography revealed a significant reduction in volume of ascites in two of seven patients. Thus, injection of these antitumor Vc gamma Vd delta T-cells can result in local control of malignant ascites in patients for whom no standard therapy apart from paracentesis is available. Adoptively transferred Vc gamma Vd delta T-cells do indeed recognize tumor cells and exert antitumor effector activity in vivo, when they access to the tumor cells.

We established CD4 T-cell clones, Mz-1B7, and Ue-21, which recognized the NY-ESO-1 121-138 peptide from peripheral blood mononuclear cells (PBMCs) of an esophageal cancer patient, E-2, immunized with an NY-ESO-1 protein and determined the NY-ESO-1 minimal epitopes. Minimal peptides recognized by Mz-1B7 and Ue-21 were NY-ESO-1125-134 and 124-134, respectively, both in restriction to DRB1*08:03. Using a longer peptide, 122-135, and five other related peptides, including either of the minimal epitopes recognized by the CD4 T-cell clones, we investigated the free peptide/DR recognition on autologous EBV-B cells as APC and peptide/DR tetramer binding. The results showed a discrepancy between them. The tetramers with several peptides recognized by either Mz-1B7 or the Ue-21 CD4 T-cell clone did not bind to the respective clone. On the other hand, unexpected binding of the tetramer with the peptide not recognized by CD4 T-cells was observed. The clone Mz-1B7 did not recognize the free peptide 122-135 on APC, but the peptide 122-135/DRB1*08:03 tetramer bound to the TCR on those cells. The failure of tetramer production and the unexpected tetramer binding could be due to a subtly modified structure of the peptide/DR tetramer from the structure of the free peptide/DR molecule. We also demonstrated that the NY-ESO-1 123-135/DRB1*08:03 tetramer detected ex vivo CD4 T-cell responses in PBMCs from patients after NY-ESO-1 vaccination in immunomonitoring. (C) 2013 Elsevier Ltd. All rights reserved.

We conducted a clinical trial of an NY-ESO-1 cancer vaccine using 4 synthetic overlapping long peptides (OLP; peptides #1, 79-108; #2, 100-129; #3, 121-150; and #4, 142-173) that include a highly immunogenic region of the NY-ESO-1 molecule. Nine patients were immunized with 0.25 mg each of three 30-mer and a 32-mer long NY-ESO-1 OLP mixed with 0.2 KE Picibanil OK-432 and 1.25 mL Montanide ISA-51. The primary endpoints of this study were safety and NY-ESO-1 immune responses. Five to 18 injections of the NY-ESO-1 OLP vaccine were well tolerated. Vaccine-related adverse events observed were fever and injection site reaction (grade 1 and 2). Two patients showed stable disease after vaccination. An NY-ESO-1-specific humoral immune response was observed in all patients and an antibody against peptide #3 (121-150) was detected firstly and strongly after vaccination. NY-ESO-1 CD4 and CD8 T-cell responses were elicited in these patients and their epitopes were identified. Using a multifunctional cytokine assay, the number of single or double cytokine-producing cells was increased in NY-ESO-1-specific CD4 and CD8 T cells after vaccination. Multiple cytokine-producing cells were observed in PD-1 (-) and PD-1 (+) CD4 T cells. In conclusion, our study indicated that the NY-ESO-1 OLP vaccine mixed with Picibanil OK-432 and Montanide ISA-51 was well tolerated and elicited NY-ESO-1-specific humoral and CD4 and CD8 T-cell responses in immunized patients.

Heat shock protein 105 (Hsp105) is one of the cancer/testis antigens, which is overexpressed in a variety of cancer cells, including urinary bladder cancer, and has been investigated as a target molecule for immunotherapy due to its immunogenicity. In this study, we assessed the expression of Hsp105 in primary bladder cancer samples from 84 patients treated with radical cystectomy, using immunohistochemical analysis, and investigated its correlation with clinicopathological characteristics and cancer-specific survival. The immunoreactivity of Hsp105 expression was evaluated as a score of 0-3, according to the intensity of the signal. The Hsp105 expression was high (score 2 or 3) in 31 cases and low (score 0 or 1) in 53 cases; however, it was not significantly correlated with age, nuclear grade, pathological tumor stage and previous intravesical Bacillus Calmette-Guérin immunotherapy. Female gender, lymphovascular invasion and lymph node metastasis were associated with low Hsp105 scores, although the differences were not statistically significant (P=0.071, 0.061 and 0.175, respectively). However, a high Hsp105 score was significantly associated with a favorable prognosis (P=0.017) and was identified as an independent prognostic factor by multivariate analysis (P=0.032; hazard ratio, 2.34). These findings suggested that the expression of Hsp105 may be a novel indicator of a favorable prognosis in bladder cancer.

Magnetic nanoparticle-mediated hyperthermia (MNHT) generates heat to a local tumor tissue of above 43 degrees C without damaging surrounding normal tissues. By applying MNHT, a significant amount of heat-shock proteins is expressed within and around the tumor tissues, inducing tumor-specific immune responses. In vivo experiments have indicated that MNHT can induce the regression of not only a local tumor tissue exposed to heat, but also distant metastatic tumors unexposed to heat. In this article, we introduce recent progress in the application of MNHT for antitumor treatments and summarize the mechanisms and processes of its biological effects during antitumor induction by MNHT. Several clinical trials have been conducted indicating that the MNHT system may add a promising and novel approach to antitumor therapy.

γδ T cells are attractive effector cells for cancer immunotherapy as they can secrete cytokines abundantly and exert potent cytotoxicity against a wide range of cancer cells. They comprise 1-5% of peripheral blood T cells, the majority expressing the Vγ9Vδ2 T cell receptor that recognizes phosphoantigens. Direct in vivo activation of Vγ9Vδ2 T cells in cancer patients as well as adoptive transfer of ex vivo expanded Vγ9Vδ2 T cells has been investigated in several clinical trials. We previously established a large-scale in vitro expansion method for Vγ9Vδ2 T cells using zoledronate and interleukin-2 (IL-2). We found that Vγ9Vδ2 T cells from patients with advanced cancer as well as from healthy donors underwent extensive proliferation under these conditions. Such cultured Vγ9Vδ2 T cells retained cytokine secretion capacity and mediated cytotoxicity against a variety of cancer cell lines. Recently, we conducted a phase I clinical study to evaluate safety and potential anti-tumor effects of re-infusing ex vivo expanded γδ T cells in patients with advanced or recurrent non-small-cell lung cancer (NSCLC) refractory to or intolerant of current conventional treatments. There were no severe adverse events related to the therapy. All patients remained alive during the study period with a median survival of 589 days and median progression-free survival of 126 days. Six patients had stable disease (SD), whereas the remaining six evaluable patients experienced progressive disease (PD) four weeks after the sixth transfer. We conclude that adoptive transfer of zoledronate-expanded γδ T cells is safe and feasible in patients with NSCLC, refractory to other treatments.

The intrahepatic immune environment is normally biased towards tolerance. Nonetheless, effective antiviral immune responses can be induced against hepatotropic pathogens. To examine the immunological basis of this paradox we studied the ability of hepatocellularly expressed hepatitis B virus (HBV) to activate immunologically naive HBV-specific CD8(+) T cell receptor (TCR) transgenic T cells after adoptive transfer to HBV transgenic mice. Intrahepatic priming triggered vigorous in situ T cell proliferation but failed to induce interferon gamma production or cytolytic effector function. In contrast, the same T cells differentiated into cytolytic effector T cells in HBV transgenic mice if Programmed Death 1 (PD-1) expression was genetically ablated, suggesting that intrahepatic antigen presentation per se triggers negative regulatory signals that prevent the functional differentiation of naive CD8(+) T cells. Surprisingly, coadministration of an agonistic anti-CD40 antibody (alpha CD40) inhibited PD-1 induction and restored T cell effector function, thereby inhibiting viral gene expression and causing a necroinflammatory liver disease. Importantly, the depletion of myeloid dendritic cells (mDCs) strongly diminished the alpha CD40 mediated functional differentiation of HBV-specific CD8(+) T cells, suggesting that activation of mDCs was responsible for the functional differentiation of HBV-specific CD8(+) T cells in alpha CD40 treated animals. These results demonstrate that antigen-specific, PD-1-mediated CD8(+) T cell exhaustion can be rescued by CD40-mediated mDC-activation.

Neutron capture therapy (NCT) is a promising non-invasive cancer therapy approach and some recent NCT research has focused on using compounds containing gadolinium as an alternative to currently used boron-10 considering several advantages that gadolinium offers compared to those of boron. In this study, we evaluated gadolinium-entrapped liposome compound as neutron capture therapy agent by in vivo experiment on colon-26 tumor-bearing mice. Gadolinium compound were injected intravenously via tail vein and allowed to accumulate into tumor site. Tumor samples were taken for quantitative analysis by ICP-MS at 2, 12, and 24 h after gadolinium compound injection. Highest gadolinium concentration was observed at about 2 h after gadolinium compound injection with an average of 40.3 mg/g of wet tumor tissue. We performed neutron irradiation at JRR-4 reactor facility of Japan Atomic Energy Research Institute in Tokaimura with average neutron fluence of 2 x 10(12) n/cm(2). The experimental results showed that the tumor growth suppression of gadolinium-injected irradiated group was revealed until about four times higher compared to the control group, and no significant weight loss were observed after treatment suggesting low systemic toxicity of this compound. The gadolinium-entrapped liposome will become one of the candidates for Gd delivery system on NCT. (C) 2013 Elsevier Masson SAS. All rights reserved.

Background: Aberrant epithelial repair is a crucial event in the airway remodeling that characterizes obliterative bronchiolitis (OB) in transplanted lungs. Recent data from experiments using epithelial cell lines and human airway tissues from lung transplant recipients suggest that epithelial to mesenchymal transition (EMT) plays an important role in OB. The aim of this study was to clarify whether EMT is involved in airway remodeling in an animal model. Methods: We performed orthotopic tracheal transplantation from BALB/c to C57BL/6 mice with from BALC/c to BALB/c mouse grafts as controls. Five allogeneic and 3 syngeneic recipients were humanely killed at predetermined postoperative days 2-12 as well as 14 and 21. Histology was evaluated using hematoxylin-eosin (H& E) staining. We studied the expression of specific markers, including E-cadherin, an epithelial marker α-smooth muscle actin (SMA), and S100A4, mesenchymal markers, and zinc finger E-box-binding homeobox 1 (ZEB1), an EMT-related transcription factor. Results: Histologic assessment of serial H& E stains of allogeneic grafts showed remarkable pseudostratified respiratory epithelium with subepithelial inflammatory cell infiltration, as well as denuded and flattened epithelium and subepithelial fibrosis. The dynamic epithelial changes occurred earlier than the subepithelial fibrosis. Immunohistochemical evaluation indicated the emergence of α-SMA- positive epithelial cells that were most prominent on day 7. The expression of E-cadherin was attenuated in α-SMA-positive epithelial cells. S100A4 was also expressed in epithelial cells. A few days before the intraepithelial expression of α-SMA, ZEB1 emerged in the nuclei of epithelial cells. Conclusions: We observed expression of an EMT-related transcription factor and mesenchymal markers along with the attenuation of epithelial marker expression in epithelial cells, several days before prominent subepithelial fibrosis formation, results that suggest epithelial cells to play an important fibrosis role in airway remodeling during epithelial to mesenchymal transition. © 2013 Elsevier Inc. All rights reserved.

Background aims. Adoptive immunotherapy is emerging as a potent anti-tumor treatment modality; V gamma 9V delta 2 T cells may represent appropriate agents for such cancer immunotherapy. To improve the currently limited success of V gamma 9V delta 2 T-cell-based immunotherapy, we examined the in vivo dynamics of these adoptively-transferred cells and hypothesized that interleukin (IL)-15 is the potential factor for V gamma 9 delta 2 T cell in vivo survival. Methods. We conducted a clinical trial of adoptive V gamma 9V delta 2 T-cell transfer therapy in six colorectal cancer patients who received pulmonary metastasectomy. Patients' peripheral blood mononuclear cells were stimulated with zoledronate (5 mu mol/L) and IL-2 (1000 IU/mL) for 14 d. Harvested cells, mostly V gamma 9V delta 2 T cells, were given intravenously weekly without additional IL-2 eight times in total. The frequency, phenotype and common gamma-chain cytokine receptor expression of V gamma 9V delta 2 T cells in peripheral blood was monitored by flow cytometry at each time point during treatment and 4 and 12 weeks after the last administration. Results. Adoptively transferred V gamma 9V delta 2 T cells expanded well without exogenous IL-2 administration or lymphodepleting preconditioning. They maintained effector functions in terms of interferon-gamma secretion and prompt release of cytotoxic granules in response to PMA/ionomycin or isopentenyl pyrophosphate-positive cells. Because they are IL-2R alpha-IL-7R alpha-IL-15R alpha-IL-2R beta(+)gamma(+)(c), it is likely that IL-2 or IL-15 is required for their maintenance. Conclusions. The persistence of large numbers of functionally active adoptively transferred V gamma 9V delta 2 T cells in the absence of exogenous IL-2 implies that an endogenous factor, such as IL-15 transpresentation, is adequate to support these cells in vivo.

Immunogenicity of a long 20-mer NY-ESO-1f peptide vaccine was evaluated in a lung cancer patient TK-f01, immunized with the peptide with Picibanil OK-432 and Montanide ISA-51. We showed that internalization of the peptide was necessary to present CD8 T-cell epitopes on APC, contrasting with the direct presentation of the short epitope. CD8 T-cell responses restricted to all five HLA class I alleles were induced in the patient after the peptide vaccination. Clonal analysis showed that B*35:01 and B*52:01-restricted CD8 T-cell responses were the two dominant responses. The minimal epitopes recognized by A*24:02, B*35:01, B*52:01 and C*12:02-restricted CD8 T-cell clones were defined and peptide/HLA tetramers were produced. NY-ESO-1 91-101 on A*24:02, NY-ESO-1 92-102 on B*35:01, NY-ESO-1 96-104 on B*52:01 and NY-ESO-1 96-104 on C*12:02 were new epitopes first defined in this study. Identification of the A*24:02 epitope is highly relevant for studying the Japanese population because of its high expression frequency (60%). High affinity CD8 T-cells recognizing tumor cells naturally expressing the epitopes and matched HLA were induced at a significant level. The findings suggest the usefulness of a long 20-mer NY-ESO-1f peptide harboring multiple CD8 T-cell epitopes as an NY-ESO-1 vaccine. Characterization of CD8 T-cell responses in immunomonitoring using peptide/HLA tetramers revealed that multiple CD8 T-cell responses comprised the dominant response.

Both innate and adaptive immunity are crucial for cancer immunosurveillance, but precise therapeutic equations to restore immunosurveillance in patients with cancer patients have yet to be developed. In murine models, alpha-galactosylceramide (alpha-GalCer)-loaded, tumor antigen-expressing syngeneic or allogeneic cells can act as cellular adjuvants, linking the innate and adaptive immune systems. In the current study, we established human artificial adjuvant vector cells (aAVC) consisting of human HEK293 embryonic kidney cells stably transfected with the natural killer T (NKT) immune cell receptor CD1d, loaded with the CD1d ligand alpha-GalCer and then transfected with antigen-encoding mRNA. When administered to mice or dogs, these aAVC-activated invariant NKT (iNKT) cells elicited antigen-specific T-cell responses with no adverse events. In parallel experiments, using NOD/SCID/IL-2r gamma c(null)-immunodeficient (hDC-NOG) mouse model, we also showed that the human melanoma antigen, MART-1, expressed by mRNA transfected aAVCs can be cross-presented to antigen-specific T cells by human dendritic cells. Antigen-specific T-cell responses elicited and expanded by aAVCs were verified as functional in tumor immunity. Our results support the clinical development of aAVCs to harness innate and adaptive immunity for effective cancer immunotherapy. Cancer Res; 73(1); 62-73. (C) 2012 AACR.

The intrahepatic immune environment is normally biased towards tolerance. Nonetheless, effective antiviral immune responses can be induced against hepatotropic pathogens. To examine the immunological basis of this paradox we studied the ability of hepatocellularly expressed hepatitis B virus (HBV) to activate immunologically naïve HBV-specific CD8⁺ T cell receptor (TCR) transgenic T cells after adoptive transfer to HBV transgenic mice. Intrahepatic priming triggered vigorous in situ T cell proliferation but failed to induce interferon gamma production or cytolytic effector function. In contrast, the same T cells differentiated into cytolytic effector T cells in HBV transgenic mice if Programmed Death 1 (PD-1) expression was genetically ablated, suggesting that intrahepatic antigen presentation per se triggers negative regulatory signals that prevent the functional differentiation of naïve CD8⁺ T cells. Surprisingly, coadministration of an agonistic anti-CD40 antibody (αCD40) inhibited PD-1 induction and restored T cell effector function, thereby inhibiting viral gene expression and causing a necroinflammatory liver disease. Importantly, the depletion of myeloid dendritic cells (mDCs) strongly diminished the αCD40 mediated functional differentiation of HBV-specific CD8⁺ T cells, suggesting that activation of mDCs was responsible for the functional differentiation of HBV-specific CD8⁺ T cells in αCD40 treated animals. These results demonstrate that antigen-specific, PD-1-mediated CD8⁺ T cell exhaustion can be rescued by CD40-mediated mDC-activation.

CD137 (4-1BB) is an important costimulatory ligand and a potent stimulator of T-cell responses. It has been used therapeutically to stimulate immunity against several solid malignancies as well as to modulate susceptibility to autoimmune disease and infection. However, clinical trials of anti-CD137 agonistic antibody have been suspended because of deleterious side effects. To overcome this problem, we fine-tuned the combination of adoptive transfer of tumor-specific cytotoxic T lymphocytes (CTL) and anti-CD137 monoclonal antibody (mAb) treatment. B16 melanoma cells (1 x 10(6)) were implanted subcutaneously in C57BL/6 mice. On day 9, CTLs (1 x 10(7)) were intravenously injected into tumor-bearing mice. Transferred CTL distributed throughout the body and infiltrated into the tumor. CD137 expression was upregulated on tumor-infiltrating CTL, but not in other tissues or other cell types. Therefore, mice received anti-CD137 mAb (100 mu g) 3 days after CTL transfer. interferon-g was produced in the tumor only by transferred CTL, not recipient-derived cells. The functional CTLs in the tumor were increased and interferon-g production per cell was augmented by anti-CD137 treatment. It was not detected in CTL found in other tissues. Consistent with this, no organ damage was observed on anti-CD137 treatment. On the basis of the spatiotemporal expression of CD137 on tumor-infiltrating CTLs, anti-CD137 mAb selectively activated these tumor-infiltrating cells, augmented their antitumor activity and greatly decreased tumor growth. Tumor-specific CTL can guide agonistic anti-CD137 mAb to the tumor and in turn, become functionally augmented. Thus, CD137 mAb therapy may become feasible again in combination with tumor-specific CTL therapy.

Lymph node (LN) structure is remodeled during immune responses, a process which is considered to play an important role in the regulation of immune function. To date, little attention has been paid to the remodeling of the medullary region, despite its proposed role as a niche for antibody-producing plasma cells. Here, we show that B cells mediate medullary remodeling of antigen-draining LNs during inflammation. This process occurs with kinetics similar to changes in plasma cell number and is accompanied by stromal renetworking which manifests as the segregation of B cells and plasma cells. Medullary remodeling depends on signaling via the lymphotoxin-beta receptor and the presence of B cells but occurs independently of T-dependent humoral responses or other immune cell subsets including T cells, monocytes and neutrophils. Moreover, reconstitution of non-cognate polyclonal B cells in B cell-deficient mice restores not only the medullary remodeling but also the antibody response by separately transferred cognate B cells, suggesting that non-cognate B cells contribute to antibody responses through medullary remodeling. We propose that non-cognate B cells mediate the expansion of the plasma cell niche in LN through medullary remodeling, thereby regulating the size of the LN plasma cell pool.

Antigens (Ag) from cancer or virus-infected cells must be internalized by dendritic cells (DCs) to be presented to CD8(+) T cells, which eventually differentiate into Ag-specific cytotoxic T lymphocytes (CTLs) that destroy cancer cells and infected cells. This pathway is termed cross-presentation and is also implicated as an essential step in triggering autoimmune diseases such as Type I diabetes. Internalized Ag locates within endosomes, followed by translocation through a putative pore structure spanning endosomal membranes into the cytosol, where it is degraded by the proteasome to generate antigen peptides. During translocation, Ag is believed to be unfolded since the pore size is too narrow to accept native Ag structure. Here, we show that paraformaldehyde-fixed, structurally inflexible Ag is less efficient in cross-presentation because of diminished translocation into the cytosol, supporting the "unfolded Ag" theory. We also show that HSP70 inhibitors block both endogenous and cross-presentation. ImageStream analysis revealed that the inhibition in cross-presentation is not due to blocking of Ag translocation because a HSP70 inhibitor rather facilitates the translocation, which is in marked contrast to the effect of an HSP90 inhibitor that blocks Ag translocation. Our results indicate that Ag translocation to the cytosol in cross-presentation is differentially regulated by HSP70 and HSP90.

We conducted a phase I clinical trial of a cancer vaccine using a 20-mer NY-ESO-1f peptide (NY-ESO-1 91-110) that includes multiple epitopes recognized by antibodies, and CD4 and CD8 T cells. Ten patients were immunized with 600 mu g of NY-ESO-1f peptide mixed with 0.2 KE Picibanil OK-432 and 1.25 ml Montanide ISA-51. Primary end points of the study were safety and immune response. Subcutaneous injection of the NY-ESO-1f peptide vaccine was well tolerated. Vaccine-related adverse events observed were fever (Grade 1), injection-site reaction (Grade 1 or 2) and induration (Grade 2). Vaccination with the NY-ESO-1f peptide resulted in an increase or induction of NY-ESO-1 antibody responses in nine of ten patients. The sera reacted with recombinant NY-ESO-1 whole protein as well as the NY-ESO-1f peptide. An increase in CD4 and CD8 T cell responses was observed in nine of ten patients. Vaccine-induced CD4 and CD8 T cells responded to NY-ESO-1 91-108 in all patients with various HLA types with a less frequent response to neighboring peptides. The findings indicate that the 20-mer NY-ESO-1f peptide includes multiple epitopes recognized by CD4 and CD8 T cells with distinct specificity. Of ten patients, two with lung cancer and one with esophageal cancer showed stable disease. Our study shows that the NY-ESO-1f peptide vaccine was well tolerated and elicited humoral, CD4 and CD8 T cell responses in immunized patients.

We previously showed that exposure to UV radiation after immunization suppresses Th1 and Th2 immune responses, leading to impaired Ab and allo-immune responses, but the impact of UV radiation after immunization on anti-tumor immune responses mediated by tumor-specific CD8(+) T cell responses remains less clear. Furthermore, the exact phenotypic and functional characteristics of regulatory T cell population responsible for the UV-induced immunosuppression still remain elusive. Using the MBL-2 lymphoma cell line engineered to express OVA as a surrogate tumor Ag, here we demonstrate that UV irradiation after tumor Ag-immunization suppresses the anti-tumor immune response in a manner dependent on the immunizing Ag. This suppression was mediated by interleukin (IL)-10 released from CD4(+)CD25(+) T cells, by which impaired the induction of cytotoxic T lymphocytes (CTL) able to kill Ag-expressing tumor cells. In addition, we generated a panel of T cell clones from UV-irradiated and non-irradiated mice, and all of the clones derived from UV-irradiated mice had a Tr1-type regulatory T cell phenotype with expression of IL-10 and c-Maf, but not Foxp3. These Tr1-type regulatory T cell clones suppressed tumor rejection in vivo as well as Th cell activation in vitro in an IL-10 dependent manner. Given that suppression of Ag-specific CTL responses can be induced in Ag-sensitized mice by UV irradiation, our results may imply that exposure to UV radiation during premalignant stage induces tumor-Ag specific Tr1 cells that mediate tumor-Ag specific immune suppression resulting in the promotion of tumor progression.

Human gamma delta T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity. (1) The majority of gamma delta T cells in peripheral blood have the V gamma 9V delta 2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions. (1)Therefore, gamma delta T cells are attractive candidate effector cells for cancer immunotherapy. V gamma 9V delta 2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis; 2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway. (3) In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of gamma delta T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and gamma delta T cells activation. (3) Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to gamma delta T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating gamma delta T cells with aminobisphosphonates. (4) Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. (5) Monocytes that accumulate IPP become antigen-presenting cells and stimulate V gamma 9V delta 2 T cells in the peripheral blood. (6) Based on these mechanisms, we developed a technique for large-scale expansion of gamma delta T cell cultures using zoledronate and interleukin-2 (IL-2).(7) Other methods for expansion of gamma delta T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP) (8) or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).(9) All of these methods allow ex vivo expansion, resulting in large numbers of gamma delta T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded gamma delta T cells display CD27(-)CD45RA(-) effector memory phenotype and thier function can be evaluated by IFN-gamma production assay.(7)

Strength of inflammatory stimuli during the early expansion phase plays a crucial role in the effector versus memory cell fate decision of CD8(+) T cells. But it is not known how early lymphocyte distribution after infection has an impact on this process. We demonstrate that the chemokine receptor CXCR3 is involved in promoting CD8(+) T cell commitment to an effector fate rather than a memory fate by regulating T cell recruitment to an antigen/inflammation site. After systemic viral or bacterial infection, the contraction of CXCR3(-/-) antigen-specific CD8(+) T cells is significantly attenuated, resulting in massive accumulation of fully functional memory CD8(+) T cells. Early after infection, CXCR3(-/-) antigen-specific CD8(+) T cells fail to cluster at the marginal zone in the spleen where inflammatory cytokines such as IL-12 and IFN-alpha are abundant, thus receiving relatively weak inflammatory stimuli. Consequently, CXCR3(-/-) CD8(+) T cells exhibit transient expression of CD25 and preferentially differentiate into memory precursor effector cells as compared with wild-type CD8(+) T cells. This series of events has important implications for development of vaccination strategies to generate increased numbers of antigen-specific memory CD8(+) T cells via inhibition of CXCR3-mediated T cell migration to inflamed microenvironments.

Lung cancer is the leading cause of cancer death worldwide, yet there are still no satisfactory protocols available for treating this disease, emphasizing the urgency for more effective therapies. Recent clinical trials have provided encouraging evidence of the benefits of certain forms of immunotherapy. Here, we summarize recent developments in the area of gamma delta T-cell therapy for lung cancer in our center. gamma delta T cells constitute 2%-10% of T lymphocytes in human blood and play a role in immune surveillance against microbial pathogens and, possibly, cancer. These T cells recognize phosphoantigens via polymorphic gamma delta T-cell antigen receptors (TCR), as well as the major histocompatibility complex (MHC) class I chain-related molecules, A and B (MICA and MICB), via nonpolymorphic NKG2D receptors in an MHC-unrestricted manner. This implies that gamma delta T cells could retain antitumor effects despite reduced expression of MHC and tumor antigens on cancer cells. Thus, clinical trials have been conducted to evaluate the safety and efficacy of gamma delta T-cell-based immunotherapies for non-Hodgkin lymphoma, multiple myeloma, and solid tumors. This review focuses on the current status of gamma delta T-cell-based immunotherapy for lung cancer.

Human gamma delta T cells can recognize and kill non-small cell lung cancer (NSCLC) cells using the V gamma 9V delta 2 T-cell receptor and/or NKG2D. We have established clinical grade large-scale ex vivo expansion of gamma delta T cells from peripheral blood mononuclear cells by culturing with zoledronate and interleukin-2 (IL-2). A phase I study was conducted to evaluate safety and potential antitumor effects of re-infusing ex vivo expanded gamma delta T cells in patients with recurrent or advanced NSCLC. Patient's peripheral blood mono-nuclear cells were stimulated with zoledronate (5 mu M) and IL-2 (1000 IU/mL) for 14 days. Harvested cells, mostly gd T cells, were given intravenously every 2 weeks without additional IL-2, a total of 6 times. The cumulative number of transferred gamma delta T cells ranged from 2.6 to 45.1 x 10(9) (median, 15.7 x 10(9)). Fifteen patients underwent adoptive immunotherapy with these gamma delta T cells. There were no severe adverse events related to the therapy. Immunomonitoring data showed that with increasing numbers of infusions, the number of peripheral gamma delta T cells gradually increased. All patients remained alive during the study period with a median survival of 589 days and median progression-free survival of 126 days. According to the Response Evaluation Criteria In Solid Tumors, there were no objective responses. Six patients had stable disease, whereas the remaining 6 evaluable patients experienced progressive disease 4 weeks after the sixth transfer. We conclude that adoptive transfer of zoledronate-expanded gamma delta T cells is safe and feasible in patients with NSCLC, refractory to other treatments.

Interleukin-18 (IL-18) produced by activated antigen-presenting cells stimulates natural killer (NK) cells, natural killer T (NKT) cells, and T cells to secrete gamma interferon (IFN-gamma). In this study, injection of a single 10-mug dose of recombinant murine IL-18 rapidly, reversibly, and noncytopathically inhibited hepatitis B virus (HBV) replication in the livers of HBV transgenic mice. Furthermore, HBV replication was inhibited by as little as 1 mug of IL-18 injected repetitively, and also by a single 0.1-mug dose of IL-18 injected together with 1 ng of IL-12, neither of which inhibited HBV replication individually, demonstrating synergy between these cytokines in this system. The antiviral effect of IL-18 was mediated by its ability to activate resident intrahepatic NK cells and NKT cells to produce IFN-gamma and by its ability to induce IFN-alpha/beta production in the liver. These results suggest that IL-18 has the potential to contribute to the control of HBV replication during self-limited infection and that it may have therapeutic value for the treatment of patients with chronic hepatitis.

Persistent hepatitis B virus (HBV) infection is characterized by a weak and narrowly focused CD8(+) T-cell response to HBV that is thought to reflect the induction of central and/or peripheral tolerance to HBV proteins in neonatal and adult onset infections, respectively. Immunotherapeutic strategies that overcome tolerance and boost these suboptimal responses may lead to viral clearance in chronically infected individuals. The present study was performed to compare the relative immunogenicities and tolerogenicities of HBV structural (envelope [ENV]) and nonstructural (polymerase [POL]) proteins at the CD8(+) cytotoxic T lymphocyte (CTL) level in transgenic mice that replicate HBV in the liver and secrete infectious virus into the blood, thus representing an excellent model of persistent HBV infection. Interestingly, the mice were tolerant to the ENV but not to the POL proteins at the CTL level. Furthermore, the POL-specific CTLs had no impact on HBV replication or liver function in vivo, even though they were readily induced and reached the liver after DNA immunization, reflecting their relatively low avidity and the low level at which the POL protein is expressed by the hepatocyte. Collectively, these results suggest that the factors that make POL less tolerogenic also make POL-specific CTLs relatively inefficient effector cells when they reach the target organ. Immunotherapeutic strategies to control HBV infection by inducing virus-specific CTL responses in chronically infected subjects should be evaluated in light of this observation.

がん免疫治療の根幹は、がん細胞を特異的に認識し傷害する腫瘍（抗原）特異的T細胞（キラーT細胞）による抗腫瘍免疫応答である。腫瘍特異的T細胞は、腫瘍に存在する遺伝子変異によって生じた変異アミノ酸を含んだネオアンチゲンを認識することが知られているが、ネオアンチゲンの多くは、パッセンジャー変異と呼ばれる個々の患者に固有の遺伝子変異産物であるため、ネオアンチゲンを同定することは容易ではない。ネオアンチゲンを同定できない大多数の患者においては、MHCテトラマーを用いた腫瘍特異的T細胞を同定、検出することが困難である。がん免疫治療開発に従事するためには、腫瘍特異的T細胞の同定と分離を可能にする新たな技術革新の導入が必要であった。 シンクサイトの研究者らは、高速に細胞の構造情報を取得するイメージング技術と、その情報をリアルタイムで処理するデータ処理技術に先端マイクロ流体細胞分取技術を融合させたGhost Cytometry (GC) 技術（Kawamura Y, et al. Science. 2018 15;360: 1246-1251）の開発に成功した。研究代表者の垣見は、このGC技術に着目し、細胞表面抗原の発現を検出するフローサイトメトリーなどの従来の技術だけでは識別できなかった腫瘍特異的T細胞（がんを攻撃するキラーT細胞）集団に対して、GC技術を用いた独自の評価を行うことで、形態を超えて機能評価を可能にするシステム開発が可能になると考えて本研究を実施した。動的ゴーストイメージング (GMI: Ghost Motion Imaging) 法によって計測された細胞画像情報を、画像に再構成せずにデータのまま活用し、機械学習によりその細胞特性を捉えて高速判別する新手法を活用して、がんを攻撃するキラーT細胞を判別する顕微鏡、セルソーターを開発することが可能になると期待される。

本年度は不可逆電気穿孔法治療（IRE）がどの程度腫瘍免疫を賦活するかを評価するために、担癌モデルマウスを用いてIRE治療後の免疫応答が腫瘍にもたらす影響について検討した。具体的な評価手法を以下に示す。EG7OVA細胞1.0×106cellsをマウスの右後肢部の皮内(n=14)に接種し、14日目に IRE を以下の条件で行った（電圧 : 750 V, パルス長 : 90 μs, パルス数 : 90）。IRE施行後28日目に再発が見られなかったマウス(IRE治療群：n=12)及び非担癌マウス(コントロール群：n=5)の左後肢部の皮内に EG7OVA細胞1.0×106cellsを再接種した（リチャレンジ）。また、IRE治療群のうち半数(n=6)に200μgのCD8抗体を3回に分けて腹腔内投与した。その後の腫瘍の増殖とマウスの生存について各群間で観察した。結果、皮内に接種しIREを行ったマウス全例(n=6)に再発は認めなかった。IRE施行28日後にそれらのマウスに腫瘍のリチャレンジを行ったところ腫瘍の形成は認めなかったが(n=6)、CD8抗体を投与したマウスには腫瘍の形成が認められた(n=4)。リチャレンジ群においてIRE治療群のうちCD8抗体非投与のマウスはコントロール群と比較して有意に生存の延長が認められた。以上より、IREによって得られた抗腫瘍免疫は長期間維持され、これにはCD8陽性T細胞の関与が考えられた。

進行性尿路上皮癌に対し免疫チェックポイント阻害薬のペムブロリズマブを投与した患者のうち、どのような患者に有効で、どのような患者に効果不良であるか調査を行っている。 すでに、ペムブロリズマブの奏効について多角的な視点から検討した結果、血中のアルブミン/グロブリン比が大きい患者は効果良好であることを見出し、論文化した（Taguchi S, Kawai T, et al. Prognostic significance of the albumin-to-globulin ratio for advanced urothelial carcinoma treated with pembrolizumab: a multicenter retrospective study. Sci Rep. 2021;11:15623）。 また、免疫関連有害事象を生じた患者は薬剤に対する免疫反応が良好な患者と考えられる。今回、ペムブロリズマブにより免疫関連有害事象を生じた患者はoverall survivalも良好であることをtime-dependent analysisによって明らかにし、論文化した（Kawai T, et al. Impact of immune-related adverse events on the therapeutic efficacy of pembrolizumab in urothelial carcinoma: a multicenter retrospective study using time-dependent analysis. J Immunother Cancer. 2022;10:e003965)。 さらに血中の各種免疫マーカーとペムブロリズマブの奏効との関係を調べ、現在論文化している最中である。

マウス肺がんモデルで、腫瘍内T細胞浸潤の多いASB-XIVと、少ないLLC1について検討を行った。ASB-XIV細胞株からDNA、RNAを抽出し、全エクソームシークエンス、RNAシークエンスを実施し、1410個のミスセンス変異を同定した。RNAシークエンスでFPKM≧1の遺伝子に絞り込み、変異を含む8-10merのエピトープのMHCクラスIへの結合予測をNetMHCpanおよびMHCflurryを用いて行った。NetMHCpanで変異エピトープのIC50が500nM以下となる87ペプチド、変異エピトープに対して野生型エピトープのIC50が10倍以上となる20ペプチド、MHCflurryでpresentation percentileが2以下となる256のエピトープのうち、NetMHCpanで予測されなかった196ペプチド、合計303ペプチドを合成した。 ASB-XIVを抗PD-1抗体、または抗CTLA-4抗体を投与して拒絶させたマウスの脾細胞をASB-XIV細胞で刺激して培養することで、ASB-XIVに反応するCD8+T細胞株を樹立した。このCD8+T細胞株をASB-XIV担がんマウスに投与すると、腫瘍が退縮した。このCD8+T細胞株に対して303個の合成ペプチドのスクリーニングを行ったところ、1つのペプチドに反応が認められた。 LLC1については、腫瘍内に浸潤するT細胞が少ないことが問題だったが、CpGを投与することにより、CD８+T細胞の浸潤が増加することが明らかとなった。しかしながらCpG単独およびCpGと抗PD-1抗体との併用ではLLC1腫瘍は退縮しなかった。 肺がん手術検体でネオアンチゲン特異的T細胞を検討するために、シークエンス用に23件の手術検体を保存し、このうち19件についてはT細胞を培養して保存した。

がん患者の腫瘍組織の中に存在している腫瘍浸潤T 細胞(Tumor InfiltratingLymphocyte: TIL)を、in vitro で活性化・増殖させた後、再び患者へ輸注するTIL療法は、非常に強力ながん免疫治療である。しかしながら、悪性黒色腫では効率良くTIL の培養に成功するが、固形がんにおいては腫瘍組織内のTIL の数が少なく、さらに腫瘍内における免疫抑制性の環境のためTIL の培養・増殖が非常に困難であることが分かっている。本研究では、ポリエチレングリコール（PEG）脂質を利用した細胞表面工学技術を免疫制御技術へ応用し、がん細胞表面を加工して、がん細胞-T 細胞間相互作用における免疫抑制シグナルを制御する。特に、がん細胞が持つMHC クラスI 分子に提示されているがん抗原を特異的に認識するTIL を選択的に増殖させるために、細胞表面工学技術を活用してがん細胞を抗原提示細胞へと改変することで、固形がんのTIL を増殖させることを目指す。 そこで、我々はここで提案する増殖方法によりTIL を増殖させ、TIL 療法として展開し、最終的には、その腫瘍特異的T 細胞受容体（TCR）の遺伝子をクローニングして、得られた遺伝子をレトロウイルスベクターに組み込み、新たに患者の末梢血リンパ球に遺伝子導入することで、患者の腫瘍特異的T リンパ球を作製して治療に用いる（TCR-T 細胞治療）ことを目指している。昨年度では、PEG脂質を用いた細胞表面技術により、マウス悪性黒色腫細胞株B16（B16F10細胞）に対して、CD80-Fc をその表面に固定化することに成功した。今年度では、そのCD80-Fcの細胞表面への固定化に関して、最適化条件を探索することに取り組み、条件を見つけることに成功した。また、実際のCTLへの細胞間相互作用による増殖刺激を検討したところ、細胞傷害性 T 細胞 (CTL) との共培養で、CTLの増殖の大きな変化は見られなかった。

がん種を問わず、一定の割合で長期生存がん患者が存在する。遺伝子変異由来の新生抗原(ネオアンチゲン)に対する免疫応答が、患者の長期生存に寄与している可能性がある。本研究では、長期生存肺がん患者を対象とし、手術(生検)時に採取された腫瘍から全エクソーム/RNAシーケンスを実施し、in silicoのアルゴリズムを用いて遺伝子変異由来のネオアンチゲンを予測する。患者の外来来院時に採血を行い、分離した末梢血単核球（PBMC）から、予測したネオアンチゲンに対する長期生存メモリーT細胞を、フローサイトメトリー、T細胞受容体（TCR）レパトア解析等で検出する。また、T細胞の認識するネオアンチゲンの特徴や腫瘍内の免疫関連遺伝子発現の検討する。さらに腫瘍浸潤リンパ球(TIL)の免疫組織化学染色による病理学的な検討を加え、早期再発症例（コントロール）と比較することで、長期生存肺がん患者の抗腫瘍免疫応答を統合的に評価する。 愛知県がんセンター倫理審査委員会で承認後、当センター呼吸器内科で抗がん剤、免疫チェックポイント阻害剤治療を受けた長期生存患者（治療開始日より、5年が経過する患者）のリストを作成し、臨床情報をまとめた。5年生存を超える症例の候補が23例存在し、現在、6カ月ごとに当センター外来を受診しているため、該当症例から採血を順次実施している。これまでに6症例から採血を実施した。新たな患者から採血を実施するとともに、過去の手術（生検）検体からDNA、RNAを抽出し、全エクソーム/RNAシーケンスを実施する予定である。

腫瘍組織内免疫担当細胞・因子を、効果細胞側と免疫抑制側に大別し、さらに主にフローサイトメトリーを用いて分画化、分画ごとの多彩な機能を解析し、絶対的な強度を付与した各分画を集合させることで、最終的にがん種ごと、症例ごとのがん免疫の強さを総合的に絶対的に評価することを目的としている。 個人間・がん種間の絶対的評価に向けて、消化器癌、呼吸器癌、婦人科癌の症例・新鮮腫瘍組織を新たに集積し、すでに集積済みの検体なども使用可能にするための環境整備を行った。①T細胞を中心とした効果細胞とCD3、CD14、CD15陽性細胞中の抑制因子フローサイトメーター解析、②さらなる亜分画化への工夫としての次世代シーケンサーの応用、③各分画の抗原発現による機能解析、④各分画の更なる機能解析のための培養系の応用；抗原刺激による効果細胞誘導能、刺激培養によるサイトカイン・細胞傷害因子などの産生、遺伝子導入標的細胞に対する直接細胞傷害能、効果細胞との共培養による抑制機能測定、⑤東京大学・垣見教授による遺伝子的解析および東京医大・佐藤准教授による免疫組織学的解析による裏付け、⑥大阪大学・牧野先生による臨床データとの比較、を実施する。 多くの症例ごと、そしてがん種ごとに解析していく事で、絶対的な評価を行うため、個々の解析方法を適応していく。それらに先駆けて、絶対的評価の検討のため、PD-1経路阻害薬などの免疫療法を受けた治験症例においてこれら解析を実施し、その臨床効果と対比しうる包括的腫瘍免疫能評価方法を確定する。Nivolumab投与症例及び抗CCR4抗体投与治験症例の末梢血及び腫瘍組織内免疫細胞を用いた上記包括的解析を進めている。ただし、総合的免疫状態の同定のための評価の確立には更なる症例の集積が必要となる。

近年卵巣癌に対する免疫療法の研究が活発に行われてきている。しかし、進行および再発卵巣癌に対する免疫チェックポイント阻害薬の臨床試験では未だ十分な成果が得られていない。これは、卵巣癌が多彩な組織型を持つだけでなく、多彩な免疫学的背景を持つことがその一因であると考えられる。本研究では、卵巣癌における個別化がん免疫療法の確立を目指すために、次世代シーケンサーを利用した免疫ゲノム解析によって得られる免疫プロファイル、ネオアンチゲン、抗原提示機能などの多面的な免疫学的指標を包括的かつ視覚化できるようなバイオマーカーであるイムノグラムの開発を目指している。がん免疫療法を成功に導くためには、ターゲット腫瘍においてCancer-Immunity Cycleにおけるどのステップが問題になっているかをよく検討することが重要である。そうすることにより、自ずと奏効する可能性が高い患者の選出や、有用な併用薬のパートナーが見つけやすくなる。ただ、このCancer-Immunity Cycleにも見られるように、多くの因子が複雑に絡みあい、腫瘍に対する免疫反応を減弱させているため、効果的な免疫療法を計画するには様々な段階での免疫の増強および免疫抑制因子の排除の両方の検討が必要である。この複雑な宿主および腫瘍の免疫相互作用をCancer-Immunity Cycleになぞらえて、レーダーチャートを利用した上で視覚化するイムノグラムという概念が提唱され、がん免疫治療への応用が期待されている。当該年度はZ-scoreを利用してイムノグラムの各因子のクラスター解析を行い、また特徴的な遺伝子変異及び臨床的ファクターも加えて評価を行い、漿液性癌、明細胞癌のいずれにおいても免疫がアクティブなグループを特定した。

本研究では、多くの患者で共通して認められる遺伝子変異として、マイクロサテライトの遺伝子変異により生じたフレームシフト（MSFS）に着目し、MSFS由来のネオアンチゲンを同定し、その免疫原性を評価し、ワクチン開発の可能性を検討した。International Cancer Genome Consortium (ICGC)、The Cancer Genome Atlas (TCGA)のデータベースを活用し、全ゲノム上でマイクロサテライトに認められる遺伝子変異の中から、フレームシフト変異を検索した。様々ながん種における変異の頻度、さらには変異ペプチドのRNA発現量をもとに、ネオアンチゲンの候補として30個の遺伝子（ACVR2A、KIAA2018、ASTE1、SEC31A、MSH3、RNF43、TBC1D23、TMEM60、TGFBR2、LTN1、CASP5、KMTC2、AIM2、LMAN1、PHACTR4、SMC6、WDTC1、BAX、GINS1、RPL22、CCDC150、RBM27、SLC35F5、AASDH、ESRP1、PRDM2、GRB14、MSH6、SLC35G2、MBD4）に着目した。患者ごとに異なるHLAに対応するため、ネオアンチゲンの探索には、25アミノ酸からなるロングペプチドを用いることとした。合成したペプチドは、フレームシフトの開始アミノ酸から20アミノ酸上流の配列からC末端のアミノ酸までの領域を10アミノ酸ずつスライドさせながらフレームシフトによって生じたアミノ酸変異の全領域をカバーするように68個のペプチドを合成した。4例のMSI-H患者が含む53例の卵巣がん患者の腫瘍の一部を採取し、次世代シーケンサー解析を実施した。今後、MSI-H患者とMSI-S患者におけるMSFS変異の有無を比較し、ネオアンチゲンの探索を行う予定である。

生体に備わる免疫応答を意図的に増幅し全身的な治療効果すなわちアブスコパル効果を惹起させることは腫瘍学上も大変斬新で魅力的であり、革新的な次世代の治療法に貢献し得る。近年癌治療の場面で最大の注目を集め続ける腫瘍免疫に着目しつつ、免疫チェックポイント阻害薬を用いて放射線照射を局所から全身治療へと発展させる新規治療戦略の開発を見据えた科学的根拠を分子細胞レベルで確立し臨床応用することが本研究の目的である。飛躍的に運用が拡がる免疫チェックポイント阻害剤だが、依然として以下の問題点が挙げられる。 I. 標的病変の良好な反応性および生命予後延長が期待できる治療患者選別のためのバイオマーカーが未だ十分に確立していない II. 放射線治療併用における安全性有効性の検証が不十分 III.アブスコパル効果誘導に対するバイオマーカーが不明 IV. 腫瘍特異的遺伝子変異由来の新生抗原（ネオアンチゲン）が未解明 これらのcriticalな問題点を解決するため、下記プロトコールの前向き臨床研究を検討した。I.放射線治療未施行例の原発巣または少数転移病巣：標的腫瘍に50Gy/5分割で SBRT II.放射線治療既施行例の照射野内再発病巣：サイズに応じて30-40Gy/5-8分割でSBRT primary endpointは非標的病変に対する治療効果＝アブスコパル効果の有無、secondary endpointは、IVR技術を用い治療前後に採取した標的・非標的病変腫瘍組織における特異的遺伝子変異の全エクソンシーケンスによる同定である。 初年度は国内外で進むphase I/IIのoligometastasis症例に対する臨床試験等のバイオマーカー探索の詳細を徹底的に調査し、二年目は免疫治療で不可欠なPD-(L)1抗体等が投与される内科・泌尿器科・頭頸部腫瘍科・乳腺外科を含む横断的協力体制を構築し研究継続している。

本研究の目的は、転移性肺腫瘍に対する新たな治療戦略の構築であった。肺転移を含む遠隔転移に対する最適な治療戦略の構築のためには、転移巣における時間空間的多様性を明らかにし、個々の腫瘍における癌微小環境および免疫逃避メカニズムを描出することが必要である。本研究では肺転移巣および原発巣の手術検体における次世代シーケンシングデータを利用し、腫瘍特異的変異やcopy number variationを解析し、clonal evolutionを推測することを予定していた。またあわせてマウスを用いた動物実験も行い、頑健な評価法・解析法を確立することを予定した。 2019年度には、転移性肺腫瘍の手術検体8例を研究用に凍結保存した（直腸癌２例、腎癌１例、甲状腺癌１例、膵癌１例、その他３例）。近年当科では腫瘍がより小さな段階でより低侵襲に切除する取り組みを進めている（触知不能な病変に対するVirtual-assisted lung mapping (VALMAP)併用肺切除等）。当初、腎癌と大腸癌で10例/年程度の転移性肺腫瘍の検体採取を見込んだが、１ｃｍ未満の小病変に対する手術が多く、本研究用検体採取に適した検体はわずかであり、採取症例数が伸びなかった。化学療法によって修飾されたあとの病変に対する手術が増加していることも影響した。そこで、マウス実験の準備も進めつつ、既存の肺癌検体に対する次世代シーケンスデータ解析を進めた。近年肺癌では気腔内進展（spread through air spaces ;STAS)が予後不良因子として注目されている。次世代シーケンスデータを用いたSTASと腫瘍微小環境の解析はこれまで報告がない。扁平上皮癌26例、腺癌65例についてそれぞれ解析を行い、STAS陽性例では免疫関連遺伝子群の発現が低い傾向にあることが認められた。得られた成果を世界肺癌学会や日本肺癌学会で報告した。

本研究の目的は、ミニブタモデルで自己細胞をもとに再生肺を構築し生体内でガス交換能を維持できるか検証することである。再生肺の構築のために我々は脱細胞化技術を用いた。レシピエントとなるブタの肺部分切除から得て培養した自家細胞を、ドナーブタから摘出し脱細胞化した肺に、配置して再生肺を構築した。得られた再生肺を用いてレシピエントに左片肺移植を行った。再生肺を移植されたブタは2時間生存し、その間の酸素ガス交換能は同種他家肺移植と同等であった。

血液透析患者40名、腎癌患者2名、膀胱癌患者2名に対して、血液検体の採取、保存を完了し、血液透析患者からはQOL調査を38名から聴取した。 QOL調査はKDQOL-SFを用い、解析可能な血液透析導入患者9名と血液透析導入1年以上経過の患者17名について、腎疾患特異的尺度と包括的尺度(SF-36)を比較した。腎疾患特異的尺度4項目、包括的尺度5項目で、透析導入患者が透析導入1年以上経過の患者に対して、有意に項目素得点の平均値が高い結果であり、血液透析導入によるQOLの低下が示唆された。

胃癌50例、食道癌58例の手術検体と末梢血検体を採取した。胃癌29例について、次世代シークエンス、フローサイトメトリー、液性因子解析を行い、腫瘍内免疫応答を多層的に解析した。「がん免疫サイクル」の概念に基づき、RNAシークエンスデータから抽出した9つの評価軸からなる「イムノグラム」を作成し、患者個々の腫瘍内免疫応答を表現した。さらにクラスター解析により胃癌を4 群に分類した。この分類は、従来の臨床分類とは相関を認めなかったが、がん抗原数や上皮間葉転換、遺伝子変異、腫瘍微小環境、腫瘍浸潤Tリンパ球の機能不全や浸潤排除の有無などが特徴づけられ、腫瘍内免疫応答の評価に有用と考えられた。

最適な癌治療を提供するには、患者ごとに最適な治療の組み合わせるのが望ましい。癌ワクチン、チェックポイント阻害剤、その他免疫制御法を適切に組み合わせることが重要である。 本研究では、まずヒト肺癌から得た次世代シーケンスデータを用いて腫瘍内免疫応答を多角的に評価する手法を確立した。その手法を用いてマウス肺癌細胞株に対する解析を行い、免疫細胞浸潤を誘導する治療が必要であることが予測された。個別化ワクチンのみでは効果が乏しかったが、免疫細胞浸潤を誘導する免疫アジュバント（補助剤）を組み合わせてマウスに投与したところ、腫瘍の増殖が抑制された。

頭頸部癌50例の手術検体と末梢血検体を採取した。腫瘍組織は細断し、一部は酵素処理等を行って新鮮腫瘍分解物（FTD）を作成した。残りは培地上に播種し、腫瘍浸潤Tリンパ球（TIL）および腫瘍細胞を培養したところ、45例中40例（85%）でTILの増殖が確認された。十分な量のFTDが得られた22例について、TILとFTDを共培養し、上清を回収してELISAでインターフェロンγ（IFNγ）産生を解析したところ、20例（91%）でIFNγ産生の上昇を認めた。 現在は、全50例について次世代シークエンスを外注で行い、またフローサイトメトリー、液性因子解析を行い、腫瘍内免疫応答の多層的な解析を行っている。

末梢血に含まれるがん抗原に対する自己抗体をバイオマーカーとして、個人差が大きいがんに対する免疫応答を的確に予測・モニタリングする技術開発に取り組んだ。150種類超の全長ヒトがん抗原の生産リソースを整備し、水溶性で全エピトープを露出するS-カチオン化抗原とluminex法を組み合わせた診断薬プロトタイプを完成した。実際にがん免疫サイクルの活性化を反映する自己抗体の変動を鋭敏に観察することに成功した。

担癌マウスモデルで免疫応答の解析が可能な4つのがん細胞株（YTN2、YTN16、LLC１、B16)をNGS解析しネオアンチゲンを同定した。DNMT阻害剤5-Azacytidine、DecitabineとHDAC阻害剤Vorinostat、Trichostatin A、Panobinostat、Valproic acidによるエピゲノムの制御により、遺伝子発現が1000倍に増加した抗原を認めた。Trichostatin Aにより、発現が認められなかった18個のネオアンチゲン中12個のネオアンチゲンの発現が誘導された。エピゲノムの制御でネオアンチゲンに対する免疫応答を増強する可能性が示唆された。

遺伝子変異由来のネオ抗原をターゲットにした腎がんの免疫療法を開発することを目的とし、5例の腎癌のエクソーム/RNAシーケンスのデータからin silicoのMHC結合予測アルゴリズムを用いて、121個のHLA-A2拘束性の候補ネオエピトープを同定した。これらのネオエピトープペプチドに対する免疫反応をまずはHLA-A2トランスジェニックマウスを用いてスクリーニングしたところ、15個のペプチドで強い反応が検出された。次に、その15個のペプチドに対するヒト正常人PBMCの免疫反応を検討した。PTEN遺伝子変異由来のネオエピトープに対して反応を認めた。

尿路上皮癌に対するγδT細胞療法の開発を目指して研究を行った。特に、臨床応用が開始された免疫チェックポイント阻害剤との併用あるいは使い分けを意識した。 末梢血中γδT細胞が1.5%以上で、CD27-CD45RAhiタイプが少ない場合、γδT細胞の増殖が確実に可能であった。γδT細胞上の免疫阻害/刺激分子を経時的に解析したところ、γδT細胞の免疫抑制にはPD-1よりもTim-3が関与しており、PD-1/PD-L1経路阻害薬との併用は可能と考えられた。膀胱癌の臨床検体を用いた解析では、γδT細胞の標的となる分子の発現は、腫瘍に浸潤するCD4・CD8陽性リンパ球の多寡により相違があることを見出した。

本研究の目的は、neoantigenをターゲットとした肺癌個別化ワクチンの開発である。そのためにまず、肺癌患者の手術検体から腫瘍特異的遺伝子変異を同定し、MHC結合予測プログラムを用いて患者ごとのneoantigen候補を予測するアルゴリズムを構築した。さらにRNAseqデータを用いて変異mRNAの発現を伴う候補に絞り込み、合成した変異ペプチドワクチンと健常人ドナーPBMCを用いて免疫原性を評価した。さらに、neoantigenワクチンを含む免疫制御法の個別化に向けて、患者ごとの癌免疫サイクルの状態を評価するためのイムノグラムを開発した。

がん細胞はそのがん化の過程で、多くの体細胞遺伝子突然変異を蓄積する。遺伝子変異産物のうち、アミノ酸変異を伴う一部のものはMHCペプチド複合体として細胞表面に提示されT細胞受容体によって認識される抗原となる。腫瘍特異的な遺伝子変異に由来するアミノ酸配列を持った抗原はネオアンチゲンと呼ばれる。ネオアンチゲンは胸腺内で発現を認めないため、中枢性の免疫寛容が誘導されず高い免疫原性を有する可能性がり、免疫治療の標的となることが期待されている。次世代シーケンサーを活用してネオアンチゲンとネオアンチゲン反応性T細胞を同定する方法を開発した。

外因性ヒストンを直接静脈投与することで、肺障害を生じる動物モデルを今回確立した。ヒストン関連肺障害を生じたドナー肺を摘出し、同系ラットに移植し、移植後肺障害、とくに血管内皮障害、微小循環障害の観点から検討した。ヒストン関連肺障害に関しては炎症マーカー(CXCL-1など）や低酸素障害マーカー（HIF-1,2など）で評価した。ヒストン投与後、移植早期の段階で（１時間程度）非常に強い組織障害と血管抵抗増大が起こっていることを観察し得た。微小循環改善に重要な因子であるトロンボモジュリンを投与したところ抗炎症効果より線溶系活性により働くことが示唆された。

パッセンジャー変異を主とする腫瘍特異的遺伝子変異に由来する固有抗原を予測するシステムを構築した。まず肺癌6例のエクソームデータに対して、複数のソフトウェアを用いてミスセンス変異を検出するアルゴリズムを構築した。続いて、腫瘍特異的変異に対してMHC結合予測プログラムを用いて15例の肺癌患者の固有抗原を予測した。固有抗原候補の大半は、患者間で共有される頻度の高い変異由来でなく、個々の患者で異なるパッセンジャー変異に由来するものであることが確認された。そして、エクソームとトランスクリプトームデータを組み合わせることで、発現した変異mRNAに由来するn固有抗原候補に効率的に絞り込む手法を確立した。

我々は、次世代シーケンスとMHCクラスＩ結合予測法を用いて、遺伝子変異由来の抗原(ネオアンチゲン)を同定するシステムを構築した。97症例の腎がんの解析において、ネオアンチゲンの数が多くHLAの発現が高い集団は予後が良好であり、腫瘍内にCD8、パーフォリン、グランザイムを高発現していた。しかし、腫瘍内ではCTLA-4、PD-1といった免疫チェックポイント分子の発現が高く、同時に免疫抑制を引き起こしていることが考えられた。将来、腎がんに対する個別化がん免疫治療を行っていく準備段階として、抗原同定システムを構築し、ネオアンチゲンの発現と腫瘍内の免疫シグネチャー、そして予後との関連を明らかにした。

我々は、次世代シーケンスとMHCクラスＩ結合予測法を用いて、遺伝子変異由来の候補固有抗原を同定するシステムをマウスの胃がんモデルで構築した。これまで29例の胃がん手術組織検体とそのペアーとなる末梢血単核球(PBMC)を採取し保存した。10例で腫瘍浸潤リンパ球（TIL）における免疫チェックポイント分子の発現及び腫瘍内の制御性T細胞の浸潤を解析し、胃がんの腫瘍局所の免疫抑制環境を検討した。また、3例で自己腫瘍に特異的に反応するTILを培養することが可能であった。将来の個別化がん免疫治療の準備段階として、固有抗原同定システムを構築し、胃がんの固有抗原同定に向けた胃がん組織検体を採取できた。

腎摘除術を施行された転移性腎細胞癌患者を対象とし、腎摘除時に採取した自己腫瘍組織を免疫源として樹状細胞(DC)ワクチンを作成し、IFNαあるいはスニチニブと併用投与する臨床試験を実施した。研究期間中に5例を組み入れた。DCワクチンの平均投与回数は10.6回で、全例DCワクチンの投与に伴う重篤な合併症はなく、安全に投与可能であった。本科研費の執行以前から開始していた患者を含めた8例について、スニチニブおよびワクチン投与前後での免疫応答の変化を解析した。DCワクチンとスニチニブの併用投与によって抑制的な免疫細胞が減少し、免疫学的な抗腫瘍効果が増強される可能性が示唆された。

難治性の疾患である胸膜中皮腫に対する抗原特異的免疫細胞治療を開発するため、γδT細胞にメソテリン特異的受容体遺伝子（MesoCAR）を導入する技術を確立した。In vitro/ in vivoでのMesoCAR+γδT細胞の抗腫瘍効果が確認された。MesoCAR+αβT細胞と比較し、遜色ない抗腫瘍効果だった。正常組織に対するoff-tumor/on-target toxicityは認められず、移植片対宿主病も誘導されなかった。MesoCAR+γδT細胞を用いた免疫治療は、胸膜中皮腫含むメソセリン陽性腫瘍に対して副作用を起こさずに抗腫瘍効果を発揮する有効な治療法となる可能性が示された。

革新的な免疫細胞治療法を開発するために、東大病院で実施された橋渡し研究で得られた患者検体と臨床情報をフィードバックして、治療モデルマウスを用いた免疫動態の解析と機能的に統合し、免疫細胞治療に於ける症例選択と治療効果判定に重要なバイオマーカーの同定を試みた。がんワクチン治療、樹状細胞治療、γδT細胞移入治療のTRにおいて、臨床効果が得られた症例を経験する一方で、抗腫瘍免疫応答が誘導されたにもかかわらず、十分な治療効果が得られない症例も数多く存在する。B16メラノーマ担癌マウスの免疫治療モデルでは、腫瘍に対する免疫応答が、免疫抑制性の環境を誘導しており、その制御が重要であることを明らかにした。

我々は、次世代シーケンスとMHCクラス Ｉ結合予測アルゴリズムを組み合わせることで、腫瘍特異的な遺伝子変異から候補固有抗原を同定するシステムを、膵がん組織を用いて構築した。また、候補固有抗原に対する免疫反応をマウス生体で検証するアッセイ法も確立した。肺がん6例（喫煙者3例と非喫煙者3例）においては、遺伝子変異（ミスセンス変異）の数は喫煙者で平均280個に対し、非喫煙者で平均75個であった。予測された候補固有抗原（エピトープ）の数は、それぞれ平均535個と140個であった。将来の個別化がん免疫治療の準備段階として、抗原同定システムを構築し、肺がんで候補固有抗原を同定することが可能であった。

本研究では、化学修飾法を用いた変性タンパク質のタンパク質可溶化技術を活用して、多くが不安定な物性のがん抗原タンパク質を全長・水溶性として調製する技術開発とリソース強化を進めた。本抗原を活用して、高感度な抗体検査試薬のプロトタイプ開発に成功し、がん免疫活性の診断薬となる可能性が示された。これらのリソース整備と周辺技術の組み合わせにより、タンパク質カチオン化技術のがん免疫治療分野への活用に向けた基盤が整った。

HSP105由来ペプチドパルス樹状細胞(DC)ワクチン治療の第一相臨床試験を行った。対象は進行・再発がん患者で、HSP105の発現が確認され、HHLA-A2またはHLA-A24を有する患者である。DCは5X106細胞/回(ステップ1)、1X107細胞/回(ステップ2)、2X107細胞/回(ステップ3)を計4回投与する用量漸増試験とした。 ステップ1ではHSP105特異的CD8陽性T細胞の検出は全例不可能であった。ステップ2では全例投与部位のDTH反応を認め、SD1例、PD2例であった。ステップ3に膵癌4例を登録予定であったが、実際の投与例は1例に留まった。膵癌症例の効果判定はPDであった。

タンパクの有する多価抗原性とペプチドの簡便性を合わせ持ち、抗原提示能に優れた長鎖複合ペプチドワクチンを用いて臨床試験を実施した。NY-ESO-1f長鎖ペプチドについて、抗原提示細胞(APC)によるエピトープ提示機構を明らかにし、がんワクチンとして用いて臨床試験を実施し、免疫効果を明らかにした。これらの知見をもとに、4種類の長鎖オーバーラップペプチドを混合したNY-ESO-1OLP長鎖複合ペプチドについても臨床試験を実施し、免疫効果を明らかにした。さらに、新しいがん抗原XAGE-1bについても同様に解析した。

以前の研究から、同種血管移植後(新鮮グラフト)には移植片が免疫応答に関連するIDOを介した抗感染能を取得している可能性が示唆されてきた。本研究では、同種移植後の移植片として新鮮グラフトのみならず凍結保存後のグラフトにおいてもMRSAの増殖を抑制する機能を獲得しており移植片局所における特異的炎症反応の存在(Tcellresponse、IFNγ・TNFαの遺伝子発現)を確認した。更に、同種移植時に発現するIFNγの刺激により抗感染性に関与すると考えられるtryptophan代謝酵素IDO(indoleamine2,3-deoxygenase)についても凍結保存グラフトで遺伝子・蛋白レベルで発現していることが確認できた。これらにより、同種移植後IFNγの刺激により移植片に発現したIDOが移植片のもつ抗感染性メカニズムがグラフトの凍結保存加工を経た後も期待できる効果であることが実証された。臨床的に観察されてきた同種凍結保存心臓弁・血管組織(所謂「ホモグラフト」)のMRSA抗感染性が科学的実証できたと考えられた。

放射線治療による抗腫瘍効果を、直接作用による腫瘍の細胞死に加えて、生体の免疫応答を介した間接的な抗腫瘍効果を含めて再評価することにより、免疫細胞治療を併用し、免疫応答を介した生体に与える効果を増幅し、全身的な治療効果が期待できる集学的な治療法を「免疫化学放射線治療」として確立することを目的に研究した。放射線治療とCTL治療を併用すると、骨髄性抑制細胞の誘導が抑制され、CTL浸潤が増強し、抗腫瘍効果を担うIFNγの産生が増加した。このような免疫反応の制御が放射線治療の効果を増強することに重要であることを明らかにした。

結腸・直腸癌の肺転移は手術切除によって根治の可能性があるが、術後再発率は高く、難治性である。肺転移完全切除例の術後に自己リンパ球(γδT細胞分画)を採取、体外で培養活性化させ、抗癌作用を高めた後に自己の体内に戻す免疫療法を行った。本治療は安全に遂行することができた。ヒストリカルコントロール群と比較し、生存率・無再発生存率に有意差は無かった。

我々は抗原特異的細胞障害性リンパ球(CTL)がどのようなメカニズムで肝細胞に到達し、肝障害を引き起こすのか検討した。HBs抗原を肝細胞に発現したHBV TgマウスにCTLsを投与することによりヒトの劇症肝炎類似の現象を誘導するマウスモデルを用いて、接着因子であるCD44が、CTLの肝細胞内への浸潤に重要であることを示し、CTLs側のCD44よりも、類洞壁内皮細胞でのCD44の発現がCTLの類洞内の浸潤に重要であることを示した。

膵癌治癒切除後症例を対象として、Gemcitabine(GEM)を用いた全身化学療法に、自己活性化γδT細胞を用いた免疫療法を加えた補助療法を施行した。研究期間中に22名のエントリーがあり、最終的に施行可能症例は11例であった。安全性は確認されたが、無再発期間はGEM単独治療と差はなく、さらなるフォローアップが必要である。

免疫療法は新たな治療法のひとつとして期待されているが、現状では十分な効果を得るには至っていない。肺癌患者において、腫瘍抗原に対するアビディティの高いCTLを樹立することは非常に困難であることが知られている。アビディティの高いCTLからTCR遺伝子をクローニングして、レトロウイルスベクターを用いて患者の末梢血のリンパ球に導入して腫瘍特異的CTLを作製し、細胞移入治療を実現することを目的として本研究を実施した。肺癌患者の腫瘍組織から肺癌細胞株F1121Lを樹立し、手術時に採取・凍結保存していた自己のリンパ節リンパ球と共培養を繰り返すことにより癌精巣抗原であるKK-LC1をHLA-B15拘束性に認識するCTLクローンH1/10を樹立した。TCRα鎖およびβ鎖をクローニングし、翻訳効率の高いlinkerである2AでTCRα鎖(Vα1)とTCRβ鎖(Vβ19)をつなぎ、PHXレトロウイルスベクターに組み込んだ。末梢血からPBMCを採取しゾレドロン酸とIL-2で刺激してγδT細胞を増殖させ、これに上記TCRαβ遺伝子のみを導入した。KK-LC1陽性のF1121LおよびをKK-LC1ペプチドをパルスしたF1121-EB-virus transformed B cellに特異的に傷害活性とサイトカイン産生を示し、この反応は抗MHC class I抗体添加により阻害された。さらにSCID mouseを用いて、TCRαβ-CD8移入γδT細胞の養子免疫モデルを作成し、抗原特異的なin vivoでの腫瘍増殖抑制効果が得られた。肺癌患者末梢血からPBMCを採取し、5μMのゾレドロン酸と1000IU/mlのIL-2を用いてγδT細胞を培養し、15名の肺癌患者にγδT細胞移入治療を実施した。この研究成果をもとに、今後肺癌特異的CTL由来のTCRを患者末梢血γδT細胞に導入し、肺癌に対する細胞治療法を開発する。

Radiation Immunology & Immunotherapy の確立を目指して、放射線治療と免疫細胞治療に関する基礎的検討と臨床研究を実施した。担癌マウスモデルを用いて、電子線照射が腫瘍と生体の免疫系に与える効果を検討した。腫瘍を取り巻く微小環境内では、Gr-1^+CD11b^+のmyeloid-derived suppressor cells (MDSC) と呼ばれる骨髄系の細胞が蓄積し、ケモカインによって制御されていることを明らかにした。 ex vivoで大量培養したγδT細胞を、骨転移に対する放射線治療後に投与する臨床研究を実施した。前立腺癌に対する抗アンドロゲン治療を受けている患者では、γδT細胞の培養が困難であったが、治療群では病勢コントロールが得られた症例を認めた。

【目的】治療困難・予後不良な肺癌再発例に対して自己活性化γδ-T 細胞(γδT)による免疫療法を試み、安全性および有効性について明らかにする。 【対象と方法】原発性肺癌、非小細胞肺癌治療後再発例、本研究に同意された方。評価可能病変を有し、除外基準を持たないことを条件とした。 【結果】腺癌8例・扁平上皮癌1例・大細胞癌1例計10例を対象とした。γδTは3-12回投与された(中央値6回)。全有害事象はGrade1のべ3回、Grade3のべ2回(細菌性肺炎・放射線肺炎)であった。いずれもγδT 治療と関連は無かった。投与後240-850日(中央値445日)観察され、最終観察時生存6、死亡4例であった。γδT投与中死亡は見られなかった。死因はいずれも肺癌再発増悪であった。RECICS 判定では5回投与後CR/PR/SD/PD=0/0/5/4であった。後観察期間では0/0/3/5判定不能2であった。CR+PR+SDの割合を病勢コントロール率とすると5回投与後では50%,後観察期間では30%であった。投与後末梢血中のVγ9-γδT 細胞数は次第に増加傾向にあった。FACT-BRM total score の経時的測定においてはGrade 3有害事象症例をのぞき、投与期間中はスコア値が安定ないし上昇し、治療期間中のQOLは良好に保たれた。 【考察】非小細胞肺癌表面に過剰発現するMICA/B0を認識するNKG2DをγδTは発現しており、isopentenyl pyrophosphate をTCR/CD3のリガンドとして認識し、癌細胞に接触・破壊する。体内に多量の自己γδT を投与した場合の安全性ならびに有効性について明らかにしたが、さらに今後はこの細胞障害活性をより効果的に体内で発現させるための方策について検討を進めたい。

本研究において、同種移植後の移植片がMRSAの増殖を抑制する機能を獲得しており移植片局所における特異的炎症反応の存在(T cell response、IFNγ・TNFα の遺伝子発現)を確認した。更に、同種移植時に発現するIFNγの刺激により抗感染性に関与すると考えられるIDOが移植片局所において遺伝子・蛋白レベルで発現していることが確認できた。移植片のIDOと抗感染性との関連については、特にIDOによるTrp代謝産物の系に注目して検証した。Trp代謝 産物のうち3-Hydroxykynurernineが抗菌活性を持つことが確認された。これらの結果は、同種移植後IFNγの刺激により移植片に発現したIDOが移植片のもつ抗感染性のメカニズムにおいて重要な役割を担っていることを示唆し、ホモグラフトのMRSA感染に対する科学的実証であると考えられた。

局所制御能の優れた放射線治療にシステミックな効果とメモリー機能を持った免疫細胞治療を併用することにより、免疫応答を介して生体に与える効果を増幅し、照射部位のみならず全身的で断続的な治療効果を発揮する革新的な癌の治療法の開発が期待できる。この理論的根拠として、局所のみの放射線治療が時として他部位の腫瘍にも影響を与える事実"abscopal effect"が50年ほど前からしられている。この興味深い現象の免疫学的な基礎実験データによる検証はほぼ皆無であったが、本研究において抗腫瘍活性を持ったエフェクター細胞としてMacrophage inflammatory protein-1α(MIP-1α,CCL3)に着目し、単独あるいは放射線照射を併用した抗腫瘍活性に関する基礎的検討を行い、免疫賦活剤と放射線を併用することで抗腫瘍作用が著しく増強されることを確かめた。これにより再現性の高い"abscopal effect"の人為的な誘導に成功した。 その機序を分子レベル、細胞レベル、個体レベルで解明することにより革新的治療法を開発して癌治療の新分野を開くことをを目的とし、前年度に明らかにした免疫担当細胞浸潤との関連につき各々にdepletion studyを施行ことでeffector cellを同定した。 また"abscopal effect"の分子生物マーカー探索においてHMGB1が深く関与していることも発見し、このタンパクが認識するTLR4活性化を介したDCの誘導がこの効果の本質である可能性が示唆された。この端緒的発見は、放射線治療や化学療法の治療効果の背景に腫瘍免疫が何らかの役割を果たしているという未知の可能性を含み、今後の飛躍的発展につながる潜在的可能性を持つものと期待される。

T 細胞受容体トランスジェニックマウス細胞を用いて反復感染応答におけるCTL 細胞群の消長を検討した。ある抗原特異的CTL 集団に着目した場合、メモリー期のみならず、エフェクター期から、抗原経験回数が異なるCTL 群が、生体内で異なる分布を示していて、個体レベルでは抗原特異的CTL 集団の維持に働いていることを明らかにした。また、Naive CD8 陽性T 細胞、一次メモリー、二次メモリーCTL に発現している転写産物(タグ)を解析した。

光増感剤と抗原を同時に樹状細胞に作用させ、エンドサイトース経路により細胞内に取り込ませた。光増感剤としては、685nmに最大吸収波長を有するデンドリマーフタロシアニン(DPc、分子量:4,903)を内包した高分子ミセルを利用した。DPc内包ミセルは、エンドサイトーシスにより細胞内に取り込まれるが、この時、光照射を行うことによってDPcより産生される一重項酸素がエンドソーム膜に障害を与え、エンドソーム内の化合物(抗原)が細胞質内に放出される仕組みである。具体的には、免疫応答の標的分子となる抗原タンパク質と共にDPc内包ミセルを樹状細胞に取り込ませた後、光刺激を加えてエンドソームに貯留している抗原を細胞質へと放出させる。本来なら、大部分の抗原はMHCクラスII経路に入り、CD4陽性T細胞を活性化する。しかし、光刺激により細胞質内に放出された抗原は、小胞体内でMHCクラスI分子に結合し、細胞表面に提示され、CD8陽性T細胞を活性化することが可能になった。C57BL/6マウスの骨髄細胞をGM-CSFとIL-4の存在下で培養し、樹状細胞に分化させて実験に用いた。樹状細胞に抗原を導入し、OT-I細胞やOT-II細胞と共培養した場合に、どちらの細胞が反応するかを観察した。樹状細胞にOVAを取り込ませた後、OT-I細胞とOT-II細胞を反応させると、樹状細胞はOT-II細胞を刺激活性化することが可能であるが、OT-I細胞を刺激する効率は非常に低かった。一方、DPc内包ミセルと光刺激処理を行った樹状細胞は、CD8陽性のOT-I細胞を効率よく刺激することが可能であった。光刺激によるエンドソームのターゲッティング技術を、抗原提示細胞内でのMHCクラスI経路とクラスII経路のターゲッティングに応用し、クロスプレゼンテーション効率を改善し、免疫応答を増強することを明らかにした。

「肝がんの再発予防を可能にする樹状細胞(DC)ワクチンの開発」を目的として本研寒を実施した。HCV肝細胞癌の再発に対し、RFA治療後に超音波ガイド下に腫瘍を直接穿刺してmaturatingDCの投与を実施した。末梢血単核細胞(PBMC)をGM-CSF(800U/ml)とIL-4(50ng/lm)の存在下に7日間培養しimmatureDCへと分化させた。OK432(0.001KE/ml)で2時間刺激を与えたmaturatingDC(1x10^6-10^8)をRFA治療直後と1週後に投与した。投与されたDCはCD14^-CD40^+CD80^+Cb86^+CD83^-であった。2例の患者に対してDCの投与を行なったところ、1例の患者で、2回目の投与後に間質性肺炎を認めた。直ちにステロイドを投与し適切に対応した。投与前後の患者のPBMCの分画をフローサイトメーターで解析したところ、間質性肺炎の発症と一致して、末梢血からCD14陽性細胞が消失した。CD14陽性の単球が肺へ浸潤したためと考えられた。患者の回復とステロイドなどの免疫応答に関わる薬剤の投与が終了した後、肺炎と樹状細胞治療との関係を明確にするために、患者の末梢血から単核細胞(PBMC)を分離し治療に用いた樹状細胞に対する反応と樹状細胞の刺激に用いたOK432に対する反応を3H-Thymidineの取り込みで検討した。治療前、治療直後、肺炎回復後のいずれのPBMCも樹状細飽に対する増殖反応を示さず、樹状細胞の投与により自己免疫反応や過敏反応の誘導は認められなかった。OK432に対してPBMCの増殖が認められたが、治療前後のPBMCの反応に有意な差は認められなかつた。抗原特異的な自己免疫反応の誘導ではなく、肝臓の腫瘍細胞内に投与した樹状細胞が、抗原非特異的な免疫応答を活性化したと考えられる。

我々は本研究のテーマである肝炎ウイルスに対する生体のダイナミックな免疫応答を解析する過程において、肝炎ウイルスに限らず、免疫の本質の一つである免疫学的メモリー(immunological memory)に関する重要な知見を明らかにすることができた。ウイルス感染を制御した個体では、ウイルス特異的CD8+T細胞がメモリー細胞に分化して、一生涯生体を守っている。これまで、メモリー細胞はあたかもstem cellのように自己増殖を繰り返す、不老不死・長命の細胞と考えられていたが、我々は「抗原特異的CTLは感染刺激の都度大きく交代する」ことを証明した。 CD8陽性T細胞はウイルスに感染した細胞を排除するのに重要な役割を果たす獲得免疫細胞のひとつで、体内にナイーブな状態で一定数存在するが、ウイルスに感染するとエフェクターCTLとして増殖したのち減少してメモリーCTLとなり、次に同一のウイルスにより感染を受けた場合にすばやく応答して感染状態の悪化を防いでいる。我々は、二度目の感染(二次感染)時に、体内に存在しているナイーブ細胞からもごく少量ながらメモリーCTLが誘導されることに着目し、一次メモリーCTLと二次メモリーCTLがどのような役割を果たしているかを検証した。一次メモリーCTLと二次メモリーCTLが共存しているマウスから取り出した細胞を別のマウスに移入し、三次感染刺激を与えて各メモリーCTLの分裂応答能やTCRレパトア推移をみた。その結果、三次感染時の抗原特異的免疫応答の主体は二次感染時に誘導されたより若いCTLであることが明らかになった。 減少してゆき、感染の都度誘導されてくるメモリーCTL(新兵)が量的にも質的にも感染防御に力強く働いている様を明らかにすることができた。

本研究では原発性非小細胞肺癌再発例に対する自己末梢血中γδTリンパ球活性化療法の安全性と抗腫瘍効果につき検討した。末梢血の単核球分画をIL-2とPamidronate存在下培養すると、γδT細胞が1000倍程度増殖し、全単核球の85-95%となった。NKG2D陽性であり、MICA/B発現非小細胞肺癌株に対する細胞障害活性をin vitroで認めた。 当施設倫理委員会の承認下、臨床治験を開始した。非小細胞肺癌術後再発例に対して活性化自己γδT細胞を容量漸増方式で2週間毎に6回投与し、安全性・抗腫瘍効果を検討した。 治療同意された8例中6例が0.45-0.9x10^9個のγδT細胞を得、適格と判断され治療、4例が治験終了。治験中止例はなかった。2例γδT細胞が十分増殖せず不適格となった。 安全性:γδT細胞療法とは関連のない有害事象(感冒症候群)が一過性に認められたが、本治療関連の有害事象はなかった。調査用紙に基づくQOL測定(N=3)では、感冒によるQOL低下の1例以外はQOL低下は認められなかった。 CTによる再発病変評価(N=4)では有意な腫瘍径縮小は本研究期間中認められなかった。 3例で6回のγδT細胞投与前後の末梢血γδT細胞のサブセット解析を行った。3例とも投与期間中末梢血γδT細胞数およびeffector活性の増加がフローサイトメトリーで示された。現状では明らかな抗腫瘍効果が認められないが、in vitroではMICA陽性と陰性の癌細胞株に対する検討ではMICA陽性細胞株(U266骨髄腫由来)に対する細胞障害活性にNKG2D-MICA/Bの関与が示された。 現在までのところ非小細胞肺癌に対する明らかな抗腫瘍効果は示されなかったが、本治験は十分量のγδT細胞が実質4回しか投与できていないことから、今後投与量・間隔を考慮し実際の抗腫瘍効果が得られるべく検討を進めたい。

この研究の基礎研究となった研究であるHCV患者のIFN-α産生能は病態の進行に伴い低下する、肝硬変から肝癌発症群は非発症群に比較してより低値である事を、"Impairment of IFN-α production capacity in patients with hepatitis C virus and the risk of the development of hepatocellular carcinoma."として完成、World J Gastroenteroolに発表した。更に、ルイ・パストゥール医学研究センターで15年にわたり蓄積されたIFN-α産生能検査と一般血液検査結果の17年のデータベースを完成させ、そこから、健常人112名、HCV患者20名を抽出、その統計学的解析を行った。HCV患者では60%が低下傾向あるいは低値を示したのに対し、健常人ではその数は13%と、明らかに肝癌のハイリスク群であるHCV患者で高値であった。更に、肝癌発症者の平均IFN-α産生能は健常人、非発症者に比べて低値であることを明らかにした。この結果は"Impairedinterferon-α production and the risk of cancer development"の論文にまとめ、J IFN＆Cytokine Research in pressである。さらにIFN-α産生能の低下の病因解明の為、患者末梢血中のIFN-α産生細胞の同定のためのFACSによるIFN-α産生細胞測定系を確立した。その結果主たるIFN-α産生細胞は、plasmacytoid dendritic cellであることが明らかにした。

造血器腫瘍の新しい分子標的療法を開発し、臨床的に効果のある治療法の開発を目指す。本研究では次世代型ABLチロシンキナーゼ阻害剤(dasatinib)とimatinibとの相乗効果の分子メカニズムをDNAマイクロアレイを用いて解析した。K562細胞をdasatinibまたはimatinibと共に培養後、DNAマイクロアレイにて誘導される遺伝子群について解析したところ、大多数の遺伝子は重複して抑制されていたが、dasatinibにて特異的に発現抑制が見られた遺伝子群の中でCDK2、CDK8を同定した。CDK2、CDK8 siRNAを用いることによりdasatinibとimatinibの相乗効果にCDK2、CDK8の関与が確認された。さらにThr315はBCR-ABLのキナーゼドメインの中心部に位置しており、ATP結合部位を覆っているため、ABLキナーゼ阻害剤に対てgatekeeperと呼ばれている。T3151変異型BCR-ABLはimatinib抵抗性Ph陽性白血病の約20%に認められる変異であり、第2世代型チロシンキナーゼ阻害剤、dasatinib及びnilotinibも無効であるため、有効な治療法は確立されていない。VE-465はATP競合型のAuroraキナーゼ阻害剤であり、BCR-ABL、Jak-2、Flt-3に対しても阻害効果を有する。本研究ではVE-465のWT BCR-ABL及びT315I BCR-ABLに対する生物活性を明らかにし、Ph陽性白血病に対する有用性を検討した。VE-465はp185BCR-ABL、T315IBCR-ABL共にキナーゼ活性をIC_<50>値2-5μMにて抑制した。VE-465、imatinib併用によりK562細胞においてアポトーシス誘導の増加、Caspase-3、PARPの活性化及びAkt、c-Mycの活性低下が確認された。VE-465はin vivoにおいてもT315IBCR-ABLキナーゼ活性を阻害し、BaF3 BCR-ABL移植マウスの生存期間を延長させた。

肝がんに対するRFA治療を受けた患者に対して免疫細胞治療を実施するために、in vitroにおける樹状細胞(DC)培養条件の最適化を検討し、肝がん患者に対する臨床試験の開始を計画した。腫瘍特異的な免疫応答を誘導するためにDCには、1.RFA治療により熱変性した腫瘍細胞をuptakeする能力、2.免疫応答を誘導するために必要なCD80, CD86などの共刺激分子の発現、3.リンパ節へのホーミングに必要なケモカイン受容体CCR7の発現、が必要である。そこで我々は、 1.immature DCをOK432で刺激して2-6時間後のDC(maturating DC)は、OK432刺激を除いても、それ以降mature DCへの成熟過程が進行し、16時間後にはCD80, CD86, CCR7などの分子を発現すること。2.肝癌細胞Huh7 cellをRFA治療と同様の加熱条件(85℃で10分間)で熱変性させた後、さまざまな成熟段階のDCとover nightで共培養すると、maturating DCは、熱変性した腫瘍細胞を効率よく取り込むこと。3.さらにmaturating DCは腫瘍の取り込みによって成熟過程を妨げられることなくmature DCへと変化することを明らかにした。 これらの結果に基づいて、肝がんRFA治療後に腫瘍局所内へ投与するDCは、GM-CSFとIL-4によって誘導した末梢血単球由来のimmature DCを、OK432を用いて2時間刺激したmaturating DCの状態で用いることに決定した。東京医科大学病院において、肝がんの治療を受けた患者を対象に臨床試験を実施するためにプロトコールを作成した。倫理委員会での承認を受け臨床試験を開始し、肝がん患者の登録を開始した。

【目的】肝炎ウイルスに対する生体(宿主)の免疫応答を解析するために、独自に新しい肝炎モデルマウスを作成し、ウイルス特異的CD8^+T細胞の誘導過程と炎症細胞の肝臓内への浸潤過程を解析した。 【方法】HBVプラスミドDNAを用いてマウスを免疫し、HBV特異的CD8^+T細胞を誘導した。HBVが組込まれたアデノウイルス(Ad-HBV)をマウスに感染させ、肝細胞内にHBVゲノムを導入し、ウイルス蛋白の発現とウイルスの複製を誘導した。HBV特異的な免疫応答の存在下で、Ad-HBVを感染させ、肝臓内で発現したHBVウイルス抗原に対する免疫応答による炎症反応を誘発した。MHC class I-dimerやIFN-_γ染色法を用いてフローサイトメーターでHBV特異的CD8^+T細胞を検出し、体内動態を詳細に検討した。肝臓に発現している遺伝子をリアルタイムPCR法で定量的に解析した。 【結果】HBV特異的CD8^+T細胞は、脾臓などのリンパ組織だけでなく、末梢血中や肝臓を含めた末梢組織中にも広く分布していた。肝臓内にHBVウイルス抗原が発現すると、ウイルス特異的なCD8^+T細胞は迅速に反応し激しい炎症反応を誘発した。炎症のピークであるday5にはHBV特異的CD8^+T細胞は脾臓や末梢血中から消失し、肝臓内に蓄積し、活発に分裂増殖していた。肝臓からウイルス抗原が消失したday14では、再び末梢血中や脾臓にHBV特異的CD8^+T細胞が最分布した。CD8^+T細胞の肝臓内への浸潤と一致して、IFN-_γなどのサイトカインや、CXCL9,CXCL10などのケモカインが強く発現していた。 【結論】HBVに対する特異的免疫応答が惹起する肝臓内の炎症反応とそれに伴うウイルス特異的なCD8^+T細胞のダイナミックな生体内の動きを明らかにした。ウイルス抗原排除後にメモリー細胞が最分布することを明らかにした。