Naozumi Harada

Regulatory T Cell–Resistant CD8+ T Cells Induced by Glucocorticoid-Induced Tumor Necrosis Factor Receptor Signaling

We previously found that a Salmonella typhimurium vector engineered to secrete soluble tumor antigen induces CD4(+) T cells resistant to CD4(+)CD25(+) regulatory T cells (Treg) and that glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) signal is involved in the development of this resistance. In this study, we address the potential of incorporating GITR ligand (GITRL) as a way to augment the immunogenicity of cancer vaccines. BALB/c mice were immunized by gene gun with plasmids encoding the mutated extracellular signal-regulated kinase 2 (mERK) with or without plasmids encoding mouse GITRL. Coadministration with GITRL during primary and secondary immunization enhanced the induction of mERK-specific CD8(+) T cells. Antibody depletion and minigene analysis suggested that GITRL directly activated CTL epitope-specific CD8(+) T cells independently of CD4(+) T cells. Immunization with plasmids encoding a CTL epitope and GITRL resulted in strong tumor inhibition in a CD8(+) T cell-dependent manner. Furthermore, CTL epitope-specific CD8(+) T cells induced by immunization with plasmids encoding CTL epitope coadministered with GITRL were refractory to suppression by CD4(+)CD25(+) Tregs compared with CD8(+) T cells induced without GITR signaling. We propose that coadministration of GITR signaling agents with tumor antigens constitutes a promising novel strategy for cancer vaccine development.

Engineering hybrid exosomes by membrane fusion with liposomes.

Exosomes are a valuable biomaterial for the development of novel nanocarriers as functionally advanced drug delivery systems. To control and modify the performance of exosomal nanocarriers, we developed hybrid exosomes by fusing their membranes with liposomes using the freeze–thaw method. Exosomes embedded with a specific membrane protein isolated from genetically modified cells were fused with various liposomes, confirming that membrane engineering methods can be combined with genetic modification techniques. Cellular uptake studies performed using the hybrid exosomes revealed that the interactions between the developed exosomes and cells could be modified by changing the lipid composition or the properties of the exogenous lipids. These results suggest that the membrane-engineering approach reported here offers a new strategy for developing rationally designed exosomes as hybrid nanocarriers for use in advanced drug delivery systems.

Peptide Vaccine Induces Enhanced Tumor Growth Associated with Apoptosis Induction in CD8+ T Cells

CD8+ CTLs play a critical role in antitumor immunity. However, vaccination with synthetic peptide containing CTL epitopes has not been generally effective in inducing protective antitumor immunity. In this study, we addressed the detailed mechanism(s) involved in this failure using a new tumor model of BALB/c transplanted tumors expressing NY-ESO-1, an extensively studied human cancer/testis Ag. Whereas peptide immunization with an H2-Dd–restricted CTL epitope derived from NY-ESO-1 (NY-ESO-1 p81–88) induced NY-ESO-181–88–specific CD8+ T cells in draining lymph nodes and spleens, tumor growth was significantly enhanced. Single-cell analysis of specific CD8+ T cells revealed that peptide immunization caused apoptosis of >80% of NY-ESO-181–88–specific CD8+ T cells at tumor sites and repetitive immunization further diminished the number of specific CD8+ T cells. This phenomenon was associated with elevated surface expression of Fas and programmed death-1. When peptide vaccination was combined with an adjuvant, a TLR9 ligand CpG, the elevated Fas and programmed death-1 expression and apoptosis induction were not observed, and vaccine with peptide and CpG was associated with strong tumor growth inhibition. Selection of appropriate adjuvants is essential for development of effective cancer vaccines, with protection of effector T cells from peptide vaccine-induced apoptosis being a prime objective.

Guanine-Rich Sequences Are a Dominant Feature of Exosomal microRNAs across the Mammalian Species and Cell Types

Exosome is an extracellular vesicle released from multivesicular endosomes and contains micro (mi) RNAs and functional proteins derived from the donor cells. Exosomal miRNAs act as an effector during communication with appropriate recipient cells, this can aid in the utilization of the exosomes in a drug delivery system for various disorders including malignancies. Differences in the miRNA distribution pattern between exosomes and donor cells indicate the active translocation of miRNAs into the exosome cargos in a miRNA sequence-dependent manner, although the molecular mechanism is little known. In this study, we statistically analyzed the miRNA microarray data and revealed that the guanine (G)-rich sequence is a dominant feature of exosome-dominant miRNAs, across the mammalian species-specificity and the cell types. Our results provide important information regarding the potential use of exosome cargos to develop miRNA-based drugs for the treatment of human diseases.

Establishment of animal models to analyze the kinetics and distribution of human tumor antigen-specific CD8 + T cells

Many patients develop tumor antigen-specific T cell responses detectable in peripheral blood mononuclear cells (PBMCs) following cancer vaccine. However, measurable tumor regression is observed in a limited number of patients receiving cancer vaccines. There is a need to re-evaluate systemically the immune responses induced by cancer vaccines. Here, we established animal models targeting two human cancer/testis antigens, NY-ESO-1 and MAGE-A4. Cytotoxic T lymphocyte (CTL) epitopes of these antigens were investigated by immunizing BALB/c mice with plasmids encoding the entire sequences of NY-ESO-1 or MAGE-A4. CD8(+) T cells specific for NY-ESO-1 or MAGE-A4 were able to be detected by ELISPOT assays using antigen presenting cells pulsed with overlapping peptides covering the whole protein, indicating the high immunogenicity of these antigens in mice. Truncation of these peptides revealed that NY-ESO-1-specific CD8(+) T cells recognized D(d)-restricted 8mer peptides, NY-ESO-181-88. MAGE-A4-specific CD8(+) T cells recognized D(d)-restricted 9mer peptides, MAGE-A4265-273. MHC/peptide tetramers allowed us to analyze the kinetics and distribution of the antigen-specific immune responses, and we found that stronger antigen-specific CD8(+) T cell responses were required for more effective anti-tumor activity. Taken together, these animal models are valuable for evaluation of immune responses and optimization of the efficacy of cancer vaccines.

Activated CD8 + T cell extracellular vesicles prevent tumour progression by targeting of lesional mesenchymal cells

Fibroblastic tumour stroma comprising mesenchymal stem cells (MSCs) and cancer-associated fibroblasts (CAFs) promotes the invasive and metastatic properties of tumour cells. Here we show that activated CD8+ T cell-derived extracellular vesicles (EVs) interrupt fibroblastic stroma-mediated tumour progression. Activated CD8+ T cells from healthy mice transiently release cytotoxic EVs causing marked attenuation of tumour invasion and metastasis by apoptotic depletion of mesenchymal tumour stromal cells. Infiltration of EV-producing CD8+ T cells is observed in neovascular areas with high mesenchymal cell density, and tumour MSC depletion is associated with preferential engulfment of CD8+ T cell EVs in this setting. Thus, CD8+ T cells have the capacity to protect tumour progression by EV-mediated depletion of mesenchymal tumour stromal cells in addition to their conventional direct cytotoxicity against tumour cells.Immune cells have an important role in tumour progression. Here, the authors show that extracellular vesicles from activated CD8+ T cells attenuate tumour progression by depletion of mesenchymal tumour stromal cells.

Epirubicin, Identified Using a Novel Luciferase Reporter Assay for Foxp3 Inhibitors, Inhibits Regulatory T Cell Activity

Forkhead box protein p3 (Foxp3) is crucial to the development and suppressor function of regulatory T cells (Tregs) that have a significant role in tumor-associated immune suppression. Development of small molecule inhibitors of Foxp3 function is therefore considered a promising strategy to enhance anti-tumor immunity. In this study, we developed a novel cell-based assay system in which the NF-κB luciferase reporter signal is suppressed by the co-expressed Foxp3 protein. Using this system, we screened our chemical library consisting of approximately 2,100 compounds and discovered that a cancer chemotherapeutic drug epirubicin restored the Foxp3-inhibited NF-κB activity in a concentration-dependent manner without influencing cell viability. Using immunoprecipitation assay in a Treg-like cell line Karpas-299, we found that epirubicin inhibited the interaction between Foxp3 and p65. In addition, epirubicin inhibited the suppressor function of murine Tregs and thereby improved effector T cell stimulation in vitro. Administration of low dose epirubicin into tumor-bearing mice modulated the function of immune cells at the tumor site and promoted their IFN-γ production without direct cytotoxicity. In summary, we identified the novel action of epirubicin as a Foxp3 inhibitor using a newly established luciferase-based cellular screen. Our work also demonstrated our screen system is useful in accelerating discovery of Foxp3 inhibitors.

Identification of an immunogenic neo-epitope encoded by mouse sarcoma using CXCR3 ligand mRNAs as sensors

ABSTRACT The CXCR3 ligands CXCL9, 10, and 11 play critical roles in the amplification of immune responses by recruiting CXCR3+ immune effector cells to the tumor site. Taking advantage of this property of CXCR3 ligands, we aimed to establish a novel approach to identify immunogenic mutated-antigens. We examined the feasibility of using CXCR3 ligand mRNAs as sensors for detection of specific immune responses in human and murine systems. We further investigated whether this approach is applicable for the identification of immunogenic mutated-antigens by using murine sarcoma lines. Rapid synthesis of CXCR3 ligand mRNAs occurred shortly after specific immune responses in both human and murine immune systems. Particularly, in CMS5 tumor-bearing mice, we detected specific immune responses to mutated mitogen-activated protein kinase 2 (ERK2), which has previously been identified as an immunogenic mutated-antigen. Furthermore, by combining this approach with whole-exome and transcriptome sequencing analyses, we identified an immunogenic neo-epitope derived from mutated staphylococcal nuclease domain-containing protein 1 (Snd1) in CMS7 tumor-bearing mice. Most importantly, we successfully detected the specific immune response to this neo-epitope even without co-administration of anti-cytotoxic T-lymphocyte protein-4 (CTLA-4), anti-programmed cell death-1 (PD-1) and anti-glucocorticoid-induced TNFR-related protein (GITR) antibodies, which vigorously augmented the immune response and consequently enabled us to detect the specific immune response to this neo-epitope by conventional IFNγ intracellular staining method. Our data indicate the potential usefulness of this strategy for the identification of immunogenic mutated-antigens. We propose that this approach would be of great help for the development of personalized cancer vaccine therapies in future.

Signal-transducing adaptor protein-2 promotes generation of functional long-term memory CD8 + T cells by preventing terminal effector differentiation

Long-surviving memory CD8+ T cells generated by stimulation with appropriate tumor-associated antigens are the most aggressive and persistent tumoricidal effectors. In this event of memory CD8+ T cell development, the signal transducer and activator of transcription (STAT) proteins function as the crucial intracellular signaling molecules, but the regulatory mechanism of STATs in CD8+ T cells is not fully understood. In this study, we report for the first time, by using murine vaccination models, that signal-transducing adaptor protein-2 (STAP2) maintains the cytotoxicity of long-lived memory CD8+ T cells by controlling a STAT3/suppressor of cytokine signaling 3 (SOCS3) cascade. Following T cell activation, STAP2 expression was transiently reduced but was subsequently recovered and augmented. Analysis using small-interfering RNA (siRNA) demonstrated that restored STAP2 expression was associated with the activation of STAT3/SOCS3 signals and maintenance of cytotoxic T lymphocytes (CTLs) secondary responses by preventing their differentiation into terminal effector cells. Notably, this STAP2-dependent memory differentiation was observed in the spleen, but not in the lymph nodes (LNs). These findings indicate an essential role for STAP2 in the generation of a high-quality memory CD8+ CTLs periphery, and suggest the therapeutic potential of STAP2 in cancer patients.

Abstract PR02: Antigen delivery targeting tumor-infiltrating macrophages leads to eradication of tumor highly resistant to immune checkpoint inhibitors

Recently, immune checkpoint inhibitors using anti-CTLA-4, anti-PD-1, and anti-PD-L1 antibodies has been emerging as novel, effective modality for the treatment of cancer. However, immune checkpoint inhibitors have been proven to be effective only for a partial group of patients who have lymphocyte infiltration or inflammation in the tumor microenvironment prior to the treatment. Although the relationship between efficacy of checkpoint inhibitors and anti-tumor cellular immune response has been revealed, these studies paid attention only for the induction of immune responses in lymph node and do not pay attention to the mechanisms regulating the anti-tumor cellular immune responses in tumor site. In this study, by using checkpoint inhibitors-sensitive or resistant murine in vivo tumors, we explored a mechanism underlying the resistance focusing on immune cells at the tumor site. We newly identified CD11b+/F4/80+ tumor-associated macrophages as a key factor highly correlated to the resistance. In the resistant tumor, tumor-associated macrophages remained immature and did not exert antigen-presenting activity. A nanogel-based antigen delivery system enabled to selectively and efficiently deliver a long peptide antigen to tumor-associated macrophages. Co-administration of the nanogel:long peptide antigen and TLR agonist activated tumor-associated macrophages and induced antigen presentation by these cells. This change in the tumor microenvironment turned the resistant tumor to treatment-sensitive, in particular, adoptive transfer of TCR-engineered T cells after the treatment with nanogel:long peptide antigen and TLR agonist resulted in the cure of resistant tumor. These results indicate that the status and function of tumor-associated macrophages have a great impact on treatment sensitivity, and that these cells might be a promising target to improve the efficacy of immunotherapy including checkpoint inhibitors and adoptive cell therapies. This abstract is also being presented as Poster B68. Citation Format: Daisuke Muraoka, Naozumi Harada, Naohiro Seo, Tae Hayashi, Keisuke Fujii, Rui Yamaguchi, Seiya Imoto, Satoru Miyano, Hideo Yagita, Kazunari Akiyoshi, Hiroshi Shiku. Antigen delivery targeting tumor-infiltrating macrophages leads to eradication of tumor highly resistant to immune checkpoint inhibitors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr PR02.