Modulation of tumor immune microenvironment by TAS-115, a multi-receptor tyrosine kinase inhibitor, promotes antitumor immunity and contributes anti-PD-1 antibody therapy.

TAS-115 is an oral multi-receptor tyrosine kinase inhibitor that strongly inhibits kinases implicated in antitumor immunity, such as colony stimulating factor 1 receptor and vascular endothelial growth factor receptor. Because these kinases are associated with the modulation of immune pathways, we investigated the immunomodulatory activity of TAS-115. An in vitro cytokine assay revealed that TAS-115 upregulated interferon γ (IFNγ) and interleukin-2 secretion by T cells, suggesting that TAS-115 activated T cells. Gene expression analysis suggested that TAS-115 promoted M1 macrophage differentiation. In in vivo experiments, although TAS-115 exerted a moderate antitumor effect in the MC38 mouse colorectal cancer model under immunodeficient conditions, this effect was enhanced under immunocompetent conditions. Furthermore, combination of TAS-115 and anti-PD-1 antibody exhibited greater antitumor activity than either treatment alone. Flow cytometry analysis showed the increase in IFNγ- and granzyme B (Gzmb)-secreting tumor-infiltrating T cells by TAS-115 treatment. The combination treatment further increased the percentage of Gzmb+CD8+ T cells and decreased the percentage of macrophages compared with either treatment alone. These results highlight the potential therapeutic effect of TAS-115 in combination with PD-1 blockade, mediated via activation of antitumor immunity by TAS-115.

Japanese cedar and cypress pollinosis updated: New allergens, cross-reactivity, and treatment.

Pollen from many tree species in the Cupressaceae family is a well-known cause of seasonal allergic diseases worldwide. Japanese cedar pollinosis and Japanese cypress pollinosis, which are caused by pollen from Japanese cedar (Cryptomeria japonica) and Japanese cypress (Chamaecyparis obtusa), respectively, are the most prevalent seasonal allergic diseases in Japan. Recently, the novel major Japanese cypress allergen Cha o 3 and the homologous Japanese cedar allergen Cry j cellulase were identified, and it was shown, for the first time, that cellulase in plants is allergenic. Although the allergenic components of pollen from both species exhibit high amino acid sequence identity, their pollinosis responded differently to allergen-specific immunotherapy (ASIT) using a standardized extract of Japanese cedar pollen. Pharmacotherapy and ASIT for Japanese cedar and cypress pollinosis have advanced considerably in recent years. In particular, Japanese cedar ASIT has entered a new phase, primarily in response to the generation of updated efficacy data and the development of new formulations. In this review, we focus on both Japanese cypress and cedar pollinosis, and discuss the latest findings, newly identified causative allergens, and new treatments. To manage pollinosis symptoms during spring effectively, ASIT for both Japanese cedar and Japanese cypress pollen is considered necessary.

TAS0314, a novel multi-epitope long peptide vaccine, showed synergistic antitumor immunity with PD-1/PD-L1 blockade in HLA-A*2402 mice.

Cancer peptide vaccines are a promising cancer immunotherapy that can induce cancer-specific cytotoxic T lymphocytes (CTLs) in tumors. However, recent clinical trials of cancer vaccines have revealed that the efficacy of the vaccines is limited. Targeting single antigens and vaccination with short peptides are partly the cause of the poor clinical outcomes. We synthesized a novel multi-epitope long peptide, TAS0314, which induced multiple epitope-specific CTLs in HLA knock-in mice. It also showed superior epitope-specific CTL induction and antitumor activity. We also established a combination treatment model of vaccination with PD-1/PD-L1 blockade in HLA-A*2402 knock-in mice, and it showed a synergistic antitumor effect with TAS0314. Thus, our data indicated that TAS0314 treatment, especially in combination with PD-1/PD-L1 blockade, is a promising therapeutic candidate for cancer immunotherapy.

Immunological effects of sublingual immunotherapy with Japanese cedar pollen extract in patients with combined Japanese cedar and Japanese cypress pollinosis.

Sublingual immunotherapy (SLIT) with Japanese cedar (JCe) pollinosis was expected to be effective for Japanese cypress (JCy) pollinosis. However, only a half of JCy pollinosis patients clinically improved. Therefore, we examined the immunological effect of SLIT for JCy pollinosis. Peripheral blood mononuclear cells (PBMCs) from patients with JCe and JCy pollinosis who did and did not receive SLIT were incubated with Cry j 1, Cha o 1 and Cha o 3 antigens. Basophil activation test (BAT) were performed. Production of IL-5 and IL-17 induced by antigens was inhibited in the SLIT group. Cry j 1-specific production of IL-10 was increased, and serum Cry j 1-specific IgE and -IgG4 were elevated. However, Cha o 1- or Cha o 3-specific production of IL-10 and specific IgG4 was not increased. Antigens-specific BAT did not decrease after SLIT. New SLIT with JCe and JCy is needed for patients with combined JCe and JCy pollinosis.

Correction: Development of a novel immunoproteasome digestion assay for synthetic long peptide vaccine design.

[This corrects the article DOI: 10.1371/journal.pone.0199249.].

Development of a novel immunoproteasome digestion assay for synthetic long peptide vaccine design.

Recently, many autologous tumor antigens have been examined for their potential use in cancer immunotherapy. However, the success of cancer vaccines in clinical trials has been limited, partly because of the limitations of using single, short peptides in most attempts. With this in mind, we aimed to develop multivalent synthetic long peptide (SLP) vaccines containing multiple cytotoxic T-lymphocyte (CTL) epitopes. However, to confirm whether a multivalent vaccine can induce an individual epitope-specific CTL, the only viable screening strategies currently available are interferon-gamma (IFN-γ enzyme-linked immunospot (ELISPOT) assays using human peripheral blood mononuclear cells, or expensive human leukocyte antigen (HLA)-expressing mice. In this report, we evaluated the use of our developed murine-20S immunoproteasome (i20S) digestion assay, and found that it could predict the results of IFN-γ ELISPOT assays. Importantly, the murine-i20S digestion assay not only predicted CTL induction, but also antitumor activity in an HLA-expressing mouse model. We conclude that the murine-i20S digestion assay is an extremely useful tool for the development of "all functional" multivalent SLP vaccines.

Identification of Cha o 3 homolog Cry j 4 from Cryptomeria japonica (Japanese cedar) pollen: Limitation of the present Japanese cedar-specific ASIT.

BACKGROUND: About one-third of the Japanese population suffers from Japanese cedar pollinosis, which is frequently accompanied by Japanese cypress pollinosis. Recently, a novel major Japanese cypress pollen allergen, Cha o 3, was discovered. However, whether a Cha o 3 homolog is present in Japanese cedar pollen remains to be determined. METHODS: Western blot analysis was performed using Cha o 3-specific antiserum. In addition, cloning of the gene encoding Cry j 4 was conducted using total cDNA from the male flower of Japanese cedar trees. Allergen potency and cross-reactivity were investigated using a T-cell proliferation assay, basophil activation test, and ImmunoCAP inhibition assay. RESULTS: A low amount of Cha o 3 homolog protein was detected in Japanese cedar pollen extract. The deduced amino acid sequence of Cry j 4 showed 84% identity to that of Cha o 3. Cross-reactivity between Cry j 4 and Cha o 3 was observed at the T cell and IgE levels. CONCLUSIONS: Cry j 4 was discovered as a counterpart allergen of Cha o 3 in Japanese cedar pollen, with a relationship similar to that between Cry j 1-Cha o 1 and Cry j 2-Cha o 2. Our findings also suggest that allergen-specific immunotherapy (ASIT) using Japanese cedar pollen extract does not induce adequate immune tolerance to Cha o 3 due to the low amount of Cry j 4 in Japanese cedar pollen. Therefore, ASIT using Cha o 3 or cypress pollen extract coupled with Japanese cedar pollen extract is required in order to optimally control allergy symptoms during Japanese cypress pollen season.

Glycoform of a newly identified pollen allergen, Cha o 3, from Chamaecyparis obtusa (Japanese cypress, Hinoki).

Cha o 3 is a newly found glycosylated allergen from Chamaecyparis obtusa (Japanese cypress) pollen. The deduced amino acid sequence of Cha o 3 indicates that this glycoallergen contains a cellulase domain and a number of putative N-glycosylation sites. However, the structures of N -glycans linked to Cha o 3 remain to be determined. In this study, therefore, we analyzed the glycoform of Cha o 3 and found that this glycoallergen carries exclusively plant complex-type N-glycans; major structures were GlcNAc2Man3Xyl1Fuc1GlcNAc2 (39%), Gal1Fuc1GlcNAc2Man3Xyl1Fuc1GlcNAc2 (14%), and Gal2Fuc2GlcNAc2Man3Xyl1Fuc1GlcNAc2 (25%). The glycoform of Cha o 3 bearing the Lea epitope is similar to those of Cry j1, Jun a 1, or Cup a 1, major glycoallergens in cedar or cypress pollens, and the predominant occurrence of GlcNAc2Man3Xyl1Fuc1GlcNAc2 is a common structural feature of glycoallergens from Cupressaceae pollens.

Generation of a Novel HLA Class I Transgenic Mouse Model Carrying a Knock-in Mutation at the ß2-Microglobulin Locus.

We generated a series of monochain HLA class I knock-in (KI) mouse strains, in which a chimeric HLA class I molecule (α1/α2 domain of HLA-A*0201, HLA-A*0301, HLA-A*2402, or HLA-A*3101 and α3 domain of H-2Db) was covalently linked with 15 aa to human ß2-microglobulin (ß2m) and introduced into the endogenous mouse ß2m locus. In homozygous KI mice, mouse ß2m gene disruption resulted in loss of the endogenous H-2 class I molecules and reduction in the peripheral CD8+ T cell population that was partially restored by monochain HLA class I expression. A gene dosage-dependent expression of HLA, similar to that in human PBMCs, was detected in heterozygous and homozygous HLA KI mice. Upon vaccination with various virus epitopes, HLA-restricted, epitope-specific CTLs were induced in HLA KI mice, similar to the response in the commonly used HLA transgenic mice. Importantly, the CTL responses induced in heterozygous KI mice were similar to those in homozygous KI mice. These results suggest that coexpression of H-2 class I does not affect HLA-restricted CTL responses in HLA KI mice, which differs from the situation reported for monochain HLA Tg × ß2m-/- mice. Furthermore, we generated double KI mice harboring two different HLA (HLA-A*2402 and HLA-A*0301) KI alleles, which showed a CTL response against both HLA-A24 and HLA-A3 epitopes when immunized with a mixture of both peptides. These results indicated that this HLA class I KI mouse model provides powerful research tools not only for the study of HLA class I-restricted CTL responses, but also for preclinical vaccine evaluation.

Inhibition of RANKL-induced osteoclastogenesis by (-)-DHMEQ, a novel NF-kappaB inhibitor, through downregulation of NFATc1.

UNLABELLED: (-)-DHMEQ, a newly designed NF-kappaB inhibitor, inhibited RANKL-induced osteoclast differentiation in mouse BMMs through downregulation of the induction of NFATc1, an essential transcription factor of osteoclastogenesis. INTRODUCTION: Bone destruction is often observed in advanced case of rheumatoid arthritis and neoplastic diseases, including multiple myeloma. Effective and nontoxic chemotherapeutic agents are expected for the suppression of these bone destructions. RANKL induces activation of NF-kappaB and osteoclastogenesis in bone marrow-derived monocyte/macrophage precursor cells (BMMs). Targeted disruption or pharmacological suppression of NF-kappaB result in impaired osteoclastogenesis, but how NF-kappaB is involved in the regulation of osteoclastogenesis is not known. MATERIALS AND METHODS: The effect of (-)-dehydroxymethylepoxyquinomicin [(-)-DHMEQ] on osteoclast differentiation was studied using a culture system of mouse BMMs stimulated with RANKL and macrophage colony-stimulating factor. The mechanism of the inhibition was studied by biochemical analysis such as immunoblotting and retroviral transfer experiments. RESULTS: (-)-DHMEQ strongly inhibited RANKL-induced NF-kappaB activation in BMMs and inhibited RANKL-induced formation of TRACP(+) multinucleated cells. Interestingly, (-)-DHMEQ specifically inhibited the RANKL-induced expression of NFATc1 but not the expressions of TRAF6 or c-fos. Inhibition of osteoclast differentiation by (-)-DHMEQ was rescued by overexpression of NFATc1, suggesting that the inhibition is not caused by a toxic effect. Moreover, pit formation assays showed that (-)-DHMEQ also inhibited the bone-resorbing activity of mature osteoclasts. CONCLUSION: The inhibition of NF-kappaB suppresses osteoclastogenesis by downregulation of NFATc1, suggesting that NFATc1 expression is regulated by NF-kappaB in RANKL-induced osteoclastogenesis. Our results also indicate the possibility of (-)-DHMEQ becoming a new therapeutic strategy against bone erosion.