The methods for expansion of human cytomegalovirus (HCMV)-specific T lymphocytes are limited due to the complex culture process, long culture duration, and human leukocyte antigen (HLA) restriction. Here, we report that in vitro stimulation with pp65 kDa phosphoprotein (pp65)-derived overlapping synthetic peptides rapidly generates large numbers of HCMV-specific cytotoxic T lymphocytes from peripheral blood mononuclear cells (PBMCs) regardless of HLA type. Treatment of PBMCs from healthy volunteers expressing HLA-A*02:01 or HLA-A*24:02 with 138 pp65 overlapping peptides (OLP) resulted in an expansion of HCMV pp65 NLVPMVATV (NLV) pentamer-specific CD8+ T lymphocytes that expressed interferon (IFN)-γ, but the pp65 NLV peptide did not generate HCMV-specific CD8+ T lymphocytes in PBMCs obtained from an HLA-A*24:02 donor due to HLA restriction. The OLP-induced T lymphocytes specific for HCMV derived from PBMCs of HLA-A*02:01- and HLA-A*24:02-expressing donors showed effective cytolytic responses against target cells loaded with OLP or the NLV epitope, but pp65 NLV peptide-induced T lymphocytes did not. Phenotypic analyses demonstrated that OLP increased the frequency of CD3+ CD8+ cells, but not CD3+ CD4+, CD14+, or CD56+ cells, in donor PBMCs. Thus, this study provides evidence that in vitro stimulation with OLP efficiently generates sufficient numbers of HCMV pp65-specific cytotoxic T lymphocytes for adoptive cell therapy. Keywords: human cytomegalovirus; cytotoxic T lymphocyte; overlapping peptides; pp65; cytotoxicity.

Increased γ−synuclein (SNCG) by radiation contributes to immunosuppressive effects via the inhibition of dendritic cell (DC) differentiation and activation, thus making it a potential target for cancer treatment. Radiotherapy (RT) is a well-established standard tumor treatment, and over half of all cancer patients will receive RT as part of their treatment plan.1 Exposure to ionizing radiation (IR) provokes several distinct cell death programs, such as apoptosis, necrosis, mitotic catastrophe, and autophagy, against tumor cells, as well as the surrounding immune cells.2 Although RT has traditionally been recognized as cytotoxic and immunosuppressive, in recent years substantial evidence has prompted the re-characterization of radiation as immunomodulatory rather than immunosuppressive. IR-induced ‘danger signals' from dying tumor cells that may contribute to incite a potent anti-tumor immune response via immunogenic cell death (ICD).3 However, the interplay between danger signaling patterns behind the trafficking of damage-associated molecular patterns (DAMPs) and their immune-sensing systems appears to be very plastic and highly dependent on the dose and fractionation of radiation, the type of radiation-induced cell death, and the experimental conditions. Thus, whether the effect of intracellular proteins released by RT could be beneficial or detrimental to the final therapy outcome remains controversial. We have recently demonstrated that single or fractionated doses of radiation induced several secretory proteins in human breast cancer cells.4 One of the interesting candidates from the previous study, SNCG, was markedly increased by a high single dose of 10 Gy but not by fractionated irradiation. Several studies have revealed that SNCG is highly expressed in several cancer types, such as the advanced stages of breast, liver, ovarian carcinomas, colon and prostate cancer, and is associated with cancer metastasis and invasiveness.5 Therefore, we aim to investigate whether the newly identified secretory SNCG derived from RT-treated dying tumor cells could subsequently elicit anti-tumorigenic immunity or a pro-tumorigenic immune response. DCs have a vital role as professional antigen-presenting cells that are able to activate naive T cells and initiate T-cell responses, acting as messengers between the innate and adaptive immune systems.6 Upon exposure to SNCG, TNF-α- or LPS-stimulated semi-mature DCs (smDCs) or mature DCs (mDCs) reduced their expression of several surface molecules such as CD40, CD80, CD86, and MHC-II that contribute to co-stimulation and antigen presentation to T cells. In addition, mDCs in the presence of SNCG significantly reduced the production of the inflammatory cytokines IL-1β, IL-6, IL-12, IL-23, IFN-γ, and TNF-α. Co-culture with SNCG-treated DCs downregulated T-cell proliferation and altered the T-cell cytokine production profile, reducing pro-inflammatory cytokine IFN-γ and IL-17 secretion and inducing the anti-inflammatory cytokines IL-4 and TGF-β.7 Owing to limitations on our ability to evaluate the in vivo quantitative and qualitative DC activation in the tumor microenvironment, we further investigated whether the soluble secretory factors from irradiated tumor cells may actually affect DC maturation. Using the Trans-well system, irradiated tumor cells inhibited the activation of LPS-stimulated DCs through a decrease in surface maturation ligands and inflammatory IL-12 and TNF-α cytokine production. SNCG derived from RT-treated dying tumor cells may moderate the stimulation of DCs, similar to smDCs, with low expression of phenotypic maturation ligands and the induction of immunosuppressive cytokines, thereby rendering the DCs incapable of efficiently interacting with T cells or eliciting fully immunogenic responses (Figure 1). Figure 1 Immunosuppressive effect of SNCG. Tumor cells exposed to radiation undergo different types of tumor cell death such as apoptosis, necrosis, mitotic catastrophe, and senescence. The dying tumor cells emitted various surface molecules and cellular components ... Despite the accumulation of emerging evidence, it still remains challenging to understand how, when, and to what extent this dynamic spectrum of DC activation drives tumor-specific anti-tumor immunity, particularly in the context of anti-cancer therapy. In this respect, the pre-existing or therapy-generated tumor microenvironments, as well as the cross-talk between dying cancer cells and DCs, mediated by soluble and vesicular factors, are crucial determinants of the DC maturation status and anti-cancer immune response. Furthermore, the DAMP spectrum can change even for the same cancer cell line depending on the type of treatment; the optimal dosing, timing and sequencing of RT, or other stimuli must be further investigated. This study indicates that SNCG, which can be released from dying irradiated breast cancer cells, might be at least partially involved in the persistence of tumor resistance against RT, and modulation of SNCG may be a promising approach for anti-cancer therapy. With emerging interest in studying the mechanisms of IR-induced ICD, it is necessary to find novel immunomodulators and analyze certain existing therapies for their potential to cause DC maturation irrespective of whether they induced ICD. This study also cautiously suggests the predictable response of DCs against radiation-induced dying cancer cells.

Recently, γ-synuclein (SNCG), which is also known as breast cancer-specific gene-1, has been demonstrated to be an adverse and aggressive marker in breast cancer. In our previous study, SNCG was significantly upregulated in irradiated human breast cancer cells. The aim of this study was to investigate whether radiation-induced, tumor-derived SNCG can influence dendritic cell (DC) function in immune systems. The phenotypical and functional changes of DCs in the presence or absence of SNCG were investigated by FACS analysis, ELISA, and real-time PCR. The ability of SNCG-treated DCs to influence T cells was also examined by coculturing with T cells. The treatment of DCs with SNCG protein inhibited the surface expression of the co-stimulatory molecules CD40 and CD86, and decreased the mRNA levels of pro-inflammatory cytokines. The SNCG-treated DCs inhibited T-cell proliferation slightly, but distinctively increased the population of regulatory T cells. In addition, the production of TGF-β from T cells was significantly increased when they were cocultured with SNCG-treated DCs. Taken together, these results demonstrate that tumor-derived SNCG contributes to immunosuppressive effects via the inhibition of DC differentiation and activation, thus making it a potential target for cancer treatment.

Peroxisome proliferator-activated receptors (PPARs) inhibit lipopolysaccharide (LPS)-primed release of high mobility group box 1 (HMGB1), a late proinflammatory mediator, but the underlying molecular mechanism is not completely understood. In this study, we demonstrated that the inhibition of HMGB1 release by PPAR- δ and - γ is associated with the deacetylase activity of SIRT1. Ligand-activated PPAR- δ and - γ inhibited LPS-primed release of HMGB1, concomitant with elevation in SIRT1 expression and promoter activity. These effects were significantly reduced in the presence of small interfering (si)RNAs against PPAR, indicating that PPAR- δ and - γ are involved in both HMGB1 release and SIRT1 expression. In addition, modulation of SIRT1 expression and activity by siRNA or chemicals correspondingly influenced the effects of PPARs on HMGB1 release, suggesting a mechanism in which SIRT1 modulates HMGB1 release. Furthermore, we showed for the first time that HMGB1 acetylated in response to LPS or p300/CBP-associated factor (PCAF) is an effective substrate for SIRT1, and that deacetylation of HMGB1 is responsible for blockade of HMGB1 release in macrophages. Finally, acetylation of HMGB1 was elevated in mouse embryonic fibroblasts from SIRT1 -knockout mice, whereas this increase was completely reversed by ectopic expression of SIRT1. These results indicate that PPAR-mediated upregulation of SIRT1 modulates the status of HMGB1 acetylation, which, in turn, has a critical role in the cellular response to inflammation through deacetylation-mediated regulation of HMGB1 release.

The Korea Occupational Safety & Health Agency (KOSHA) is developing an Integrated Risk Management System (IRMS) to support the quantification and management of risk from chemical plants. The IRMS system includes the development of methodology, software tools and database necessary for quantitative risk assessment, which are consequence analysis software, graphical display of results on a geometric map, reliability analysis software, component reliability database, and equipment and hazardous material information databases. An overview of the IRMS will be presented in another paper, "GIS-based IRMS." The quantification of a risk consists of two major parts: one is a deterministic analysis, such as the consequence analysis of an explosion of flammable material, and the other is a probabilistic part such as the frequency analysis of an explosion or a reliability analysis of the protection system. This paper describes the development work in a probabilistic part of the IRMS.

Selective cleavage of thiazolidines such as (I) is carried out in MeCN with CaCO3 as acid scavenger.