Quality Verification with a Cluster-Controlled Manufacturing System to Generate Monocyte-Derived Dendritic Cells.

Dendritic cell (DC) vaccines for cancer immunotherapy have been actively developed to improve clinical efficacy. In our previous report, monocyte-derived DCs induced by interleukin (IL)-4 with a low-adherence dish (low-adherent IL-4-DCs: la-IL-4-DCs) improved the yield and viability, as well as relatively prolonged survival in vitro, compared to IL-4-DCs developed using an adherent culture protocol. However, la-IL-4-DCs exhibit remarkable cluster formation and display heterogeneous immature phenotypes. Therefore, cluster formation in la-IL-4-DCs needs to be optimized for the clinical development of DC vaccines. In this study, we examined the effects of cluster control in the generation of mature IL-4-DCs, using cell culture vessels and measuring spheroid formation, survival, cytokine secretion, and gene expression of IL-4-DCs. Mature IL-4-DCs in cell culture vessels (cluster-controlled IL-4-DCs: cc-IL-4-DCs) displayed increased levels of CD80, CD86, and CD40 compared with that of la-IL-4-DCs. cc-IL-4-DCs induced antigen-specific cytotoxic T lymphocytes (CTLs) with a human leukocyte antigen (HLA)-restricted melanoma antigen recognized by T cells 1 (MART-1) peptide. Additionally, cc-IL-4-DCs produced higher levels of IFN-γ, possessing the CTL induction. Furthermore, DNA microarrays revealed the upregulation of BCL2A1, a pro-survival gene. According to these findings, the cc-IL-4-DCs are useful for generating homogeneous and functional IL-4-DCs that would be expected to promote long-lasting effects in DC vaccines.

Interferon-α-Induced Dendritic Cells Generated with Human Platelet Lysate Exhibit Elevated Antigen Presenting Ability to Cytotoxic T Lymphocytes.

Given the recent advancements of immune checkpoint inhibitors, there is considerable interest in cancer immunotherapy provided through dendritic cell (DC)-based vaccination. Although many studies have been conducted to determine the potency of DC vaccines against cancer, the clinical outcomes are not yet optimal, and further improvement is necessary. In this study, we evaluated the potential ability of human platelet lysate (HPL) to produce interferon-α-induced DCs (IFN-DCs). In the presence of HPL, IFN-DCs (HPL-IFN-DCs) displayed high viability, yield, and purity. Furthermore, HPL-IFN-DCs displayed increased CD14, CD56, and CCR7 expressions compared with IFN-DCs produced without HPL; HPL-IFN-DCs induced an extremely higher number of antigen-specific cytotoxic T lymphocytes (CTLs) than IFN-DCs, which was evaluated with a human leukocyte antigen (HLA)-restricted melanoma antigen recognized by T cells 1 (MART-1) peptide. Additionally, the endocytic and proteolytic activities of HPL-IFN-DCs were increased. Cytokine production of interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-α was also elevated in HPL-IFN-DCs, which may account for the enhanced CTL, endocytic, and proteolytic activities. Our findings suggest that ex-vivo-generated HPL-IFN-DCs are a novel monocyte-derived type of DC with high endocytic and proteolytic activities, thus highlighting a unique strategy for DC-based immunotherapies.

Dendritic Cells Pre-Pulsed with Wilms' Tumor 1 in Optimized Culture for Cancer Vaccination.

With recent advances in cancer vaccination therapy targeting tumor-associated antigens (TAAs), dendritic cells (DCs) are considered to play a central role as a cell-based drug delivery system in the bioactive immune environment. Ex vivo generation of monocyte-derived DCs has been conventionally applied in adherent manufacturing systems with separate loading of TAAs before clinical use. We developed DCs pre-pulsed with Wilms' tumor (WT1) peptides in low-adhesion culture maturation (WT1-DCs). Quality tests (viability, phenotype, and functions) of WT1-DCs were performed for process validation, and findings were compared with those for conventional DCs (cDCs). In comparative analyses, WT1-DCs showed an increase in viability and recovery of the DC/monocyte ratio, displaying lower levels of IL-10 (an immune suppressive cytokine) and a similar antigen-presenting ability in an in vitro cytotoxic T lymphocytes (CTLs) assay with cytomegalovirus, despite lower levels of CD80 and PD-L2. A clinical study revealed that WT1-specific CTLs (WT1-CTLs) were detected upon using the WT1-DCs vaccine in patients with cancer. A DC vaccine containing TAAs produced under an optimized manufacturing protocol is a potentially promising cell-based drug delivery system to induce acquired immunity.

Genomic Insights into the Evolution of the Nicotine Biosynthesis Pathway in Tobacco.

In tobacco (Nicotiana tabacum), nicotine is the predominant alkaloid. It is produced in the roots and accumulated mainly in the leaves. Jasmonates play a central signaling role in damage-induced nicotine formation. The genome sequence of tobacco provides us an almost complete inventory of structural and regulatory genes involved in nicotine pathway. Phylogenetic and expression analyses revealed a series of structural genes of the nicotine pathway, forming a regulon, under the control of jasmonate-responsive ETHYLENE RESPONSE FACTOR (ERF) transcription factors. The duplication of NAD and polyamine metabolic pathways and the subsequent recruitment of duplicated primary metabolic genes into the nicotine biosynthesis regulon were suggested to be the drivers for pyridine and pyrrolidine ring formation steps early in the pathway. Transcriptional regulation by ERF and cooperatively acting MYC2 transcription factors are corroborated by the frequent occurrence of cognate cis-regulatory elements of the factors in the promoter regions of the downstream structural genes. The allotetraploid tobacco has homologous clusters of ERF genes on different chromosomes, which are possibly derived from two ancestral diploids and include either nicotine-controlling ERF189 or ERF199 A large chromosomal deletion was found within one allele of the nicotine-controlling NICOTINE2 locus, which is part of one of the ERF gene clusters, and which has been used to breed tobacco cultivars with a low-nicotine content.

p185(HER-2/neu) and p21(CIP1/WAF1) expression in primary tumors and lymph node metastases in non-small cell lung cancer.

p185(HER-2/neu), a tyrosine kinase receptor, is one of the target molecules for cancer therapy, and its expression may reduce the sensitivity of tumor cells to anti-cancer drugs. p21(CIP1/WAF1) is a cyclin-dependent kinase inhibitor, and its expression may also be involved in chemoresistance. Non-small cell lung cancer (NSCLC) is a potentially systemic disease, and systemic therapies play an important role in its treatment. However, there have been no studies comparing the expression of these molecules between primary and metastatic tumors. We investigated the expression of p185(HER-2/neu) and p21(CIP1/WAF1) in 57 paired samples of primary NSCLC tumors and corresponding lymph node metastases by immunohistochemistry. Expression of each of p185(HER-2/neu) and p21(CIP1/WAF1) was highly correlated between primary tumors and lymph node metastases, and similar correlations were also obtained when adenocarcinoma and squamous cell carcinoma cases were analyzed individually. However we failed to detect any correlation between p185(HER-2/neu) and p21(CIP1/WAF1) expression. Our results suggested that expression of both p185(HER-2/neu) and p21(CIP1/WAF1) is concordant between primary and metastatic tumors.

An anti-GD2 monoclonal antibody enhances apoptotic effects of anti-cancer drugs against small cell lung cancer cells via JNK (c-Jun terminal kinase) activation.

Small cell lung cancer (SCLC) cell lines specifically express ganglioside GD2, and anti-GD2 monoclonal antibodies (mAbs) caused suppression of cell growth and induced apoptosis of SCLC cells with single use. Here, enhancement of the cytotoxic effects of various anti-cancer drugs with an anti-GD2 mAb was demonstrated. The cytotoxicity of all six drugs examined was markedly enhanced, i.e. 2.4 - 7.8-fold increase of cell sensitivity in terms of IC(50). In particular, the combination of cisplatin (CDDP) with an anti-GD2 mAb resulted in prominent enhancement of cytotoxicity even in low - moderate GD2-expressing lines. The anti-GD2 mAb induced weak activation of c-Jun terminal kinase (JNK) in SCLC cells, and all anti-cancer drugs also induced its activation to various degrees. When CDDP and an anti-GD2 mAb were used together, significantly stronger JNK activation was observed corresponding to the cytotoxic effects, suggesting that synergistic phosphorylation of JNK with two reagents induced prominent apoptosis. The essential role of JNK in the induction of SCLC apoptosis with CDDP and anti-GD2 mAb was confirmed by experiments with a JNK inhibitor, curcumin. These results suggest that anti-GD2 mAbs would be very efficient in combination with anti-cancer drugs, both to achieve SCLC-specific cytotoxicity and to enhance its magnitude.

[Successful use of immunosuppressive agents for the treatment of progressive rheumatoid interstitial lung disease: a case report].

Interstitial pneumonia (IP) is sometimes a fatal complication of rheumatoid arthritis (RA). We describe a patient with progressive rheumatoid interstitial pneumonia, who responded to intravenous intermittent cyclophosphamide (IV-CY) and cyclosporine (CsA). A 62-year-old man with rheumatoid arthritis was admitted to this hospital because of dyspnea. Examinations on admission revealed that he had active RA with vasculitis and IP Initially, he responded to high-dose corticosteroid therapy. A lung biopsy performed after initial corticosteroid therapy revealed diffuse interstitial pneumonia with marked infiltrations of macrophages into the air spaces. On corticosteroid therapy with prednisolone 30 mg/day, the IP became exacerbated and was refractory to the current high-dose steroid treatment. He responded to intravenous cyclophosphamide, but his IP remained unstable. After CsA treatment was started, a clinical remission was obtained. In this case, CsA was the most effective agent tried. Clinical and pathological considerations led us to speculate that activated alveolar macrophages played a crucial role in the pathogenesis of steroid-resistant IP in this patient, and that the clinical remission induced by CsA may have been due to its inhibitory effect on alveolar macrophages.