A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy.

Clinical successes demonstrated by chimeric antigen receptor T-cell immunotherapy have facilitated further development of T-cell immunotherapy against wide variety of diseases. One approach is the development of "off-the-shelf" T-cell sources. Technologies to generate T-cells from pluripotent stem cells (PSCs) may offer platforms to produce "off-the-shelf" and synthetic allogeneic T-cells. However, low differentiation efficiency and poor scalability of current methods may compromise their utilities. Here we show improved differentiation efficiency of T-cells from induced PSCs (iPSCs) derived from an antigen-specific cytotoxic T-cell clone, or from T-cell receptor (TCR)-transduced iPSCs, as starting materials. We additionally describe feeder-free differentiation culture systems that span from iPSC maintenance to T-cell proliferation phases, enabling large-scale regenerated T-cell production. Moreover, simultaneous addition of SDF1α and a p38 inhibitor during T-cell differentiation enhances T-cell commitment. The regenerated T-cells show TCR-dependent functions in vitro and are capable of in vivo anti-tumor activity. This system provides a platform to generate a large number of regenerated T-cells for clinical application and investigate human T-cell differentiation and biology.

Enhancing T Cell Receptor Stability in Rejuvenated iPSC-Derived T Cells Improves Their Use in Cancer Immunotherapy.

Limited T cell availability and proliferative exhaustion present major barriers to successful T cell-based immunotherapies and may potentially be overcome through the use of "rejuvenated" induced pluripotent stem cells derived from antigen-specific T cells (T-iPSCs). However, strict antigen specificity is essential for safe and efficient T cell immunotherapy. Here, we report that CD8αß T cells from human T-iPSCs lose their antigen specificity through additional rearrangement of the T cell receptor (TCR) α chain gene during the CD4/CD8 double positive stage of in vitro differentiation. CRISPR knockout of a recombinase gene in the T-iPSCs prevented this additional TCR rearrangement. Moreover, when CD8αß T cells were differentiated from monocyte-derived iPSCs that were transduced with an antigen-specific TCR, they showed monoclonal expression of the transduced TCR. TCR-stabilized, regenerated CD8αß T cells effectively inhibit tumor growth in xenograft cancer models. These approaches could contribute to safe and effective regenerative T cell immunotherapies.

Analysis of circulating tumor cells derived from advanced gastric cancer.

Studies in circulating tumor cells (CTCs) have proceeded to be accepted as prognostic markers in several types of cancers. But they are still limited because many are mainly from enumeration of CTCs. Here, we tried to evaluate the tumorigenicity of CTCs from advanced gastric cancer patients (n = 42). Peripheral blood mononuclear cells (PBMC) from the patients were separated into CD45 negative and positive fractions and both were subcutaneously injected into immunodeficient mice. Within 5 months nine tumor-like-structures from six patients but not from healthy volunteers were established. They were durable for passages and all had been confirmed human origin. Eight of the nine tumor-like-structures were from nonauthorized CTC containing cells expressing CD45 and B-cell markers. On the contrary, one of them was developed from CD45(-) PBMC fraction of a patient with bone marrow metastasis reflecting authorized CTCs. Histopathology showed common features with that of original gastric tumor. The cells isolated from the tumor-like-structure expressed EpCAM and CEA further supporting they were from the original tumor. Moreover the cells were CD44 positive to varying degree and a limiting dilution study showed that the CD44(+/high) fraction had tumorigenicity. The CD44 was dominantly in the form of CD44 variant 8-10. The CD44(+/high) cells had higher expression of the glutamate/cysteine transporter xCT compared with the CD44(-/low) cells. Our results showed the existence of tumor-initiating cells in blood of advanced gastric cancer patients and they could be a therapeutic target and prospective tool for further investigations.

Use of Sato's curved laryngoscope and an insulated-tip knife for endoscopic incisional therapy of esophageal web.

We experienced two cases of esophageal web accompanying severe stricture that were treated by endoscopic incisions with an insulated-tip knife (IT-knife). With attention paid to the mucosa at the stricture, the lesion was incised with an IT-knife without complications. Sato's curved laryngoscope was used even in cervical esophageal lesions and an excellent field was secured.

HER2 and HER3 cooperatively regulate cancer cell growth and determine sensitivity to the novel investigational EGFR/HER2 kinase inhibitor TAK-285.

The human epidermal growth factor receptor (HER) family plays a major role in cancer cell proliferation. Overexpression of these receptors occurs in various cancers, including breast cancer, and correlates with shorter time to relapse and lower overall survival. We recently reported that TAK-285, an orally bioavailable small molecule inhibitor of HER kinases, is not a p-glycoprotein substrate and penetrates the blood-brain barrier, suggesting favorable activity for the treatment of brain metastases. To identify the determinants of sensitivity to TAK-285, we examined the relationship between the IC50 values of TAK-285 for cell growth inhibition and the expression of candidate genes that are involved in the HER family signaling pathway and trastuzumab resistance in a panel of human breast cancer cell lines, other types of cancer cells, and non-transformed cells in vitro. These analyses showed an inverse correlation between sensitivity to TAK-285 (IC50 values) and HER2 or HER3 expression. HER3 was highly phosphorylated in TAK-285-sensitive cells, where TAK-285 treatment reduced HER3 phosphorylation level. Because HER3 does not possess kinase activity and a selective inhibitor of HER2 but not of an epidermal growth factor receptor reduced the phospho-HER3 level, HER3 was suggested to be trans-phosphorylated by HER2. HER3 knockdown using small interfering RNA (siRNA) inhibited cancer cell growth in TAK-285-sensitive cells but not in TAK-285-insensitive cells. These results suggest that HER2 and HER3 mainly regulate cancer cell growth in TAK-285-sensitive cells and that phospho-HER3 could be used as a potential molecular marker to select patients most likely to respond to TAK-285.

Antitumor Activity of TAK-285, an Investigational, Non-Pgp Substrate HER2/EGFR Kinase Inhibitor, in Cultured Tumor Cells, Mouse and Rat Xenograft Tumors, and in an HER2-Positive Brain Metastasis Model.

Breast cancer therapy has improved following the development of drugs with specific molecular targets, exemplified by inhibitors of human epidermal growth factor receptor-2 (HER2) or epidermal growth factor receptor (EGFR) such as trastuzumab and lapatinib. However, these drugs have little effect on brain metastasis due to the combined effects of poor penetration of the blood-brain barrier and their removal from the central nervous system (CNS) by the p-glycoprotein (Pgp) drug efflux pump. We investigated the effects of TAK-285, a novel, investigational, dual EGFR/HER2 inhibitor that has been shown to penetrate the CNS and has comparable inhibitory efficacy to lapatinib which is a known Pgp substrate. Tested against a panel of 96 kinases, TAK-285 showed specificity for inhibition of HER family kinases. Unlike lapatinib, TAK-285 is not a substrate for Pgp efflux. In mouse and rat xenograft tumor models, TAK-285 showed antitumor activity against cancers that expressed HER2 or EGFR. TAK-285 was as effective as lapatinib in antitumor activity in a mouse subcutaneous BT-474 breast cancer xenograft model. TAK-285 was examined in a model of breast cancer brain metastasis using direct intracranial injection of BT-474-derived luciferase-expressing cells and showed greater inhibition of brain tumor growth compared to animals treated with lapatinib. Our studies suggest that investigational drugs such as TAK-285 that have strong antitumor activity and are not Pgp substrates may be useful in the development of agents with the potential to treat brain metastases.

Regulation of autophagy and its associated cell death by "sphingolipid rheostat": reciprocal role of ceramide and sphingosine 1-phosphate in the mammalian target of rapamycin pathway.

The role of "sphingolipid rheostat" by ceramide and sphingosine 1-phosphate (S1P) in the regulation of autophagy remains unclear. In human leukemia HL-60 cells, amino acid deprivation (AA(-)) caused autophagy with an increase in acid sphingomyleinase (SMase) activity and ceramide, which serves as an autophagy inducing lipid. Knockdown of acid SMase significantly suppressed the autophagy induction. S1P treatment counteracted autophagy induction by AA(-) or C(2)-ceramide. AA(-) treatment promoted mammalian target of rapamycin (mTOR) dephosphorylation/inactivation, inducing autophagy. S1P treatment suppressed mTOR inactivation and autophagy induction by AA(-). S1P exerts biological actions via cell surface receptors, and S1P(3) among five S1P receptors was predominantly expressed in HL-60 cells. We evaluated the involvement of S1P(3) in suppressing autophagy induction. S1P treatment of CHO cells had no effects on mTOR inactivation and autophagy induction by AA(-) or C(2)-ceramide. Whereas S1P treatment of S1P(3) overexpressing CHO cells resulted in activation of the mTOR pathway, preventing cells from undergoing autophagy induced by AA(-) or C(2)-ceramide. These results indicate that S1P-S1P(3) plays a role in counteracting ceramide signals that mediate mTOR-controlled autophagy. In addition, we evaluated the involvement of ceramide-activated protein phosphatases (CAPPs) in ceramide-dependent inactivation of the mTOR pathway. Inhibition of CAPP by okadaic acid in AA(-)- or C(2)-ceramide-treated cells suppressed dephosphorylation/inactivation of mTOR, autophagy induction, and autophagy-associated cell death, indicating a novel role of ceramide-CAPPs in autophagy induction. Moreover, S1P(3) engagement by S1P counteracted cell death. Taken together, these results indicated that sphingolipid rheostat in ceramide-CAPPs and S1P-S1P(3) signaling modulates autophagy and its associated cell death through regulation of the mTOR pathway.

Regulation of cell migration by sphingomyelin synthases: sphingomyelin in lipid rafts decreases responsiveness to signaling by the CXCL12/CXCR4 pathway.

Sphingomyelin synthase (SMS) catalyzes the formation of sphingomyelin, a major component of the plasma membrane and lipid rafts. To investigate the role of SMS in cell signaling and migration induced by binding of the chemokine CXCL12 to CXCR4, we used mouse embryonic fibroblasts deficient in SMS1 and/or SMS2 and examined the effects of SMS deficiency on cell migration. SMS deficiency promoted cell migration through a CXCL12/CXCR4-dependent signaling pathway involving extracellular signal-regulated kinase (ERK) activation. In addition, SMS1/SMS2 double-knockout cells had heightened sensitivity to CXCL12, which was significantly suppressed upon transfection with the SMS1 or SMS2 gene or when they were treated with exogenous sphingomyelin but not when they were treated with the SMS substrate ceramide. Notably, SMS deficiency facilitated relocalization of CXCR4 to lipid rafts, which form platforms for the regulation and transduction of receptor-mediated signaling. Furthermore, we found that SMS deficiency potentiated CXCR4 dimerization, which is required for signal transduction. This dimerization was significantly repressed by sphingomyelin treatment. Collectively, our data indicate that SMS-derived sphingomyelin lowers responsiveness to CXCL12, thereby reducing migration induced by this chemokine. Our findings provide the first direct evidence for an involvement of SMS-generated sphingomyelin in the regulation of cell migration.

Sphingomyelin synthase 1-generated sphingomyelin plays an important role in transferrin trafficking and cell proliferation.

Transferrin (Tf) endocytosis and recycling are essential for iron uptake and the regulation of cell proliferation. Tf and Tf receptor (TfR) complexes are internalized via clathrin-coated pits composed of a variety of proteins and lipids and pass through early endosomes to recycling endosomes. We investigated the role of sphingomyelin (SM) synthases (SMS1 and SMS2) in clathrin-dependent trafficking of Tf and cell proliferation. We employed SM-deficient lymphoma cells that lacked SMSs and that failed to proliferate in response to Tf. Transfection of SMS1, but not SMS2, enabled these cells to incorporate SM into the plasma membrane, restoring Tf-mediated proliferation. SM-deficient cells showed a significant reduction in clathrin-dependent Tf uptake compared with the parental SM-producing cells. Both SMS1 gene transfection and exogenous short-chain SM treatment increased clathrin-dependent Tf uptake in SM-deficient cells, with the Tf being subsequently sorted to Rab11-positive recycling endosomes. We observed trafficking of the internalized Tf to late/endolysosomal compartments, and this was not dependent on the clathrin pathway in SM-deficient cells. Thus, SMS1-mediated SM synthesis directs Tf-TfR to undergo clathrin-dependent endocytosis and recycling, promoting the proliferation of lymphoma cells.

Effect of dibutyryl cyclic adenosine monophosphate on the gene expression of plasminogen activator inhibitor-1 and tissue factor in adipocytes.

INTRODUCTION: Hypertrophic adipocytes in obese states express the elevated levels of plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF). An increase in the intracellular concentration of cyclic adenosine monophosphate (cAMP) promotes triglyceride hydrolysis and may improve dysregulation of adipocyte metabolism. Here, we investigate the effect of dibutyryl-cAMP (a phosphodiesterase-resistant analog of cAMP) on the gene expression of PAI-1 and TF in adipocytes. MATERIALS AND METHODS: Differentiated 3T3-L1 adipocytes were treated with dibutyryl-cAMP and agents that would be expected to elevate intracellular cAMP, including cilostazol (a phosphodiesterase inhibitor with anti-platelet and vasodilatory properties), isoproterenol (a beta adrenergic agonist) and forskolin (an adenylyl cyclase activator). The levels of PAI-1 and TF mRNAs were measured using real-time quantitative reverse transcription-PCR. RESULTS AND CONCLUSIONS: The treatment of adipocytes with dibutyryl-cAMP resulted in the inhibition of both lipid accumulation and TF gene expression. However, PAI-1 gene expression was slightly but significantly increased by dibutyryl-cAMP. On the other hand, cilostazol inhibited the expression of PAI-1 without affecting lipid accumulation. When the adipocytes were treated with cilostazol in combination with isoproterenol or forskolin, the inhibitory effect of cilostazol on PAI-1 gene expression was counteracted, thus suggesting that inhibition by cilostazol may not be the result of intracellular cAMP accumulation by phosphodiesterase inhibition. These results suggest the implication of cAMP in regulation of the gene expression of TF and PAI-1 in adipocytes. Our findings will serve as a useful basis for further research in therapy for obesity-associated thrombosis.

Radionuclide imaging study of long-term pulmonary function after lobectomy in children with congenital cystic lung disease.

PURPOSE: We evaluated the long-term pulmonary function after lobectomy for congenital cystic lung disease, in both infants and children, using radionuclide imaging (RI). METHODS: We performed a retrospective review of 93 patients who underwent resection of cystic lung lesions between 1974 and 2001. The results of postoperative lung volume/perfusion scintigraphy at 1 (n = 64), 5 (n = 32), and 10 years (n = 18) after surgery (V1, 5, 10/Q1, 5, 10) and mean transit time (MTT-a marker for air-trapping) at 1, 5, and 10 years after surgery (MTT1, 5, 10) were compared with respect to age at operation, preoperative infection, underlying disease, and type of surgery. RESULTS: Patients who were younger than 1 year at the time of surgery showed a significantly lower MTT5 (1.09 +/- 0.08) and MTT10 (1.15 +/- 0.11) than patients who were older than 1 year at the time of surgery (MTT5, 1.49 +/- 0.67; MTT10, 1.54 +/- 0.33). The noninfected group had significantly higher Q10 and lower MTT10 values (P < .05) compared to the infected group. No significant differences were observed between patients with single lobe vs multiple lobe resection. CONCLUSIONS: The optimal age for surgery in patients with congenital cystic lung disease appears to be less than 1 year.

Modified slide tracheoplasty for congenital tracheal stenosis.

Slide tracheoplasty has become a standard procedure to treat long-segment congenital tracheal stenosis because it is a reasonable and simple technique. Slide tracheoplasty does not affect the carina during long-segment congenital tracheal stenosis management, and thus lesions of the carina, such as stenosis and tracheobronchomalacia can become important causes of extubation failure after surgery. In this manusript, we describe the effectiveness of our modified slide tracheoplasty, which includes reconstruction of the carina. We have performed this technique on three patients, all of whom were extubated without developing any respiratory symptoms.

Immunotherapy of ovarian cancer with cell wall skeleton of Mycobacterium bovis Bacillus Calmette-Guérin: effect of lymphadenectomy.

The significance of lymphadenectomy in surgery for various kinds of cancer has been widely debated, particularly in the gynecological field. The cell wall skeleton of Mycobacterium bovis Bacillus Calmette-Guérin (BCG-CWS) has been used as an effective adjuvant for immunotherapy of a variety of cancer patients. Here we tested the immunological importance of lymph nodes in treatment of ovarian cancer patients with BCG-CWS. After surgical removal of tumors, 73 ovarian cancer patients were intracutaneously inoculated with BCG-CWS in the antigen-unloaded state in the upper arm at 4-week intervals at a dosage of 2-200 microg. Significant correlation of lymphadenectomy and reduced survival of patients was observed (stages I, II, III, IV; hazard ratio 2.38, 95% confidence interval 1.02-5.12, Cox regression model). Lymphadenectomy also compromised with induction of interferon-gamma. In view of the importance of the role of lymph nodes in stimulation of Toll-like receptors by BCG-CWS, it is suggested that lymph nodes should be kept as much as possible to preserve the patient's immunity against cancer. Application of these results to surgery for other cancers should be considered.

Self-interaction of soluble and surface-bound beta2-glycoprotein I and its enhancement by lupus anticoagulants.

Antiphospholipid antibodies found in antiphospholipid syndrome are autoantibodies to phospholipid-binding proteins, such as beta2-glycoprotein I (beta2GPI). We have previously reported that among these antibodies, the so-called lupus anticoagulants (LAs) augment beta2GPI binding to the phospholipid membrane surface, which is associated with the pathological action of LAs. However, the molecular mechanisms underlying this augmentation are uncertain. Here we show that beta2GPI, which is monomeric in solution, self-interacts at the interface of soluble and surface-bound molecules. In addition, this self-interaction is enhanced by LA-positive, but not LA-negative, anti-beta2GPI monoclonal antibodies. This study suggests that beta2GPI self-interaction upon surface binding could be involved in the LA-induced potentiation of beta2GPI binding to the phospholipid surface.

A redundancy-based measure of dissimilarity among probability distributions for hierarchical clustering criteria.

We introduce novel dissimilarity into a probabilistic clustering task to properly measure dissimilarity among multiple clusters when each cluster is characterized by a subpopulation in the mixture model. This measure of dissimilarity is called redundancy-based dissimilarity among probability distributions. From aspects of both source coding and a statistical hypothesis test, we shed light on several of the theoretical reasons for the redundancy-based dissimilarity among probability distributions being a reasonable measure of dissimilarity among clusters. We also elucidate a principle in common for the measures of redundancy-based dissimilarity and Ward's method in terms of hierarchical clustering criteria. Moreover, we show several related theorems that are significant for clustering tasks. In the experiments, properties of the measure of redundancy-based dissimilarity are examined in comparison with several other measures.

A strategy for efficient cross-presentation of CTL-epitope peptides leading to enhanced induction of in vivo tumor immunity.

The activation of antitumor cytotoxic T-lymphocytes (CTLs) depends on how efficiently the relevant tumor antigen peptides are delivered into the major histocompatibility complex (MHC) class I presentation pathway in antigen presenting cells (APCs). An elegant approach to promote the peptide-MHC class I association has been described for enhanced peptide transportation into the endoplasmic reticulum (ER) by adding an ER insertion signal sequence (Eriss). Nevertheless, this approach does not appear potent enough to induce in vivo tumor protective immunity. Herein, we present a novel peptide-vaccine strategy based on the combined utilization of Eriss and fusogenic liposomes (FLs) capable of directly introducing encapsulated CTL-epitope peptides into the MHC class I pathway of APCs. APCs pulsed with free peptides, FL-encapsulated peptides, or FL-encapsulated Eriss-conjugated peptides exhibited comparable levels of antigen-presenting activity at early phases after pulsing. Interestingly, whereas in the first two methods the APC ability began to decline 40 to 60 h after pulsing, FL-encapsulated Eriss(+) peptides allowed APCs to retain peptide-presentation activity for at least 140 h. This advantage of FL-encapsulated Eriss(+) peptides correlated with the induction of more potent antitumor immunity compared with soluble Eriss(+) or Eriss(-) peptides or FL-encapsulated Eriss(-) peptides when they were administered in vivo. Thus, Eriss-conjugated CTL-epitope peptides encapsulated in FLs provide a highly efficient tumor-vaccine to enhance the induction of in vivo tumor immunity.

Vaccine efficacy of fusogenic liposomes containing tumor cell-lysate against murine B16BL6 melanoma.

Recent advances in tumor immunology have facilitated the development of cancer immunotherapy targeting tumor-associated antigens (TAAs). However, because TAAs were identified in only a few types of human cancer, novel vaccine strategies that utilize tumor cell-lysate (TCL), including unidentified TAAs as an antigen source, are needed. Herein, we describe the utility of fusogenic liposomes (FLs) as TCL-delivery carriers for both ex vivo dendritic cell-based vaccination and in vivo direct immunization in the murine B16BL6 melanoma model. As a result, both in vivo direct immunization and ex vivo immunization induced anti-B16 melanoma prophylactic effects. Ex vivo dendritic cell (DC)-mediated vaccination strategy exert more potent anti-tumor effect than direct immunization. Our results suggest that this flexible system is a promising approach for the development of versatile cancer immunotherapy regimes.

Analysis of treatment outcome for children with recurrent or metastatic hepatoblastoma.

For better total survival rate of children with hepatoblastoma, the therapeutic strategy for recurrent or metastatic hepatoblastoma should be improved. From 1991 to 1999, 134 cases of hepatoblastoma were treated by surgery and combination chemotherapy of cisplatin (CDDP) and THP-Adriamycin (THP-ADR) based on the JPLT-1 protocol. In 114 non-metastatic cases, 90 primary liver tumors were resected completely by partial hepatectomy, but 12 recurrences were observed in the liver (4 cases) and the lungs (8 cases). Distant metastases on the diagnosis were observed in 20 cases. The treatment outcome of these 12 recurrent and 20 metastatic tumors was analyzed. In four recurrent liver tumors, surgical resection was performed in all four cases, and all the patients were alive and well. In eight recurrent lung tumors, surgical resection was performed completely in six cases with unilateral lung disease, and five of the six patients were alive and well. In stage IV tumors, the survival rate of the patients having primary tumors within two hepatic sections was significantly higher than that of the patients having primary tumors over three hepatic sections. Active surgical intervention to lung metastases and a more intensive chemotherapy to facilitate complete resection of primary hepatic tumor could improve survival rate of children with refractory hepatoblastoma.