Actin filament assembly in nonmuscle cells is regulated by the actin polymerization machinery, including the Arp2/3 complex and formins. However, little is known about the regulation of actin ...assembly in muscle cells, where straight actin filaments are organized into the contractile unit sarcomere. Here, we show that Fhod3, a myocardial formin that localizes to thin actin filaments in a striated pattern, regulates sarcomere organization in cardiomyocytes. RNA interference-mediated depletion of Fhod3 results in a marked reduction in filamentous actin and disruption of the sarcomeric structure. These defects are rescued by expression of wild-type Fhod3 but not by that of mutant proteins carrying amino acid substitution for conserved residues for actin assembly. These findings suggest that actin dynamics regulated by Fhod3 are critical for sarcomere organization in striated muscle cells.
Vaccination with irradiated granulocyte macrophage colony-stimulating factor (GM-CSF)-transduced autologous tumor cells (GVAX) has been shown to induce therapeutic antitumor immunity. However, its ...effectiveness is limited. We therefore attempted to improve the antitumor effect by identifying little-known key pathways in GM-CSF-sensitized dendritic cells (GM-DC) in tumor-draining lymph nodes (TDLN). We initially confirmed that syngeneic mice subcutaneously injected with poorly immunogenic Lewis lung carcinoma (LLC) cells transduced with Sendai virus encoding GM-CSF (LLC/SeV/GM) remarkably rejected the tumor growth. Using cDNA microarrays, we found that expression levels of type I interferon (IFN)-related genes, predominantly expressed in plasmacytoid DCs (pDC), were significantly upregulated in TDLN-derived GM-DCs and focused on pDCs. Indeed, mouse experiments demonstrated that the effective induction of GM-CSF-induced antitumor immunity observed in immunocompetent mice treated with LLC/SeV/GM cells was significantly attenuated when pDC-depleted or IFNα receptor knockout (IFNAR(-/-)) mice were used. Importantly, in both LLC and CT26 colon cancer-bearing mice, the combinational use of imiquimod with autologous GVAX therapy overcame the refractoriness to GVAX monotherapy accompanied by tolerability. Mechanistically, mice treated with the combined vaccination displayed increased expression levels of CD86, CD9, and Siglec-H, which correlate with an antitumor phenotype, in pDCs, but decreased the ratio of CD4(+)CD25(+)FoxP3(+) regulatory T cells in TDLNs. Collectively, these findings indicate that the additional use of imiquimod to activate pDCs with type I IFN production, as a positive regulator of T-cell priming, could enhance the immunologic antitumor effects of GVAX therapy, shedding promising light on the understanding and treatment of GM-CSF-based cancer immunotherapy.
Erythropoiesis is the process of proliferation, differentiation, and maturation of erythroid cells. Understanding these steps will help to elucidate the basis of specific diseases associated with ...abnormal production of red blood cells. In this study, we continued our efforts to identify genes involved in erythroid proliferation. Lentivirally transduced UT-7/Epo erythroleukemic cells expressing ribosomal protein L11 (RPL11) or retinol dehydrogenase 11 (RDH11) could proliferate in the absence of erythropoietin, and their cell-cycle profiles revealed G0 /G1 prolongation and low percentages of apoptosis. RPL11-expressing cells proliferated more rapidly than the RDH11-expressing cells. The antiapoptotic proteins BCL-XL and BCL-2 were expressed in both cell lines. Unlike the parental UT-7/Epo cells, the expression of hemoglobins (Hbs) in the transduced cells had switched from adult to fetal type. Several signal transduction pathways, including STAT5, were highly activated in transduced cells; furthermore, expression of the downstream target genes of STAT5, such as CCND1 , was upregulated in the transduced cells. Taken together, the data indicate that RPL11 and RDH11 accelerate erythroid cell proliferation by upregulating the STAT5 signaling pathway with phosphorylation of Lyn and cyclic AMP response element-binding protein (CREB).
BLT1 is a high-affinity receptor for leukotriene B4 (LTB4) that is a potent lipid chemoattractant for myeloid leukocytes. The role of LTB4/BLT1 axis in tumor immunology, including cytokine-based ...tumor vaccine, however, remains unknown. We here demonstrated that BLT1-deficient mice rejected subcutaneous tumor challenge of GM-CSF gene-transduced WEHI3B (WGM) leukemia cells (KO/WGM) and elicited robust antitumor responses against second tumor challenge with WEHI3B cells. During GM-CSF–induced tumor regression, the defective LTB4/BLT1 signaling significantly reduced tumor-infiltrating myeloid-derived suppressor cells, increased the maturation status of dendritic cells in tumor tissues, enhanced their CD4+ T-cell stimulation capacity and migration rate of dendritic cells that had phagocytosed tumor-associated antigens into tumor-draining lymph nodes, suggesting a positive impact on GM-CSF–sensitized innate immunity. Furthermore, KO/WGM mice displayed activated adaptive immunity by attenuating regulatory CD4+ T subsets and increasing numbers of Th17 and memory CD44hiCD4+ T subsets, both of which elicited superior antitumor effects as evidenced by adoptive cell transfer. In vivo depletion assays also revealed that CD4+ T cells were the main effectors of the persistent antitumor immunity. Our data collectively underscore a negative role of LTB4/BLT1 signaling in effective generation and maintenance of GM-CSF–induced antitumor memory CD4+ T cells.
Abstract
Development of a novel therapeutic modality targeting cancer stem cells (CSCs) holds great promise for the eventual eradication of cancer. It was demonstrated that CSCs shared antigenic ...similarities with embryonic stem (ES) cells and the vaccination using ES cells could generate antitumor immunity. However, the use of ES cells raises potential immunological and ethical issues. Recently, by the forced ectopic expression of defined transcription factors, autologous somatic cells were successfully reprogrammed to induced pluripotent stem (iPS) cells that closely resemble ESCs. We hypothesized that novel cell vaccines using mouse iPS cells genetically engineered to express the immunostimulatory cytokine of GM-CSF would cross-react CSC cells and induce long term antitumor immunity against poorly immunogenic syngeneic LLC mouse lung cancer cells. Our results of in vitro assays demonstrated that non-transmissible recombinant Sendai virus-mediated mouse GM-CSF gene transfer to iPSCs (iPS/GM-CSF) was effective to produce abundant GM-CSF in vitro and iPS/GM-CSF cells maintained their stemness in terms of morphology and antigenicity as evidenced by the expression of SSEA-1,Oct3/4 and alkaline phosphatase compared with unmodified iPS cells. Prophylactic iPSCs vaccine studies revealed that wild-type female mice subcutaneously vaccinated with irradiated iPS (ir.iPS) cells on weeks 1, 2, and 3 before the tumor challenge with LLC cells significantly suppressed the LLC tumor growth compared with untreated mice (p<0.05). Importantly, mice carrying pre-established LLC tumor treated with ir.iPS/GM-CSF cells showed significantly suppressed tumor growth compared with mice treated with ir.iPS/GFP cells (p<0.05). No serious adverse events were observed without any organ damages of liver and kidney. Furthermore, the antitumor effects observed in mice treated with ir.iPS/ GM- CSF cells were significantly abrogated when CD4+ T or CD8+ T cells were depleted, showing their capacity to incite T cells-mediated antitumor immunity. Lastly, we performed a cDNA microarray analysis to identify commonly upregulated genes of the putative CSCs-associated antigens as a target of iPS cells-based vaccines among LLC cells, iPS vaccine cells. Several sperm- or cell surface- specific antigens were predominantly expressed and shared between the 2 distinctive cell fractions.
Taken together, our results demonstrated that iPS cells-based vaccine could induce both prophylactic and therapeutic antitumor immunity in syngeneic mouse models, and suggested that this novel vaccine strategy may be a promising modality for cancer immunotherapy.
Citation Format: Hiroyuki Inoue, Ayumi Watanabe, Megumi Narusawa, Chika Sakamoto, Takafumi Hiramoto, Shohei Miyamoto, Makoto Inoue, Koichi Takayama, Mamoru Hasegawa, Yoichi Nakanishi, Tomoki Todo, Kenzaburo Tani. Therapeutic vaccination with GM-CSF gene-transduced iPS cells induces potent T cells-mediated antitumor immunity. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2814. doi:10.1158/1538-7445.AM2014-2814
Development of a novel therapeutic modality targeting cancer stem cells (CSCs) holds great promise for the eventual eradication of cancer. It was demonstrated that CSCs shared antigenic similarities ...with embryonic stem (ES) cells and the vaccination using ES cells could generate antitumor immunity. However, the use of ES cells raises potential immunological and ethical problematic issues. Recently, by the forced ectopic expression of defined transcription factors, autologous somatic cells were successfully reprogrammed into induced pluripotent stem (iPS) cells that closely resemble ESCs. We hypothesized that novel cell vaccines using mouse iPS cells genetically engineered to express the immunostimulatory cytokine of GM-CSF would cross-react CSC cells to induce antitumor immunity against poorly immunogenic syngeneic LLC mouse lung cancer cells, which would resolve such problematic issues. Our results of in vitro assays demonstrated that non-transmissible recombinant Sendai virus-mediated mouse GM-CSF gene transfer to iPSCs (iPS/GM-CSF) was effective to produce abundant GM-CSF in vitro and iPS/GM-CSF cells maintained their stemness in terms of morphology and antigenicity as evidenced by the expression of SSEA-1,Oct3/4 and alkaline phosphatase compared with unmodified iPS cells. Prophylactic iPSCs vaccine studies revealed that wild-type female mice subcutaneously vaccinated with irradiated iPS (ir.iPS) cells on weeks 1, 2, and 3 before the tumor challenge with LLC cells significantly suppressed the LLC tumor growth compared with untreated mice (p<0.05). Additionally, mice vaccinated with ir.iPS/GM-CSF cells significantly inhibited the tumor growth compared with mice treated with ir.iPS/GFP cells (p<0.05), showing that genetic manipulation of iPS cells with GM-CSF encoding gene potentiated the antitumor effect. Of note, no serious adverse events were observed with lack of liver and kidney dysfunctions as evidenced by biochemical analysis. Furthermore, therapeutic vaccinations with repeated ir.iPS/GM-CSF cells significantly inhibited the pre-established LLC tumor growth compared with untreated mice (p<0.05), with unaltered body weight. To address the effectors of the observed antitumor effects by ir.iPS/GM-CSF cells, we performed in vivo depletion experiments. The antitumor effects observed in mice treated with iPS/GM cells were significantly abrogated when CD4+ T cells- or CD8+ T cells were depleted, showing that iPS cells-based vaccines effectively generated T cells-mediated antitumor immunity. Lastly, we performed a cDNA microarray analysis to detect comparably expressed genes for the putative CSCs-associated antigens as a target of iPS cells-based vaccines on LLC cells, iPS cells, ir.iPS cells and ir.iPS/GM-CSF cells. Several sperm- or cell surface- specific antigens were predominantly expressed and shared between LLC cells and these iPS cell fractions, indicating that LLC cells and iPS cells may share CSCs-associated antigens. In conclusion, our results collectively demonstrate iPS cells-based vaccine can induce both prophylactic and therapeutic antitumor immunity in syngeneic mouse models, and indicate that this novel vaccine strategy may eliminate CSCs that shared antigenic similarities as a promising modality for cancer immunotherapy.
No relevant conflicts of interest to declare.
Vaccination with irradiated granulocyte macrophage-colony stimulating factor (GM-CSF)-transduced autologous tumor cells (GVAX) has been shown to induce therapeutic antitumor immunity presumably ...through the activated maturation of myeloid dendritic cells (DCs). However, its effectiveness is limited, and little is known about the biological properties related to GM-CSF-sensitized DCs (GM-DCs) in tumor-draining lymph nodes (TDLNs). We therefore attempted to enhance the antitumor effect of GVAX therapy by identifying the key pathways in GM-DCs in TDLNs. We initially confirmed that syngeneic mice subcutaneously (s.c.) injected with poorly immunogenic Lewis lung carcinoma (LLC) cells transduced with Sendai virus encoding GM-CSF (LLC/SeV/GM) significantly rejected the tumor growth. Using cDNA microarrays, we obtained a large number of gene expression data from CD86+ GM-DCs and control DCs in TDLNs, and found that the expressions of type I interferons (IFNs)-related genes, including IRF7 and TLR7, known to be abundantly expressed in plasmacytoid DCs (pDCs), were upregulated in GM-DCs. Indeed, to further activate pDCs, mice s.c. challenged with LLC/SeV/GM cells in combination with a TLR7 ligand, imiquimod, but not a TLR4 ligand, LPS, significantly suppressed the tumor growth compared with mice treated with LLC/SeV/GM cells alone. In contrast, pDCs-depleted mice challenged with LLC/SeV/GM cells facilitated the tumor growth, strongly suggesting that pDCs are essential immune subpopulation in exerting GM-CSF-initiated antitumor effects.
Furthermore, the additional use of imiquimod overcame the refractoriness of therapeutic vaccines with irradiated LLC/SeV/GM cells in mice with pre-established LLC tumors. Moreover, similar improvement of GVAX therapy was also observed in a mouse model of CT26 colorectal cancer. Mechanistically, mice treated with the combined vaccination displayed increased cell ratio of PDCA-1+ pDCs and expression levels of CD86, CD9, which correlate with an antitumor phonetype, and Siglec-H, which promote CD8+ T cell proliferation, in TDLNs. Indeed, allogeneic MLR test showed that bone marrow-derived pDCs matured by in vitro culture with GM-CSF plus imiquimod elicited a superior capacity of CD8+ T cell proliferation compared with those with GM-CSF only. On the other hand, the ratio of CD4+CD25+FoxP3+ regulatory T cells (Tregs) was decreased in TDLNs mice treated with the combined vaccination.
These findings collectively elucidated that pDCs play positive roles in GM-CSF-initiated antitumor immunity and that further activation of pDC by imiquimod targeting TLR7-IRF7 dependent type I IFNs pathway enhance the antitumor effects of GM-CSF-based tumor vaccination.
No relevant conflicts of interest to declare.
Abstract 3246
GM-CSF (Granulocyte macrophage-colony stimulating factor) gene-transduced leukemia cell vaccine has been reported to be promising approach to enhance antitumor immune response in ...leukemia patients. The enhancement of this specific antitumor immunity is considered to be clinically beneficial to maintain complete remission for long time after chemotherapy without stem cell transplantion. Leukotriene B4 (LTB4) is an extremely potent lipid inflammatory mediator derived from membrane phospholipids, known to recruit and activate leukocytes including neutrophils, mediated by the same class of receptors, the GPCR (G-protein coupled receptor) superfamily, BLT1 and BLT2. So far, the role of LTB4 in tumor immunology is not well known. Previously, we demonstrated that the GM-CSF gene transduction into murine monocytic leukemia cell line of WEHI3B (WGM) eliminated the tumorigenicity in subcutaneous challenge model using wild type (WT) BALB/c mice. At day 50 after the challenge, we rechallenged WEHI3B cells into the opposite flank of both WT and BLT1-KO mice which had rejected WGM cells (WT/WGM, KO/WGM, respectively). Intriguingly, more KO/WGM mice re-rejected the rechallenged WEHI3B cells and showed significantly prolonged survival compared with WT/WGM mice.
To clarify this unique mechanism of the long-lasting antitumor effects observed in KO/WGM mice, we performed following immunological assays. Our in vivo immune cell depletion assays (NK, CD4+ T, CD8+ T cell) showed that KO/WGM mice treated with anti-CD4 antibody displayed rather enhanced tumor growth compared with WT/WGM mice. Thus, we next compared the rates of different subsets of memory T cells, so called memory stem T cell subsets (TSCM), central memory T cell subsets (TCM) and effector memory T cell subsets (TEM) in TDLNs (Tumor draining lymph nodes) between WT/WGM mice and KO/WGM mice at the day 46 after tumor challenge. Expectedly, results showed that TDLNs harvested from KO/WGM mice exhibited higher subset ratios of CD44+CD62L+ (TCM) cell to CD4+ T cells as well as CD44+CD62L− or (TEM) cell to CD4+ T cells than those from WT/WGM mice. In the case of use of CD122 for memory marker, similar results were obtained. Of note, TDLNs from KO/WGM mice exhibited increased ratios of CD44+CD122+CD62L− (TSCM) cell. In addition, the cell numbers of immunosuppressive CD3+ CD4+ PD-1+, CD3+ CD4+ GITR+ and CD3+ CD4+ CTLA4+ cells were reduced in both TDLNs and spleen derived from KO/WGM mice compared with those from WT/WGM mice. Furthermore, our results of CBA (Cytometric Bead Array) assay using splenocytes harvested from day 2 to day 15 showed that the Th2 cytokine production level of IL-4 and IL-5 from splenocyte harvested from KO/WGM mice were higher than those from WT/WGM mice. In regard to IL-2 and IFN-g (Th1 cytokine), the production level at both day 7 and day10 from splenocytes harvested from KO/WGM mice were also higher than those from WT/WGM mice, implicating that loss of LTB4/BLT1 signaling promotes systemic activation of both tumor antigens specific Th1 and Th2 CD4+ T cell subsets. Finally, our flow cytometric analyses demonstrated that exogenously GM-CSF-driven upregulated MFI of CD40+, CD80+ and CD86+ DCs in TDLNs from KO/WGM mice were further significantly increased compared with those from WT/WGM mice (p<0.05), and more numerous number of CD86+ DCs that had phagocytosed TAAs of WEHI3B cells was detected in TDLNs harvested from KO/WGM mice than that from WT/WGM mice.
In conclusion, we for the first time demonstrate that loss of LTB4/BLT1 axis can sustain long-lasting memory CD4+ T cell-dependent antitumor immunity against syngeneic tumor cells long after GM-CSF triggering tumor rejection, allowing us to expect the possibility that the strategy of blocking LTB4/BLT1signaling may be useful to maintain antitumor immunity induced by GM-CSF gene-transduced leukemia cell vaccine in clinical settings.
No relevant conflicts of interest to declare.
Because blood cells can be obtained with relatively easy and safe procedure, they have been routinely used for transfusion and transplantation purposes. And they are now considered as attractive cell ...sources for developing new gene therapies including cancer therapy using various immune cells, and regenerative therapy using hematopoietic stem cells or induced pluripotent stem cells (iPS) cells. For example, chimeric antigen receptor modified autologous T cells have been considered as effective therapy for various cancers. And iPS cells have been easily established from peripheral T cells for the purpose of treating various diseases. However, in spite of these possibilities, the development of the safer and more efficient genetic modification methods of hematopoietic cells is imminent. In this study, we developed the novel measles viral (MV) vector which enables us to transduce multiple genes into immune cells. The wild type measles virus is one of the aerosol-transmitted viruses and has strong infectious capacity to immune cells, and epithelial cells via signaling lymphocyte activation molecule (SLAM) or nectin-4. First, we modified the wild type measles virus genome to non-transmissible and non-lytic, and equipped with the ability of transducing multiple genes, at most six genes, into target cells. Briefly, the intrinsically non-segmented wild type virus genome was divided into two segments and point mutations were introduced into the virus genes encoding hemagglutinin and the matrix protein. Moreover, as the fusion protein gene was removed from the virus genome, the virus could not replicate in neighbor cells. We examined the gene transduction efficiency of the gene modified measles virus (H8-Fd-MV vector) into hematopoietic cells. We first constructed the H8-Fd-MV vector with GFP gene and transduced into hematopoietic progenitor cells and immune cells from human cord blood and peripheral blood. We observed that almost all of HPCs from cord blood (99.7% in floating cells expressed CD34), T cells (99.9% in CD3+ cells), and B cells (98.2% in CD19+ cells) from peripheral blood expressed GFP at two days after the transduction. Especially, to express GFP gene in human peripheral T cells, it was not necessary to pre-stimulate them with CD3/CD28 beads (99.6% in stimulating T cells (72.9% in SLAM+ cells) v.s. 82.6 % in non-stimulating cells (37.4% in SLAM+ cells)). T cells from cord blood showed almost all naïve phenotype (CD4+ cells: 93.2±1.8% in CD45RA+CCR7+ cells, 1.7±1.1% in CD45RA+CCR7- cells; CD8+ cells: 41.6±5.7% in CD45RA+CCR7+ cells, and 41.9±11.0% in CD8+CD45RA+CCR7- cells) and T cells transduced by MV vector expressed GFP more (CD4+ cells: 80.3±13.7%, and CD8+ cells: 82.5±8.5%) than those transduced by Sendai viral vector (CD4+ cells: 15.5±0.7%, and CD8+ cells: 17.4±5.4%). These data suggested that H8-Fd-MV vector could transduce GFP gene efficiently into various T cell lineages including naïve T cells, which had been difficult to be transduced with classical gene transduction methods. We next generated H8-Fd-MV vector for expressing 6 genes (OCT4, SOX2, KLF4, L-MYC, PIN1, and GFP) and transduced into stimulated T cells. After 3 days, GFP+ T cells expressed all of these 6 genes. We also detected that more than 50% of the stimulated T cells with IL-2 expressed GFP at 14 days after the transduction. After 27 days from transfection, embryonic stem cell (ES cell)-like colonies were picked up and analyzed the character. These cells showed ES cell morphology over 20 times passages and expressed pluripotent marker (NANOG, OCT4, Tra-1-60, Tra-1-81). We also found T cell receptor rearrangements in these cells. Embryoid bodies from these cells expressed three germ line markers in vitro. We next examined the hematopoietic differentiation of these cells using coculture system with murine embryonic aorta-gonad-mesonephros region-derived stromal cell line (AGM-3 cells). The co-cultured cells harvested at day 12 expressed CD34 and CD45. These data suggested that we established iPS cells from terminal differentiated T cells using H8-Fd-MV vector for expressing reprogramming factor.
These results indicated that multiple genes were expressed efficiently in immune cells using H8-Fd-MV vector. Highly efficient transduction ability of MV vector for T cells would enable us to develop new gene therapy targeting cancer using gene modified T cells as well as organ regeneration using iPS cells.
No relevant conflicts of interest to declare.
Abstract 1477
Poster Board I-500
Human embryonic stem (ES) cells differentiate into three lineages in vitro and in vivo as mouse ES cells. They are therefore highly promising source of various ...cells/tissues in the regenerative medicine. The current protocols, however, remain to be optimized for the induction of the cells/tissues required. We have recently reported that the lentiviral transduction of TAL1/SCL gene to ES cells derived from the common marmoset, a small nonhuman primate, enables efficient differentiation into hematopoietic progenitor cells even in the absence of stromal cells (Kurita et al. Stem Cells.24:2014-22, 2006). Such culture condition without any stromal cells is considered to facilitate clinical application of ES cell-derived cell/tissues therapy in the regenerative medicine. The present study addressed whether the strategy is also effective in human ES cells. First, we determined optimal culture conditions to induce multilineage hematopoietic differentiation in a human ES cell line, khES-1, kindly provided by Dr. Nakatsuji, Kyoto University, Japan, as assessed by the expression of Brachyury, Flk1 and CD34. We found that the addition of BMP4 and VEGF augmented hematopoietic differentiation of embryoid bodies, and determined optimal concentrations of the cytokines. We established four human ES cell lines stably expressing TAL1/SCL gene by lentiviral transduction. The TAL1/SCL transduction further increased the hematopoietic differentiation under the optimal culture condition as assessed by the expression of CD34, CD235a and CD133. We also observed increased number of hematopoietic progenitor cells derived from two of the TAL1/SCL expressing human ES cell lines by colony-forming assays. Hematopoietic differentiation of the TAL1/SCL expressing ES cells in vivo is also being investigated by transplantation into irradiated immune deficient mice. These results suggest that the combination of optimal culture conditions and lentiviral TAL1/SCL gene transduction is a highly effective strategy to obtain hematopoietic stem cells from human ES cells in the absence of any stromal cells.
No relevant conflicts of interest to declare.
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