EXTH-15. INCREASING THE TRAFFICKING OF DENDRITIC CELLS VIA CXC CHEMOKINE SIGNALING PATHWAY LEADS TO IMPROVED ANTI-TUMOR EFFICACY OF DENDRITIC CELL VACCINES

Abstract Dendritic cell (DC) vaccine efficacy is directly related to the efficiency of DC migration to the lymph node after delivery to the patient. In this research we discovered increasing cell migration by utilizing sarcosine improved anti-tumor efficacy. We hypothesized that sarcosine induced cell migration was due to chemokine or cytokine signaling. METHODS To generate DC vaccines, DCs were harvested from bone marrow of wild type C57BL/6 mice and electroporated with OVA-mRNA. Human DCs were isolated from PBMCs. DCs were treated with sarcosine at 20mM. OT-I T cells were isolated from transgenic mice and injected intravenously into B16F10-OVA tumor bearing mice. Following T cell transfer, DC vaccines were injected intradermal. In vitro migration was analyzed via transwell migration assay. In vivo migration was evaluated by flow-cytometry and immunofluorescence microscopy. Gene expression in RNA was investigated in DCs via RT-PCR and Nanostring. RESULTS Sarcosine significantly increases human and murine DC migration in vitro. In vivo murine model, sarcosine-loaded DCs had significantly increased migration to both the lymph nodes and spleen after intradermal delivery. B16F10-OVA tumor bearing mice were treated with the sarcosine-loaded DC vaccines resulted in a significant survival advantage over control and naïve DC vaccines. Gene expression in RNA was investigated in DCs. CXCR2,CXCL3 and CXCL1 were found to be upregulated in sarcosine-loaded DCs. Further metabolic analysis demonstrated the upregulation of cyclooxygenase-1 and Pik3cg. In vitro DC migration in presence of CXCR2 neutralizing antibody showed sarcosine induced migration was abrogated by adding the CXCR2 neutralizing antibody in both human and murine DCs. Animals that treated with sarcosine-loaded DC showed significantly better tumor control compares to the animals receiving anti-CXCR2 antibody one hour before DC injection. CONCLUSION Sarcosine increases the migration of murine and human DCs via the CXC chemokine pathway. This platform can be utilized to improve existing DC vaccine strategies.

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Single cell RNA sequencing of calvarial and long bone endocortical cells

10.1101/849224 ◽

2019 ◽

Cited By ~ 1

Author(s):

Ugur M. Ayturk ◽

Joseph P. Scollan ◽

Alexander Vesprey ◽

Christina M. Jacobsen ◽

Paola Divieti Pajevic ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Cultured Cells ◽

Neutralizing Antibody ◽

Long Bone ◽

Appendicular Skeleton ◽

Rna Seq ◽

Isolated Cells

ABSTRACTSingle cell RNA-seq (scRNA-seq) is emerging as a powerful technology to examine transcriptomes of individual cells. We determined whether scRNA-seq could be used to detect the effect of environmental and pharmacologic perturbations on osteoblasts. We began with a commonly used in vitro system in which freshly isolated neonatal mouse calvarial cells are expanded and induced to produce a mineralized matrix. We used scRNA-seq to compare the relative cell type abundances and the transcriptomes of freshly isolated cells to those that had been cultured for 12 days in vitro. We observed that the percentage of macrophage-like cells increased from 6% in freshly isolated calvarial cells to 34% in cultured cells. We also found that Bglap transcripts were abundant in freshly isolated osteoblasts but nearly undetectable in the cultured calvarial cells. Thus, scRNA-seq revealed significant differences between heterogeneity of cells in vivo and in vitro. We next performed scRNA-seq on freshly recovered long bone endocortical cells from mice that received either vehicle or Sclerostin-neutralizing antibody for 1 week. Bone anabolism-associated transcripts were also not significantly increased in immature and mature osteoblasts recovered from Sclerostin-neutralizing antibody treated mice; this is likely a consequence of being underpowered to detect modest changes in gene expression, since only 7% of the sequenced endocortical cells were osteoblasts, and a limited portion of their transcriptomes were sampled. We conclude that scRNA-seq can detect changes in cell abundance, identity, and gene expression in skeletally derived cells. In order to detect modest changes in osteoblast gene expression at the single cell level in the appendicular skeleton, larger numbers of osteoblasts from endocortical bone are required.

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Reinforcement of Cancer Immunotherapy by Adoptive Transfer of Cblb-Deficient Cytotoxic T Lymphocytes Combined with a Dendritic Cell Vaccine

Blood ◽

10.1182/blood.v116.21.957.957 ◽

2010 ◽

Vol 116 (21) ◽

pp. 957-957

Author(s):

Christina Lutz-Nicoladoni ◽

Patrizia Stoizner ◽

Magdalena Pircher ◽

Stephanie Wallner ◽

Anna Maria Wolf ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Dendritic Cell ◽

Cancer Immunotherapy ◽

Cytotoxic T Lymphocytes ◽

Adoptive Transfer ◽

Ex Vivo ◽

Dc Vaccine

Abstract Abstract 957 Introduction: Various approaches to induce immunological rejection of tumors including transfer of autologous tumor infiltrating lymhocytes (TIL) after ex vivo clonal expansion or application of ex vivo transduced antigen specific T cell (TCR) transgenic T cells have been elaborated. In general, adoptive T cell transfer (ATC) has been combined with lympho-depleting agents (e.g. cyclophosphamide). However, the therapeutic efficacy of these cancer immunotherapy approaches is limited due to insufficient in vivo activation, expansion and survival of transferred effector immune cells, which is mainly due to suppressive mileu signals and immune evasion mechanisms induced by TGF-β. The E3 ubiquitin ligase Cbl-b is a key regulator of T cell activation and is assumed to confer TGF-β resistance. Thus we performed a proof-of-concept study evaluating Cbl-b targeting as “intracellular adjuvant” strategy to improve ATC for cancer immunotherapy. Material and Methods: We first tested the in vitro sensitivity of CTL towards TGF-β mediated immuno-suppressive cues and then in vivo evaluated the anti-tumor reactivity of cblb-deficient cytotoxic T lymphocytes (CTL) in murine tumor models alone or in combination with a dendritic cell (DC) vaccine. Results: Cblb-deficient CTL are hyper-responsive to TCR/CD28-stimulation in vitro and protected from the negative cues induced by TGF-β as determined by quantification fo IFN-g secretion and quantification of their proliferative capacity. Unexpectedly, adoptive transfer of polyclonal, non TCR-transgenic cblb-deficient CD8+ CTL, however, is not sufficient to reject B16ova or EG7 tumors in vivo, which is in clear contrast to previous reports using lymphopenic animals receiving adoptively transferred TCR-transgenic T cells. Thus, we next evaluated in vivo re-activation of adoptively transferred cblb-deficient T cells by a DC vaccine (i.e. SIINFEKL-pulsed DC). In strict contrast to ATC monotherapy, this approach now markedly delays tumor outgrowth and significantly increase survival rates, which is paralleled by an increased CTL infiltration rate to the tumor site and an enrichment of ova-specific and IFN-g-secreting CTL in the draining lymph nodes. Moreover, compared to wild-type CTL, cblb-deficient mice vaccinated with the DC vaccine show an increased cytolytic activity in vivo. Conclusions: In summary, we provide experimental evidence that genetic inactivation of cblb in polyclonal, non-TCR transgenic adoptively transferred CTL might serve as a novel “adjuvant approach”, suitable to augment the effectiveness of anti-cancer immunotherapies using ATC in immune-competent recipients. Disclosures: No relevant conflicts of interest to declare.

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Anti-HMGB1 Neutralizing Antibody Ameliorates Neutrophilic Airway Inflammation by Suppressing Dendritic Cell-Mediated Th17 Polarization

Mediators of Inflammation ◽

10.1155/2014/257930 ◽

2014 ◽

Vol 2014 ◽

pp. 1-11 ◽

Cited By ~ 17

Author(s):

Fang Zhang ◽

Gang Huang ◽

Bo Hu ◽

Li-Ping Fang ◽

E-Hong Cao ◽

...

Keyword(s):

Dendritic Cell ◽

Airway Inflammation ◽

High Mobility ◽

Neutralizing Antibody ◽

Th17 Response ◽

Neutrophilic Inflammation ◽

Allergic Lung Inflammation ◽

Hmgb1 Expression

We demonstrate that high mobility group box 1 protein (HMGB1) directs Th17 skewing by regulating dendritic cell (DC) function. First, ourin vitrostudies reveal that recombinant HMGB1 (rHMGB1) activates myeloid DCs to produce IL-23in vitro, and rHMGB1-activated DCs prime naïve lymphocytes to produce the Th17 cytokine IL-17A. Second, we demonstrate that anti-HMGB1 neutralizing antibody attenuates HMGB1 expression, neutrophilic inflammation, airway hyperresponsiveness, and Th17-related cytokine secretionin vivoby using a murine model of neutrophilic asthma induced by ovalbumin (OVA) plus lipopolysaccharide (LPS). Furthermore, anti-HMGB1 neutralizing antibody decreases the number of Th17 cells in lung cells and suppresses the production of IL-23 by lung CD11C+APCs. Finally, we show that intranasal adoptive transfer of rHMGB1-activated DCs was sufficient to restore lung neutrophilic inflammation and the Th17 response in a DC-driven model of asthma, whereas the transfer of rHMGB1 plus anti-HMGB1-treated mDCs significantly reduced these inflammation phenotypes. These data suggest, for the first time, that HMGB1 drives the DC-polarized Th17-type response in allergic lung inflammation and that blocking HMGB1 may benefit the attenuation of neutrophilic airway inflammation in asthma.

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Prostate tumor-induced stromal reprogramming generates Tenascin C that promotes prostate cancer metastasis through YAP/TAZ inhibition

10.1101/2021.11.08.467778 ◽

2021 ◽

Author(s):

Sue-Hwa Lin ◽

Yu-Chen Lee ◽

Song-Chang Lin ◽

Guoyu Yu ◽

Ming Zhu ◽

...

Keyword(s):

Prostate Cancer ◽

Cell Migration ◽

Cancer Metastasis ◽

Neutralizing Antibody ◽

Metastatic Potential ◽

In Vivo Studies ◽

Hybrid Cells ◽

Tenascin C

Metastatic prostate cancer (PCa) in bone induces bone-forming lesions that enhance PCa progression. How tumor-induced bone formation enhances PCa progression is not known. We have previously shown that PCa-induced bone originates from endothelial cells (EC) that have undergone endothelial-to-osteoblast (EC-to-OSB) transition by tumor-secreted BMP4. Here, we show that EC-to-OSB transition leads to changes in the tumor microenvironment that increases the metastatic potential of PCa cells. We found that conditioned medium (CM) from EC-OSB hybrid cells increases the migration, invasion and survival of PC3-mm2 and C4-2B4 PCa cells. Quantitative mass spectrometry (iTRAQ) identified Tenascin C (TNC) as one of the major proteins secreted from EC-OSB hybrid cells. TNC expression in tumor-induced osteoblasts was confirmed by immunohistochemistry of MDA-PCa118b xenograft and human bone metastasis specimens. Mechanistically, BMP4 increases TNC expression in EC-OSB cells through the Smad1-Notch/Hey1 pathway. How TNC promotes PCa metastasis was next interrogated by in vitro and in vivo studies. In vitro studies showed that a TNC neutralizing antibody inhibits EC-OSB-CM-mediated PCa cell migration and survival. TNC knockdown decreased, while addition of recombinant TNC or TNC overexpression increased migration and anchorage-independent growth of PC3 or C4-2b cells. When injected orthotopically, PC3-mm2-shTNC clones decreased metastasis to bone, while C4-2b-TNC overexpressing cells increased metastasis to lymph nodes. TNC enhances PCa cell migration through α5β1 integrin-mediated YAP/TAZ inhibition. These studies elucidate that tumor-induced stromal reprogramming generates TNC that enhances PCa metastasis and suggest that TNC may be a target for PCa therapy.

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Vimentin provides target search efficiency and mechanical resilience for dendritic cell migration

10.1101/2020.12.18.423401 ◽

2020 ◽

Author(s):

Luiza Da Cunha Stankevicins ◽

M. Reza Shaebani ◽

Doriane Vesperini ◽

Marta Urbanska ◽

Daniel A. D. Flormann ◽

...

Keyword(s):

Dendritic Cells ◽

Cell Migration ◽

Dendritic Cell ◽

Cell Stiffness ◽

Search Efficiency ◽

Mechanical Stiffness ◽

Target Search ◽

Dendritic Cell Migration

AbstractDendritic cells use amoeboid migration to pass through confined tissues to reach the lymph nodes, and this homing function is crucial for immune responses. The underlying mechanisms for this type of migration remain unknown. As vimentin intermediate filaments regulate adhesion-dependent migration, we analyzed whether they have a similar effect on amoeboid migration. We show that lack of vimentin impairs amoeboid migration in vitro in confined environments, and blocks lymph-node homing in mice in vivo. Importantly, we show that vimentin-deficient dendritic cells have a lower coupling factor between cell speed and persistence and reduced target search efficiency (e.g., finding a pathogen, or another cell). These data show that the characteristics of vimentin in its dynamic regulation of cell stiffness and load-bearing, and also elastic capacity, appear to explain the coupling between their migratory ability and search efficiency. Taken together, these data show that vimentin provides the specific mechano-dynamics required for dendritic cell migration and for efficient target searching.Summary statementVimentin contributes to the mechanical stiffness of cells required for amoeboid cell migration through confined spaces, and improves cell-search efficiency. Vimentin-deficient cells migrate more slowly and their migration speed is less coupled to persistence compared to control cells.

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IFN-g-induced Jmjd3-Zeb1 axis contributes to an aggressive phenotype in lung adenocarcinoma

10.21203/rs.3.rs-75572/v1 ◽

2020 ◽

Author(s):

Jianjian Yang ◽

Xue Wang ◽

Bing Huang ◽

Rong Liu ◽

Hui Xiong ◽

...

Keyword(s):

Gene Expression ◽

Cell Proliferation ◽

Cell Migration ◽

Expression Patterns ◽

Gene Expression Patterns ◽

Ifn Γ ◽

Zeb1 Expression

Abstract Background Active IFN-γ signaling is a common feature of tumors responding to PD-1 checkpoint blockade. IFN-γ exhibits both anti- and pro-tumor activities. Therefore, identifying the pro-tumor effects of IFN-γ and their underlying molecular mechanisms could be a critical step for developing therapeutic strategies to maximize the anti-tumor efficacy of immunotherapies. Methods Western blot, immunofluorescence, and quantitative real-time PCR assays were used to evaluate the expression of ZEB1 and EMT associated biomarkers. Trans-well assay was used to examine the role of IFN-γ on cancer cell migration in vitro. Murine tumor xenograft models were performed to examine the effect of IFN-γ on cancer cell metastasis in vivo. Colony formation assay was performed to detect the role of ZEB1 in cell proliferation. RNA-seq was performed to analyze the EMT-associated gene expression patterns in response to IFN-g treatment. Loss-of-function analysis and chromatin immunoprecipitation were used to reveal the mechanism underlying ZEB1 induction by IFN-γ. Results we demonstrate that the treatment of lung adenocarcinoma cells with IFN-γ leads to a rapid increase of ZEB1 expression and a significant change in epithelial-mesenchymal-transition (EMT)-associated gene expression patterns. Moreover, functional analysis shows that IFN-γ promotes cell migration in vitro and metastasis in vivo. Mechanistically, IFN-γ-induced JMJD3 significantly reduces H3K27 trimethylation in the promoter of the ZEB1 gene, thereby activating ZEB1 transcription. Inhibition of JMJD3 abrogates IFN-γ-induced ZEB1 expression. We previously demonstrated that IFN-γ-mediated anti-tumor response, including suppression of cell proliferation and induction of CXCL9 and CXCL10 expression, is STAT1-IRF1 dependent. The knockdown of ZEB1 diminishes IFN-γ-mediated cell migration and metastasis, but it does not affect STAT1 and IRF1 expression and has no effect on cell proliferation as well as the induction of CXCL9 and CXCL10 expression. Conclusion Inhibition or downregulation of ZEB1 may prevent the pro-tumor activity of IFN-γ while retaining its anti-tumor function. The study expands our understanding of IFN-γ-mediated signaling and helps to identify therapeutic targets to improve current immunotherapies.

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Irisin directly stimulates osteoclastogenesis and bone resorption in vitro and in vivo

eLife ◽

10.7554/elife.58172 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Eben G Estell ◽

Phuong T Le ◽

Yosta Vegting ◽

Hyeonwoo Kim ◽

Christiane Wrann ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Bone Resorption ◽

Osteoclast Differentiation ◽

Neutralizing Antibody ◽

Mouse Bone Marrow ◽

Differential Gene

Irisin, a skeletal-muscle secreted myokine, facilitates muscle-bone crosstalk and skeletal remodeling in part by its action on osteoblasts and osteocytes. In this study, we investigated whether irisin directly regulates osteoclasts. In vitro, irisin (2–10 ng/mL) increased osteoclast differentiation in C57BL/6J mouse bone marrow progenitors; however, this increase was blocked by a neutralizing antibody to integrin αVβ5. Irisin also increased bone resorption on several substrates in situ. RNAseq revealed differential gene expression induced by irisin including upregulation of markers for osteoclast differentiation and resorption, as well as osteoblast-stimulating ‘clastokines’. Forced expression of the irisin precursor Fndc5 in transgenic C57BL/6J mice resulted in lower bone mass at three ages and greater in vitro osteoclastogenesis from Fndc5-transgenic bone marrow progenitors. This study demonstrates that irisin acts directly on osteoclast progenitors to increase differentiation and promote bone resorption, supporting the tenet that irisin not only stimulates bone remodeling but may also be an important counter-regulatory hormone.

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Irisin Directly Stimulates Osteoclastogenesis and Bone Resorption In Vitro and In Vivo

10.1101/2020.05.09.085977 ◽

2020 ◽

Author(s):

Eben G. Estell ◽

Phuong T. Le ◽

Yosta Vegting ◽

Hyeonwoo Kim ◽

Roland Baron ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Bone Resorption ◽

Osteoclast Differentiation ◽

Neutralizing Antibody ◽

Low Bone Mass ◽

Differential Gene

AbstractThe myokine irisin facilitates muscle-bone crosstalk and skeletal remodeling in part by its action on osteoblasts and osteocytes. In the current study we investigated whether irisin also directly regulates osteoclasts. In vitro, irisin (2-10 ng/mL) increased osteoclast differentiation in C57BL/6J bone marrow progenitors; this increase was blocked by a neutralizing antibody to integrin αVβ5. Irisin also increased resorption on several substrates in situ. RNAseq revealed differential gene expression induced by irisin including upregulation of markers for osteoclast differentiation and resorption, as well as osteoblast-stimulating ‘clastokines’. In vivo, forced expression of the irisin precursor Fndc5 in murine muscle resulted in low bone mass and increased number of osteoclasts. Taken together, our work demonstrates that irisin acts directly on cultured osteoclast progenitors to increase differentiation and promote bone resorption. These actions support the tenet that irisin not only stimulates bone remodeling but may also be an important counter-regulatory hormone during exercise.

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