GLI2, An Effector of the Hedgehog Pathway, Is a Novel Regulator of IL6 Oncogenic Function In the Tumor Microenvironment

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 613-613
Author(s):  
Sherine F. Elsawa ◽  
Luciana L. Almada ◽  
Steven Ziesmer ◽  
Thomas E. Witzig ◽  
Stephen M. Ansell ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
B Cell ◽  
Plasma Cell ◽  
Stromal Cells ◽  
Hedgehog Signaling ◽  
Promoter Activity ◽  
Cell Plasma ◽  
Signaling Axis

Abstract Abstract 613 Uncontrolled immunoglobulin (Ig) secretion by malignant cells is a characteristic feature of lymphoplasmacytic malignancies and associates with significant morbidity. IL6 has been shown to promote Ig secretion by malignant B cells, however, the mechanisms mediating this cellular event remain elusive. Here, we identified a CCL5 induced pathway that upregulates IL6 secretion in B cell/plasma cell malignancies including Waldenström macroglobulinemia (WM). Characterization of this mechanism revealed that activation of the CCL5 pathway increases IL6 expression and promoter activity in stromal cells (HS-5, Saka and primary stromal cells from WM patients). Conversely, stable RNAi knockdown of the CCL5 receptor CCR3 in stromal cells led to significantly reduced IL6 promoter activity and expression. Further analysis using a combination of bioinformatics analysis, genetic analysis and chromatin immunoprecipitation assays (ChIP) identified GLI2 as the mediator of this CCL5-IL6 functional interaction. CCL5 induces GLI2 expression via activation of the PI3K/AKT pathway. Constitutively active PI3K and AKT mutants increase expression of GLI2 through the canonical NFkB pathway. ChIP assay showed p65 binds to the GLI2 promoter and increases GLI2 levels in stromal cells. Dominant negative molecules antagonizing these pathways effectively blocked CCR3 induction of GLI2 expression. Interestingly, this CCL5-induced pathway was independent of the Hedgehog signaling. Blockade of this cascade using pharmacological or genetic tools did not affect CCL5 activation of GLI2 in stromal cells. To validate the biological significance of this newly identified CCL5-GLI2-IL6 signaling axis in the regulation of IgM secretion, we used an in vitro coculture system and an in vivo mouse model. Coculture of primary WM stromal cells, HS-5 or Saka cells with primary WM malignant cells or BCWM.1, an IgM producing cell line, led to a significant increase in IL6 and IgM secretion. Similar results were obtained in vivo using athymic nu/nu mice subcutaneously injected with BCWM.1 and HS-5 cells. We then confirmed the requirement of GLI2 in vitro and in vivo. We infected HS-5 cells with a lentivirus containing shRNA targeting GLI2 or a scramble control and cocultured them with BCWM.1 cells. Knockdown of GLI2 decreased IL6 and IgM secretion in coculture. This was confirmed in vivo by infecting HS-5 cells or primary WMsc with lentivirus targeting GLI2 or scrambled shRNA and coinjecting with BCWM.1 cells into athymic nu/nu mice. Mice injected with cells lacking GLI2 had lower IgM levels in the serum and tumors compared with mice injected with HS-5 cells with an intact GLI2, further supporting the hypothesis that CCL5 requires an intact GLI2 to increase IL6 and consequently IgM. Finally, we confirmed the importance of this phenomenon in other B cell/plasma cell malignancies such as MGUS and MM. Primary stromal cultures from these neoplasms were infected with lentivirus targeting GLI2 or scrambled shRNA and cocultured with BCWM.1 cells. Similar to WM, there was a significant decrease in IL6 and IgM secretion in the absence of GLI2 suggesting the role of this molecule is conserved in other B cell/plasma cell malignancies. In summary, our data identifies a novel role for GLI2, an effector of the Hedgehog signaling pathway, in mediating the interaction between CCL5 and IL6 in the tumor microenvironment that modulates Ig secretion by malignant B cells. Therefore therapies targeting this signaling axis in the tumor microenvironment might provide efficacy in patients with Ig-mediated diseases. Disclosures: No relevant conflicts of interest to declare.

Fetal liver pro-B and pre-B lymphocyte clones: expression of lymphoid-specific genes, surface markers, growth requirements, colonization of the bone marrow, and generation of B lymphocytes in vivo and in vitro

1992 ◽  
Vol 12 (2) ◽  
pp. 518-530
Author(s):  
R Palacios ◽  
J Samaridis
Keyword(s):  
B Cell ◽  
B Lymphocytes ◽  
Stromal Cells ◽  
B Lymphocyte ◽  
Germ Line ◽  
Fetal Liver ◽  
Rag 1 ◽  
B Cell Precursors

We describe here the development and characterization of the FLS4.1 stromal line derived from 15-day fetal liver of BALB/c embryos and defined culture conditions that efficiently support the cloning and long-term growth of nontransformed B-220+ 14-day fetal liver cells at two stages of B-cell development, namely, pro-B lymphocytes (immunoglobulin [Ig] genes in germ line configuration) and pre-B cells (JH-rearranged genes with both light-chain Ig genes in the germ line state). All B-cell precursor clones require recombinant interleukin-7 (rIL-7) and FLS4.1 stromal cells for continuous growth in culture, but pro-B lymphocyte clones can also proliferate in rIL-3. None proliferate in rIL-1, rIL-2, rIL-4, rIL-5, rIL-6, or leukemia inhibitory factor. FLS4.1 stromal cells synthesize mRNA for Steel factor but not for IL-1 to IL-7; all pro-B and pre-B clones express c-Kit, the receptor for Steel factor, and a c-Kit-specific antibody inhibits the enhanced proliferative response of fetal liver B-220+ B-cell precursors supported by FLS4.1 stromal cells and exogenous rIL-7 but does not affect that promoted by rIL-7 alone. Northern (RNA) blot analysis of the expression of the MB-1, lambda 5, Vpre-B, c mu, RAG-1, and RAG-2 genes in pro-B and pre-B clones show that transcription of the MB-1 gene precedes IgH gene rearrangement and RNA synthesis from c mu, RAG-1, RAG-2, lambda 5, and Vpre-B genes. All clones at the pre-B-cell stage synthesize mRNA for c mu, RAG-1, and RAG-2 genes; transcription of the lambda 5 and Vpre-B genes seems to start after D-to-JH rearrangement in B-cell precursors, indicating that the proteins encoded by either gene are not required for B-cell progenitors to undergo D-to-JH gene rearrangement. These findings mark transcription of the MB-1 gene as one of the earliest molecular events in commitment to develop along the B-lymphocyte pathway. Indeed, both pro-B and pre-B clones can generate in vitro and in vivo B lymphocytes but not T lymphocytes; moreover, these clones do not express the CD3-gamma T-cell-specific gene, nor do they have rearranged gamma, delta, or beta T-cell antigen receptor genes.

scholarly journals IMMU-28. TARGETING IMMUNOSUPPRESSIVE MYELOID DERIVED SUPPRESSOR CELLS VIA MIF/CD74 SIGNALING AXIS TO ATTENUATE GBM GROWTH

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi125-vi125
Author(s):  
Tyler Alban ◽  
Defne Bayik ◽  
Balint Otvos ◽  
Matthew Grabowski ◽  
Manmeet Ahluwalia ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Tumor Growth ◽  
Immune Cell ◽  
Suppressor Cells ◽  
Myeloid Derived Suppressor Cells ◽  
Signaling Axis ◽  
Immunosuppressive Microenvironment ◽  
And Function

Abstract The immunosuppressive microenvironment in glioblastoma (GBM) enables persistent tumor growth and evasion from tumoricidal immune cell recognition. Despite a large accumulation of immune cells in the GBM microenvironment, tumor growth continues, and evidence for potent immunosuppression via myeloid derived suppressor cells (MDSCs) is now emerging. In agreement with these observations, we have recently established that increased MDSCs over time correlates with poor prognosis in GBM, making these cells of interest for therapeutic targeting. In seeking to reduce MDSCs in GBM, we previously identified the cytokine macrophage migration inhibitory factor (MIF) as a possible activator of MDSC function in GBM. Here, using a novel in vitro co-culture system to reproducibly and rapidly create GBM-educated MDSCs, we observed that MIF was essential in the generation of MDSCs and that MDSCs generated via this approach express a repertoire of MIF receptors. CD74 was the primary MIF receptor in monocytic MDSCs (M-MDSC), which penetrate the tumor microenvironment in preclinical models and patient samples. A screen of MIF/CD74 interaction inhibitors revealed that MN-166, a clinically relevant blood brain barrier penetrant drug, which is currently fast tracked for FDA approval, reduced MDSC generation and function in vitro. This effect was specific to M-MDSC subsets expressing CD74, and appeared as reduced downstream pERK signaling and MCP-1 secretion. In vivo, MN-166 was able reduce tumor-infiltrating MDSCs, while conferring a significant increase in survival in the syngeneic glioma model GL261. These data provide proof of concept that M-MDSCs can be targeted in the tumor microenvironment via MN-166 to reduce tumor growth and provide a rationale for future clinical assessment of MN-166 to reduce M-MDSCs in the tumor microenvironment. Ongoing studies are assessing the effects of MDSC inhibition in combination with immune activating approaches, in order to inhibit immune suppression while simultaneously activating the immune system.

scholarly journals Gli1+ mesenchymal stromal cells modulate epithelial metaplasia in lung fibrosis

2019 ◽  
Author(s):  
Monica Cassandras ◽  
Chaoqun Wang ◽  
Jaymin Kathiriya ◽  
Tatsuya Tsukui ◽  
Peri Matatia ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Hedgehog Signaling ◽  
List Type ◽  
Basal Cells ◽  
Human Lungs ◽  
Metaplastic Epithelium ◽  
Organ Fibrosis

AbstractOrgan fibrosis is often accompanied by aberrant epithelial reprogramming, culminating in a transformed barrier composed of scar and metaplastic epithelium. Understanding how the scar promotes an abnormal epithelial response could better inform strategies to reverse the fibrotic damage. Here we show that Gli1+ mesenchymal stromal cells (MSCs), previously shown to contribute to myofibroblasts in the scar, promote metaplastic differentiation of airway progenitors into KRT5+ basal cells in vitro and in vivo. During fibrotic repair, Gli1+ MSCs integrate hedgehog activation to promote metaplastic KRT5 differentiation by upregulating BMP antagonism in the progenitor niche. Restoring the balance towards BMP activation attenuated metaplastic KRT5+ differentiation while promoting adaptive alveolar differentiation. Finally, fibrotic human lungs demonstrate altered BMP activation in the metaplastic epithelium. These findings show that Gli1+ MSCs integrate hedgehog signaling as a rheostat to control BMP activation in the progenitor niche to determine regenerative outcome in fibrosis.HighlightsGli1+ MSCs are required for metaplastic airway progenitor differentiation into KRT5+ basal cells.Hedgehog activation of MSCs promotes KRT5 differentiation of airway progenitors by suppressing BMP activation.Restoring BMP activation attenuates metaplastic KRT5 differentiationMetaplastic KRT5+ basal cells in human fibrotic lungs demonstrate altered BMP activation.

Emetine Elicits Apoptosis of Intractable B-Cell Lymphoma Cells with MYC Rearrangement through Inhibition of Glycolytic Metabolism

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3019-3019
Author(s):  
Tomohiro Aoki ◽  
Kazuyuki Shimada ◽  
Akihiko Sakamoto ◽  
Keiki Sugimoto ◽  
Takanobu Morishita ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Glucose Metabolism ◽  
Tumor Cells ◽  
Drug Screening ◽  
Stromal Cells ◽  
Research Funding ◽  
B Cell Lymphoma ◽  
Lymphoma Cells

Abstract Background: Despite remarkable advances of initial treatment in diffuse large B-cell lymphoma (DLBCL), the prognosis of the disease with MYC rearrangement remains poor with a median overall survival of less than 1 year. The application of intensive or targeting treatment failed to show a benefit for the disease, an innovative approach should be thus required to overcome the obstacle of MYC rearrangement. Recent findings revealed that the close interaction of tumor cells with stromal cells in its microenvironment is involved in resistance to chemotherapy, and that tumor microenvironment has been shed light on a potential attractive therapeutic target. Purpose: To overcome poor prognoses of intractable DLBCL with MYC rearrangement, we explored an effective drug targeting tumor microenvironment through the high-throughput drug screening (Sugimoto et al. Sci Rep. 2015). Material and methods: Allpatient samples were experimentally used with written informed consent. To perform drug screening against primary patient lymphoma cells with intractable clinical course,we firstly developed co-culture system of lymphoma cells and stromal cells, which allowed us to culture them in vitro.For this, isolated stromal cells derived from human lymph node were prepared. Then 3,440 compounds mainly containing known pharmacologically active substance or off-patent drugs were screened to identify effective drugs for patient lymphoma cells. The efficacy and mechanism of action of the drug were confirmed by subsequent in vitro and in vivo analyses. Results: Two patient tumor cells with MYC/BCL2 rearrangement were used for the drug screening. Both patients developed refractory diseases within 1 year after diagnosis. In the screening analyses, primary lymphoma cells obtained from lymph node for patient (Pt) #1 were used, and tumor cells from PDX mouse model for Pt #2 were used to validate the result of Pt #1. The both tumor cells could not survive in in vitro monoculture, while the both lymphoma cells could remarkably survive longer in co-culture with stromal cells. Then we performed drug screening against primary tumor cells from Pt #1. Ninety-nine compounds with the viability of tumor cells less than 0.5 were identified, and we validated cell death of these 99 compounds against the other lymphoma cells from Pt #2. Among 10 compounds identified as potentially effective for the both tumor cells, we picked out emetine, which induced cell death against the both cells with an IC50 of 312 nM and 506 nM, respectively. Regarding the effect of emetine on stromal cells, the proliferation and survival was not affected in the concentration of 2 µM emetine whose concentration was used for the screening. However, stromal cells pretreated 0.5 µM emetine decreased a support potential to tumor cells resulting from decreased ATP production and glutathione in tumor cells. In terms of the effect of emetine on tumor cells, the drug induced a G2/M arrest in tumor cells, which resulted in induction of apoptosis. Based on previous finding that emetine suppresses HIF-1a expression, which is one of key regulators glucose metabolisms, we investigated the expression in tumor cells under the treatment of emetine. HIF-1a expression was suppressed in tumor cells as expected; we subsequently analyzed the status of glucose metabolism in tumor cells. The expression of key enzymes including HK2, PDK1, and LDHA were suppressed and ATP production and GLUT1 expression were also suppressed. The serial cascade of the alteration of glucose metabolism including the decreased mitochondrial membrane potential, the alteration of pentose phosphate pathway, and the reduction of NADPH and glutathione leading to the accrual of reactive oxygen species (ROS) was observed under the presence of emetine. In in vivo analyses, significant growth inhibition was observed under the emetine treatment (Figure A and B). Conclusions: Emetine identified by the drug screening is clearly effective for patient lymphoma cells with intractable clinical course in vitro and in vivo. Subsequent analyses regarding the mechanism of action of emetine revealed that the drug affected the both tumor cells and stromal cells in tumor microenvironment through the inhibition of glucose metabolism. Further investigations of the translation to clinic should be warranted. Disclosures Sugimoto: Otsuka Pharmaceutical Co., Ltd.: Employment. Kiyoi:Nippon Shinyaku Co., Ltd.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Phizer Japan Inc.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; MSD K.K.: Research Funding; Alexion Pharmaceuticals: Research Funding; Novartis Pharma K.K.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Toyama Chemikal Co.,Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; AlexionpharmaLLC.: Research Funding; JCR Pharmaceutlcals Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Celgene Corporation: Consultancy; Zenyaku Kogyo Co.LTD.: Research Funding; Kyowa-Hakko Kirin Co.LTD.: Research Funding; Chugai Pharmaceutical Co. LTD.: Research Funding.

scholarly journals Fetal liver pro-B and pre-B lymphocyte clones: expression of lymphoid-specific genes, surface markers, growth requirements, colonization of the bone marrow, and generation of B lymphocytes in vivo and in vitro.

1992 ◽  
Vol 12 (2) ◽  
pp. 518-530 ◽  
Author(s):  
R Palacios ◽  
J Samaridis
Keyword(s):  
B Cell ◽  
B Lymphocytes ◽  
Stromal Cells ◽  
B Lymphocyte ◽  
Germ Line ◽  
Fetal Liver ◽  
Rag 1 ◽  
B Cell Precursors

We describe here the development and characterization of the FLS4.1 stromal line derived from 15-day fetal liver of BALB/c embryos and defined culture conditions that efficiently support the cloning and long-term growth of nontransformed B-220+ 14-day fetal liver cells at two stages of B-cell development, namely, pro-B lymphocytes (immunoglobulin [Ig] genes in germ line configuration) and pre-B cells (JH-rearranged genes with both light-chain Ig genes in the germ line state). All B-cell precursor clones require recombinant interleukin-7 (rIL-7) and FLS4.1 stromal cells for continuous growth in culture, but pro-B lymphocyte clones can also proliferate in rIL-3. None proliferate in rIL-1, rIL-2, rIL-4, rIL-5, rIL-6, or leukemia inhibitory factor. FLS4.1 stromal cells synthesize mRNA for Steel factor but not for IL-1 to IL-7; all pro-B and pre-B clones express c-Kit, the receptor for Steel factor, and a c-Kit-specific antibody inhibits the enhanced proliferative response of fetal liver B-220+ B-cell precursors supported by FLS4.1 stromal cells and exogenous rIL-7 but does not affect that promoted by rIL-7 alone. Northern (RNA) blot analysis of the expression of the MB-1, lambda 5, Vpre-B, c mu, RAG-1, and RAG-2 genes in pro-B and pre-B clones show that transcription of the MB-1 gene precedes IgH gene rearrangement and RNA synthesis from c mu, RAG-1, RAG-2, lambda 5, and Vpre-B genes. All clones at the pre-B-cell stage synthesize mRNA for c mu, RAG-1, and RAG-2 genes; transcription of the lambda 5 and Vpre-B genes seems to start after D-to-JH rearrangement in B-cell precursors, indicating that the proteins encoded by either gene are not required for B-cell progenitors to undergo D-to-JH gene rearrangement. These findings mark transcription of the MB-1 gene as one of the earliest molecular events in commitment to develop along the B-lymphocyte pathway. Indeed, both pro-B and pre-B clones can generate in vitro and in vivo B lymphocytes but not T lymphocytes; moreover, these clones do not express the CD3-gamma T-cell-specific gene, nor do they have rearranged gamma, delta, or beta T-cell antigen receptor genes.

scholarly journals Histone methyltransferase DOT1L controls state-specific identity during B cell differentiation

2019 ◽  
Author(s):  
Muhammad Assad Aslam ◽  
Mir Farshid Alemdehy ◽  
Eliza Mari Kwesi-Maliepaard ◽  
Marieta Caganova ◽  
Iris N. Pardieck ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Cell Fate ◽  
Plasma Cell ◽  
Plasma Cells ◽  
B Cell Differentiation ◽  
Humoral Immune

AbstractDifferentiation of naïve peripheral B cells into terminally differentiated plasma cells is characterized by epigenetic alterations, yet the epigenetic mechanisms that control B cell fate remain unclear. Here we identified a central role for the histone H3K79 methyltransferase DOT1L in controlling B cell differentiation. Murine B cells lacking Dot1L failed to establish germinal centers (GC) and normal humoral immune responses in vivo. In vitro, activated B cells showed aberrant differentiation and prematurely acquired plasma cell features. Mechanistically, combined epigenomics and transcriptomics analysis revealed that DOT1L promotes expression of a pro-proliferative, pro-GC program. In addition, DOT1L supports the repression of an anti-proliferative, plasma cell differentiation program by maintaining expression of the H3K27 methyltransferase Ezh2, the catalytic component of Polycomb Repressor Complex 2 (PRC2). Our findings show that DOT1L is a central modulator of the core transcriptional and epigenetic landscape in B cells, establishing an epigenetic barrier that warrants B cell naivety and GC B cell differentiation.

scholarly journals A Selective Culture System for Generating Terminal Deoxynucleotidyl Transferase-Positive Lymphoid CellsIn Vitro. V. Detection of Stage-Specific Pro-B-Cell Stimulating Activity in Medium Conditioned by Mouse Bone Marrow Stromal Cells

1993 ◽  
Vol 3 (3) ◽  
pp. 181-195 ◽  
Author(s):  
Sean D. Mckenna ◽  
Irving Goldschneider
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Stromal Cells ◽  
Culture System ◽  
Lymphoid Cells ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Cell Phenotype ◽  
B Lineage

The selectivein vitrogeneration of rat, mouse, and human terminal deoxynucleotidyl transferase-positive (TdT+lymphoid cells in our long-term xenogeneic bone marrow (BM) culture system is characterized by physical interaction between the developing lymphocytes and mouse BM-adherent stromal cells and macrophages. In the present study, experiments in which micropor)us membrane culture inserts were inoculated with rat BM cells demonstrated that although the generation of primitive B-lineage lymphoid cells requires the presence of a mouse BM feeder layer, cognitive recognition events are not necessary. Similarly, cell-free (and serum-free) medium conditioned with mouse BM (but not thymus or spleen) adherent cells and stromal-cell lines therefrom supported the proliferation of early rat lymphoid cells in a dose-dependent manner. Double immunofluorescence for incorporated bromo-deoxyuridine (BrdU) and early B-lineage markers of rat BM lymphoid cells maintained in culture inserts or conditioned medium (CM), and studies of their in vitro andin vivodevelopmental potentials, indicated that the lymphoproliferative response resulted from the selective stimulation of lymphoid stem and/or progenitor cells. The most primitive of these target cells had a HIS24+HIS50-TdT-cμ-sIg-, pre-pro-B-cell phenotype. Whereas this subset normally constitutes less than 2% of B-lineage BM cellsin vivo, it comprises more than 25% of total lymphoid cellsin vitro. In addition, the number of TdT+cells, predominantly of the early pro-B-cell phenotype (HIS24+HIS50-TdT-cμ-sIg-), was increased approximately tenfold above input levels. Based on these and previous findings, a schematic model is proposed for the developmental pathway of early B-lineage cells in rat BM from the level of the committed (possibly common) lymphoid stem cell to that of the pre-B-cell.

scholarly journals Adipose-derived stromal cells modulating composite allotransplant survival is correlated with B cell regulation in a rodent hind-limb allotransplantation model

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Chien-Chang Chen ◽  
Rong-Fu Chen ◽  
Jheng-Syuan Shao ◽  
Yun-Ting Li ◽  
Yu-Chi Wang ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Stromal Cells ◽  
Hind Limb ◽  
Cell Regulation ◽  
Allograft Survival ◽  
Short Term ◽  
Immunoregulatory Cytokines

Abstract Background Our previous studies demonstrated that adipose-derived mesenchymal stromal cells (ASCs) have immunomodulatory effects that prolong allograft survival in a rodent hind-limb allotransplant model. In this study, we investigated whether the effects of immunomodulation by ASCs on allograft survival are correlated with B cell regulation. Methods B cells isolated from splenocytes were cocultured with ASCs harvested from adipose tissue from rodent groin areas for in vitro experiments. In an in vivo study, hind-limb allotransplantation from Brown-Norway to Lewis rats was performed, and rats were treated with ASCs combined with short-term treatment with anti-lymphocyte serum (ALS)/cyclosporine (CsA) as immunosuppressants. Peripheral blood and transplanted tissue were collected for further analysis. Result An in vitro study revealed that ASCs significantly suppressed lipopolysaccharide-activated B cell proliferation and increased the percentage of Bregs. The levels of immunoregulatory cytokines, such as TGF-β1 and IL-10, were significantly increased in supernatants of stimulated B cells cocultured with ASCs. The in vivo study showed that treatment with ASCs combined with short-term ALS/CsA significantly reduced the B cell population in alloskin tissue, increased the proportion of circulating CD45Ra+/Foxp3+ B cells, and decreased C4d expression in alloskin. Conclusion ASCs combined with short-term immunosuppressant treatment prolong allograft survival and are correlated with B cell regulation, C4d expression and the modulation of immunoregulatory cytokines.

Proteinkinase C-β Inhibitors Mitigate Microenvironment-Mediated Survival Of CLL Cells and Sensitise Malignant B Cells To Cytotoxic Drugs

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 669-669
Author(s):  
Gloria Lutzny ◽  
Zhoulei Li ◽  
Madlen Oelsner ◽  
Jolanta Slawska ◽  
Thomas Kocher ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Stromal Cells ◽  
Cytotoxic Drugs ◽  
B Cell Lymphomas ◽  
Cytotoxic Effects ◽  
Pkc Β ◽  
Apoptotic Proteins

Abstract Heterotypic interactions between bone marrow stromal cells (BMSCs) and malignant B cells contribute to apoptosis-resistance of tumour cells, based on the provision of anti-apoptotic factors by stromal cells. For this reason, interference with the microenvironment may offer an alternative approach to chemotherapy to target B cell lymphomas/ leukaemias. However, the success of such treatments critically relies on specific targets expressed in the lymphoma microenvironment. We have previously reported that monoclonal B cells from patients with CLL, MCL und ALL activate and reprogram BMSCs. This activation of stromal cells is an essential prerequisite for microenvironment-mediated survival of malignant B cells and requires the induction and activation of protein-kinase C-β in stromal cells in vitro and in vivo. This is underscored by a complete resistance of PKC-β knockout mice to adoptively transferred B cell lymphomas from TCL1 transgenic mice (Lutzny et al. Cancer Cell. 2013 Jan 14; 23(1):77-92.). Analyses of primary CLL cells co-cultured on human or mouse BMSCs indicate that stromal cells protect malignant B cells from apoptosis primarily by enhancing the expression of the anti-apoptotic proteins Mcl1, XIAP, BclXL and Bcl2A1. Knockdown of Mcl1 expressed in CLL cells further demonstrates that its expression is crucial for stroma-mediated survival even in the presence of BMSCs. Since BMSC-mediated survival and propagation of CLL depends on the kinase activity of PKC-β, we hypothesized that pharmacological inhibition of the activation of stromal cells using small molecule inhibitors against PKC-β may display anti-leukemic effects. Here we report that the PKC-β inhibitor enzastaurin enhances the cytotoxic effects of standard chemotherapeutic drugs and of the BCL2-inhibitor ABT737. Dose-response analyses indicate that enzastaurin potentiates the cytotoxic effects of ABT737 in a synergistic manner, rapidly causing caspase-mediated apoptosis of malignant B cells. This drug-sensitising effect of enzastaurin is mediated by inhibition of PKC-β induced and expressed in activated stromal cells and not related to a direct cytotoxic effect on malignant B cells: PKC-β deficient BMSCs fail to maintain high expression levels of the anti-apoptotic proteins Mcl1, XIAP and Bcl2A1 in CLL cells, which show a significantly enhanced sensitivity to ABT737 compared to CLL cells cultured on PKC-β proficient stromal cells. These data provide evidence that PKC-β inhibitors can therapeutically be used to abolish microenvironment-mediated survival of malignant B cells. In order to assess whether drug combinations of enzastaurin and BCL2 inhibitors demonstrate synergistic anti-lymphoma effects in vivo, we adoptively transferred malignant B cells from diseased TCL1 mice into wild-type recipient mice. Tumour-bearing mice were treated with enzastaurin, ABT199 or a combination of both drugs. Response to treatment, assessed by PET-CT scan, indicates that the combination of enzastaurin and ABT199 is superior to single agent treatment. These data indicate that malignant B cells can be sensitized to cytotoxic drugs by inhibiting the tumour-promoting effects of the lymphoma microenvironment with PKC-β inhibitors. Our preclinical in vitro and in vivo experiments justify testing this drug combination as a new approach to leukaemia/ lymphoma therapy in a phase I/II clinical trial. Disclosures: No relevant conflicts of interest to declare.

A CD47xCD19 Bispecific Antibody That Remodels the Tumor Microenvironment for Improved Killing and Provokes a Memory Immune Response to Cancer B Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 44-44
Author(s):  
Walter G. Ferlin ◽  
Xavier Chauchet ◽  
Vanessa Buatois ◽  
Susana Salgado-Pires ◽  
Limin Shang ◽  
...  
Keyword(s):  
B Cells ◽  
Tumor Microenvironment ◽  
Tumor Growth ◽  
B Cell ◽  
Cell Lymphoma ◽  
Treatment Strategies ◽  
B Cell Lymphoma ◽  
Tumor Killing

Abstract Up-regulation of CD47 in hematological and solid cancers correlates with poor clinical prognosis. CD47 interaction with SIRPα provides a ‘don't eat me’ signal that allows healthy cells to limit elimination by immune cells, in particular macrophages. Although tumor-associated macrophages (TAMs) are often considered pro-tumorigenic, several studies report a high phagocytic potential and tumoricidal function in the presence of therapeutic antibodies (Ab). Therefore, targeting the CD47-SIRPα pathway in the tumor microenvironment is an attractive approach to maximize the tumor killing potential of TAMs to boost tumor destruction. However, clinical development of monoclonal Abs to CD47 is likely to be hindered by the ubiquitous expression of CD47 leading to rapid drug elimination and toxicity including anemia. To address these concerns, we have created NI-1701, a bispecific Ab that drives efficacious binding only to CD19+B cells by pairing a high affinity anti-CD19 targeting arm to an anti-CD47 arm of optimized affinity.. In addition to in vitro data demonstrating that the bispecific Ab, NI-1701, effectively kills CD19+ human tumor B cells through ADCP (antibody-dependent cellular phagocytosis) and antibody-dependent cell-mediated cytotoxicity (ADCC), we have observed significant tumor killing in vivo, as either a monotherapy or in a combination approach. NI-1701 controlled sub-cutaneously implanted Raji cell tumor growth in NOD/SCID mice in a manner dependent on the co-ligation of both CD19 and CD47. Examination of the excised tumors revealed that NI-1701 reshaped the tumor microenvironment by enhancing the tumoricidal activity of macrophages (i.e., more macrophages engulfing tumor cells), by promoting an antitumor M1-like phenotype, and reducing the proportion of CD11b+Gr1+myeloid-derived suppressor cells (MDSCs). Extending these findings to a disseminated in vivo model, NI-1701 eliminated tumor cells from the peripheral blood, bone marrow and liver in mice transplanted either with the B-Acute Lymphocytic Leukemia (B-ALL) cell line NALM-6 or with primary cells from B-ALL patients. Furthermore, NI-1701 also abrogated tumor growth more efficiently than the BTK inhibitor ibrutinib in a Diffuse Large B-Cell Lymphoma (DLBCL) patient-derived xenograft (PDX) mouse model. As combination therapies are gaining traction as successful treatment strategies in the clinic, we next tested the effect of blocking CD47 biology in combination with clinically validated molecules. Interestingly, in NOD/SCID mice implanted with Raji cells, NI-1701 was shown to be more efficacious at controlling tumor cell growth than Rituximab. A combination of NI-1701 and Rituximab was shown to act synergistically at controlling tumor growth and leading to tumor regression in some mice. Finally, in a syngeneic re-challenge model, using bispecific reagents targeting CD47 blockade to the A20 murine B-cell lymphoma, we observed the induction of a durable and protective anti-tumor response when combined with a single administration of cyclophosphamide. Importantly, in vitro safety studies demonstrate a favorable binding profile of NI-1701 to B cells compared with erythrocytes, no evidence of platelet activation or aggregation and no haemagglutination at and above anticipated therapeutic concentrations. Single and multiple dose studies in non-human primates demonstrated favorable elimination kinetics and no effects on hematological parameters (e.g., red blood cell and platelet counts) up to 100mg/kg, the highest dose tested. Taken together, we describe a novel bispecific approach that balances a safe yet effective blockade of CD47 with a high selectivity for a B cell associated antigen resulting in impressive tumor cell killing in a range of preclinical models. The effects on both the reshaping of the tumor microenvironment and the induction of long term tumor immunity provide further evidence that manipulation of myeloid lineage cells (e.g., macrophages and dendritic cells) is a promising approach for the next frontier in immune-oncology treatment strategies. NI-1701 is in preclinical enabling studies in preparation for a Phase I clinical study in patients with CD19+ B cell malignancies, planned for early 2017. Disclosures Ferlin: Novimmune S.A.: Employment, Equity Ownership. Chauchet:Novimmune S.A.: Employment. Buatois:Novimmune S.A.: Employment. Salgado-Pires:Novimmune S.A.: Employment. Shang:Novimmune S.A.: Employment. Dheilly:Novimmune S.A.: Employment. Masternak:Novimmune S.A.: Employment. Johnson:Novimmune S.A.: Employment. DiPersio:Incyte Corporation: Research Funding. Kosco-Vilbois:Novimmune S.A.: Employment. Fischer:Novimmune S.A.: Employment.

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