A20 Cells Research Articles

Abstract 2752: Novel inhibitor of HSP90- PU-H71 exhibits significant activity in Ewing sarcoma pre-clinical models.

Abstract Ewing sarcoma is characterized by multiple deregulated pathways that mediate cell survival and proliferation. Heat shock protein-90 (HSP90) is a critical component of the multi-chaperone complexes that regulate the disposition and activity of a large number of proteins responsible for the assembly and regulation of cell-signaling systems. We tested the efficacy of PU-H71, a novel purine scaffold HSP90 inhibitor developed at Memorial Sloan-Kettering Cancer Center in several Ewing sarcoma cell lines and primary cultures established from patient samples. We investigated its effect on cell proliferation and cell viability on multiple Ewing cell lines, benign epithelial, endothelial and mesenchymal stromal cells and its effect on colony forming assay using CD34 hematopoietic stem cells . We performed cell cycle analysis, clonogenic assay, immunoblot analysis and in vivo experiments in NOD scid gamma (NSG) mice using A673 cell line transduced with GFP luciferase for bioimaging. We also established a metastatic model by calculating the mean fluorescence intensity (MFI) in various organs. Our results show that there is a significant therapeutic window in its activity against Ewing cell lines and benign cells. PU-H71 treatment resulted in arrest in G2 phase and diminished S phase in Ewing cell lines. We also noted that many of the deregulated pathway proteins including IGF1R, AKT, ERK, MYC, hTERT and EWS-FLI1 are stabilized by HSP90 and exposure to PU-H71 resulted in depletion of the above proteins. We also noted an increase in cleaved PARP and proapoptotic proteins and a decrease in antiapoptotic proteins on immunoblot analysis. Based on earlier PK and MTD studies, we treated mice at a dose of 75mg/kg by intraperitoneal injection, three times a week for 4 weeks. Our results indicated that Ewing's sarcoma tumor growth was significantly inhibited and metastatic burden was significantly decreased in the mice injected with PU-H71 compared to the mice injected with vehicle, based on MFI, weight and size of the tumors. Immunohistochemical staining showed that CD31 expression was significantly reduced in PU-H71 treated mice tumors. Our results suggest that PU-H71 has the ability to affect multiple critical oncogenic pathways simultaneously resulting in decreased tumor growth and metastases and has a strong rationale for clinical evaluation in Ewing sarcoma. Citation Format: Srikanth R. Ambati, Eloisi Caldas Lopes, Ullas Mony, Kohji Kosugi, Andre Moreira, Paul Meyers, Gabriela Chiosis, Malcolm Moore. Novel inhibitor of HSP90- PU-H71 exhibits significant activity in Ewing sarcoma pre-clinical models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2752. doi:10.1158/1538-7445.AM2013-2752

HMG-CoA reductase inhibitors induce apoptosis of lymphoma cells by promoting ROS generation and regulating Akt, Erk and p38 signals via suppression of mevalonate pathway

Statins, the inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, are widely used cholesterol-lowering drugs. Convincing evidence indicates that statins stimulate apoptotic cell death in several types of proliferating tumor cells in a cholesterol-lowering-independent manner. The objective here was to elucidate the molecular mechanism by which statins induce lymphoma cells death. Statins (atorvastatin, fluvastatin and simvastatin) treatment enhanced the DNA fragmentation and the activation of proapoptotic members such as caspase-3, PARP and Bax, but suppressed the activation of anti-apoptotic molecule Bcl-2 in lymphoma cells including A20 and EL4 cells, which was accompanied by inhibition of cell survival. Both increase in levels of reactive oxygen species (ROS) and activation of p38 MAPK and decrease in mitochondrial membrane potential and activation of Akt and Erk pathways were observed in statin-treated lymphoma cells. Statin-induced cytotoxic effects, DNA fragmentation and changes of activation of caspase-3, Akt, Erk and p38 were blocked by antioxidant (N-acetylcysteine) and metabolic products of the HMG-CoA reductase reaction, such as mevalonate, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). These results suggests that HMG-CoA reductase inhibitors induce lymphoma cells apoptosis by increasing intracellular ROS generation and p38 activation and suppressing activation of Akt and Erk pathways, through inhibition of metabolic products of the HMG-CoA reductase reaction including mevalonate, FPP and GGPP.

CpG oligodeoxynucleotide induces apoptosis and cell cycle arrest in A20 lymphoma cells via TLR9-mediated pathways

Recent studies have suggested that the anti-cancer activity of CpG-oligodeoxynucleotides (CpG-ODNs) is owing to their immunomodulatory effects in tumor-bearing host. The purpose of this study is to investigate the directly cytotoxic activity of KSK-CpG, a novel CpG-ODN with an alternative CpG motif, against A20 and EL4 lymphoma cells in comparison with previously used murine CpG motif (1826-CpG). To evaluate the potential cytotoxic effects of KSK-CpG on lymphoma cells, cell viability assay, confocal microscopy, flow cytometry, DNA fragmentation, Western blotting, and reverse transcription-polymerase chain reaction (RT-PCR) analysis were used. We found that KSK-CpG induced direct cytotoxicity in A20 lymphoma cells, but not in EL4 lymphoma cells, at least in part via TLR9-mediated pathways. Apoptotic cell death was demonstrated to play an important role in CpG-ODNs-induced cytotoxicity. In addition, both mitochondrial membrane potential decrease and G1-phase arrest were involved in KSK-CpG-induced apoptosis in A20 cells. The activities of apoptotic molecules such as caspase-3, PARP, and Bax were increased, but the activation of p27 Kip1 and ERK were decreased in KSK-CpG-treated A20 cells. Furthermore, autocrine IFN-γ partially contributed to apoptotic cell death in KSK-CpG-treated A20 cells. Collectively, our findings suggest that KSK-CpG induces apoptotic cell death in A20 lymphoma cells at least in part by inducing G1-phase arrest and autocrine IFN-γ via increasing TLR9 expression, without the need for immune system of tumor-bearing host. This new understanding supports the development of TLR9-targeted therapy with CpG-ODN as a direct therapeutic agent for treating B lymphoma.

Effect of shRNA targeting mouse CD99L2 gene in a murine B cell lymphoma in vitro and in vivo

Mouse CD99 antigen-like 2 (mCD99L2) has previously been confirmed to be expressed in murine Blymphoma (A20) cells by our group. The present study aimed to establish a mCD99L2‑downregulated A20 cell line and to investigate the effect of shRNA targeting mCD99L2 in A20 cells invitro and invivo. Four pLenti6/mCD99L2 expression vectors containing the mCD99L2 shRNA-expressing cassette were constructed, transfected into A20 cells and stable mCD99L2-downregulated A20 subclones, termed A20-mCD99L2- cells, were established and identified by quantitative PCR and western blot analysis. Light and transmission electron microscopy, MTT assay, flow cytometry and immunofluorenscence labeling were used to observe the morphological, biological and phenotypic characteristics invitro. Some of the A20-mCD99L2- cells exhibited H/RS‑cell like morphology, a decreased proliferative ability, a prolonged G2phase and increased CD30 and CD15 expression. Upon injecting cells into nude or immunocompetent BALB/c mice, tumorigenesis, tumor growth, morphology and phenotypes invivo were observed. A20-mCD99L2- cells induced tumors in nude and BALB/c mice, but with less potency in the latter compared with the controls. Similar morphological, biological and phenotypic characteristics were observed in the A20-mCD99L2- cell-induced tumors as those invitro. Several cytokines including CD30T, IL-12p40/p70, IL-3, IFN-γ, CXCL16, MIP-1α and CD40 were upregulated following mCD99L2 downregulation when detected using antibody arrays. The results from western blot analysis indicated that the regulation of mCD99L2 expression may involve the activated nuclear factor-κB pathway in the murine Blymphoma cells. The present study provides data for further investigation into the mCD99L2 gene in tumor cells.

Autophagy contributes to apoptosis in A20 and EL4 lymphoma cells treated with fluvastatin

Convincing evidence indicates that statins stimulate apoptotic cell death in several types of proliferating tumor cells in a cholesterol-lowering-independent manner. However, the relationship between apoptosis and autophagy in lymphoma cells exposed to statins remains unclear. The objective of this study was to elucidate the potential involvement of autophagy in fluvastatin-induced cell death of lymphoma cells. We found that fluvastatin treatment enhanced the activation of pro-apoptotic members such as caspase-3 and Bax, but suppressed the activation of anti-apoptotic molecule Bcl-2 in lymphoma cells including A20 and EL4 cells. The process was accompanied by increases in numbers of annexin V alone or annexin V/PI double positive cells. Furthermore, both autophagosomes and increases in levels of LC3-II were also observed in fluvastatin-treated lymphoma cells. However, apoptosis in fluvastatin-treated lymphoma cells could be blocked by the addition of 3-methyladenine (3-MA), the specific inhibitor of autophagy. Fluvastatin-induced activation of caspase-3, DNA fragmentation, and activation of LC3-II were blocked by metabolic products of the HMG-CoA reductase reaction, such as mevalonate, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). These results suggest that autophagy contributes to fluvastatin-induced apoptosis in lymphoma cells, and that these regulating processes require inhibition of metabolic products of the HMG-CoA reductase reaction including mevalonate, FPP and GGPP.

Examination of an immunoregulator in a syngeneic animal model

Event Abstract Back to Event Examination of an immunoregulator in a syngeneic animal model Giovanna Merchand-Reyes1, Miguel A. Becerril-Garcia1, Isabel Gracia-Mora2, Sergio Estrada-Parra1, Iris Estrada-Garcia1, Sonia M. Perez-Tapia1 and Claudia Sandoval-Montes1* 1 Instituto Politecnico Nacional, Immunology, Mexico 2 Universidad Nacional Autonoma de Mexico, Mexico Dialyzable leukocyte extracts (DLE), described by Lawrence in the mid-20th century, have been used widely as an adjuvant therapy for various diseases due to their immunoregulatory activity. Clinical studies on DLE in combination with conventional therapy against cancer have demonstrated that they are beneficial, reducing metastases and the side effects of chemotherapy and increasing life span. Nevertheless, their mechanism of action in cancer remains poorly understood. Diffuse large B-cell lymphoma is a highly aggressive neoplasm that is resistant to many therapies, prompting the development of new therapies, particularly those that increase the immune response. This study examined the effects of DLE in A20 cell lymphoma in a syngeneic murine model. In vitro, the proliferation and cell cycle progression of A20 cells were unaltered on incubation with DLE of human origin. In vivo, treatment with various doses of DLE in mice that were subcutaneously isotransplanted with B cell lymphoma effected less tumor growth than in untreated mice but was insufficient to control the tumor. Mice that received combination therapy of cyclophosphamide and DLE experienced less tumor growth than those that were treated with either agent alone. Further, combination therapy delayed tumor recurrence resulted in fewer cases of recurrence than cyclophosphamide. In conclusion, DLE enhance the response to conventional treatment in a syngeneic murine tumor model. Acknowledgements We thank to the Consejo Nacional de Ciencia y Tecnologia, Mexico (scholarship number 252826), the SIP Project 20120870 at the Instituto Politecnico Nacional, Mexico, and the Proyecto Factor de Transferencia, Instituto Politecnico Nacional, Mexico (Project IB-11-006) References Donnou, S., Galand, C., Touitou, V., Sautes-Fridman, C., Fabry, Z. and Fisson, S. (2012). Murine models of B-cell lymphomas: promising tools for designing cancer therapies. Adv Hematol, 2012, 701704. doi: 10.1155/2012/701704 Elpek, K. G., Lacelle, C., Singh, N. P., Yolcu, E. S. and Shirwan, H. (2007). CD4+CD25+ T regulatory cells dominate multiple immune evasion mechanisms in early but not late phases of tumor development in a B cell lymphoma model. J Immunol, 178, 6840-8. doi: 178/11/6840 [pii] Pineda, B., Estrada-Parra, S., Pedraza-Medina, B., Rodriguez-Ropon, A., Perez, R. and Arrieta, O (2005). Interstitial transfer factor as adjuvant immunotherapy for experimental glioma. J Exp Clin Cancer Res, 24, 575-83. Keywords: B cell lymphoma, Animal Models, immunomodulators, combined therapy, dialyzable leukocyte extract Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune-mediated disease pathogenesis Citation: Merchand-Reyes G, Becerril-Garcia MA, Gracia-Mora I, Estrada-Parra S, Estrada-Garcia I, Perez-Tapia SM and Sandoval-Montes C (2013). Examination of an immunoregulator in a syngeneic animal model. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01159 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 16 Jul 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Claudia Sandoval-Montes, Instituto Politecnico Nacional, Immunology, Mexico City, 11340, Mexico, claudiaqfb@yahoo.com.mx Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Giovanna Merchand-Reyes Miguel A Becerril-Garcia Isabel Gracia-Mora Sergio Estrada-Parra Iris Estrada-Garcia Sonia M Perez-Tapia Claudia Sandoval-Montes Google Giovanna Merchand-Reyes Miguel A Becerril-Garcia Isabel Gracia-Mora Sergio Estrada-Parra Iris Estrada-Garcia Sonia M Perez-Tapia Claudia Sandoval-Montes Google Scholar Giovanna Merchand-Reyes Miguel A Becerril-Garcia Isabel Gracia-Mora Sergio Estrada-Parra Iris Estrada-Garcia Sonia M Perez-Tapia Claudia Sandoval-Montes PubMed Giovanna Merchand-Reyes Miguel A Becerril-Garcia Isabel Gracia-Mora Sergio Estrada-Parra Iris Estrada-Garcia Sonia M Perez-Tapia Claudia Sandoval-Montes Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Role of Mitochondrial NCX on CXCL12-Induced Chemotaxis in A20 B Lymphocytes

Lymphocyte chemotaxis plays important roles in immunological reactions. It has been reported that mitochondria redistribute at the uropod during T lymphocyte migration triggered by a chemokine ligand (CXCL12). However, how mitochondria are involved in lymphocyte chemotaxis has been unclear. We studied roles of mitochondria Ca2+ handling proteins, Na-Ca exchange (NCXm) and Ca2+ uniporter, on CXCL12-induced chemotaxis in A20 B lymphocytes. CXCL12 (100 ng/ml) increased transwell migration of A20 cells from 4.6 ± 0.5 % (non-stimulated cells) to 12.6 ± 0.6 % (P < 0.05). This increase was dose-dependently inhibited by CGP-37157 (an inhibitor of NCXm), but not affected by Ru360 (an inhibitor of mitochondrial Ca2+ uniporter). Knock-down of a gene of NCXm (NCLX) by siRNA reduced NCLX protein expression to 52 ± 9 % and inhibited transwell migration similarly to CGP-37157. In the 8 hrs observation of cell migration under microscope, mean displacement of NCLX siRNA cells without CXCL12 was larger (21.4 ± 1.5 μm) than control siRNA cells (13.5 ± 2.2 μm, P < 0.05), and applying CXCL12 did not increase mean displacement and percentage of directional migration in NCLX siRNA cells. Intracellular Ca2+ measured by fura-2 was higher in NCLX siRNA cells (fura-2 ratio 0.49 ± 0.01) than the control siRNA cells (0.45 ± 0.01, P < 0.05) in the absence of CXCL12. After 2 hrs CXCL12 stimulation, the intracellular Ca2+ increased in control siRNA but not in NCLX siRNA cells. Treating A20 cells with BAPTA-AM (an intracellular Ca2+ chelator) suppressed the cell migration. Our results suggest that NCLX-mediated Ca2+ signaling is associated with CXCL12-induced chemotaxis in B lymphocytes.

CD28 Aptamers as Powerful Immune Response Modulators

CD28 is one of the main costimulatory receptors responsible for the proper activation of T lymphocytes. We have isolated two aptamers that bind to the CD28 receptor. As a monomer, one of them interfered with the binding of CD28 to its ligand (B7), precluding the costimulatory signal, whereas the other one was inactive. However, dimerization of any of the anti-CD28 aptamers was sufficient to provide an artificial costimulatory signal. No antibody has featured a dual function (i.e., the ability to work as agonist and antagonist) to date. Two different agonistic structures were engineered for each anti-CD28 aptamer. One showed remarkably improved costimulatory properties, surpassing the agonistic effect of an anti-CD28 antibody. Moreover, we showed in vivo that the CD28 agonistic aptamer is capable of enhancing the cellular immune response against a lymphoma idiotype and of prolonging survival of mice which receive the aptamer together with an idiotype vaccine. The CD28 aptamers described in this work could be used to modulate the immune response either blocking the interaction with B7 or enhancing vaccine-induced immune responses in cancer immunotherapy.

Rapid Screening of Novel Agents for Combination Therapy in Sarcomas

For patients with sarcoma, metastatic disease remains very difficult to cure, and outcomes remain less than optimal. Treatment options have not largely changed, although some promising gains have been made with single agents in specific subtypes with the use of targeted agents. Here, we developed a system to investigate synergy of combinations of targeted and cytotoxic agents in a panel of sarcoma cell lines. Agents were investigated alone and in combination with varying dose ratios. Dose-response curves were analyzed for synergy using methods derived from Chou and Talalay (1984). A promising combination, dasatinib and triciribine, was explored in a murine model using the A673 cell line, and tumors were evaluated by MRI and histology for therapy effect. We found that histone deacetylase inhibitors were synergistic with etoposide, dasatinib, and Akt inhibitors across cell lines. Sorafenib and topotecan demonstrated a mixed response. Our systematic drug screening method allowed us to screen a large number of combinations of sarcoma agents. This method can be easily modified to accommodate other cell line models, and confirmatory assays, such as animal experiments, can provide excellent preclinical data to inform clinical trials for these rare malignancies.

Cytokine-Mediated Programmed Proliferation of Virus-Specific CD8+ Memory T Cells

During infection, CD8(+) Tcells not only respond to antigenic signals through their Tcell receptor (TCR) but also incorporate inflammatory signals from cytokines produced in the local infected microenvironment. Transient TCR-mediated stimulation will result in programmed proliferation that continues despite removal of the antigenic stimulus, but it remains unclear whether brief exposure to specific cytokines will elicit similar effects. Here, we have demonstrated that brief stimulation of memory Tcells with interleukin-12 (IL-12) and interleukin-18 (IL-18) results in tightly regulated programmed proliferation, in addition to acquisition of enhanced virus-specific cytokine production and cytolytic activity. CD8(+) Tcells briefly exposed to IL-12 and IL-18 invitro showed improved antiviral activity invivo, as demonstrated by increased proliferation and reduced viremia. These results indicate that even transitory exposure to inflammatory cytokines can provide a selective advantage to infiltrating CD8(+) Tcells by triggering a developmental program that is initiated prior to direct contact with virus-infected cells.

Interruption of IFNγR Signaling Results in Altered T Cell Trafficking in Vivo and Abrogation of GvHD While Maintaining a Robust Gvl Response

Abstract 455Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment for patients with relapsed/refractory leukemia, and marrow failure states such as myelodysplasia and aplastic anemia. However, allo-HSCT is complicated by allogeneic donor T cell-mediated graft-versus-host disease (GvHD) which can be life-threatening especially in recipients of unrelated or HLA-mismatched hematopoietic stem cell products. These same alloreactive donor T cells also mediate a beneficial graft-versus-leukemia (GvL) effect. Thus, the clinical goal in allo-HSCT is to minimize GvHD while maintaining GvL. Recent studies have suggested that this might be achieved by infusing regulatory T cells (Tregs) which in some preclinical models suppress GvHD-causing alloreactive donor T cells but have only limited effects on GvL-promoting alloreactive donor T cells. Unfortunately, Tregs exist in low frequency in the peripheral blood, are costly to purify and expand, and after expansion are difficult to isolate due to the lack of cell surface markers, all of which prevent their routine use in the clinic. Thus, alternative therapeutic approaches that do not require Tregs are needed.We have found that interferon gamma receptor deficient (IFNγR−/−) allogeneic donor T cells induce significantly less GvHD in both a MHC fully-mismatched (B6 (H-2b) → Balb/c (H-2d)) and a minor-mismatched (B6 (H-2b) → B6×129(H-2b)) allo-HSCT models compared to WT T cells. In addition, IFNγR−/− donor T cells maintain a beneficial GvL effect, which has been examined in both systemic leukemia and solid tumor models using luciferase-expressing A20 cells derived from Balb/c. We find that IFNγR−/− T cells migrate primarily to the spleen while WT T cells to GI tract and peripheral lymph nodes (LNs) using bioluminescence imaging (BLI), suggesting that altered T cell trafficking of IFNγR−/− T cells to GvHD target organs might be the major reason for the reduced GvHD. We further demonstrate that the IFNγR-mediated signaling in alloreactive donor T cells is required for expression of CXCR3 which has been implicated in trafficking of T cells to areas of inflammation and target organs, commonly known to be the sites of GvHD. Indeed, CXCR3−/− T cells recapitulate the reduced GvHD potential of IFNγR−/− T cells. In addition, forced overexpression of CXCR3 in IFNγR−/− T cells via retroviral transduction partially rescues the GvHD defect observed in IFNγR−/− T cells. We next examine if inhibition of IFNγR signaling using a small molecule inhibitor can recapitulate the anti-GVHD effects seen in IFNγR−/− T cells. We find that INCB018424, an inhibitor of JAK1/JAK2 which are the mediators of IFNγR signaling, blocks CXCR3 expression in vitro. Most importantly, in vivo administration of INCB018424 after allo-HSCT alters T cell trafficking and significantly reduces GvHD. Thus, the IFNγR signaling pathway represents a promising therapeutic target for future efforts to mitigate GvHD while maintaining GvL after allo-HSCT. Moreover, this pathway can be exploited in other diseases besides GvHD such as those from organ transplantation, chronic inflammatory diseases and autoimmune diseases. Disclosures:DiPersio:genzyme: Honoraria.

Myeloid Suppressive Cells Mobilized by GM-CSF in Non-Tumor Bearing Mice Are Dependent On Interferon Gamma for Function

Abstract 832Myeloid-derived-suppressor cells (MDSCs) are enriched in tumors, and exist to a lesser extent in the blood, spleen and bone marrow of tumor bearing mice. Monocytic MDSCs (monoMDSCs) suppress CD8+ T-cells via expression of Arginase 1 (ARG1) and inducible nitric oxide synthase (iNOS). Tumor derived factors are critical to the maintenance of MDSCs and preventing differentiation to mature macrophages and dendritic cells. GM-CSF is a hematopoietic cytokine that can be secreted by tumors and promotes MDSC generation. Cells phenotypically similar to MDSCs can be isolated from blood of normal individuals but lack suppressive function. Hematopoietic peripheral blood stem cell mobilization with G-CSF and GM-CSF enriches for cells phenotypically similar to MDSCs. There is limited data on the role and function of these cells isolated from non-tumor bearing, normal individuals. Recent evidence suggests that graft-versus-host disease (GvHD) can be abrogated in mice by ex vivo expanded, bone marrow derived, MDSCs generated in the presence of GM-CSF, G-CSF and IL-13 (Highfill et al. Blood 2010 116:5738). It remains to be shown whether phenotypic MDSCs identified in non-tumor bearing mice are capable of immune suppression; and, the mechanism by which an immature myeloid cell becomes a functional MDSC remains unknown. We have observed an increase (up to 8 fold) in a population of cells phenotypically resembling monoMDSCs (CD11b+/Ly6C+/Ly6G-) in the spleens and blood of mice mobilized with pegylated-murine-GM-CSF (peg-mGM-CSF). We hypothesized that this population of cells would have suppressive function similar to MDSCs in vitro and in vivo, and may have the potential to abrogate graft-versus-host disease (GvHD). To investigate the function of MDSCs found in spleens of C57/Bl6 (B6) mice treated with peg-mGM-CSF we performed CFSE based anti-CD3/CD28 antibody stimulated T-cell proliferation assays, mixed leukocyte reactions and transwell assays. We observed that CD11b+Ly6C+Ly6G- cells isolated from spleens of mice treated with peg-mGM-CSF have potent suppressive function in vitro that is contact dependent and abrogated by blocking ARG1 or iNOS. This suppressive effect was lost in APC stimulated MLRs using B6 T-cells and Balb/C stimulators (confirmed in two separate experiments). Furthermore, the in vivo potential of these putative MDSCs to abrogate murine GvHD was investigated using a B6 to Balb/C donor leukocyte infusion GvHD model. Adoptive transfer of purified splenic CD11b+Ly6C+Ly6G- cells from peg-mGM-CSF mobilized B6 donors along with an equivalent number of congenic T-cells failed to abrogate GvHD. We investigated timing of MDSC infusion in the B6 to Balb/C GvHD model and found no improvement in weight loss, GvHD score or survival in mice receiving 5×105 monoMDSCs IV on day 1, 6 or 10 after transplant compared to T-cells alone control (n = 5 – 10/group, Log rank, p= NS). To address in vivo function further in a bioluminescent imaging (BLI) tumor model. Balb/C recipients were injected SC with A20 cells mixed +/− monoMDSCs at a 1:10 ratio after lethal irradiation and T-cell deplete bone marrow on day 0. Donor T-cells were infused at day +11. The rate of tumor growth measured by photon flux was the same between subcutaneous tumors either with or without monoMDSCs. (two separate experiments, 5 mice/group). This in vivo data suggested that a critical factor present in vitro might be lacking or insufficient in vivo. To investigate the critical factor(s) present in vitro we performed T-cell proliferation assays in the presence of blocking antibodies against IFNy, TNFalpha, IL-10, GM-CSF and CD154. Only neutralization of IFNy resulted in negation of the suppressive effects of these cells. To investigate the source of IFNy production we used transgenic IFNy knockout mice as T-cell and MDSC donors. Proliferation of IFNy deficient T-cells was suppressed efficiently by wild-type (WT) MDSCs, and, neutralizing IFNy using a blocking antibody negated suppression. This suggested IFNy production by a cell within the putative MDSC sorted population might be critical for MDSC function. IFNy deficient peg-mGM-CSF mobilized CD11b+Ly6C+Ly6G- spleen cells failed to suppress WT or IFNy deficient T-cell proliferation. These results suggest a critical role for IFNy production by CD11b+Ly6C+Ly6G- myeloid cells in maintaining their suppressive phenotype in vitro and perhaps in vivo. Disclosures:No relevant conflicts of interest to declare.

The NLRP4-DTX4 axis: a key suppressor of TBK1 and innate antiviral signaling

The NLRP4-DTX4 axis: a key suppressor of TBK1 and innate antiviral signaling

Murine Gammaherpesvirus 68 LANA Acts on Terminal Repeat DNA To Mediate Episome Persistence

Murine gammaherpesvirus 68 (MHV68) ORF73 (mLANA) has sequence homology to Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA). LANA acts on the KSHV terminal repeat (TR) elements to mediate KSHV episome maintenance. Disruption of mLANA expression severely reduces the ability of MHV68 to establish latent infection in mice, consistent with the possibility that mLANA mediates episome persistence. Here we assess the roles of mLANA and MHV68 TR (mTR) elements in episome persistence. mTR-associated DNA persisted as an episome in latently MHV68-infected tumor cells, demonstrating that the mTR elements can serve as a cis-acting element for MHV68 episome maintenance. In some cases, both control vector and mTR-associated DNAs integrated into MHV68 episomal genomes. Therefore, we also assessed the roles of mTRs as well as mLANA in the absence of infection. DNA containing both mLANA and mTRs in cis persisted as an episome in murine A20 or MEF cells. In contrast, mTR DNA never persisted as an episome in the absence of mLANA. mLANA levels were increased when mLANA was expressed from its native promoters, and episome maintenance was more efficient with higher mLANA levels. Increased numbers of mTRs conferred more efficient episome maintenance, since DNA containing mLANA and eight mTR elements persisted more efficiently in A20 cells than did DNA with mLANA and two or four mTRs. Similar to KSHV LANA, mLANA broadly associated with mitotic chromosomes but relocalized to concentrated dots in the presence of episomes. Therefore, mLANA acts on mTR elements to mediate MHV68 episome persistence.

Atypical fluoroquinolone gold(III) chelates as potential anticancer agents: Relevance of DNA and protein interactions for their mechanism of action

Quinolones are known for their antimicrobial and antitumor activities. Gold(III) compounds constitute an emerging class of biologically active substances, of special interest as potential anticancer agents. In this work three gold(III) complexes of the fluoroquinolones antimicrobial agents norfloxacin (NOR), levofloxacin (LEVO) and sparfloxacin (SPAR) were prepared and characterized with physicochemical and spectroscopic techniques. In these complexes, NOR, LEVO and SPAR act as bidentate neutral ligands bound to gold(III) through the nitrogen atoms of the piperazine ring, which is an unusual mode of coordination for this class of compounds. Two chloride ions occupy the remaining coordination sites.The cytotoxic activity of the fluoroquinolones and their gold(III) complexes was tested against the A20 (murine lymphoma), B16-F10 (murine melanoma) and K562 (human myeloid leukemia) tumor cell lines as well as the L919 (murine lung fibroblasts) and MCR-5 (human lung fibroblasts) normal cells lines. All complexes were more active than their corresponding free ligands. Complex [AuCl2(LEVO)]Cl was selected for DNA fragmentation and cell cycle analysis.Spectroscopic titration with calf-thymus DNA (CT DNA) showed that the complexes can bind weakly to CT DNA, probably by an external contact (electrostatic or groove binding). The complexes exhibit good binding propensity to bovine serum albumin (BSA) having relatively high binding constant values.

Natural killer cells can exert a graft-vs-tumor effect in haploidentical stem cell transplantation for pediatric solid tumors

Little progress has been made with regard to the survival of children with metastatic and refractory solid tumors. Preliminary data from haploidentical stem cell transplantation (haplo-SCT) suggested a clinically beneficial allograft-vs-tumor effect associated with natural killer cell (NK) donor-recipient mismatch. We hypothesized that interaction between activatory receptors on NK cells and their ligands on tumor cells could be also important. To evaluate the NK-cell-mediated allograft-vs-tumor effect, we conducted a pilot study of haplo-SCT on six children with refractory solid tumors. Our specific goal for this study was NKG2D-major histocompatibility complex class I-related chain A interaction. Tasks include specific immunoassays that support haplo-SCT in refractory solid tumors. Patients suffered from neuroblastoma (n = 1), Ewing sarcoma (n = 2), a desmoplastic tumor (n = 1), nasopharyngeal carcinoma (n = 1), and embryonal rhabdomyosarcoma (n = 1). Pretransplantation disease status showed progressive disease in 2 patients, partial remission in 2 patients, and complete remission in 2 patients. NK-cell mismatch was present in three donor-recipients. Ligands for NKG2D receptors, major histocompatibility complex class I-related chain A and UL16 binding protein 2 were overexpressed in six of six and four of six tumors, respectively. NK cells led early immune reconstitution. After haplo-SCT, three patients were in complete remission, one patient showed partial remission, and two patients were in stable disease. With a median follow-up of 14 months, three patients were alive and in complete remission, and three patients had died; two due to progressive disease and one of transplant-related toxicity. Blocking NKG2D-major histocompatibility complex class I-related chain A interaction in vitro reduced NK-cell cytotoxicity. Our preliminary results suggest a beneficial effect from haplo-SCT in refractory solid tumors.

Effects of ICOSLG expressed in mouse hematological neoplasm cell lines in the GVL reaction

As a member of the B7 family, inducible co-stimulator ligand (ICOSLG) expressed on tumor cell has been reported to have an important role in tumor immunity. In this study, we sought to determine whether the expression of ICOSLG in mouse hematological malignancy cells influences GVL reaction after mouse allogeneic BMT. In our study, we analyzed the expression of ICOSLG in six mice hematological malignancy cell lines for the first time, and found that FBL3, A20 and P388 cells expressed high levels of ICOSLG. Then, we chose A20 cells as targets to construct a GVL model and study the effects on the GVL reaction by silencing the ICOSLG gene. The survival was analyzed. We found that in GVL model, mortality of interference groups was significantly delayed compared with the control group (P=0.0005). Our results indicate that knockdown of ICOSLG of mouse leukemic cells may significantly enhance GVL effect after allogeneic BMT.

Proinsulin C-peptide activates α-enolase: implications for C-peptide–cell membrane interaction

Proinsulin C-peptide shows beneficial effects on microvascular complications of Type 1 diabetes. However, the possible occurrence of membrane C-peptide receptor(s) has not been elucidated. The aim of this study was to identify and characterize membrane proteins to which C-peptide binds. The enzyme α-enolase was co-immunoprecipitated with C-peptide after chemical cross-linking to HL-60 cell surface proteins and identified by mass spectrometry. Recombinant α-enolase activity was modulated by C-peptide, with a significant decrease in K(m) for 2-phosphoglycerate without affecting V(max). The enzyme modulation by C-peptide was abolished when C-terminal basic lysine residue (K434) of the enzyme was replaced by neutral alanine or acidic glutamate, but not with basic arginine. The enzyme modulation by C-peptide was reproduced with the C-peptide fragments containing glutamate corresponding to position 27 (E27) of the full-length C-peptide. Addition of a lysine analogue to the assay and A31 cell culture abrogated the enzyme modulation and MAP kinase activation by C-peptide, respectively. The results indicate that C-peptide has the capacity to activate α-enolase through a specific interaction between E27 of the peptide and K434 of the enzyme. Since α-enolase plays a role as a cell surface receptor for plasminogen, it may conceivably also serve as a receptor for C-peptide in vivo.

Macrophage Inflammatory Protien-2/CXCR2 Blockade Attenuates Acute Graft-versus-Host Disease while Preserving Graft-versus-Leukemia Activity (126.16)

Abstract Graft-versus-host disease (GVHD) is the principal complication after allogeneic bone marrow transplantation (BMT). Strategies that effectively separate the beneficial GVL effects from GVHD toxicity are necessary for enhanced survival after BMT. GVHD mice were induced by infusion of both donor BM cells and spleen cells into BU-CY conditioned recipient female BALB/c mice. Tumor-bearing GVHD mice were also made adding A20 cells. A total of 31 cytokines and chemokines were measured in the liver, intestine, and skin in GVHD mice by Luminex Multiplex assay. Four cytokines, G-CSF, KC, MIP-2, and IL-23, were commonly unregulated in target organs at the initial GVHD stage and were reduced by neutralizing MIP2/CXCR2 axis ligation. CXCR2 blockade on donor T cells and systemic administration of anti-MIP-2 neutralizing antibodies to BMT recipients caused a significant decrease in both systemic GVHD and target organ histopathology. GVL responses (BCL2 expression or B220+/CD19+ cells in BM and PB) after MIP-2/CXCR2 blockade were largely preserved and mortality was significantly delayed compared to control allogeneic BMT recipients. These data suggest that blocking the MIP-2/CXCR2 axis represents a novel strategy to separate the toxicity of GVHD from the beneficial GVL effects after allogeneic BMT.

Expression of ICOSLG on Mouse Hematologic Neoplasm Cell Lines and Their Influence on Cytotoxicity in Allogeneic Mixed Lymphocyte Reactions

In this study, we sought to determine whether the expression of inducible co-stimulator ligand (ICOSLG) on mouse hematologic neoplasm cells influences cytotoxicity. Using flow cytometry, we examined the expression of ICOSLG in mouse hematologic neoplasm cell lines for the first time and used FBL3/A20 as targets to study. Effectors and targets were incubated at effector:target (E:T) ratios ranging from 5:1 to 50:1 for 10 h. Enzyme-linked immunosorbent assay was used to assess the expression of cytokines associated with type 2 T helper cells. We found that FBL3, A20 and P388 cells expressed ICOSLG. At E:T ratios ranging from 20:1 to 30:1, the cytotoxic activity of alloreactive T cells was significantly increased when ICOSLG was blocked (p < 0.05). We also found that ICOSLG blockade was associated with decreased expression of interleukin 4 and interleukin 10. Our results indicate that ICOSLG blockade may enhance cytotoxity in allogeneic mixed lymphocyte–hematologic neoplasm cell reactions.