The oncolytic features of several naturally oncolytic viruses have been shown on Glioblastoma Multiforme cell lines and in xenotransplant models. However, orthotopic glioma studies in immunocompetent animals are lacking. Here we investigated Newcastle disease virus (NDV) in the orthotopic, syngeneic murine GL261 model. Seven days after tumor induction, mice received NDV intratumorally. Treatment significantly prolonged median survival and 50% of animals showed long-term survival. We demonstrated immunogenic cell death (ICD) induction in GL261 cells after NDV infection, comprising calreticulin surface exposure, release of HMGB1 and increased PMEL17 cancer antigen expression. Uniquely, we found absence of secreted ATP. NDV-induced ICD occurred independently of caspase signaling and was blocked by Necrostatin-1, suggesting the contribution of necroptosis. Autophagy induction following NDV infection of GL261 cells was demonstrated as well. In vivo, elevated infiltration of IFN-c 1 T cells was observed in NDV-treated tumors, along with reduced accumulation of myeloid derived suppressor cells. The importance of a functional adaptive immune system in this paradigm was demonstrated in immunodeficient Rag2 2/2 mice and in CD8 1 T cell depleted animals, where NDV slightly prolonged survival, but failed to induce long-term cure. Secondary tumor induction with GL261 cells or LLC cells in mice surviving long-term after NDV treatment, demonstrated the induction of a long-term, tumor-specific immunological memory response by ND virotherapy. For the first time, we describe the therapeutic activity of NDV against GL261 tumors, evidenced in an orthotopic mouse model. The therapeutic effect relies on the induction of ICD in the tumor cells, which primes adaptive antitumor immunity.Glioblastoma Multiforme (GBM) is a WHO grade IV neoplasm and the most frequent primary brain tumor in adults. 1 Current multimodal treatment consists of maximal surgical resection of the tumor, radiochemotherapy and maintenance chemotherapy. Despite great advances in these fields, the prognosis of GBM patients remains dismal and relapse

Multiply-transfused individuals are at higher risk for BM rejection. We show that whereas allosensitization resulted in the priming of both cellular and humoral immunity, preformed antibody was the major barrier to engraftment. The generation of cross-reactive alloantibody led to rejection of BM of a different MHC-disparate strain. Imaging studies indicated that antibody-mediated rejection was very rapid (< 3 hours) in primed recipients, while T-cell-mediated rejection in nonprimed mice took more than 6 days. Antibody-

The immunomodulator FTY720 (FTY) has been shown to be beneficial in experimental models of organ transplantation and autoimmunity. We show that FTY significantly inhibited but did not prevent graftversus-host disease (GVHD) in lethally irradiated or nonirradiated allogeneic recipients. Although most studies implicate prevention of lymphocyte egress from lymphoid organs as the primary mechanism of action, our data indicate that FTY effects on the host are more likely to be responsible for GVHD inhibition. IntroductionDespite advances in the field, graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality in patients undergoing bone marrow transplantation (BMT). In this study, we examine the effect of FTY720 (FTY), a potent immunomodulator, in a murine model of GVHD.FTY, derived from a metabolite of the fungus Isaria sinclairii, is a high-affinity agonist for 4 of the 5 known sphingosine 1-phosphate receptors (S1PRs). S1P 1 is expressed on all cell types, while S1P 3 is found in endothelial cells, S1P 4 is found primarily on lymphoid cells, and S1P 5 is expressed on the white matter of the central nervous system. [1][2][3][4] These receptors are critically involved in cell survival, cytoskeletal rearrangements, cell motility, and cell migration. [1][2][3][4] FTY induces internalization of the receptor, rendering the cells unresponsive to serum lipid S1P that is produced by platelets. 1,5,6 It is generally accepted that FTY exerts its immunomodulatory effects primarily by sequestering lymphocytes within secondary lymphoid organs, thereby denying them the ability to recirculate to peripheral sites of inflammation. FTY is also thought to act on endothelial cells by enhancing the adherens junction assembly, thereby strengthening the endothelial barrier. 7 More recent data indicate that in addition to exhibiting profound effects on lymphocyte migration and endothelial barrier integrity, FTY also modulates dendritic cell (DC) trafficking and function. [8][9][10][11] Accumulating data in animal studies indicate that FTY is a promising immunosuppressive agent for the treatment of various autoimmunities, promotion of engraftment in several models of solid organ transplantation (reviewed in Brinkmann, 1 Brinkmann and Lynch, 2 Chiba, 3 Yopp et al, 4 and Brinkmann et al 12 ), and inhibition of GVHD. [13][14][15] Although the complete mechanism of FTY is not completely understood, most studies implicate an important role for lymph node (LN) trapping as a mechanism of action. It is generally accepted that sequestration of effector T cells in secondary lymphoid organs is associated with a decrease in T-cell migration and infiltration to sites of inflammation, solid organ grafts, or GVHD target organs, thereby ameliorating autoimmunity, solid organ graft rejection, or GVHD.Our studies indicate that FTY inhibits but does not prevent GVHD. In contrast to other studies, we did not find that FTY sequestered effector T cells to secondary lymphoid organs, thereby reducing their migration to GVHD target or...

IntroductionCytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG ODNs) are synthetic oligodeoxynucleotides with unmethylated DNA sequences that mimic viral and bacterial DNA and are recognized by Toll-like receptor 9 (TLR9), a pattern-recognition receptor, expressed in certain innate immune cells, including dendritic cell (DC) subsets, macrophages, monocytes, neutrophils, and natural killer (NK) cells, and also in activated CD4 ϩ T cells and B cells. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] CpG ODNs are known to trigger innate immune activation, resulting in the expression of costimulatory molecules, resistance to apoptosis, and the secretion of T helper 1 (Th1)-promoting chemokines and cytokines, including macrophage inflammatory protein-1, IFN-inducible protein-10, and other IFNinducible proteins. 9,[18][19][20][21][22][23][24] This innate immune activation typically is followed by the generation of potent adaptive immune responses. Immunostimulatory CpG ODNs have been shown to protect against a broad range of viruses, bacteria, intracellular parasites, prions, immune disorders, and tumors in numerous animal and human models of disease (reviewed in Krieg 9,25 ).Our previous studies showed that CpG ODNs were highly effective in reducing tumor-related mortality in murine recipients of syngeneic bone marrow transplants and acute myeloid leukemia. 26 However, although CpG ODNs increased the graft-versusleukemia effect of delayed lymphocyte infusion (DLI) in allogeneic bone marrow transplantation (BMT) recipients, resulting in enhanced survival, CpG ODNs also increased DLI-induced graftversus-host disease (GVHD) as indicated by clinical appearance and weight curves. 26 The present study was undertaken to directly determine the effects of CpG ODNs on donor antihost alloresponses that can culminate in GVHD and also on host antidonor alloresponses that can culminate in allogeneic BM graft rejection. We found that CpG ODNs, administered at the time of transplantation, accelerated GVHD lethality by TLR9 ligation of host antigen-presenting cells (APCs). In addition, we demonstrated that CpG ODNs promoted allogeneic BM rejection by TLR9 ligation of donor BM APCs. CpG ODN-mediated BM rejection occurred independently of host TLR9 ligation. An important and unexpected result of these studies was the finding that donor BM APCs modulate the strength of the rejection process. These data give new insight into the mechanisms of GVHD and allogeneic BM rejection and suggest that donor BM APCs may be a potential therapeutic target for the enhancement of alloengraftment in BMT. MethodsMice BALB/c (H2 d ) and C57BL/6 (H2 b ; termed B6) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) or the National Institutes of Submitted September 19, 2007; accepted June 27, 2008. Prepublished online as Blood First Edition paper, July 9, 2008; DOI 10.1182 DOI 10. /blood-2007 An Inside Blood analysis of this article appears at the front of this issue.The publication costs of this article were defrayed in...

The RAS-RAF-MEK-ERK pathway is deregulated in over 90% of malignant melanomas and targeting MEK as central kinase of this pathway is currently tested in clinical trials. However, dose-limiting side effects are observed, and MEK inhibitors that sufficiently reduce ERK activation in patients show a low clinical response. Apart from dose-limitations, a reason for the low response to MEK targeting drugs is thought to be the up-regulation of counteracting signalling cascades as a direct response to MEK inhibition. Therefore, understanding the biology of melanoma cells and the effects of MEK inhibition on these cells will help to identify new combinatorial approaches that are more potent and allow for lower concentrations of drug being used. We have discovered that in melanoma cells MEK inhibition by selumetinib (AZD6244, ARRY-142886) or PD184352 while efficiently suppressing proliferation stimulates increased invasiveness. Inhibition of MEK suppresses actin-cortex contraction and increases integrin-mediated adhesion. Most importantly, and surprisingly MEK inhibition results in a significant increase in MMP-2 and MT1-MMP expression. All together MEK inhibition in melanoma cells induces a ‘mesenchymal’ phenotype that is characterised by protease driven invasion. This mode of invasion is dependent on integrin-mediated adhesion, and because SRC kinases are main regulators of this process, the SRC kinase inhibitor saracatinib (AZD0530) completely abolished the MEK inhibitor induced invasion. Moreover, the combination of saracatinib and selumetinib effectively suppressed the growth and invasion of melanoma cells in a 3D environment, suggesting that combined inhibition of MEK and SRC is a promising approach to improve the efficacy of targeting the ERK/MAP kinase pathway in melanoma.