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Hemophagocytic lymphohistiocytosis (HLH) comprises an emerging spectrum of inherited and noninherited disorders of the immune system characterized by the excessive production of cytokines, including interferon-γ and interleukins 2, 6, and 10 (IL-2, IL-6, and IL-10). The Janus kinases (JAKs) transduce signals initiated following engagement of specific receptors that bind a broad array of cytokines, including those overproduced in HLH. Based on the central role for cytokines in the pathogenesis of HLH, we sought to examine whether the inhibition of JAK function might lessen inflammation in murine models of the disease. Toward this end, we examined the effects of JAK inhibition using a model of primary (inherited) HLH in which perforin-deficient (Prf1(-∕-)) mice are infected with lymphocytic choriomeningitis virus (LCMV) and secondary (noninherited) HLH in which C57BL/6 mice receive repeated injections of CpG DNA. In both models, treatment with the JAK1/2 inhibitor ruxolitinib significantly lessened the clinical and laboratory manifestations of HLH, including weight loss, organomegaly, anemia, thrombocytopenia, hypercytokinemia, and tissue inflammation. Importantly, ruxolitinib treatment also significantly improved the survival of LCMV-infectedPrf1(-∕-)mice. Mechanistic studies revealed that in vivo exposure to ruxolitinib inhibited signal transducer and activation of transcription 1-dependent gene expression, limited CD8(+)T-cell expansion, and greatly reduced proinflammatory cytokine production, without effecting degranulation and cytotoxic function. Collectively, these findings highlight the JAKs as novel, druggable targets for mitigating the cytokine-driven hyperinflammation that occurs in HLH. These observations also support the incorporation of JAK inhibitors such as ruxolitinib into future clinical trials for patients with these life-threatening disorders.

Oncolytic viruses (OVs) do more than simply infect and kill host cells. The accepted mechanism of action for OVs consists of a primary lytic phase and a subsequent antitumor and antiviral immune response. However, not all cells are subject to the direct effects of OV therapy, and it is becoming clear that OVs can also impact uninfected cells in the periphery. This review discusses the effects of OVs on uninfected neighboring cells, so-called bystander effects, and implications for OV therapies alone or in combination with other standard of care chemotherapy.

High Mobility Group Box 1 (HMGB1) is a multifunctional protein that plays various roles in the processes of inflammation, cancer, and other diseases. Many reports document abundant HMGB1 release following infection with oncolytic viruses (OVs). Further, other groups including previous reports from our laboratory highlight the synergistic effects of OVs with chemotherapy drugs. Here, we show that virus-free supernatants have varying cytotoxic potential, and HMGB1 is actively secreted by two established fibroblast cell lines (NIH 3T3 and 3T6-Swiss albino) following HSV1716 infection in vitro. Further, pharmacologic inhibition or genetic knock-down of HMGB1 reveals a role for HMGB1 in viral restriction, the ability to modulate bystander cell proliferation, and drug sensitivity in 3T6 cells. These data further support the multifactorial role of HMGB1, and suggest it could be a target for modulating the efficacy of oncolytic virus therapies alone or in combination with other frontline cancer treatments.

Dendritic cells (DCs) are antigen presenting cells capable of inducing specific immune responses against microbial infections, transplant antigens, or tumors. DCs have been shown to possess a high plasticity showing different phenotypes in response to their microenvironment. For example, tumor-associated DCs can acquire an angiogenic phenotype thus promoting tumor growth. Further, DCs cultured in vitro under different conditions are able to upregulate the expression of endothelial markers and to express angiogenic factors. Indeed, it has been shown that soluble factors such as VEGF of PGE-2, that are present in the microenvironment of several tumors, affect the biology of these cells. We hypothesize that in addition to soluble factors the adhesion to different substrates will also define the phenotype and function of DCs. Herewith we demonstrate that murine myeloid(m) DCs upregulate endothelial markers such as VE-Cadherin, and to a lesser extent TIE-2, and decrease their immune capabilities when cultured on solid surfaces as compared with the same cells cultured on ultra-low binding (ULB) surfaces. On the other hand, the expression of angiogenic molecules at the level of RNA was not different among these cultures. In order to further investigate this phenomenon we used the murine ID8 model of ovarian cancer which can generate solid tumors when cancer cells are injected subcutaneously or a malignant ascites when they are injected intraperitoneally. This model gave us the unique opportunity to investigate DCs in suspension or attached to solid surfaces under the influence of the same tumor cells. We were able to determine that DCs present in solid tumors showed higher levels of expression of endothelial markers and angiogenic molecules but were not able to respond to inflammatory stimuli at the same extent as DCs recovered from ascites. Moreover, mDCs cultured on ULB surfaces in the presence of tumor factors do not expressed endothelial markers. Taking into account all these data we consider that tumor factors might be responsible for inducing angiogenic properties in DCs, but that in some settings the expression of endothelial markers such as VE-Cadherin and TIE-2 might be a function of attachment to solid surfaces and independent of the angiogenic properties of these cells.

Ovarian cancer (OC) is an aggressive disease that affects approximately 1 in 70 women and has a poor prognosis (

Aurora A Kinase (AURKA) inhibition with the investigational agent alisertib results in abnormal mitotic progression and the induction of apoptosis in multiple tumor cell lines. Cell cycle arrest is a strategy employed by a variety of viruses, including herpes simplex virus (HSV), to bolster viral reproduction. We previously demonstrated the anti-tumor efficacy of oncolytic HSV and alisertib as monotherapies in models of malignant peripheral nerve sheath tumor (MPNST). We then proceeded to test alisertib and the oncolytic HSV Seprehvir in combination, finding a synergistic anti-tumor effect between the two therapies. Considering this, we hypothesized that alisertib potentiates Seprehvir infection, leading to more rapid and robust viral proliferation with concomitant tumor regression. However, our efforts to quantify enhanced viral infection with combination therapy have uncovered no significant differences in Seprehvir viral growth kinetics witih or without alisertib therapy. Therefore, the synergistic tumor response of Seprehvir combined with alisertib in vitro or in vivo is not due to augmented viral infection. We then hypothesized that Seprehvir infection potentiates the anti-tumor effect of alisertib. In line with this hypothesis, there is a signficant decrease in tumor cell proliferation in alisertib-treated tumor cells that are exposed to soluble factors released by Seprehvir-infected cells. Furthermore, by UV inactivation of Seprehvir conditioned media, we demonstrate that these soluble death signals are not replication competent virus released by infected cells. Taken together, our results suggest a novel mechanism through which Seprehvir-infected cells release a paracrine signal to surrounding cells promoting cell death and increasing the anti-tumor activity of alisertib therapy.

Many clinical trials have been carried out or are in progress to assess the therapeutic potential of dendritic-cell- (DC-) based vaccines on cancer patients, and recently the first DC-based vaccine for human cancer was approved by the FDA. Herewith, we describe the general characteristics of DCs and different strategies to generate effective antitumor DC vaccines. In recent years, the relevance of the tumor microenvironment in the progression of cancer has been highlighted. It has been shown that the tumor microenvironment is capable of inactivating various components of the immune system responsible for tumor clearance. In particular, the effect of the tumor microenvironment on antigen-presenting cells, such as DCs, does not only render these immune cells unable to induce specific immune responses, but also turns them into promoters of tumor growth. We also describe strategies likely to increase the efficacy of DC vaccines by reprogramming the immunosuppressive nature of the tumor microenvironment.

e20035 Background: High dose interferon-alpha-2b (IFNa) is the only approved adjuvant therapy after surgery for high-risk melanoma lesions. The side-effects of standard year-long, high dose IFNa th...

Background Dendritic cells (DCs) are antigen presenting cells capable of inducing specific immune responses against microbial infections, transplant antigens, or tumors. Interestingly, microenvironment conditions such as those present in tumor settings might induce a DC phenotype that is poorly immunogenic and with the capability of promoting angiogenesis. We hypothesize that this plasticity may be caused not only by the action of specific cytokines or growth factors but also by the properties of the surfaces with which they interact, such as extracellular matrix (ECM) components. Results Herewith we studied the effect of different surfaces and soluble factors on the biology of DCs. To accomplish this, we cultured murine myeloid(m) DCs on surfaces coated with fibronectin, collagen I, gelatin, and Matrigel using poly-D-lysine and polystyrene as non-biological surfaces. Further, we cultured these cells in the presence of regular DC medium (RPMI 10% FBS) or commercially available endothelial medium (EGM-2). We determined that mDCs could be kept in culture up to 3 weeks in these conditions, but only in the presence of GM-CSF. We were able to determine that long-term DC cultures produce an array of angiogenic factors, and that some of these cultures still retain the capability to induce T cell responses. Conclusions Altogether these data indicate that in order to design DC-based vaccines or treatments focused on changing the phenotype of DCs associated with diseases such as cancer or atherosclerosis, it becomes necessary to fully investigate the microenvironment in which these cells are present or will be delivered.