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Gross histological examination of B16/F10 tumors treated with MYXV

Efficacy of vPD1 therapy in the Lewis Lung Carcinoma model

Legends for supplemental figures.

Elimination of PDL1 expression from B16/F10 tumor cells.

Oncolytic virotherapy represents an attractive option for the treatment of a variety of aggressive or refractory tumors. While this therapy is effective at rapidly debulking directly injected tumor masses, achieving complete eradication of established disease has proven difficult. One method to overcome this challenge is to use oncolytic viruses to induce secondary antitumor immune responses. Unfortunately, while the initial induction of these immune responses is typically robust, their subsequent efficacy is often inhibited through a variety of immunoregulatory mechanisms, including the PD1/PDL1 T-cell checkpoint pathway. To overcome this inhibition, we generated a novel recombinant myxoma virus (vPD1), which inhibits the PD1/PDL1 pathway specifically within the tumor microenvironment by secreting a soluble form of PD1 from infected cells. This virus both induced and maintained antitumor CD8+ T-cell responses within directly treated tumors and proved safer and more effective than combination therapy using unmodified myxoma and systemic αPD1 antibodies. Localized vPD1 treatment combined with systemic elimination of regulatory T cells had potent synergistic effects against metastatic disease that was already established in secondary solid organs. These results demonstrate that tumor-localized inhibition of the PD1/PDL1 pathway can significantly improve outcomes during oncolytic virotherapy. Furthermore, they establish a feasible path to translate these findings against clinically relevant disease. Cancer Res; 77(11); 2952–63. ©2017 AACR.

Immunological response of mice eradicated of B16/F10 tumors by vPD1

Efficacy and Specificity of antibody depletion in vivo.

Effects of immune subset depletion on the efficacy of vGFP therapy

Expression of PDL1 on B16/F10 tumor cells in vivo following MYXV treatment.

Potassium (K(+)) is one of the most abundant cations in the human body. Under normal conditions, the vast majority of K(+) is found within cells, and the extracellular [K(+)] is tightly regulated to within 3.0 to 5.0 mM. However, it has recently been shown that high levels of localized necrosis can increase the extracellular concentration of K(+) to above 50 mM. This raises the possibility that elevated extracellular K(+) might influence a variety of biological processes that occur within regions of necrotic tissue. For example, K(+) has been shown to play a central role in the replication cycles of numerous viral families, and in cases of lytic infection, localized regions containing large numbers of necrotic cells can be formed. Here, we show that the replication of the model poxvirus myxoma virus (MYXV) is delayed by elevated levels of extracellular K(+). These increased K(+) concentrations alter the cellular endocytic pathway, leading to increased phagocytosis but a loss of endosomal/lysosomal segregation. This slows the release of myxoma virus particles from the endosomes, resulting in delays in genome synthesis and infectious particle formation as well as reduced viral spread. Additionally, mathematical modeling predicts that the extracellular K(+) concentrations required to impact myxoma virus replication can be reached in viral lesions under a variety of conditions. Taken together, these data suggest that the extracellular [K(+)] plays a role in determining the outcomes of myxoma infection and that this effect could be physiologically relevant during pathogenic infection. IMPORTANCE Intracellular K(+) homeostasis has been shown to play a major role in the replication of numerous viral families. However, the potential impact of altered extracellular K(+) concentrations is less well understood. Our work demonstrates that increased concentrations of extracellular K(+) can delay the replication cycle of the model poxvirus MYXV by inhibiting virion release from the endosomes. Additionally, mathematical modeling predicts that the levels of extracellular K(+) required to impact MYXV replication can likely be reached during pathogenic infection. These results suggest that localized viral infection can alter K(+) homeostasis and that these alterations might directly affect viral pathogenesis.

Current imaging approaches used to monitor tumor progression can lack the ability to distinguish true progression from pseudoprogression. Simultaneous metabolic 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) positron emission tomography (PET) and magnetic resonance imaging (MRI) offers new opportunities to overcome this challenge by refining tumor identification and monitoring therapeutic responses to cancer immunotherapy. In the current work, spatial and quantitative analysis of tumor burden were performed using simultaneous [18F]FDG-PET/MRI to monitor therapeutic responses to a novel silicified cancer cell immunotherapy in a mouse model of disseminated serous epithelial ovarian cancer. Tumor progression was validated by bioluminescence imaging of luciferase expressing tumor cells, flow cytometric analysis of immune cells in the tumor microenvironment, and histopathology. While PET demonstrated the presence of metabolically active cancer cells through [18F]FDG uptake, MRI confirmed cancer-related accumulation of ascites and tissue anatomy. This approach provides complementary information on disease status without a confounding signal from treatment-induced inflammation. This work provides a possible roadmap to facilitate accurate monitoring of therapeutic responses to cancer immunotherapies.

Oncolytic virotherapy relies on the induction of anti-tumor immune responses to achieve therapeutic efficacy. The factors that influence the induction of these responses, however, are not well understood. To begin to address this lack of knowledge, we asked how decreasing the susceptibility of malignant cells to direct viral infection would impact the induction of immune responses and therapeutic efficacy caused by oncolytic myxoma virus treatment. To accomplish this, we used CRISPR-Cas9 genome editing to remove the essential sulfation enzyme N-deacetylase/N-sulfotransferase-1 from B16/F10 murine melanoma cells. This eliminates the negative cell surface charges associated with glycosaminoglycan sulfation, which reduces a cell’s susceptibility to infection with the myxoma virus by ∼3- to 10-fold. With the use of these cells as a model of reduced susceptibility to oncolytic infection, our data demonstrate that 3- to 10-fold reductions in in vivo infection do not hinder the ability of the oncolytic myxoma virus to induce anti-tumor immunity and do not lower the overall efficacy of localized treatment. Additionally, our data show that in mice bearing multiple distinct tumor masses, the choice to treat a less-susceptible tumor mass does not reduce the overall therapeutic impact against either the injected or noninjected lesion. Taken together, these data suggest that minor changes in the susceptibility of malignant cells to direct oncolytic infection do not necessarily influence the overall outcomes of treatment., Graphical Abstract, Our work highlights that tumors do not have to be highly susceptible to direct viral infection to be treatable by oncolytic therapy. Instead, even minimal viral replication within the tumor can be sufficient to effectively delay tumor growth by activating endogenous immune surveillance.

T-cell immunoglobulin and mucin domain 3 (TIM3) is emerging as a potential target for antibody-based checkpoint blockade. However, the efficacy of TIM3 blockade in combination with other treatment modalities, has not been extensively studied. In the current work we combined TIM3 blockade with myxoma virus-based oncolytic virotherapy (OV). Our results demonstrate that myxoma virus's ability to initiate an immense antitumor immune response complements the ability of TIM3 blockade to shift the tumor microenvironment to a more proinflammatory state. As a result, the combination of TIM3 blockade and OV is able to completely eradicate established disease, while neither monotherapy is effective. These data represent the first demonstration that OV can enhance the efficacy of TIM3 blockade and suggest that this treatment may need to be incorporated into more aggressive, combinatorial regimens in order to fulfill its potential as an immunotherapeutic.

BackgroundOncolytic virotherapy (OV) represents a method to treat a variety of solid tumors by inducing antitumor immune responses. While this therapy has been extremely efficacious in preclinical models, translating these successes into human patients has proven challenging. One of the major reasons for these failures is the existence of immune-regulatory mechanisms, which dampen the efficacy of virally induced antitumor immunity. Unfortunately, the full extent of these immune-regulatory pathways remains unclear.MethodsTo address this issue, we generated a doubly recombinant, oncolytic myxoma virus which expresses both a soluble fragment of programmed cell death protein 1 (PD1) and an interleukin 12 (IL-12) fusion protein (vPD1/IL-12 (virus-expressing PD1 and IL-12)). We then tested the molecular impact and therapeutic efficacy of this construct in multiple models of disseminated disease to identify novel pathways, which are associated with poor therapeutic outcomes.ResultsOur results demonstrate that vPD1/IL-12 causes robust inflammation during therapy including inducing high levels of tumor necrosis factor (TNF). Surprisingly, although expression of TNF has generally been assumed to be beneficial to OV, the presence of this TNF appears to inhibit therapeutic efficacy by reducing intratumoral T-cell viability. Likely because of this, disruption of the TNF pathway, either through genetic knockout or antibody-based blockade, significantly enhances the overall outcomes of vPD1/IL-12-based therapy that allows for the generation of complete cures in normally non-responsive models.ConclusionsThese data suggest that some aspects of OV-induced inflammation might represent a double-edged sword during therapy and that specific blockade of TNF might enhance the efficacy of these treatments.

BackgroundAdoptive T cell transfer (ACT) therapy improves outcomes in patients with advanced malignancies, yet many individuals relapse due to the infusion of T cells with poor function or persistence. Toll-like receptor (TLR) agonists can invigorate antitumor T cell responses when administered directly to patients, but these responses often coincide with toxicities. We posited that TLR agonists could be repurposed ex vivo to condition T cells with remarkable potency in vivo, circumventing TLR-related toxicity.MethodsIn this study we investigated how tumor-specific murine CD8+T cells and human tumor infiltrating lymphocytes (TILs) are impacted when expanded ex vivo with the TLR9 agonist CpG.ResultsHerein we reveal a new way to reverse the tolerant state of adoptively transferred CD8+T cells against tumors using TLR-activated B cells. We repurposed the TLR9 agonist, CpG, commonly used in the clinic, to bolster T cell—B cell interactions during expansion for ACT. T cells expanded ex vivo from a CpG-treated culture demonstrated potent antitumor efficacy and prolonged persistence in vivo. This antitumor efficacy was accomplished without in vivo administration of TLR agonists or other adjuvants of high-dose interleukin (IL)-2 or vaccination, which are classically required for effective ACT therapy. CpG-conditioned CD8+T cells acquired a unique proteomic signature hallmarked by an IL-2RαhighICOShighCD39lowphenotype and an altered metabolic profile, all reliant on B cells transiently present in the culture. Likewise, human TILs benefitted from expansion with CpG ex vivo, as they also possessed the IL-2RαhighICOShighCD39lowphenotype. CpG fostered the expansion of potent CD8+T cells with the signature phenotype and antitumor ability via empowering a direct B–T cell interaction. Isolated B cells also imparted T cells with the CpG-associated phenotype and improved tumor immunity without the aid of additional antigen-presenting cells or other immune cells in the culture.ConclusionsOur results demonstrate a novel way to use TLR agonists to improve immunotherapy and reveal a vital role for B cells in the generation of potent CD8+T cell-based therapies. Our findings have immediate implications in the clinical treatment of advanced solid tumors.

Introduction Cancer has become one of the most critical health issues of modern times. To overcome the ineffectiveness of current treatment options, research is being done to explore new therapeutic modalities. One such novel treatment is oncolytic virotherapy (OV) which uses tumor tropic viruses to specifically target and kill malignant cells. While OV has shown significant promise in recent clinical trials, the therapeutic use of viruses poses a number of unique challenges. In particular, obtaining effective viral spread throughout the tumor microenvironment remains problematic. Previous work has suggested this can be overcome by forcing oncolytic viruses to induce syncytia formation. Methods In the current work, we generated a series of recombinant myxoma viruses expressing exogenous fusion proteins from other viral genomes and examined their therapeutic potential in vitro and in vivo. Results Similar to previous studies, we observed that the expression of these fusion proteins during myxoma infection induced the formation of multinucleated syncytia which increased viral spread and lytic potential compared to non-fusogenic controls. Contrary to expectations, however, the treatment of established tumors with these viruses resulted in decreased therapeutic efficacy which corresponded with reduced viral persistence. Discussion These findings indicate that enhanced viral spread caused by syncytia formation can actually reduce the efficacy of OV and supports a number of previous works suggesting that the in vitro properties of viruses frequently fail to predict their in vivo efficacy.

Polyamines are known to play a significant role in cancer progression and treatment using difluoromethylornithine (DFMO), an inhibitor of polyamine biosynthesis, has shown some clinical promise. It is interesting to note that, while DFMO is directly cytostatic in vitro, recent work has suggested that it achieves its antitumor efficacy in vivo by enhancing adaptive antitumor immune responses. On the basis of these data, we hypothesized that DFMO might act as an immune sensitizer to increase tumor responsiveness to checkpoint blockade. To test this hypothesis, we treated tumors with DFMO, in either the presence or absence of additional PD-1 blockade, and subsequently analyzed their immunological and therapeutic responses. Our data demonstrates that treatment with DFMO significantly enhances both the viability and activation status of intratumoral CD8+ T cells, most likely through an indirect mechanism. When combined with PD-1 blockade, this increased viability resulted in unique proinflammatory cytokine profiles and transcriptomes within the tumor microenvironment and improved therapeutic outcomes. Taken together, these data suggest that DFMO might represent a potential immunomodulatory agent that can enhance current PD-1-based checkpoint therapies.

Expression of PDL1 on the surface of tumor cells can blunt the efficacy of many cancer immunotherapies. For example, our lab has previously shown that tumors derived from malignant cells incapable of expressing PDL1 are highly susceptible to immunotherapy induced by oncolytic virus treatment while tumors derived from PDL1 capable cells are highly resistant. In patient biopsies, however, expression of PDL1 on malignant cells is often not uniform with some cells expressing PDL1 while others do not. Importantly, how this partial PDL1 positivity influences the outcomes of immunotherapy remains largely unknown. In the current work, we expand on our previous findings by generating partially PDL1 positive tumors in immune competent animals and asking what percentage of tumor cells must express PDL1 for a tumor to become functionally resistant to oncolytic treatment. Our results indicate that the responsiveness of partially PDL1+ tumors correlates linearly with the percentage of PDL1 capable cells present at the initiation of treatment. Additionally, we observe that tumors which relapse after treatment display a significant increase in the numbers of PDL1 capable cells present suggesting that specific editing of mixed tumors might play a role in disease relapse. These data indicate that varying levels of PDL1 expression can play a significant role in the outcomes of oncolytic immunotherapy and challenges the concept that tumors should be viewed as simply PDL1+ or PDL1−. Electronic supplementary material The online version of this article (10.1186/s40425-018-0496-6) contains supplementary material, which is available to authorized users.

IL-12 (p35/p40) and IL-23 (p19/p40) signal through IL-12R (IL-12Rβ2/β1) and IL-23R (IL-23Rα/IL-12Rβ1), respectively, which can promote pathogenic T lymphocyte activation, differentiation, and function in graft-versus-host disease (GVHD). With the use of murine models of allogeneic hematopoietic cell transplantation (HCT), we found that IL-12Rβ1 on donor T cells was dispensable to induce acute GVHD development in certain circumstances, while IL-23Rα was commonly required. This observation challenges the current paradigm regarding IL-12Rβ1 as a prerequisite to transmit IL-23 signaling. We hypothesized that p19/EBI3 (IL-39) may have an important role during acute GVHD. With the use of gene transfection and immunoprecipitation approaches, we verified that p19 and EBI3 can form biological heterodimers. We found that IL-39 levels in recipient serum positively correlated with development of acute GVHD in experimental models and in clinical settings, thereby implicating IL-39 in the pathogenesis of acute GVHD. Furthermore, we observed that human T cells can signal in response to IL-39. In chronic GVHD, IL-23Rα and IL-12Rβ1 were similarly required for donor T cell pathogenicity, and IL-39 levels were not significantly different from controls without GVHD. Collectively, we identify a novel cytokine, IL-39, as a pathogenic factor in acute GVHD, which represents a novel potential therapeutic target to control GVHD and other inflammatory disorders.

Surgical removal of tumors remains a front-line therapy for many types of cancer. However, this treatment often fails to eradicate disease due to either recurrence of the original tumor or development of distant micrometastases. To address these challenges, patients are often given non-curative treatments presurgery with the intent of improving surgical outcomes. These treatments, collectively known as neoadjuvant therapies, have traditionally focused on the presurgical use of chemotherapeutics. Recently, however, a variety of immunotherapies have also been identified as potentially effective in the neoadjuvant setting. One of these immunotherapies is oncolytic virotherapy, whose clinical use has exploded with the Food and Drug Administration approval of Talimogene Laherparepvec. This review summarizes both the preclinical and clinical literature examining the use of oncolytic virotherapy in the neoadjuvant setting for different types of cancers and discusses some of the major questions that still need to be addressed in order for this unique use of immunotherapy to become clinically viable.

Poxviruses are large enveloped viruses that replicate exclusively in the cytoplasm. Like all viruses, their replication cycle begins with virion adsorption to the cell surface. Unlike most other viral families, however, no unique poxviral receptor has ever been identified. In the absence of a unique receptor, poxviruses are instead thought to adhere to the cell surface primarily through electrostatic interactions between the positively charged viral envelope proteins and the negatively charged sulfate groups on cellular glycosaminoglycans (GAGs). While these negatively charged GAGs are an integral part of all eukaryotic membranes, their specific expression and sulfation patterns differ between cell types. Critically, while poxviral binding has been extensively studied using virally centered genetic strategies, the impact of cell-intrinsic changes to GAG charge has never been examined. Here we show that loss of heparin sulfation, accomplished by deleting the enzyme N-Deacetylase and N-Sulfotransferase-1 (NDST1) which is essential for GAG sulfation, significantly reduces the binding affinity of both vaccinia and myxoma viruses to the cell surface. Strikingly, however, while this lowered binding affinity inhibits the subsequent spread of myxoma virus, it actually enhances the overall spread of vaccinia by generating more diffuse regions of infection. These data indicate that cell-intrinsic GAG sulfation plays a major role in poxviral infection, however, this role varies significantly between different members of the poxviridae.

Myxoma virus is a member of Leporipoxviridae whose tropism is tightly restricted to lagomorphs. In susceptible Oryctolagus rabbits, the virus causes a highly lethal disease known as myxomatosis, which begins as a localized infection but rapidly disseminates throughout the animal, leading to immune compromise, mucosal infections, multiorgan failure, and death. In a research setting, myxoma infection of susceptible Oryctolagus cuniculus rabbits is used as a model of poxviral disease progression and represents one of only a few means to study the pathogenesis of this viral family in a native host species. However, the rapid progression of myxomatosis makes accurate prediction of humane endpoints critical to limiting animal pain and distress and preventing death as an endpoint. Here we present case studies of myxomatosis at 2 institutions and offer a refined scoring system to reliably track the course of disease in susceptible rabbits infected with myxoma virus.

Multiple myeloma (MM) is a clonal malignancy of plasma cells that is newly diagnosed in ~30,000 patients in the US each year. While recently developed therapies have improved the prognosis for MM patients, relapse rates remain unacceptably high. To overcome this challenge, researchers have begun to investigate the therapeutic potential of oncolytic viruses as a novel treatment option for MM. Preclinical work with these viruses has demonstrated that their infection can be highly specific for MM cells and results in impressive therapeutic efficacy in a variety of preclinical models. This has led to the recent initiation of several human trials. This review summarizes the current state of oncolytic therapy as a therapeutic option for MM and highlights a variety of areas that need to be addressed as the field moves forward.

BackgroundThe use of immunotherapies such as immune checkpoint blockade or adoptive cell therapy has substantially improved outcomes for many patients with advanced malignancies. However, a majority of these patients still do not respond or relapse. Thus, extensive efforts are being made to improve these therapies. We and others have demonstrated that adoptive cell therapy can be improved by the co-administration of TLR agonists intratumorally. TLR agonists have also been administered to patients alongside a number of other immunotherapies, but often induce toxic side effects that may be exacerbated by cell therapy. We hypothesized that TLR agonists could alternatively be used in ex vivo cell culture to propagate a more potent cell therapy product.MethodsTo address our question, we used a transgenic mouse model of tumor-infiltrating lymphocyte (TIL) therapy, pmel-1, in which CD8+ T cells express a TCR specific for a melanoma/melanocyte antigen. We activated CD8+ pmel-1 T cells with APCs and the Toll-like receptor 9 agonist, CpG. Cell therapy efficacy was determined against mice bearing established B16F10 melanoma. Mechanisms underlying TLR-improved T cell products were determined using ex vivo cell depletion strategies and blocking antibodies.ResultsCpG-expanded pmel-1 T cell products were much more effective against B16F10 melanoma in vivo than traditionally expanded T cells; they conferred more durable antitumor responses and improved survival of mice. CD8+ T cells generated in the presence of CpG also had heightened engraftment and persistence in the mice. We found that CpG did not act directly on T cells in culture, but on B cells, as depletion of B cells alone (not DCs, macrophages, NK cells, or CD4 cells) ablated the effects of CpG. B cells in CpG-treated cultures expressed a unique cytokine profile and upregulated several costimulatory markers on their cell surface, so we next questioned whether CpG improved the B cell/T cell axis via a direct or indirect (soluble) factor. Together, cell supernatant transfer experiments and costimulatory blockade experiments revealed that the direct interaction between B and T cells was required for the CpG-mediated improvement of the T cell product.ConclusionsOur findings reveal a novel role for B cells in the generation of potent CD8+ T cell therapies against an aggressive solid tumor. These findings highlight a unique way B cells can become powerful APCs for generating effective antitumor CD8+ T cells and can be directly translated to improve cell therapy products for patients with advanced malignancies.Ethics ApprovalAll animal procedures performed at the Medical University of South Carolina or Emory University were approved by each university’s Institutional Animal Care & Use Committee, protocol number 0488 or 201900225, respectively.

BackgroundOncolytic therapy uses live-replicating viruses to improve the immunological status of treated tumors. Critically, while these viruses are known to self-amplify in vivo, clinical oncolytic therapies still appear to display a strong dose dependence and the mechanisms mediating this dose dependence are not well understood.MethodsTo explore this apparent contradiction, we investigated how the initial dose of oncolytic myxoma virus affected the subsequent ability of treatment to alter the immunological status of tumors as well as synergize with programmed cell death protein 1 (PD1) blockade.ResultsOur results indicate that, due to viral self-amplification in vivo, the overall load of myxoma virus rapidly normalizes within treated tumors despite up to 3-log differences in inoculating dose. Because of this, therapeutic efficacy in the absence of checkpoint blockade is largely dose independent. Despite this rapid normalization, however, treatment with high or low doses of myxoma virus induces distinct immunological changes within treated tumors. Critically, these changes appear to be durably programmed based on the initial oncolytic dose with low-dose treatment failing to induce immunological improvements despite rapidly achieving equivalent viral burdens. Finally, due to the distinct immunological profiles induced by high and low myxoma virus doses, oncolytic efficacy resulting from combination with PD1 blockade therapy displays a strong dose dependence.ConclusionsTaken together, these data suggest that the ability of oncolytic myxoma virus to immunologically reprogram treated tumors is dependent on initial viral dose. Additionally, this work could provide a possible mechanistic explanation for clinical results observed with other oncolytic viruses.

Poxviruses are large, DNA viruses whose protein capsid is surrounded by one or more lipid envelopes. Embedded into these lipid envelopes are three conserved viral proteins which are thought to mediate binding of virions to target cells. While the function of these proteins has been studied in vitro , their specific roles during the pathogenesis of poxviral disease remain largely unclear. Here we present data demonstrating that the putative chondroitin binding protein M083 from the leporipoxvirus myxoma virus is a significant virulence factor during infection of susceptible Oryctolagus rabbits. Removal of M083 results in a reduced capacity of virus to spread beyond the regional lymph nodes and completely eliminates infection-mediated mortality. In vitro , removal of M083 results in only minor intracellular replication defects but causes a significant reduction in the ability of myxoma virus to spread from infected epithelial cells onto primary lymphocytes. We hypothesize that the physiological role of M083 is therefore to mediate the spread of myxoma virus onto rabbit lymphocytes, allowing these cells to disseminate virus throughout infected rabbits. IMPORTANCE Poxviruses represent both a class of human pathogens and potential therapeutic agents for the treatment of human malignancy. Understanding the basic biology of these agents is therefore significant to human health in a variety of ways. While the mechanisms mediating poxviral binding have been well studied in vitro , how these mechanisms impact poxviral pathogenesis in vivo remains unclear. The current study advances our understanding of how poxviral binding impacts viral pathogenesis by demonstrating that the putative chondroitin binding protein M083 plays a critical role during the pathogenesis of myxoma virus in susceptible Oryctolagus rabbits by impacting viral dissemination through changes in the transfer of virions onto primary splenocytes.

Glioblastoma (GB) is the most common and highly aggressive adult brain cancer. Dysfunction of receptor tyrosine kinases, such as ALK, c-Met and VEGF have all been implicated in the progression of this malignancy and resistance to therapy. Traditional therapeutic strategies targeting ALK/c-Met or VEGF have not yet given satisfactory outcomes. Myxoma virus (MYXV) is a novel oncolytic virus that has been shown to replicate in GB models. Here, we investigated the efficacy of MYXV as monotherapy, or in combination with 1 Gy radiation plus c-Met/ALK inhibitor crizotinib and VEGF inhibitor bevacizumab in different cell lines (GL261, T9, and U118) surgically obtained from GB patients. We investigated the molecular mechanisms of apoptotic death by Western blots. Colorimetric assay demonstrated that treatment with MYXV plus radiation with crizotinib or bevacizumab caused apoptosis due to an increase in activation of caspase-3 and decrease in expression of Bcl-2 proteins in GB cells and tissues. Most importantly, MYXV plus radiation significantly enhanced the anti-cancer effect of crizotinib, bevacizumab and temozolomide, in GB cell lines as well as in fresh tumor biopsies from GB patients. Our studies also suggest that induction of apoptosis by MYXV does not depend on the functional status of the GB cell lines or in the GB tissues. Further studies in different animal models of GB are warranted in the future to determine whether MYXV in combination with radiation, crizotinib or bevacizumab may be useful as anti-cancer agent for the management of human GBs in vivo.

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is an effective means by which to treat a wide variety of diseases resulting from hematological dysfunction. However, the development of graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality post transplantation. The IL-12 family of cytokines is comprised of IL-12, IL-23, IL-27, IL-35, and potentially IL-39. IL-12 family members are unique in that each cytokine and cognate receptor is comprised of heterodimers in which either the a or b subunit is shared among the others. IL-12 (p35+p40) and IL-23 (p19+p40) have well documented proinflammatory functions responsible for Th1 differentiation and Th17 stabilization, respectively, and play critical roles in GVHD development. IL-12R and IL-23R share a β-chain (IL-12Rβ1) yet use distinct α-chains to mediate their respective receptor signaling. While both IL-12R and IL-23R are widely implicated in inflammatory disorders, the role of IL-12Rβ1 in this context remains much less defined. We therefore studied the impact of eliminating the common IL-12Rβ1 chain or the unique IL-23Rα chain in T cells on GVHD using murine models of allogeneic bone marrow transplantation (BMT). In agreement with previous publications, we found a pathogenic role for IL-23Rα on donor T cells in aGVHD. Strikingly, a similar effect was not seen for IL-12Rβ1 (Figure 1A, B). These data suggest that that IL-23Rα contributes to GVHD pathogenesis via a pathway independent of IL-12Rβ1. To confirm that functional differences existed between T cells deficient for IL-23Rα or IL-12Rβ1 in GVHD, we assessed cytokine profiles of these T cells in target organs 14 days post-BMT. We found that, while production of IFNγ and IL-17 in the spleen was similarly decreased in both cohorts, GM-CSF production by CD4+ T cells was reduced exclusively in T cells deficient for IL-23Rα. Further, a significant reduction of IFNγ and GM-CSF in target organs, such as the liver and gut, was only observed in T cells deficient for IL-23Rα. The newest member of the IL-12 family, IL-39, was recently shown to contribute to SLE pathogenesis; this cytokine has been described to be composed of IL-23p19 and EBI3. Given the cognate receptor for IL-39 includes IL-23Rα and gp130, we hypothesized that IL-39 may play a role in aGVHD as this would explain why IL-12Rβ1 is dispensable. To validate that p19 and EBI3 can form a heterodimer, we transfected SV40 cells with vectors containing control, IL-23p19, EBI3 or both cDNAs. We detected IL-39 heterodimers only in the supernatant of cells transfected with both IL-23p19 and EBI3 via ELISA (Figure 1C). Furthermore, we observed significantly increased levels of IL-39 in allogeneic recipients at day 14 post BMT compared to naïve mice or recipients of BM alone in two models of aGVHD (Figure 1D). This may implicate IL-39 in the GVHD development. Taken together, our studies indicate that IL-23Rα plays an essential role, whereas IL-12Rβ1 is dispensable, for donor T cells to induce aGVHD. Our proposed model is that in the absence of IL-12Rβ1, IL-39 could transmit IL-23Rα signaling, hypothetically by forming a heterodimer with gp130. This new finding indicates that IL-23Rα and IL-39 are potential therapeutic targets for controlling aGVHD in the clinic. Figure 1. Effect of IL-12R 𝛃 1 in aGVHD and the potential role of IL-39 Lethally irradiated BALB/c mice were transplanted with 5x106 TCD-BM alone or plus 1x106 purified T cells from WT B6, IL-12R𝛃1KO, or IL-23RαKO mice. Survival (A) and body weight loss (B) are shown. Supernatant from SV40 cells transiently transfected with vectors containing control, IL-23p19, EBI3 or both cDNAs. p19 and EBI3 heterodimers were detected via ELISA (C). Serum was collected from naïve or lethally irradiated mice transplanted with 5x106 TCD-BM alone or plus 1x106 purified T cells. Formation of p19 and EBI3 heterodimers were tested in serum at 14 days post BMT via ELISA (D). Disclosures No relevant conflicts of interest to declare.