Fusogenic vesicular stomatitis virus combined with natural killer T cell immunotherapy controls metastatic breast cancer.

BACKGROUND: Metastatic breast cancer is a leading cause of cancer death in woman. Current treatment options are often associated with adverse side effects and poor outcomes, demonstrating the need for effective new treatments. Immunotherapies can provide durable outcomes in many cancers; however, limited success has been achieved in metastatic triple negative breast cancer. We tested whether combining different immunotherapies can target metastatic triple negative breast cancer in pre-clinical models. METHODS: Using primary and metastatic 4T1 triple negative mammary carcinoma models, we examined the therapeutic effects of oncolytic vesicular stomatitis virus (VSVΔM51) engineered to express reovirus-derived fusion associated small transmembrane proteins p14 (VSV-p14) or p15 (VSV-p15). These viruses were delivered alone or in combination with natural killer T (NKT) cell activation therapy mediated by adoptive transfer of α-galactosylceramide-loaded dendritic cells. RESULTS: Treatment of primary 4T1 tumors with VSV-p14 or VSV-p15 alone increased immunogenic tumor cell death, attenuated tumor growth, and enhanced immune cell infiltration and activation compared to control oncolytic virus (VSV-GFP) treatments and untreated mice. When combined with NKT cell activation therapy, oncolytic VSV-p14 and VSV-p15 reduced metastatic lung burden to undetectable levels in all mice and generated immune memory as evidenced by enhanced in vitro recall responses (tumor killing and cytokine production) and impaired tumor growth upon rechallenge. CONCLUSION: Combining NKT cell immunotherapy with enhanced oncolytic virotherapy increased anti-tumor immune targeting of lung metastasis and presents a promising treatment strategy for metastatic breast cancer.

Pilot study of jadomycin B pharmacokinetics and anti-tumoral effects in zebrafish larvae and mouse breast cancer xenograft models.

Despite numerous therapeutic options, multidrug resistance (MDR) remains an obstacle to successful breast cancer therapy. Jadomycin B, a natural product derived from Streptomyces venezuelae ISP5230, maintains cytotoxicity in MDR human breast cancer cells. Our objectives were to evaluate the pharmacokinetics, toxicity, anti-tumoral, and anti-metastatic effects of jadomycin B in zebrafish larvae and mice. In a zebrafish larval xenograft model, jadomycin B significantly reduced the proliferation of human MDA-MB-231 cells at or below its maximum tolerated dose (40 µm). In female Balb/C mice, a single intraperitoneal dose (6 mg/kg) was rapidly absorbed with a maximum serum concentration of 3.4 ± 0.27 µm. Jadomycin B concentrations declined biphasically with an elimination half-life of 1.7 ± 0.058 h. In the 4T1 mouse mammary carcinoma model, jadomycin B (12 mg/kg every 12 h from day 6 to 15 after tumor cell injection) decreased primary tumor volume compared to vehicle control. Jadomycin B-treated mice did not exhibit weight loss, nor significant increases in biomarkers of impaired hepatic (alanine aminotransferase) and renal (creatinine) function. In conclusion, jadomycin B demonstrated a good safety profile and provided partial anti-tumoral effects, warranting further dose-escalation safety and efficacy studies in MDR breast cancer models.

Mice Lacking γδ T Cells Exhibit Impaired Clearance of Pseudomonas aeruginosa Lung Infection and Excessive Production of Inflammatory Cytokines.

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic and life-threatening infections in immunocompromised patients. A better understanding of the role that innate immunity plays in the control of P. aeruginosa infection is crucial for therapeutic development. Specifically, the role of unconventional immune cells like γδ T cells in the clearance of P. aeruginosa lung infection is not yet well characterized. In this study, the role of γδ T cells was examined in an acute mouse model of P. aeruginosa lung infection. In the absence of γδ T cells, mice displayed impaired bacterial clearance and decreased survival, outcomes which were associated with delayed neutrophil recruitment and impaired recruitment of other immune cells (macrophages, T cells, natural killer cells, and natural killer T [NKT] cells) into the airways. Despite reduced NKT cell recruitment in the airways of mice lacking γδ T cells, NKT cell-deficient mice exhibited wild-type level control of P. aeruginosa infection. Proinflammatory cytokines were also altered in γδ T cell-deficient mice, with increased production of interleukin-1ß, interleukin-6, and tumor necrosis factor. γδ T cells did not appear to contribute significantly to the production of interleukin-17A or the chemokines CXCL1 and CXCL2. Importantly, host survival could be improved by inhibiting tumor necrosis factor signaling with the soluble receptor construct etanercept in γδ cell-deficient mice. These findings demonstrate that γδ T cells play a protective role in coordinating the host response to P. aeruginosa lung infection, both in contributing to early immune cell recruitment and by limiting inflammation.

Leishmania donovani Lipophosphoglycan Increases Macrophage-Dependent Chemotaxis of CXCR6-Expressing Cells via CXCL16 Induction.

CXCL16 is a multifunctional chemokine that is highly expressed by macrophages and other immune cells in response to bacterial and viral pathogens; however, little is known regarding the role of CXCL16 during parasitic infections. The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis. Even though chemokine production is a host defense mechanism during infection, subversion of the host chemokine system constitutes a survival strategy adopted by the parasite. Here, we report that L. donovani promastigotes upregulate CXCL16 synthesis and secretion by bone marrow-derived macrophages (BMDM). In contrast to wild-type parasites, a strain deficient in the virulence factor lipophosphoglycan (LPG) failed to induce CXCL16 production. Consistent with this, cell treatment with purified L. donovani LPG augmented CXCL16 expression and secretion. Notably, the ability of BMDM to promote migration of cells expressing CXCR6, the cognate receptor of CXCL16, was augmented upon L. donovani infection in a CXCL16- and LPG-dependent manner. Mechanistically, CXCL16 induction by L. donovani required the activity of AKT and the mechanistic target of rapamycin (mTOR) but was independent of Toll-like receptor signaling. Collectively, these data provide evidence that CXCL16 is part of the inflammatory response elicited by L. donovani LPG in vitro Further investigation using CXCL16 knockout mice is required to determine whether this chemokine contributes to the pathogenesis of visceral leishmaniasis and to elucidate the underlying molecular mechanisms.

Selective Cannabinoid 2 Receptor Agonists as Potential Therapeutic Drugs for the Treatment of Endotoxin-Induced Uveitis.

(1) Background: The cannabinoid 2 receptor (CB2R) is a promising anti-inflammatory drug target and development of selective CB2R ligands may be useful for treating sight-threatening ocular inflammation. (2) Methods: This study examined the pharmacology of three novel chemically-diverse selective CB2R ligands: CB2R agonists, RO6871304, and RO6871085, as well as a CB2R inverse agonist, RO6851228. In silico molecular modelling and in vitro cell-based receptor assays were used to verify CB2R interactions, binding, cell signaling (ß-arrestin and cAMP) and early absorption, distribution, metabolism, excretion, and toxicology (ADMET) profiling of these receptor ligands. All ligands were evaluated for their efficacy to modulate leukocyte-neutrophil activity, in comparison to the reported CB2R ligand, HU910, using an in vivo mouse model of endotoxin-induced uveitis (EIU) in wild-type (WT) and CB2R-/- mice. The actions of RO6871304 on neutrophil migration and adhesion were examined in vitro using isolated neutrophils from WT and CB2R-/- mice, and in vivo in WT mice with EIU using adoptive transfer of WT and CB2R-/- neutrophils, respectively. (3) Results: Molecular docking studies indicated that RO6871304 and RO6871085 bind to the orthosteric site of CB2R. Binding studies and cell signaling assays for RO6871304 and RO6871085 confirmed high-affinity binding to CB2R and selectivity for CB2R > CB1R, with both ligands acting as full agonists in cAMP and ß-arrestin assays (EC50s in low nM range). When tested in EIU, topical application of RO6871304 and RO6871085 decreased leukocyte-endothelial adhesion and this effect was antagonized by the inverse agonist, RO6851228. The CB2R agonist, RO6871304, decreased in vitro neutrophil migration of WT neutrophils but not neutrophils from CB2R-/-, and attenuated adhesion of adoptively-transferred leukocytes in EIU. (4) Conclusions: These unique ligands are potent and selective for CB2R and have good immunomodulating actions in the eye. RO6871304 and RO6871085, as well as HU910, decreased leukocyte adhesion in EIU through inhibition of resident ocular immune cells. The data generated with these three structurally-diverse and highly-selective CB2R agonists support selective targeting of CB2R for treating ocular inflammatory diseases.

Reconstitution models to evaluate natural killer T cell function in tumor control.

Natural killer T (NKT) cells are glycolipid-reactive T lymphocytes that function in immunosurveillance and immune regulation. However, reduced tumor control in NKT cell-deficient Jα18(-/-) mice may be confounded by an overall reduction in T-cell receptor (TCR) repertoire diversity in these animals. Mechanistic studies are also hindered by a lack of tools to target molecules specifically in NKT cells. To address these issues, we developed protocols to expand functional NKT cells and stably reconstitute them in Jα18(-/-) mice. In vivo delivery of α-galactosylceramide (α-GalCer)-loaded dendritic cells expanded NKT cells in wild-type mice without skewing CD4 or TCR Vß expression profiles. Expanded NKT cells exhibited enhanced cytokine responses upon re-stimulation with glycolipid or CD3 ligation. Adoptive transfer of recently expanded wild-type or interferon (IFN)-γ(-/-) NKT cells protected recipient Jα18(-/-) mice from B16 melanoma metastasis without the need for additional glycolipid stimulation. However, NKT cell reconstitution in recipient Jα18(-/-) mice was short lived. Long-term reconstitution was only achieved when expanded NKT cells were transferred into sublethally irradiated recipients. Thirty days after transfer, NKT cell numbers, phenotype and α-GalCer-induced cytokine responses were equivalent to naive wild-type mice. Jα18(-/-) recipients reconstituted with wild-type or IFN-γ(-/-) NKT cells were both protected from B16 melanoma metastasis following α-GalCer treatment, and NK cell transactivation was intact in mice reconstituted with IFN-γ(-/-) NKT cells. These studies validate the use of reconstitution protocols to investigate the mechanisms of NKT cell immune function, demonstrating that NKT cell-derived IFN-γ and the altered TCR repertoire in Jα18(-/-) mice do not impact NKT cell-mediated antitumor responses.

The reversible P2Y12 inhibitor ticagrelor inhibits metastasis and improves survival in mouse models of cancer.

Tumor cells use activated platelets to promote their proliferation and metastatic potential. Because platelet activation is largely mediated through ADP engagement of purinergic P2Y12 receptors on platelets, we investigated the potential of the reversible P2Y12 inhibitor ticagrelor, a clinical agent used in the prevention of cardiovascular and cerebrovascular events, to inhibit tumor adhesion and metastasis. In B16-F10 melanoma intravenous and intrasplenic metastasis models, mice treated with a clinical dose of ticagrelor (10 mg/kg) exhibited marked reductions in lung (84%) and liver (86%) metastases. Furthermore, ticagrelor treatment improved survival compared to saline-treated animals. A similar effect was observed in a 4T1 breast cancer model, with reductions in lung (55%) and bone marrow (87%) metastases following ticagrelor treatment. In vitro, B16-F10 cells exhibited decreased interaction with platelets from ticagrelor-treated mice compared to saline-treated mice, an effect similar to that observed with blockade of glycoprotein IIbIIIa. Similarly, B16-F10 cells co-incubated with platelets from ticagrelor-treated mice exhibited reduced adhesion to endothelial monolayers compared to those co-incubated with platelets from saline-treated animals, an effect also observed in vivo. Interestingly, pretreatment of endothelial monolayers with ticagrelor did not result in reduced tumor cell adhesion. These findings support a role for P2Y12-mediated platelet activation in promoting metastases, and provide proof-of-concept for the clinical use of ticagrelor in the prevention of tumor metastasis.

CCR4 and CXCR3 play different roles in the migration of T cells to inflammation in skin, arthritic joints, and lymph nodes.

CCR4 and CXCR3 are expressed on several T-cell subsets in inflamed tissues, yet their role in tissue-specific recruitment is unclear. We examined the contributions of CCR4 and CXCR3 to T-cell recruitment into inflamed joints in collagen-induced arthritis, antigen-draining lymph nodes (LNs) and dermal inflammatory sites (poly I:C, LPS, concanavalin A, and delayed type hypersensitivity), using labeled activated T cells from CXCR3(-/-), CCR4(-/-), and WT mice. Both CXCR3 and CCR4 deficiency reduced the development of arthritis, but did not affect Th1-cell recruitment to the inflamed joints. Accumulation in inflamed LNs was highly CXCR3 dependent. In contrast, CCR4-deficient Th1 cells had an increased accumulation in these LNs. Migration to all four dermal inflammatory sites by activated Th1 and T cytotoxic cells and memory CD4(+) T cells was partially CXCR3-dependent, but Treg-cell migration was independent of CXCR3. The subset of cells expressing CCR4 has skin-migrating properties, but CCR4 itself is not required for the migration. Thus, migration into these inflamed tissues is CCR4-independent, and partially dependent on CXCR3, except for Treg cells, which require neither receptor. CCR4 may therefore affect retention of T cells in different tissues rather than trafficking out of the blood.

Experimental TLR4 inhibition improves intestinal microcirculation in endotoxemic rats.

INTRODUCTION: Toll like receptor 4 (TLR4) represents a critical cellular link for endotoxin-induced pathology. The aim of this study was to evaluate the potential role of TLR4 inhibition on the intestinal microcirculation during experimental endotoxemia. MATERIALS AND METHODS: The intestinal microcirculation was studied by intravital microscopy in four groups of Lewis rats (n=10 per group): healthy controls (CON group), endotoxemic animals (15mg/kg lipopolysaccharide, LPS group), endotoxemic animals treated with a TLR4 antagonist (1mg/kg CRX-526, LPS+CRX526 group), and controls treated with CRX-526 (C-CRX526 group). Plasma samples were obtained for cytokine measurements at the end of the experiments. RESULTS: Endotoxemia significantly increased leukocyte adhesion in intestinal submucosal venules (e.g., V1 venules: CON 20.4±6.5n/mm(2), LPS 237.5±36.2n/mm(2), p<0.05) and reduced capillary perfusion of the intestinal wall (e.g., longitudinal muscular layer: CON 112.5±5.9cm/cm(2), LPS 71.3±11.0cm/cm(2), p<0.05) at 2h. TLR4 inhibition significantly reduced endotoxemia-associated leukocyte adhesion (V1 venules: 104.3±7.8n/mm(2)) and improved capillary perfusion (longitudinal muscular layer: 111.0±12.3cm/cm(2)). Cytokine release was not significantly affected. CONCLUSIONS: The TLR4 pathway may be a target in clinical Gram-negative sepsis since administration of the TLR4 antagonist CRX-526 improved intestinal microcirculation parameters in experimental endotoxemia.

Pseudomonas aeruginosa in chronic lung disease: untangling the dysregulated host immune response.

Pseudomonas aeruginosa is a highly adaptable opportunistic pathogen capable of exploiting barriers and immune defects to cause chronic lung infections in conditions such as cystic fibrosis. In these contexts, host immune responses are ineffective at clearing persistent bacterial infection, instead driving a cycle of inflammatory lung damage. This review outlines key components of the host immune response to chronic P. aeruginosa infection within the lung, beginning with initial pathogen recognition, followed by a robust yet maladaptive innate immune response, and an ineffective adaptive immune response that propagates lung damage while permitting bacterial persistence. Untangling the interplay between host immunity and chronic P. aeruginosa infection will allow for the development and refinement of strategies to modulate immune-associated lung damage and potentiate the immune system to combat chronic infection more effectively.

IFN regulatory factor 3 contributes to the host response during Pseudomonas aeruginosa lung infection in mice.

Pseudomonas aeruginosa is a major opportunistic pathogen. However, host defense mechanisms involved in P. aeruginosa lung infection remain incompletely defined. The transcription factor IFN regulatory factor 3 (IRF3) is primarily associated with host defense against viral infections, and a role of IRF3 in P. aeruginosa infection has not been reported previously. In this study, we showed that IRF3 deficiency led to impaired clearance of P. aeruginosa from the lungs of infected mice. P. aeruginosa infection induced IRF3 translocation to the nucleus, activation of IFN-stimulated response elements (ISRE), and production of IFN-beta, suggesting that P. aeruginosa activates the IRF3-ISRE-IFN pathway. In vitro, macrophages from IRF3-deficient mice showed complete inhibition of CCL5 (RANTES) and CXCL10 (IP-10) production, partial inhibition of TNF, but no effect on CXCL2 (MIP-2) or CXCL1 (keratinocyte-derived chemokine) in response to P. aeruginosa stimulation. In vivo, IRF3-deficient mice showed complete inhibition of CCL5 production and partial or no effects on production of other cytokines and chemokines in the bronchoalveolar lavage fluids and lung tissues. Profiling of immune cells in the airways revealed that neutrophil and macrophage recruitment into the airspace was reduced, whereas B cell, T cell, NK cell, and NKT cell infiltration was unaffected in IRF3-deficient mice following P. aeruginosa lung infection. These data suggest that IRF3 regulates a distinct profile of cytokines and chemokines and selectively modulates neutrophil and macrophage recruitment during P. aeruginosa infection. Thus, IRF3 is an integral component in the host defense against P. aeruginosa lung infection.

A critical role for mast cells and mast cell-derived IL-6 in TLR2-mediated inhibition of tumor growth.

Several TLR agonists are effective in tumor immunotherapy, but their early innate mechanisms of action, particularly those of TLR2 agonists, are unclear. Mast cells are abundant surrounding solid tumors where they are often protumorigenic and enhance tumor angiogenesis. However, antitumor roles for mast cells have also been documented. The impact of mast cells may be dependent on their activation status and mediator release in different tumors. Using an orthotopic melanoma model in wild-type C57BL/6 and mast cell-deficient Kit(W-sh/W-sh) mice and a complementary Matrigel-tumor model in C57BL/6 mice, mast cells were shown to be crucial for TLR2 agonist (Pam(3)CSK(4))-induced tumor inhibition. Activation of TLR2 on mast cells reversed their well-documented protumorigenic role. Tumor growth inhibition after peritumoral administration of Pam(3)CSK(4) was restored in Kit(W-sh/W-sh) mice by local reconstitution with wild-type, but not TLR2-deficient, mast cells. Mast cells secrete multiple mediators after Pam(3)CSK(4) activation, and in vivo mast cell reconstitution studies also revealed that tumor growth inhibition required mast cell-derived IL-6, but not TNF. Mast cell-mediated anticancer properties were multifaceted. Direct antitumor effects in vitro and decreased angiogenesis and recruitment of NK and T cells in vivo were observed. TLR2-activated mast cells also inhibited the growth of lung cancer cells in vivo. Unlike other immune cells, mast cells are relatively radioresistant making them attractive candidates for combined treatment modalities. This study has important implications for the design of immunotherapeutic strategies and reveals, to our knowledge, a novel mechanism of action for TLR2 agonists in vivo.

Cannabinoid receptor 2 activation reduces intestinal leukocyte recruitment and systemic inflammatory mediator release in acute experimental sepsis.

INTRODUCTION: Cannabinoid receptor 2 (CB2R) expression is upregulated during sepsis. However, there are conflicting results regarding the effects of CB2R modulation in the hyperinflammatory phase of the disease. The aim of this study was therefore to investigate the effects of CB2R manipulation on leukocyte activation within the intestinal microcirculation in two acute experimental sepsis models. METHODS: In the endotoxemia model we studied four groups of Lewis rats: controls, lipopolysaccharide (LPS), LPS + CB2R agonist HU308 (2.5 mg/kg), and LPS + CB2R antagonist AM630 (2.5 mg/kg). In the colon ascendens stent peritonitis (CASP)-induced sepsis model we also studied four groups: sham group, CASP and CASP + CB2R agonist (HU308, 2.5 or 10 mg/kg). Intravital microscopy was performed 2 hours following LPS/placebo administration or 16 hours following CASP/sham surgery to quantify intestinal leukocyte recruitment. Additionally, hemodynamic monitoring, histological examinations and measurements of inflammatory mediators were performed. RESULTS: HU308 administration significantly reduced intestinal leukocyte adhesion in both acute sepsis models. The systemic levels of inflammatory mediators were significantly reduced by 10 mg/kg HU308 treatment in CASP animals. CONCLUSION: CB2R activation reduces leukocyte activation and systemic release of inflammatory mediators in acute experimental sepsis. Drugs targeting the CB2R pathway may have therapeutic potential in sepsis.

Natural killer T cell immunotherapy combined with IL-15-expressing oncolytic virotherapy and PD-1 blockade mediates pancreatic tumor regression.

BACKGROUND: Pancreatic cancer is one of the leading causes of cancer death, with a 5-year -year survival rate of less than 10%. This results from late detection, high rates of metastasis, and resistance to standard chemotherapies. Furthermore, chemotherapy and radiation are associated with significant morbidity, underscoring the need for novel therapies. Recent clinical studies have shown that immunotherapies can provide durable outcomes in cancer patients, but successes in pancreatic cancer have been limited. It is likely that novel and combined therapies will be needed to achieve clinical benefits. METHODS: Using experimental mouse models of pancreatic ductal adenocarcinoma, we examined natural killer T (NKT) cell activation therapy in combination with a recombinant oncolytic vesicular stomatitis virus (VSVΔM51) engineered to express the cytokine IL-15 (VSV-IL-15). Panc02 pancreatic ductal carcinoma cells were implanted subcutaneously or orthotopically into syngeneic C57BL/6 mice. Mice were then treated with VSV expressing green fluorescent protein (VSV-GFP) or VSV-IL-15 and/or NKT cell activation therapy via delivery of α-GalCer-loaded DCs. We further assessed whether the addition of PD-1 blockade could increase the therapeutic benefit of our combination treatment. Three days after NKT cell activation, some groups of mice were treated with anti-PD-1 antibodies weekly for 3 weeks. RESULTS: VSV-GFP and VSV-IL-15 mediated equal killing of human and mouse pancreatic cancer lines in vitro. In vivo, VSV-IL-15 combined with NKT cell activation therapy to enhance tumor regression and increase survival time over individual treatments, and was also superior to NKT cell therapy combined with VSV-GFP. Enhanced tumor control was associated with increased immune cell infiltration and anti-tumor effector functions (cytotoxicity and cytokine production). While ineffective as a monotherapy, the addition of blocking PD-1 antibodies to the combined protocol sustained immune cell activation and effector functions, resulting in prolonged tumor regression and complete tumor clearance in 20% of mice. Mice who cleared the initial tumor challenge exhibited reduced tumor growth uponon rechallenge, consistent with the formation of immune memory. CONCLUSION: TThese results demonstrate that NKT cell immunotherapy combined with oncolytic VSV-IL-15 virotherapy and PD-1 blockade enhances tumor control and presents a promising treatment strategy for targeting pancreatic cancer.

Mitochondrial damage-associated molecular patterns trigger arginase-dependent lymphocyte immunoregulation.

Tissue damage leads to loss of cellular and mitochondrial membrane integrity and release of damage-associated molecular patterns, including those of mitochondrial origin (mitoDAMPs). Here, we describe the lymphocyte response to mitoDAMPs. Using primary cells from mice and human donors, we demonstrate that natural killer (NK) cells and T cells adopt regulatory phenotypes and functions in response to mitoDAMPs. NK cell-mediated cytotoxicity, interferon gamma (IFN-γ) production, T cell proliferation, and in vivo anti-viral T cell activation are all interrupted in the presence of mitoDAMPs or mitoDAMP-rich irradiated cells in in vitro and in vivo assays. Mass spectrometry analysis of mitoDAMPs demonstrates that arginase and products of its enzymatic activity are prevalent in mitoDAMP preparations. Functional validation by arginase inhibition and/or arginine add-back shows that arginine depletion is responsible for the alteration in immunologic polarity. We conclude that lymphocyte responses to mitoDAMPs reflect a highly conserved mechanism that regulates inflammation in response to tissue injury.

Role of activated protein C and its receptor in inhibition of tumor metastasis.

Engagement of endothelial protein C receptor (EPCR) by activated protein C (aPC) decreases expression of endothelial adhesion molecules implicated in tumor-endothelium interactions. We examined the role of the aPC/EPCR pathway on tumor migration and metastasis. In vitro, B16-F10 melanoma cells showed decreased adhesion to and transmigration through endothelium treated with recombinant human aPC (rhaPC). In murine B16-F10 metastasis models, transgenic EPCR overexpressing (Tie2-EPCR) mice exhibited marked reductions in liver (50%) and lung (92%) metastases compared with wild-type (WT) animals. Intravital imaging showed reduced B16-F10 entrapment within livers of Tie2-EPCR compared with WT mice. A similar reduction was observed in WT mice treated with rhaPC. Strikingly, rhaPC treatment resulted in a 44% reduction in lung metastases. This was associated with decreased lung P-selectin and TNF-alpha mRNA levels. These findings support an important role for the aPC/EPCR pathway in reducing metastasis via inhibition of tumor cell adhesion and transmigration.

Enhanced tumor metastasis in response to blockade of the chemokine receptor CXCR6 is overcome by NKT cell activation.

Invariant NKT (iNKT) cells can induce potent antitumor responses in vivo. However, the mechanisms that regulate the effects of iNKT cells are unclear. The chemokine receptor CXCR6, and its ligand CXCL16, have been shown to play critical roles in iNKT cell homeostasis and activation. Thus we investigated the role of CXCR6 in protection against experimental metastasis of B16-F10 melanoma (B16) and Lewis lung carcinoma (LLC) cells to the liver and lungs. Wild-type and CXCR6(-/-) mice exhibited no differences in tumor cell metastasis to the lungs. However, metastasis of LLC and B16 tumor cells to the liver was enhanced in CXCR6(-/-) mice. Liver metastasis was also increased in wild-type mice treated with a CXCL16 neutralizing Ab. As Ab treatments did not alter iNKT cell numbers, this implicates a direct role for CXCR6/CXCL16 in regulating antitumor immunity. Cytokine induction was significantly attenuated in CXCR6(-/-) mice upon systemic iNKT cell activation with the glycolipid Ags alpha-galactosylceramide (alpha-GalCer), alpha-C-GalCer (a Th1 polarizing derivative), or OCH (a Th2 polarizing derivative). Despite differences in the levels of cytokine production, liver and lung metastasis were inhibited significantly in both wild-type and CXCR6(-/-) mice treated with glycolipids. Single doses of alpha-GalCer, alpha-C-GalCer, or OCH were sufficient to prevent liver metastasis and subsequent doses failed to elicit optimal cytokine responses. Our findings implicate a role for CXCR6 in natural immunosurveillance against liver metastasis. However, CXCR6 deficiency could be overcome by systemic iNKT cell activation, demonstrating that even suboptimal iNKT cell activation can protect against metastasis.

The Current Landscape of NKT Cell Immunotherapy and the Hills Ahead.

NKT cells are a specialized subset of lipid-reactive T lymphocytes that play direct and indirect roles in immunosurveillance and anti-tumor immunity. Preclinical studies have shown that NKT cell activation via delivery of exogenous glycolipids elicits a significant anti-tumor immune response. Furthermore, infiltration of NKT cells is associated with a good prognosis in several cancers. In this review, we aim to summarize the role of NKT cells in cancer as well as the current strategies and status of NKT cell immunotherapy. This review also examines challenges and future directions for improving the therapy.

Natural killer T cell immunotherapy combined with oncolytic vesicular stomatitis virus or reovirus treatments differentially increases survival in mouse models of ovarian and breast cancer metastasis.

BACKGROUND: Oncolytic viruses reduce tumor burden in animal models and have generated promising results in clinical trials. However, it is likely that oncolytic viruses will be more effective when used in combination with other therapies. Current therapeutic approaches, including chemotherapeutics, come with dose-limiting toxicities. Another option is to combine oncolytic viruses with immunotherapeutic approaches. METHODS: Using experimental models of metastatic 4T1 breast cancer and ID8 ovarian peritoneal carcinomatosis, we examined natural killer T (NKT) cell-based immunotherapy in combination with recombinant oncolytic vesicular stomatitis virus (VSV) or reovirus. 4T1 mammary carcinoma cells or ID8 ovarian cancer cells were injected into syngeneic mice. Tumor-bearing mice were treated with VSV or reovirus followed by activation of NKT cells via the intravenous administration of autologous dendritic cells loaded with the glycolipid antigen α-galactosylceramide. The effects of VSV and reovirus on immunogenic cell death (ICD), cell viability and immunogenicity were tested in vitro. RESULTS: VSV or reovirus treatments followed by NKT cell activation mediated greater survival in the ID8 model than individual therapies. The regimen was less effective when the treatment order was reversed, delivering virus treatments after NKT cell activation. In the 4T1 model, VSV combined with NKT cell activation increased overall survival and decreased metastatic burden better than individual treatments. In contrast, reovirus was not effective on its own or in combination with NKT cell activation. In vitro, VSV killed a panel of tumor lines better than reovirus. VSV infection also elicited greater increases in mRNA transcripts for proinflammatory cytokines, chemokines, and antigen presentation machinery compared with reovirus. Oncolytic VSV also induced the key hallmarks of ICD (calreticulin mobilization, plus release of ATP and HMGB1), while reovirus only mobilized calreticulin. CONCLUSION: Taken together, these results demonstrate that oncolytic VSV and NKT cell immunotherapy can be effectively combined to decrease tumor burden in models of metastatic breast and ovarian cancers. Oncolytic VSV and reovirus induced differential responses in our models which may relate to differences in virus activity or tumor susceptibility.