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Ovarian cancer associated ascites contains soluble inhibitory factors. The ascites was cleared of cellular debris by centrifugation as previously described (18). (A, B) The ascites was filtered 0.2 micron filter and fractionated- f1: 50. (C) The ascites was treated with 50 μg/ml proteinase K at 37{degree sign}C for 1 hour and then incubated at 95{degree sign}C for 10min to heat inactivate the enzyme. The antigen presenting cells were treated with the clarified supernantants for 4 h at 37{degree sign}C, then washed extensively with PBS, and cocultured with NKT hybridomas for 20-24 h at 37{degree sign}C. Cytokine released (IL-2) was measured as an indication of NKT cell activation and was measured by standard sandwich ELISA.

The source of GD3 impacts CD1d-mediated antigen presentation.(A) LCD1d cells were treated with GD3 from different sources for 4 hours, then washed extensively and cocultured with NKT cell hybridomas. IL-2 was measured, as an indication of NKT cell activation, by standard cytokine ELISA. (B) Pretreatment of a-GalCer loaded aAPC with GD3. a-GalCer loaded aAPC were incubated for 4hr with either medium, GD3, GD2, or ovarian cancer-associated ascites. The aAPC were washed extensively and then cocultured with a Va14+ NKT cell hybridoma, N38-3C3. NKT cell recognition of CD1d was assessed by measuring IL-2 production in the supernatants by ELISA.

PDF file - 225K, Schematic of Ganglioside GD3 and other glycolipids used or referred to in this paper

Natural killer T (NKT) cells play a pivotal role as a bridge between the innate and the adaptive immune response and are instrumental in the regulation of homeostasis. In this review, we discuss the potential for NKT cells to serve as biodrugs in viral infections and in cancer. NKT cells are being investigated for their use as a prognostic biomarker, an immune adjuvant, and as a form of cellular therapy. Historically, the clinical utility of NKT cells was hampered by their low frequency in the blood, discrepancies in nomenclature, and challenges with ex vivo expansion. However, recent advances in the field have permitted the development of several NKT cell-based preclinical and clinical strategies. These new developments pave the way for the successful implementation of NKT cell-based approaches for the treatment of human disease.

Natural killer T (NKT) cells rapidly respond to antigenic stimulation with cytokine production and direct cytotoxicity. These innate-like characteristics arise from their differentiation into mature effector cells during thymic development. A subset of mature NKT cells remain thymic resident, but their activation and function remain poorly understood. We examined the roles of CD28 and CTLA-4 in driving the activation of thymic resident NKT cells. In contrast to studies with peripheral NKT cells, the proliferation of thymic NKT cells was significantly impaired when CD28 engagement was blocked, but unaffected by CTLA-4 activation or blockade. Within NKT subsets, however, stage 3 NKT cells, marked by higher NK1.1 expression, were significantly more sensitive to the loss of CD28 signals compared to NK1.1− stage 2 NKT cells. In good agreement, CD28 blockade suppressed NKT cell cytokine secretion, lowering the ratio of IFN-γ:IL-4 production by NK1.1+ NKT cells. Intriguingly, the activation-dependent upregulation of the master transcription factor PLZF did not require CD28-costimulation in either of the thymic NKT subsets, underlining a dichotomy between requirements for early activation vs subsequent proliferation and effector function by these cells. Collectively, our studies demonstrate the ability of CD28 co-stimulation to fine tune subset-specific responses by thymic resident NKT cells and contextually shape the milieu in this primary lymphoid organ.

Background: The development of chemoresistance to paclitaxel and carboplatin represents a major therapeutic challenge in ovarian cancer, a disease frequently characterized by malignant ascites and extrapelvic metastasis. Microtentacles (McTNs) are tubulin-based projections observed in detached breast cancer cells. In this study, we investigated whether ovarian cancers exhibit McTNs and characterized McTN biology. Methods: We used an established lipid-tethering mechanism to suspend and image individual cancer cells. We queried a panel of immortalized serous (OSC) and clear cell (OCCC) cell lines as well as freshly procured ascites and human ovarian surface epithelium (HOSE). We assessed by Western blot β-tubulin isotype, α-tubulin post-translational modifications and actin regulatory proteins in attached/detached states. We studied clustering in suspended conditions. Effects of treatment with microtubule depolymerizing and stabilizing drugs were described. Results: Among cell lines, up to 30% of cells expressed McTNs. Four McTN morphologies (absent, symmetric-short, symmetric-long, tufted) were observed in immortalized cultures as well as ascites. McTN number/length varied with histology according to metastatic potential. Most OCCC overexpressed class III ß-tubulin. OCCC/OSC cell lines exhibited a trend towards more microtubule-stabilizing post-translational modifications of α-tubulin relative to HOSE. Microtubule depolymerizing drugs decreased the number/length of McTNs, confirming that McTNs are composed of tubulin. Cells that failed to form McTNs demonstrated differential expression of α-tubulin- and actin-regulating proteins relative to cells that form McTNs. Cluster formation is more susceptible to microtubule targeting agents in cells that form McTNs, suggesting a role for McTNs in aggregation. Conclusions: McTNs likely participate in key aspects of ovarian cancer metastasis. McTNs represent a new therapeutic target for this disease that could refine therapies, including intraperitoneal drug delivery.

NK cell suppression of T cells is a key determinant of viral pathogenesis and vaccine efficacy. This process involves perforin-dependent elimination of activated CD4+ T cells during the first 3 days of infection. Although this mechanism requires cell-cell contact, NK cells and T cells typically reside in different compartments of lymphoid tissues at steady state. Here, we showed that NK cell suppression of T cells is associated with transient accumulation of NK cells within T cell-rich sites of the spleen during lymphocytic choriomeningitis virus infection. The chemokine receptor CXCR3 was required for this relocation and suppression of antiviral T cells. Accordingly, NK cell migration was mediated by type I IFN-dependent promotion of CXCR3 ligand expression. In contrast, adenoviral vectors that weakly induced type I IFN and did not stimulate NK cell inhibition of T cells also did not promote measurable redistribution of NK cells to T cell zones. Exogenous IFN rescued NK cell migration during adenoviral vector immunization. Thus, type I IFN and CXCR3 were critical for properly positioning NK cells to constrain antiviral T cell responses. Development of strategies to curtail migration of NK cells between lymphoid compartments may enhance vaccine-elicited immune responses.

Francisella tularensis (Ft) is a Gram-negative, facultative intracellular bacterium that is a Tier 1 Select Agent of concern for biodefense for which there is no licensed vaccine. A subfamily of 9 Francisella phagosomal transporter (fpt) genes belonging to the Major Facilitator Superfamily of transporters was identified as critical to pathogenesis and potential targets for attenuation and vaccine development. We evaluated the attenuation and protective capacity of LVS derivatives with deletions of the fptA and fptF genes in the C57BL/6J mouse model of respiratory tularemia. LVSΔfptA and LVSΔfptF were highly attenuated with LD50 values of &gt, 20 times that of LVS when administered intranasally and conferred 100% protection against lethal challenge. Immune responses to the fpt mutant strains in mouse lungs on day 6 post-infection were substantially modified compared to LVS and were associated with reduced organ burdens and reduced pathology. The immune responses to LVSΔfptA and LVSΔfptF were characterized by decreased levels of IL-10 and IL-1β in the BALF versus LVS, and increased numbers of B cells, αβ and γδ T cells, NK cells, and DCs versus LVS. These results support a fundamental requirement for FptA and FptF in the pathogenesis of Ft and the modulation of the host immune response.

Francisella tularensis (Ft) is a Gram-negative, facultative intracellular bacterium that is a Tier 1 Select Agent of concern for biodefense for which there is no licensed vaccine. A subfamily of 9 Francisella phagosomal transporter (fpt) genes belonging to the Major Facilitator Superfamily of transporters was identified as critical to pathogenesis and potential targets for attenuation and vaccine development. We evaluated the attenuation and protective capacity of LVS derivatives with deletions of the fptA and fptF genes in the C57BL/6J mouse model of respiratory tularemia. LVSΔfptA and LVSΔfptF were highly attenuated with LD50 values of >20 times that of LVS when administered intranasally and conferred 100% protection against lethal challenge. Immune responses to the fpt mutant strains in mouse lungs on day 6 post-infection were substantially modified compared to LVS and were associated with reduced organ burdens and reduced pathology. The immune responses to LVSΔfptA and LVSΔfptF were characterized by decreased levels of IL-10 and IL-1β in the BALF versus LVS, and increased numbers of B cells, αβ and γδ T cells, NK cells, and DCs versus LVS. These results support a fundamental requirement for FptA and FptF in the pathogenesis of Ft and the modulation of the host immune response.

NKT cells are primed lymphocytes that rapidly secrete cytokines and can directly kill cancerous cells. Given the critical role NKT cells play in cancer immune surveillance, we sought to investigate the effect of mutations in Brca1, specifically a conditional deletion of exon 11, on type I invariant NKT cell development. We observed a significant reduction in invariant NKT cells in both primary lymphoid and peripheral organs in Brca1 mutant mice compared with wild-type C57BL/6. However, the original Brca1 mutant strain was on a mixed background containing FVB/N. We determined that strain differences, rather than mutations in Brca1, led to the observed loss in NKT cells. Importantly, we found that whereas FVB/N mice lack Vβ8, there was a striking increase in the total number of thymic type I CD1d–α-galactosylceramide tetramer positive NKT cells and skewing of the NKT cell population to NKT2 compared with C57BL/6 mice. Collectively, our data demonstrate the profound effect genetics can have on NKT cell subset differentiation.

Committing to a differentiation pathway means leaving alternative pathways behind. Adoue et al. (2019) report that the H3K9-methyltrasferase Setdb1 plays a role in inhibiting the Th1 program in committed Th2 cells, and mechanistically, its role might relate to the selective targeting of endogenous retroviruses adjacent to Th1 enhancers.

Natural killer T (NKT) cells are a unique subset of lymphocytes that recognize lipid antigens in the context of the non-classical class I MHC molecule, CD1d, and serve as a link between the innate and adaptive immune system through their expeditious release of cytokines. Whereas NKT have well-established roles in mitigating a number of human diseases, herein, we focus on their role in cancer. NKT cells have been shown to directly and indirectly mediate anti-tumor immunity and manipulating their effector functions can have therapeutic significances in treatment of cancer. In this review, we highlight several therapeutic strategies that have been used to harness the effector functions of NKT cells to target different types of solid tumors. We also discuss several barriers to the successful utilization of NKT cells and summarize effective strategies being developed to harness the unique strengths of this potent population of T cells. Collectively, studies investigating the therapeutic potential of NKT cells serve not only to advance our understanding of this powerful immune cell subset, but also pave the way for future treatments focused on the modulation of NKT cell responses to enhance cancer immunotherapy.

Natural killer T (NKT) cells rapidly respond to antigenic stimulation with cytokine production and direct cytotoxicity. These innate-like characteristics arise from their differentiation into mature effector cells during thymic development. A subset of mature NKT cells remain thymic resident, but their activation and function remain poorly understood. We examined the roles of CD28 and CTLA-4 in driving activation of these thymic resident NKT cells. Unlike previous studies with peripheral NKT cells, the proliferation of thymic NKT cells was significantly impaired when CD28 engagement was blocked - but unaffected with CTLA-4 activation or blockade. Within NKT cell subsets, however, stage 3 NKT cells marked by higher NK1.1 expression were significantly more sensitive to the loss of CD28 signals compared to NK1.1 negative stage 2 NKT cells. In concordance with this, CD28 blockade suppressed all NKT cell cytokines, lowering the ratio of IFN-γ:IL-4 production by NK1.1 positive NKT cells. Intriguingly, the activation-dependent upregulation of the master transcription factor PLZF did not require CD28 co-stimulation in either NK1.1 positive or negative thymic NKT subsets – underlining a dichotomy between requirements for early activation and the subsequent proliferation and effector function of these cells. Collectively, our studies demonstrate the ability of CD28 co-stimulation to fine tune subset-specific responses by thymic resident NKT cells and contextually shape the milieu in this primary lymphoid organ.

Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin&rsquo, s lymphoma. Despite being responsive to combination chemotherapy, median survival remains around 5 years due to high rates of relapse. Sphingolipid metabolism regulates MCL survival and proliferation and we found that sphingosine-1-phosphate (S1P) is upregulated in MCL cells. Therapeutic targeting of the S1P1 receptor or knockdown of sphingosine kinase 1 (SK1), the enzyme responsible for generating S1P, in human MCL cells results in a significant increase in Natural Killer T (NKT) cell activation. NKT cells recognize glycolipid antigens presented on CD1d and can reduce MCL tumor burden in vivo. Lipidomic studies identified cardiolipin, which has been reported to bind to CD1d molecules, as being upregulated in SK1 knockdown cells. We found that the pretreatment of antigen presenting cells with cardiolipin leads to increased cytokine production by NKT cell hybridomas. Furthermore, the ability of cardiolipin to activate NKT cells was dependent on the structure of its acyl chains. Collectively, these studies delineate novel pathways important for immune recognition of malignant cells and could lead to the development of new treatments for lymphoma.

Tumors develop multiple mechanisms of immune evasion as they progress, with some cancer types being inherently better at ‘hiding’ than others. With an increased understanding of tumor immune surveillance, immunotherapy has emerged as a promising treatment strategy for breast cancer, despite historically being thought of as an immunologically silent neoplasm. Some types of cancer, such as melanoma, bladder, and renal cell carcinoma, have demonstrated a durable response to immunotherapeutic intervention, however, breast neoplasms have not shown the same efficacy. The causes of breast cancer’s immune silence derive from mechanisms that diminish immune recognition and others that promote strong immunosuppression. It is the mechanisms of immune evasion in breast cancers that are poorly defined. Thus, further characterization is critical for the development of better therapies. This brief review will seek to provide insight into the possible causes of weak immunogenicity and immune suppression mediated by breast cancers and highlight current immunotherapies being used to restore immune responses to breast cancer.

Natural killer T (NKT) cells are innate-like lymphocytes that bridge the gap between the innate and adaptive immune responses. Like innate immune cells, they have a mature, effector phenotype that allows them to rapidly respond to threats, compared to adaptive cells. NKT cells express T cell receptors (TCRs) like conventional T cells, but instead of responding to peptide antigen presented by MHC class I or II, NKT cell TCRs recognize glycolipid antigen in the context of CD1d. NKT cells are subdivided into classes based on their TCR and antigen reactivity. This review will focus on type I iNKT cells that express a semi invariant Vα14Jα18 TCR and respond to the canonical glycolipid antigen, α-galactosylceramide. The innate-like effector functions of these cells combined with their T cell identity make their developmental path quite unique. In addition to the extrinsic factors that affect iNKT cell development such as lipid:CD1d complexes, co-stimulation, and cytokines, this review will provide a comprehensive delineation of the cell intrinsic factors that impact iNKT cell development, differentiation, and effector functions - including TCR rearrangement, survival and metabolism signaling, transcription factor expression, and gene regulation.

Invariant natural killer T (iNKT) cells are an integral component of the immune system and play an important role in antitumor immunity. Upon activation, iNKT cells can directly kill malignant cells as well as rapidly produce cytokines that stimulate other immune cells, making them a front line defense against tumorigenesis. Unfortunately, iNKT cell number and activity are reduced in multiple cancer types. This anergy is often associated with upregulation of co-inhibitory markers such as programmed death-1. Similar to conventional T cells, iNKT cells are influenced by the conditions of their activation. Conventional T cells receive signals through the following three types of receptors: (1) T cell receptor (TCR), (2) co-stimulation molecules, and (3) cytokine receptors. Unlike conventional T cells, which recognize peptide antigen presented by MHC class I or II, the TCRs of iNKT cells recognize lipid antigen in the context of the antigen presentation molecule CD1d (Signal 1). Co-stimulatory molecules can positively and negatively influence iNKT cell activation and function and skew the immune response (Signal 2). This study will review the background of iNKT cells and their co-stimulatory requirements for general function and in antitumor immunity. We will explore the impact of monoclonal antibody administration for both blocking inhibitory pathways and engaging stimulatory pathways on iNKT cell-mediated antitumor immunity. This review will highlight the incorporation of co-stimulatory molecules in antitumor dendritic cell vaccine strategies. The use of co-stimulatory intracellular signaling domains in chimeric antigen receptor-iNKT therapy will be assessed. Finally, we will explore the influence of innate-like receptors and modification of immunosuppressive cytokines (Signal 3) on cancer immunotherapy.

Epithelial ovarian cancer (EOC) is the seventh most common cancer diagnosis among women worldwide and has the highest mortality rate of all gynecologic malignancies (1). In the United States, EOC is the fifth most common cause of cancer deaths among women (2). Despite improvements in treatment, 5-year survival rates of patients with advanced ovarian cancer remain less than 50% (3,4) because the majority of patients present with late stage disease (5). Thus, the identification of novel biomarkers and the development of innovative therapeutic approaches are urgently needed. It has been well established that evading immune surveillance is critical for tumor growth and metastasis, and other groups have shown that vascular endothelial growth factor (VEGF) expression is inversely correlated with survival in ovarian cancer patients (6,7). Therefore, we investigated the effects of ovarian cancer-associated VEGF on CD1d-mediated antigen presentation to natural killer T (NKT) cells (8). We found that inhibition of VEGF production by ovarian cancer cell lines led to a reduction in the expression of the immunosuppressive ganglioside GD3 and restored NKT cell activation. Thus, we identified a novel link between immunosuppressive ganglioside shedding and VEGF production by ovarian cancers.

Natural killer (NK) cells of the innate immune system and natural killer T (NKT) cells, which have roles in both the innate and adaptive responses, are unique lymphocyte subsets that have similarities in their functions and phenotypes. Both cell types can rapidly respond to the presence of tumor cells and participate in immune surveillance and antitumor immune responses. This has incited interest in the development of novel cancer therapeutics based on NK and NKT cell manipulation. Chimeric antigen receptors (CARs), generated through the fusion of an antigen-binding region of a monoclonal antibody or other ligand to intracellular signaling domains, can enhance lymphocyte targeting and activation toward diverse malignancies. Most of the CAR studies have focused on their expression in T cells; however, the functional heterogeneity of CAR T cells limits their therapeutic potential and is associated with toxicity. CAR-modified NK and NKT cells are becoming more prevalent because they provide a method to direct these cells more specifically to target cancer cells, with less risk of adverse effects. This review will outline current NK and NKT cell CAR constructs and how they compare to conventional CAR T cells, and discuss future modifications that can be explored to advance adoptive cell transfer of NK and NKT cells.

Sialic acids on glycoconjugates play a pivotal role in many biological processes. In the airways, sialylated glycoproteins and glycolipids are strategically positioned on the plasma membranes of epithelia to regulate receptor-ligand, cell-cell, and host-pathogen interactions at the molecular level. We now demonstrate, for the first time, sialidase activity for ganglioside substrates in human airway epithelia. Of the four known mammalian sialidases, NEU3 has a substrate preference for gangliosides and is expressed at mRNA and protein levels at comparable abundance in epithelia derived from human trachea, bronchi, small airways, and alveoli. In small airway and alveolar epithelia, NEU3 protein was immunolocalized to the plasma membrane, cytosolic, and nuclear subcellular fractions. Small interfering RNA-induced silencing of NEU3 expression diminished sialidase activity for a ganglioside substrate by >70%. NEU3 immunostaining of intact human lung tissue could be localized to the superficial epithelia, including the ciliated brush border, as well as to nuclei. However, NEU3 was reduced in subepithelial tissues. These results indicate that human airway epithelia express catalytically active NEU3 sialidase.

Natural killer T (NKT) cells are a unique subset of CD1d-restricted T lymphocytes that express characteristics of both T cells and natural killer cells. NKT cells mediate tumor immune-surveillance; however, NKT cells are numerically reduced and functionally impaired in lymphoma patients. Many hematologic malignancies express CD1d molecules and co-stimulatory proteins needed to induce anti-tumor immunity by NKT cells, yet most tumors are poorly immunogenic. In this study, we sought to investigate NKT cell responses to B cell lymphoma. In the presence of exogenous antigen, both mouse and human NKT cell lines produce cytokines following stimulation by B cell lymphoma lines. NKT cell populations were examined ex vivo in mouse models of spontaneous B cell lymphoma, and it was found that during early stages, NKT cell responses were enhanced in lymphoma-bearing animals compared to disease-free animals. In contrast, in lymphoma-bearing animals with splenomegaly and lymphadenopathy, NKT cells were functionally impaired. In a mouse model of blastoid variant mantle cell lymphoma, treatment of tumor-bearing mice with a potent NKT cell agonist, α-galactosylceramide (α-GalCer), resulted in a significant decrease in disease pathology. Ex vivo studies demonstrated that NKT cells from α-GalCer treated mice produced IFN-γ following α-GalCer restimulation, unlike NKT cells from vehicle-control treated mice. These data demonstrate an important role for NKT cells in the immune response to an aggressive hematologic malignancy like mantle cell lymphoma.

Invariant natural killer T (iNKT) cells are a specialized subset of T cells contributing to both, the innate and adaptive immune responses. In contrast to conventional T lymphocytes they recognize lipid antigens. The aim of the project is to establish a novel model system, to study iNKT-TCR - ligand interaction. An iNKT reporter cell line (JE6-1

Ovarian cancer is the deadliest gynecologic malignancy worldwide and mortality rates have remained steady despite advances in treatment. A major obstacle for current therapies is overcoming tumor immunosuppression, therefore targeting inhibitory factors produced in the tumor microenvironment could enhance antitumor responses. Natural Killer T (NKT) cells, a subset of T cells that recognize glycolipid antigens presented by CD1d, are important mediators of antitumor immune responses. We have previously shown that ovarian cancers shed the ganglioside GD3, which binds with high affinity to CD1d, thereby suppressing NKT cell activation. In addition to GD3, we have identified vascular endothelial growth factor (VEGF) as another immunosuppressive factor secreted by ovarian cancers. We found that VEGF inhibition results in decreased GD3 expression in human ovarian cancer cell lines and restoration of NKT cell responses. In addition, we found that treatment with VEGF can directly impair NKT cell functional responses. To therapeutically target the VEGF-GD3 signaling cascade, mouse and human ovarian cancer cell lines were treated with triptolide, a phytochemical which has demonstrated potent antitumor activity in vitro and in vivo. Treatment with triptolide reduces VEGF secretion by ovarian cancer cell lines and results in a concomitant decrease in the expression of GD3 synthase. In addition, pre-treatment of ovarian cancer cells with triptolide improves the ability of NKT cells to recognize and lyse the cancer cells. Thus, we have identified a novel therapeutic strategy that can be used to block the production of immunosuppressive factors, thereby restoring NKT cell-mediated antitumor immune responses in ovarian cancer.

Increasing evidence suggests that gut flora play an important role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Our previous studies show that hepatic natural killer T (NKT) cells play a significant role in the pathogenesis of NAFLD. In this study, we explore the mechanism by which modification of gut flora leads to the alteration of hepatic NKT cells and improvement of steatosis. Mice were fed a high-fat (HF) diet to induce NAFLD. Some of them also received different doses of mixed-strain probiotics (VSL#3); single-strain probiotic (Bifidobacterium infantis) or antibiotics. Animal weight, glucose tolerance, liver steatosis and hepatic NKT cells were assessed. Lipid extracts from probiotics were tested for their ability to activate NKT cells. Toll-like receptor 4 (TLR4) knockout mice were also evaluated for their responses to HF diet. High-dose VSL#3 was more effective than low-dose VSL#3 and B. infantis for the improvement of hepatic NKT cell depletion and steatosis. The lipids extracted from VSL#3 stimulated NKT cells both in vivo and in vitro. In contrast, lipids from B. infantis decreased α-GalCer-mediated NKT cell activation in vitro, but were able to stimulate NKT cells. TLR4 knockout mice have a similar response to HF-diet-induced NKT cell depletion and obesity. These results suggest that alterations in the gut flora have profound effects on hepatic NKT cells and steatosis, which are both strain-specific and dose-dependent, but not through TLR4 signalling. Furthermore, these data suggest that probiotics may contain bacterial glycolipid antigens that directly modulate the effector functions of hepatic NKT cells.

Natural killer T (NKT) cells are a unique subset of lymphocytes that bridge the innate and adaptive immune system. NKT cells possess a classic αβ T cell receptor (TCR) that is able to recognize self and foreign glycolipid antigens presented by the nonclassical class I major histocompatibility complex (MHC) molecule, CD1d. Type I NKT cells (referred to as invariant NKT cells) express a semi-invariant Vα14Jα18 TCR in mice and Vα24Jα18 TCR in humans. Type II NKT cells are CD1d-restricted T cells that express a more diverse set of TCR α chains. The two types of NKT cells often exert opposing effects especially in tumor immunity, where type II cells generally suppress tumor immunity while type I NKT cells can enhance anti-tumor immune responses. In this review, we focus on the role of NKT cells in cancer. We discuss their effector and suppressive functions, as well as describe preclinical and clinical studies utilizing therapeutic strategies focused on harnessing their potent anti-tumor effector functions, and conclude with a discussion on potential next steps for the utilization of NKT cell-targeted therapies for the treatment of cancer.

Histone deacetylases (HDACs) are a family of enzymes that influence expression of genes implicated in tumor initiation, progression, and anti-tumor responses. In addition to their canonical role in deacetylation of histones, HDACs regulate many non-canonical targets, such as Signal Transducer and Activator of Transcription 3 (STAT3). We hypothesize that tumors use epigenetic mechanisms to dysregulate CD1d-mediated antigen presentation, thereby impairing the ability of natural killer T (NKT) cells to recognize and destroy malignant cells. In this study, we pre-treated CD1d-expressing tumor cells with HDAC inhibitors (HDACi) and assessed CD1d-dependent NKT cell responses to mantle cell lymphoma (MCL). Pre-treatment with Trichostatin-A, a pan-HDACi, rapidly enhanced both CD1d- and MHC class II-mediated antigen presentation. Similarly, treatment of MCL cells with other HDACi resulted in enhanced CD1d-dependent NKT cell responses. The observed changes are due, at least in part, to an increase in both CD1D mRNA and CD1d cell surface expression. Mechanistically, we found that HDAC2 binds to the CD1D promoter. Knockdown of HDAC2 in tumor cells resulted in a significant increase in CD1d-mediated antigen presentation. In addition, treatment with HDACi inhibited STAT3 and STAT3-regulated inflammatory cytokine secretion by MCL cells. We demonstrated that MCL-secreted IL-10 inhibits CD1d-mediated antigen presentation and pre-treatment with TSA abrogates secretion of IL-10 by MCL. Taken together, our studies demonstrate the efficacy of HDACi in restoring anti-tumor responses to MCL through both cell-intrinsic and cell-extrinsic mechanisms and strongly implicate a role for HDACi in enhancing immune responses to cancer.

Natural killer T (NKT) cells serve as a bridge between the innate and adaptive immune systems, and manipulating their effector functions can have therapeutic significances in the treatment of autoimmunity, transplant biology, infectious disease, and cancer. NKT cells are a subset of T cells that express cell-surface markers characteristic of both natural killer cells and T cells. These unique immunologic cells have been demonstrated to serve as a link between the innate and adaptive immune systems through their potent cytokine production following the recognition of a range of lipid antigens, mediated through presentation of the major histocompatibility complex (MHC) class I like CD1d molecule, in addition to the NKT cell's cytotoxic capabilities upon activation. Although a number of glycolipid antigens have been shown to complex with CD1d molecules, most notably the marine sponge derived glycolipid alpha-galactosylceramide (α-GalCer), there has been debate as to the identity of the endogenous activating lipid presented to the T-cell receptor (TCR) via the CD1d molecule on antigen-presenting cells (APCs). This review aims to survey the use of pharmacological agents and subsequent structure-activity relationships (SAR) that have given insight into the binding interaction of glycolipids with both the CD1d molecules as well as the TCR and the subsequent immunologic response of NKT cells. These studies not only elucidate basic binding interactions but also pave the way for future pharmacological modulation of NKT cell responses.

Acute myeloid leukemia (AML) is a genetically heterogeneous disease where malignant clonal proliferation of immature myeloid cells in the bone marrow leads to disruption of normal hematopoiesis and bone marrow failure. Most treatment improvements have consisted of limited chemotherapeutic interventions, and morbidity and mortality remain unacceptably high. While patients unfit for intensive chemotherapy have the options of epigenetic treatments with DNA methyltransferase inhibitors (DNMTis), and the preclinical research underpinning these treatments is robust and promising, clinical response rates have been poor. Therefore, we have derived a unique treatment strategy combining poly-ADP ribose polymerase inhibitors (PARPis) with DNMTis, and we have previously demonstrated efficacy of this combination in AML in both in vitroand in vivomodels. This combination increases the amount and retention time of PARP1 at laser-induced DNA damage sites, increases DNA DSBs and tumor cytotoxicity, blunts cell self-renewal, and leads to strong anti-tumor responses. These results form the basis for our ongoing Phase I/II clinical trial of this combination therapy in patients with relapsed/refractory AML. To identify molecular pathways underpinning this combination, genome-wide expression microarray analysis was performed on RNA extracted from multiple BRCA-intact sporadic AML cell lines treated with single agents or drug combination. Interestingly, combination drug treatment, more than either single treatment alone, up regulated genes in immune response pathways, including interferons, natural killer cell-mediated cytotoxicity and Toll-like receptor signaling pathway genes. In both AML cell lines (N=10) and primary samples (N=8), we used qPCR to validate these expression increases, with particular focus on interferon type 1-inducible genes, including IFI6, IFI27, IFI30, IRF7, ISG15, and OASL. These interferon-inducible genes were similarly increased in selected AML clinical trial samples post combination treatment. Interferon stimulation studies showed that treatment of multiple cell lines with IFN-β similarly up regulates our genes of interest, and suggests that the immune system plays a pivotal role in mediating the effects of our combination treatment. To functionally validate this immune involvement in vivo, we use an immune competent mouse model that spontaneously develops AML. The bone marrow cells collected from leukemic FLT3/ITD;NHD13 mice are transplanted into sublethally irradiated syngeneic recipients where they undergo clonal leukemic expansion and expression of specific surface markers (CD45.1 vs CD45.2) allow for the tracking of both host and transplanted blood cells. After validating that our combination treatment increases survival and reduces tumor burden in this in vivo model, we isolated mononuclear cells from spleens and livers and found that mice in the combination therapy group had increased T cell populations with increased PD1, CD28 and 4-1BB expression. Tal single agent and combination therapy treated mice also had decreased myeloid-derived suppressor cells and increased antigen presenting cells, specifically dendritic cells, monocytes and macrophages. Finally, upon ex vivo stimulation, T cells from mice receiving combination therapy produced significantly more IFN-γ and GM-CSF than mice from other treatment groups. These results indicate that our combination therapy results in immune activation, and suggest the potential to target the immune system in order to induce anti-tumor effects, enhance efficacy of our combination treatment and improve clinical outcomes in AML. Disclosures Lapidus: KinaRx, LLC: Other: Founder and Scientific Advisor.

Dietary fatty acids are major contributors to the development and progression of insulin resistance and nonalcoholic fatty liver disease (NAFLD). Dietary fatty acids also alter hepatic NKT cells that are activated by antigens presented by CD1d. In the current study, we examine the mechanism of dietary fatty acid induced hepatic NKT cell deficiency and its causal relationship to insulin resistance and NAFLD. We discover that dietary saturated fatty acids (SFA) or monounsaturated fatty acids (MUFA), but not polyunsaturated fatty acids (PUFA), cause hepatic NKT cell depletion with increased apoptosis. Dietary SFA or MUFA also impair hepatocyte presentation of endogenous, but not exogenous, antigen to NKT cells, indicating alterations of the endogenous antigen processing or presenting pathway. In vitro treatment of normal hepatocytes with fatty acids also demonstrates impaired ability of CD1d to present endogenous antigen by dietary fatty acids. Furthermore, dietary SFA and MUFA activate the NFkappaB signaling pathway and lead to insulin resistance and hepatic steatosis. In conclusion, both dietary SFA and MUFA alter endogenous antigen presentation to hepatic NKT cells and contribute to NKT cell depletion, leading to further activation of inflammatory signaling, insulin resistance, and hepatic steatosis.

Purpose: Natural killer T (NKT) cells recognize lipid antigen presented by CD1 molecules. NKT cells can both directly, through cytotoxicity, and indirectly, through activation of other effector cells, mediate antitumor immunity. It has been shown, however, that tumor-associated lipids are frequently shed into the tumor microenvironment, which can mediate immunosuppressive activity. Given that ovarian cancer–associated ascites has been reported to have increased levels of gangliosides, we examined the effect of tumor-associated and other ascites on CD1d-mediated antigen presentation to NKT cells.Experimental Design: To investigate the effects of ascites on NKT cell activation, we pretreated CD1d-expressing cells with the ascites and measured their ability to stimulate cytokine production in NKT cells. To determine whether antigen processing or editing was necessary, CD1d-immunoglobulin–based artificial antigen presenting cells (aAPC) were also incubated with ascites. In addition, to examine specificity, we analyzed whether ascites fluid could influence the activation of classic CD8+ T cells.Results: Pretreatment of CD1d-expressing cells with ascites from the majority of patients inhibited the ability of the cells to stimulate/activate NKT cells in a dose-dependent manner. Ascites treatment also partially blocked the ability of α-galactosylceramide–loaded CD1d-immunoglobulin–based aAPC to activate NKT cells. In addition, our data show that treatment with ascites does not inhibit HLA-A2–mediated activation of classic CD8+ T cells.Conclusions: Together, these data suggest that ovarian and other cancers may have developed immune evasion mechanisms specifically targeting the CD1/NKT cell system.

One of the mechanisms by which malignancies can induce immune suppression is through the production of cytokines that affect the maturation and differentiation of inflammatory cells in the tumor microenvironment. Semaphorin 4D (Sema4D) is a proangiogenic cytokine produced by several malignancies, which has been described in the regulation of the immune system. In the present study, we examined the role of human head and neck squamous cell carcinoma (HNSCC)–secreted Sema4D on myeloid cell differentiation. CD33+ cells cultured in HNSCC cell line–derived conditioned medium differentiated into myeloid derived suppressor cells (MDSC) (CD33+CD11b+HLA-DR−/low). The addition of anti-Sema4D Ab to HNSCC conditioned medium significantly reduced the expansion of the MDSC population. Similarly, knockdown of Sema4D in an HNSCC cell line resulted in a loss of MDSC function as shown by a decrease in the production of the immune-suppressive cytokines arginase-1, TGF-β, and IL-10 by MDSC, concomitant with recovery of T cell proliferation and IFN-γ production following stimulation of CD3/CD28. Importantly, CD33+ myeloid and T cells cultured in conditioned medium of HNSCC cells in which Sema4D was knocked down promoted antitumor inflammatory profile, through recovery of the effector T cells (CD4+T-bet+ and CD8+T-bet+), as well as a decrease in regulatory T cells (CD4+CD25+FOXP3+). We also showed that Sema4D was comparable to GM-CSF in its induction of MDSC. Collectively, this study describes a novel immunosuppressive role for Sema4D in HNSCC through induction of MDSC, and it highlights Sema4D as a therapeutic target for future studies to enhance the antitumorigenic inflammatory response in HNSCC and other epithelial malignancies.

Purpose: Natural killer T (NKT) cells are important mediators of antitumor immune responses. We have previously shown that ovarian cancers shed the ganglioside GD3, which inhibits NKT-cell activation. Ovarian cancers also secrete high levels of VEGF. In this study, we sought to test the hypothesis that VEGF production by ovarian cancers suppresses NKT-cell–mediated antitumor responses. Experimental Design: To investigate the effects of VEGF on CD1d-mediated NKT-cell activation, a conditioned media model was established, wherein the supernatants from ovarian cancer cell lines (OV-CAR-3 and SK-OV-3) were used to treat CD1d-expressing antigen-presenting cells (APC) and cocultured with NKT hybridomas. Ovarian cancer–associated VEGF was inhibited by treatment with bevacizumab and genistein; conditioned medium was collected, and CD1d-mediated NKT-cell responses were assayed by ELISA. Results: Ovarian cancer tissue and ascites contain lymphocytic infiltrates, suggesting that immune cells traffic to tumors, but are then inhibited by immunosuppressive molecules within the tumor microenvironment. OV-CAR-3 and SK-OV-3 cell lines produce high levels of VEGF and GD3. Pretreatment of APCs with ascites or conditioned medium from OV-CAR-3 and SK-OV-3 blocked CD1d-mediated NKT-cell activation. Inhibition of VEGF resulted in a concomitant reduction in GD3 levels and restoration of NKT-cell responses. Conclusions: We found that VEGF inhibition restores NKT-cell function in an in vitro ovarian cancer model. These studies suggest that the combination of immune modulation with antiangiogenic treatment has therapeutic potential in ovarian cancer. Clin Cancer Res; 22(16); 4249–58. ©2016 AACR.

Vaccinia virus (VV) has been most commonly used as the vaccine to protect individuals against the causative agent of smallpox (variola virus), but it also uses a number of strategies meant to evade or blunt the host's antiviral immune response. Natural killer T (NKT) cells are a subset of immunoregulatory CD1d-restricted T lymphocytes believed to bridge the innate and adaptive immune responses. It is shown here that the VV-encoded molecules, B1R and H5R, play a role in the ability of VV to inhibit CD1d-mediated antigen presentation to NKT cells. These are the first poxvirus-encoded molecules identified that can play such a role in the evasion of an important component of the innate immune response.

Immunity to collagen V [col(V)] contributes to lung 'rejection.' We hypothesized that ischemia reperfusion injury (IRI) associated with lung transplantation unmasks antigenic col(V) such that fresh and well-healed lung grafts have differential susceptibility to anti-col(V)-mediated injury; and expression of the autoimmune cytokines, IL-17 and IL-23, are associated with this process. Adoptive transfer of col(V)-reactive lymphocytes to WKY rats induced grade 2 rejection in fresh isografts, but induced worse pathology (grade 3) when transferred to isograft recipients 30 days post-transplantation. Immunhistochemistry detected col(V) in fresh and well-healed isografts but not native lungs. Hen egg lysozyme-reactive lymphocytes (HEL, control) did not induce lung disease in any group. Col(V), but not HEL, immunization induced transcripts for IL-17 and IL-23 (p19) in the cells utilized for adoptive transfer. Transcripts for IL-17 were upregulated in fresh, but not well-healed isografts after transfer of col(V)-reactive cells. These data show that IRI predisposes to anti-col(V)-mediated pathology; col(V)-reactive lymphocytes express IL-17 and IL-23; and anti-col(V)-mediated lung disease is associated with local expression of IL-17. Finally, because of similar histologic patterns, the pathology of clinical rejection may reflect the activity of autoimmunity to col(V) and/or alloimmunity.

A critical component of the host’s innate immune response involves lipid Ag presentation by CD1d molecules to NK T cells. In this study we used murine CD1d1-transfected L (L-CD1) cells to study the effect of viruses on CD1d-mediated Ag presentation to NKT cells and found that an infection with vesicular stomatitis and vaccinia (but not lymphocytic choriomeningitis) virus inhibited murine CD1d1-mediated Ag presentation. This was under the reciprocal control of the MAPKs, p38 and ERK, and was due to changes in the intracellular trafficking of CD1d1. The reciprocal regulation of CD1d1-mediated Ag presentation by MAPK suggests that the targeting of these pathways is a novel means of immune evasion by viruses.

Dendritic cells (DCs) are central to the integration of innate and adaptive immunity. In contrast to B and T lymphocytes, DCs have retained many of the pattern recognition receptors and are thus uniquely able to sense stimuli such as tissue damage, necrosis, and bacterial and viral infection. Also, immature DCs respond to danger signals in the environment, which leads to their maturation, upon which DCs differentiate and acquire the ability to direct the development of the primary immune response. The ability of lung DCs to elicit specific CD4 and CD8 T lymphocyte responses have made them attractive targets for vaccine development strategies in the treatment and prevention of diseases such as allograft rejection responses, allergy, and asthma, as well as autoimmune disease and cancer.

Invariant natural killer T (iNKT) cells constitute an important subset of T cells that can both directly and indirectly mediate anti-tumor immunity. However, cancer patients have a reduction in both iNKT cell number and function, and these deficits limit the potential clinical application of iNKT cells for cancer therapy. To overcome the problem of limited iNKT cell numbers, we investigated whether iNKT cells can be generated in vitro from bone marrow-derived adult hematopoietic stem-progenitor cells (HSPC). Our data demonstrate that co-culture of HSPC with OP9-DL1 stromal cells, results in a functional CD3(+) T cell population. These T cells can be further differentiated into iNKT cells by secondary culture with CD1d-Ig-based artificial antigen-presenting cells (aAPC). Importantly, these in vitro-generated iNKT cells are functional, as demonstrated by their ability to proliferate and secrete IFN-γ and GM-CSF following stimulation.

Semaphorin 4D in human head and neck cancer: A promising predictive biomarker for the peri-tumoral stromal phenotype The search for biomarkers that can predict the tumor stromal phenotype in cancer patients is a challenge in the field. Semaphorin 4D (Sema4D), known for its various effects in the nervous, vascular and immune system, plays an important role in regulating pro and anti-inflammatory responses. It is over-expressed in Head and Neck cancer (HNC) as well as in other epithelial malignancies. We have recently described that HNC-associated Sema4D modulates the inflammatory profile to an immune-suppressive phenotype by inducing the expansion of myeloid derived suppressor cells. The purpose of this study was to determine the prognostic value of Sema4D as a biomarker for immune suppression in human HNC tissue and sera. Immunohistochemistry showed Sema4D+ve/high tumors to correlate positively with Stage III disease (p=0.014), and nodal metastasis (p=0.117). Sema4D+ve/high tumors correlated directly with dense fibrotic peri-tumoral stroma (p=0.0001) and inversely with tumor-associated inflammatory cells (TAIs) (p=0.0006). Knockdown of Sema4D in HNC cell lines resulted in significant reduction of TGF-β1 production (p=0.0016). Sema4D+ve/high tumor cells inversely correlated with the programmed death ligand 1 (PDL-1) positive tumors. Finally, Sema4D was detected in sera of HNC patients at higher levels compared to healthy donors (p References: 1. Younis RH, Han KL and Webb TJ. Human Head and Neck Squamous Cell Carcinoma-Associated Semaphorin 4D Induces Expansion of Myeloid-Derived Suppressor Cells. J Immunol. 2016; 196(3):1419-1429. 2. Basile JR, Castilho RM, Williams VP and Gutkind JS. Semaphorin 4D provides a link between axon guidance processes and tumor-induced angiogenesis. Proc Natl Acad Sci U S A. 2006; 103(24):9017-9022. 3. Gajewski TF. The Next Hurdle in Cancer Immunotherapy: Overcoming the Non-T-Cell-Inflamed Tumor Microenvironment. Semin Oncol. 2015; 42(4):663-671. 4. Fisher TL, Reilly CA, Winter LA, Pandina T, Jonason A, Scrivens M, Balch L, Bussler H, Torno S, Seils J, Mueller L, Huang H, Klimatcheva E, Howell A, Kirk R, Evans E, et al. Generation and preclinical characterization of an antibody specific for SEMA4D. MAbs. 2016; 8(1):150-162. Citation Format: Rania H. Younis, Roshanak Derakhshandeh, Ahmed Sultan, Haiyan Chen, Kyu Lee Han, Tonya J. Webb. Semaphorin 4D in human head & neck cancer: A promising predictive biomarker for the peri-tumoral stromal phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3672. doi:10.1158/1538-7445.AM2017-3672

Ovarian cancer is the fifth leading cause of cancer deaths among women, thereby accounting for more deaths than any other cancer of the female reproductive system. It is known that ovarian cancer tissue and ascites contain lymphocytic infiltrates, suggesting that immune cells traffic to tumors, but are then inhibited by immunosuppressive molecules within the tumor microenvironment. Moreover, vascular endothelial growth factor (VEGF) expression is inversely correlated with survival in ovarian cancer patients. Since avoiding detection by the host’s immune system is crucial for the growth and metastasis of cancer, we investigated the effects of ovarian cancer-associated VEGF on CD1d-mediated antigen presentation to natural killer T (NKT) cells. Pretreatment of antigen presenting cells with ascites or conditioned medium from OV-CAR-3 and SK-OV-3 ovarian cancer cell lines suppressed CD1d-mediated NKT cell activation. Ovarian cancer-associated ascites and conditioned medium from ovarian cancer cell lines contain high levels of VEGF and the ganglioside GD3. Importantly, inhibiting VEGF production by ovarian cancer cell lines led to a reduction in GD3 expression and restored NKT cell activation. To elucidate the mechanisms by which VEGF induces GD3 shedding by ovarian cancer cells, we are examining signaling through the VEGF-receptor, assessing its impact in vivo using the ID8-VEGF ovarian cancer mouse model, as well as measuring levels in human blood and ascites. Collectively, these studies identify novel mechanisms through which VEGF impairs anti-tumor immune responses and thus have the potential to enhance therapeutic strategies for the treatment of ovarian cancer.