CD1d-mediated Antigen Presentation Research Articles

Studying the cellular basis of small bowel enteropathy using high-parameter flow cytometry in mouse models of primary antibody deficiency.

Primary immunodeficiencies are heritable defects in immune system function. Antibody deficiency is the most common form of primary immunodeficiency in humans, can be caused by abnormalities in both the development and activation of B cells, and may result from B-cell-intrinsic defects or defective responses by other cells relevant to humoral immunity. Inflammatory gastrointestinal complications are commonly observed in antibody-deficient patients, but the underlying immune mechanisms driving this are largely undefined. In this study, several mouse strains reflecting a spectrum of primary antibody deficiency (IgA-/-, Aicda-/-, CD19-/- and JH -/-) were used to generate a functional small-bowel-specific cellular atlas using a novel high-parameter flow cytometry approach that allows for the enumeration of 59 unique cell subsets. Using this cellular atlas, we generated a direct and quantifiable estimate of immune dysregulation. This estimate was then used to identify specific immune factors most predictive of the severity of inflammatory disease of the small bowel (small bowel enteropathy). Results from our experiments indicate that the severity of primary antibody deficiency positively correlates with the degree of immune dysregulation that can be expected to develop in an individual. In the SI of mice, immune dysregulation is primarily explained by defective homeostatic responses in T cell and invariant natural killer-like T (iNKT) cell subsets. These defects are strongly correlated with abnormalities in the balance between protein (MHCII-mediated) versus lipid (CD1d-mediated) antigen presentation by intestinal epithelial cells (IECs) and intestinal stem cells (ISCs), respectively. Multivariate statistical approaches can be used to obtain quantifiable estimates of immune dysregulation based on high-parameter flow cytometry readouts of immune function. Using one such estimate, we reveal a previously unrecognized tradeoff between iNKT cell activation and type 1 immunity that underlies disease in the small bowel. The balance between protein/lipid antigen presentation by ISCs may play a crucial role in regulating this balance and thereby suppressing inflammatory disease in the small bowel.

Abstract 6635: Lipid and glycolipid antigen presentation faults as a mechanism of resistance to immune checkpoint therapy in poorly immunogenic melanomas

Abstract Introduction: Cold tumors are characterized by lacking infiltration of antitumor T cells. These tumors are universally refractory to immune checkpoint therapies (ICT). While poor T cell infiltration has been partly ascribed to low tumor mutational burden (TMB), TMB doesn’t necessarily exclude immunogenic responses nor ensure a response to ICT. Mechanisms of T cell exclusion in tumors with sufficient TMB may be caused by molecular failure of the antigen processing and presentation (APP), which impacts T cell generation. It was previously described that loss of heterozygosity (LOH) of an essential component of the MHC class I molecule, the beta-2-microglobulin (B2M), is associated with low antigen presentation, tumor T cell infiltration, and ICT resistance in melanoma. Here we provide further insight into APP and ICT response in melanoma. Methods: Transcriptomic analysis and visualization of genomic data from the GDC-TCGA skin cutaneous melanoma (SKCM) study were performed using the Xena platform. Four anti-PD1 metastatic melanoma cohorts were normalized with transcripts per million units. STRING protein-protein interaction analysis was performed in the human database with 11 nodes and 39 edges. Nanostring transcriptomic data from metastatic uveal melanoma (mUM) was revisited from our previous publications for Spearman’s correlational and group analysis of APP signatures amongst ICT responders and non-responders. Results: In an expanded analysis of 4 normalized anti-PD1 treated metastatic melanoma cohorts (n=272), we confirmed that transcriptomic levels of B2M are significantly reduced in non-responders. Moreover, STRING protein-protein interaction analysis of B2M revealed CD1D as a potential molecular partner of B2M. We, therefore, observed that CD1D transcriptomic expression is not only reduced in anti-PD1 poor responders but is a survival prognosticator in primary tumors from the GDC-TCGA study. Interestingly, CD1D expression is also significantly reduced in poorly immunogenic mUM tumors, a cancer type regarded as universally refractory to ICT. Conclusions: These findings suggest that APP faults in ICT-resistant tumors are not restricted to faults in peptide presentation to T lymphocytes (via B2M) but may also impact the presentation of tumor-associated lipid and glycolipid antigens via CD1d, which can impact antitumor responses of natural killer T cells (NKT). Adjuvant therapies that restore MHC-I and CD1d-mediated antigen presentation may unleash the response to ICT via NKT. Understanding CD1d expression faults locally and systemically may provide valuable prognostication information likely to impact how cancer is treated by optimally selecting patients for ICT and validating novel therapeutic targets for immunotherapy development. Citation Format: Carlos Rogerio Figueiredo. Lipid and glycolipid antigen presentation faults as a mechanism of resistance to immune checkpoint therapy in poorly immunogenic melanomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6635.

Cancer Immunotherapeutic Potential of NKTT320, a Novel, Invariant, Natural Killer T Cell-Activating, Humanized Monoclonal Antibody.

Invariant natural killer T cells (iNKTs) directly kill tumor cells and trans-activate the anti-tumor functions of dendritic cells (DC), natural killer (NK) cells, and T and B cells. As such, iNKTs serve as a powerful tool for use in cell-based cancer immunotherapy. iNKT cell activation commonly requires engagement of the invariant T cell receptor (iTCR) by CD1d presenting glycolipid antigens. However, transformed cells often down-regulate CD1d expression, which results in a reduction of iNKT cell anti-tumor functions. One approach to circumvent this critical barrier to iNKT cell activation is to develop an agonistic antibody that binds directly to the iTCR without the requirement for CD1d-mediated antigen presentation. To this end, we have characterized the iNKT cell stimulatory properties of NKTT320, a novel, recombinant, humanized, monoclonal antibody that binds selectively and with high affinity to human iTCRs. Strikingly, immobilized NKTT320 mediated robust iNKT cell activation (upregulation of CD25 and CD69) and proliferation (carboxyfluorescein succinimidyl ester (CFSE) dilution), as well as Th1 and Th2 cytokine production. Additionally, iNKTs stimulated by plate-bound NKTT320 exhibited increased intracellular levels of granzyme B and degranulation (exposure of CD107 on the cell surface). Furthermore, both soluble and immobilized NKTT320 induced iNKT cell-mediated activation of bystander immune cells, suggesting that this novel anti-iTCR antibody facilitates both direct and indirect iNKT cell cytotoxicity. These studies are significant, as they provide a framework by which iNKT cell anti-cancer functions could be enhanced for therapeutic purposes.

Inhibition of PI3K promotes anti-tumor immune responses to Mantle Cell Lymphoma

Abstract Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin’s lymphoma that is characterized by a chromosomal translocation resulting in overexpression of Cyclin D1, dysregulation of the cell cycle, and constitutive activation of phosphatidylinositol 3 kinase (PI3K). MCL patients also have decreased natural killer T (NKT) cells – an innate-like lymphocyte population involved in initiating the anti-tumor immune response. We hypothesize that constitutive activation of PI3K in MCL alters CD1d-mediated antigen processing and presentation, ultimately resulting in decreased anti-tumor NKT cell responses. To investigate the role of PI3K on MCL survival and immunogenicity in vitro and in vivo, we utilized a panel of PI3K inhibitors (PI3Ki). Pretreatment of human MCL cell lines with micromolar concentrations of PI3Ki resulted in increased apoptosis and enhanced immunogenicity. While dual PI3K/mTOR inhibitor Dactolisib (BEZ-235), completely abrogates NKT/T cell responses of in vitro, stimulated peripheral blood mononuclear cells, PI3K-specific inhibition allows immune cells to function. In a murine model of MCL, PI3Ki-treated animals had decreased tumor burden and increased NKT/T cell responses to ex vivo stimulation with the most significant effect seen in the Buparlisib (BKM-120)-treated group. To elucidate the mechanism, MCL cells were treated in vitro with BKM-120 and flow cytometric analysis indicated increased CD1d expression, but decreased MHC class I expression. These results suggest that selectively targeting PI3K can reduce toxicity, as well as have a significant impact on tumor burden and immune function.

Neurofibromin 1 Impairs Natural Killer T-Cell-Dependent Antitumor Immunity against a T-Cell Lymphoma.

Neurofibromin 1 (NF1) is a tumor suppressor gene encoding a Ras GTPase that negatively regulates Ras signaling pathways. Mutations in NF1 are linked to neurofibromatosis type 1, juvenile myelomonocytic leukemia and Watson syndrome. In terms of antitumor immunity, CD1d-dependent natural killer T (NKT) cells play an important role in the innate antitumor immune response. Generally, Type-I NKT cells protect (and Type-II NKT cells impair) host antitumor immunity. We have previously shown that CD1d-mediated antigen presentation to NKT cells is regulated by cell signaling pathways. To study whether a haploinsufficiency in NF1 would affect CD1d-dependent activation of NKT cells, we analyzed the NKT-cell population as well as the functional expression of CD1d in Nf1+/− mice. Nf1+/− mice were found to have similar levels of NKT cells as wildtype (WT) littermates. Interestingly, however, reduced CD1d expression was observed in Nf1+/− mice compared with their WT littermates. When inoculated with a T-cell lymphoma in vivo, Nf1+/− mice survived longer than their WT littermates. Furthermore, blocking CD1d in vivo significantly enhanced antitumor activity in WT, but not in Nf1+/− mice. In contrast, a deficiency in Type-I NKT cells increased antitumor activity in Nf1+/− mice, but not in WT littermates. Therefore, these data suggest that normal NF1 expression impairs CD1d-mediated NKT-cell activation and antitumor activity against a T-cell lymphoma.

Targeting the VEGF/GD3 immunosuppressive axis in ovarian cancer

Abstract 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.

ATP Binding Cassette Transporter ABCA7 Regulates NKT Cell Development and Function by Controlling CD1d Expression and Lipid Raft Content

ABCA7 is an ABC transporter expressed on the plasma membrane, and actively exports phospholipid complexes from the cytoplasmic to the exocytoplasmic leaflet of membranes. Invariant NKT (iNKT) cells are a subpopulation of T lymphocytes that recognize glycolipid antigens in the context of CD1d-mediated antigen presentation. In this study, we demonstrate that ABCA7 regulates the development of NKT cells in a cell-extrinsic manner. We found that in Abca7−/− mice there is reduced expression of CD1d accompanied by an alteration in lipid raft content on the plasma membrane of thymocytes and antigen presenting cells. Together, these alterations caused by absence of ABCA7 negatively affect NKT cell development and function.

Cross-talk between iNKT cells and monocytes triggers an atheroprotective immune response in SLE patients with asymptomatic plaque.

Accelerated atherosclerosis is a complication of the autoimmune rheumatic disease systemic lupus erythematosus (SLE). We questioned the role played by invariant natural killer T (iNKT) cells in this process because they not only are defective in autoimmunity but also promote atherosclerosis in response to CD1d-mediated lipid antigen presentation. iNKT cells from SLE patients with asymptomatic plaque (SLE-P) had increased proliferation and interleukin-4 production compared with those from SLE patients with no plaque. The anti-inflammatory iNKT cell phenotype was associated with dyslipidemia and was driven by altered monocyte phospholipid expression and CD1d-mediated cross-talk between iNKT cells and monocytes but not B cells. Healthy iNKT cells differentiated in the presence of healthy monocytes and SLE-P serum polarized macrophages toward an anti-inflammatory M2 phenotype. Conversely, patients with clinical cardiovascular disease had unresponsive iNKT cells and increased proinflammatory monocytes. iNKT cell function could link immune responses, lipids, and cardiovascular disease in SLE patients and, together with serum lipid taxonomy, help predict preclinical atherosclerosis in SLE patients.

Targeted attack: mechanisms by which ovarian cancers suppress the immune system.

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.

The Rho-ROCK pathway as a new pathological mechanism of innate immune subversion in chronic myeloid leukaemia.

CD1d-restricted invariant natural killer T (iNKT) cells are believed to play a key role in cancer immune surveillance, and are functionally deficient in patients with chronic myeloid leukaemia (CML). Herein, we have hypothesized that this defect might originate from BCR-ABL-dependent dysfunctions in myeloid dendritic cells (mDCs). Indeed, flow cytometry and confocal microscopy revealed that cell surface expression of CD1d was downregulated in CML mDCs, relative to healthy donor (HD) controls. The decreased cell surface display of CD1d could not be ascribed to defective mDC differentiation, as attested by normal expression of HLA-DR and the CD86 maturation marker. On the other hand, reduced membrane expression was not associated with decreased intracytoplasmic levels of CD1d or its mRNA transcripts, consistent with intracellular retention. In vitro treatment of CML mDCs with the Rho-associated protein kinase (ROCK) inhibitor Y-27632 partially restored both cell surface CD1d expression and CD1d-mediated antigen presentation, whereas it had no effect on HD mDCs. An inhibitor of BCR-ABL tyrosine kinase (TK), imatinib mesylate (IM), had no such activity. Similar recovery of CD1d expression occurred with fasudil, another ROCK inhibitor that is commonly used in clinical trials. Our data support the conclusion that BCR-ABL-dependent ROCK, but not TK, is involved in CD1d downregulation. We propose that ROCK, which is most likely activated by the DH/PH domain of BCR-ABL, mediates iNKT-cell immune subversion in CML patients by downregulating CD1d expression on CML mDCs. Our study reveals the ROCK-mDC axis as a new potential target to restore immune surveillance in patients with CML, offering new therapeutic perspectives for CML treatment. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Histone deacetylase inhibitors enhance CD1d-dependent NKT cell responses to lymphoma.

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.

Exposure to poly-dispersed single walled carbon nanotubes (AF-SWCNTs) causes inhibition of CD1d mediated lipid antigen presentation pathway in professional and non-professional antigen presenting cells (APCs)

Exposure to poly-dispersed single walled carbon nanotubes (AF-SWCNTs) causes inhibition of CD1d mediated lipid antigen presentation pathway in professional and non-professional antigen presenting cells (APCs)

Blocking of CD1d Decreases Trypanosoma cruzi-Induced Activation of CD4-CD8- T Cells and Modulates the Inflammatory Response in Patients With Chagas Heart Disease.

The control of inflammatory responses to prevent the deadly cardiac pathology in human Chagas disease is a desirable and currently unattained goal. Double-negative (DN) T cells are important sources of inflammatory and antiinflammatory cytokines in patients with Chagas heart disease and those with the indeterminate clinical form of Chagas disease, respectively. Given the importance of DN T cells in immunoregulatory processes and their potential as targets for controlling inflammation-induced pathology, we studied the involvement of CD1 molecules in the activation and functional profile of Trypanosoma cruzi-specific DN T cells. We observed that parasite stimulation significantly increased the expression of CD1a, CD1b, CD1c, and CD1d by CD14(+) cells from patients with Chagas disease. Importantly, among the analyzed molecules, only CD1d expression showed an association with the activation of DN T cells, as well as with worse ventricular function in patients with Chagas disease. Blocking of CD1d-mediated antigen presentation led to a clear reduction of DN T-cell activation and a decrease in the expression of interferon γ (IFN-γ) by DN T cells. Thus, our results showed that antigen presentation via CD1d is associated with activation of DN T cells in Chagas disease and that CD1d blocking leads to downregulation of IFN-γ by DN T cells from patients with Chagas heart disease, which may be a potential target for preventing progression of inflammation-mediated dilated cardiomyopathy.

A VP22-Null HSV-1 Is Impaired in Inhibiting CD1d-Mediated Antigen Presentation

CD1d-restricted T (natural killer T [NKT]) cells are important for controlling a herpes simplex virus (HSV) infection. One of the mechanisms of immune evasion by HSV is to downregulate CD1d-mediated activation of NKT cells. VP22 is an HSV-1-encoded protein responsible for reorganizing the host cell's cytoskeletal network and viral spreading. We have previously shown that modification of the cytoskeleton can alter CD1d-mediated antigen presentation. In this study, we found that an HSV-1 lacking VP22 (ΔUL49) was impaired in its ability to inhibit CD1d-mediated antigen presentation compared with the wild-type (WT) virus; this was reversed by a repair virus (UL49R) in CD1d-expressing cells. We further demonstrated that CD1d recycling was inhibited by infection with WT and UL49R, but not the ΔUL49 virus. Ectopic expression of VP22 in CD1d-expressing cells complemented the VP22-deficient virus in inhibiting antigen presentation. Moreover, inhibiting viral protein synthesis rescued VP22-dependent inhibition of CD1d antigen presentation. In conclusion, our findings suggest that VP22 is required (but not sufficient) for the inhibition of CD1d-mediated antigen presentation by an HSV-1 infection.

Alterations in cellular metabolism modulate CD1d-mediated NKT-cell responses.

Natural killer T (NKT) cells play a critical role in the host's innate immune response. CD1d-mediated presentation of glycolipid antigens to NKT cells has been established; however, the mechanisms by which NKT cells recognize infected or cancerous cells remain unclear. 5(')-AMP activated protein kinase (AMPK) is a master regulator of lipogenic pathways. We hypothesized that activation of AMPK during infection and malignancy could alter the repertoire of antigens presented by CD1d and serve as a danger signal to NKT cells. In this study, we examined the effect of alterations in metabolism on CD1d-mediated antigen presentation to NKT cells and found that an infection with lymphocytic choriomeningitis virus rapidly increased CD1d-mediated antigen presentation. Hypoxia inducible factors (HIF) enhance T-cell effector functions during infection, therefore antigen presenting cells pretreated with pharmacological agents that inhibit glycolysis, induce HIF and activate AMPK were assessed for their ability to induce NKT-cell responses. Pretreatment with 2-deoxyglucose, cobalt chloride, AICAR and metformin significantly enhanced CD1d-mediated NKT-cell activation. In addition, NKT cells preferentially respond to malignant B cells and B-cell lymphomas express HIF-1α. These data suggest that targeting cellular metabolism may serve as a novel means of inducing innate immune responses.

HDAC inhibitors enhance CD1d-dependent NKT cell responses to lymphoma

Abstract Histone deacetylases (HDACs) influence expression of genes implicated in tumor initiation, progression and anti-tumor responses. We hypothesize that tumors use epigenetic mechanisms to dysregulate CD1d-mediated antigen processing and presentation, which leads to a functional impairment in the ability of Natural killer T (NKT) cells to recognize and destroy cancerous cells. In this study, we treated CD1d-expressing tumor cells with HDAC inhibitors (HDACi) to assess CD1d-dependent NKT cell responses to B cell lymphoma. Consistent with previous studies, we found that treatment with Trichostatin A, a pan-HDACi, enhanced both CD1d and MHC class II-mediated antigen presentation. Similarly, treatment of B cell lymphomas with other HDACi, Panobinostat and Vorinostat, resulted in enhanced CD1d-dependent NKT cell responses. Mechanistically, we found that HDAC2 binds to the CD1d promoter and knockdown of HDAC2 in tumor cells enhanced their immunogenicity. The observed changes are due at least in part to a dose-dependent increase in both CD1d mRNA and CD1d cell surface expression. In addition, treatment with HDCAi resulted in a decrease in inflammatory cytokine secretion by B cell lymphomas. To examine the therapeutic potential of HDACi, we treated human peripheral blood mononuclear cells with TSA and Panobinostat. Treatment with these HDACi did not suppress T cell activation; conversely, treatment with a novel HDAC4 inhibitor MC1568 suppressed T cell responses. Taken together, our studies demonstrate the efficacy of HDACi in restoring anti-tumor responses to B cell lymphomas through both cell-intrinsic and extrinsic factors and strongly implicate a role for select HDACi in cancer immunotherapeutic strategies.

STAT3 promotes CD1d-mediated lipid antigen presentation by regulating a critical gene in glycosphingolipid biosynthesis.

Cytokines that regulate the immune response signal through the Janus kinase / signal transducer and activation of transcription (JAK/STAT) pathway, but whether this pathway can regulate CD1d-mediated lipid antigen presentation to natural killer T (NKT) cells is unknown. Here, we found that STAT3 promotes antigen presentation by CD1d. Antigen-presenting cells (APCs) in which STAT3 expression was inhibited exhibited markedly reduced endogenous lipid antigen presentation to NKT cells without an impact on exogenous lipid antigen presentation by CD1d. Consistent with this observation, in APCs where STAT3 was knocked down, dramatically decreased levels of UDP glucose ceramide glucosyltransferase (UGCG), an enzyme involved in the first step of glycosphingolipid biosynthesis, were observed. Impaired lipid antigen presentation was reversed by ectopic expression of UGCG in STAT3-silenced CD1d(+) APCs. Hence, by controlling a fundamental step in CD1d-mediated lipid antigen presentation, STAT3 signalling promotes innate immune responses driven by CD1d.

Abstract 4294: Cancer immunotherapeutic potential of NKTT320, a novel human invariant natural killer T-cell activating monoclonal antibody

Abstract Invariant natural killer T cells (iNKTs) directly kill tumor cells and trans-activate the anti-tumor functions of dendritic cells (DC), natural killer (NK), T and B cells. As such, iNKTs serve as a powerful tool for use in cell-based cancer immunotherapy. iNKT cell activation commonly requires engagement of the invariant T cell receptor (iTCR) by CD1d presenting glycolipid antigen. However, transformed cells often down-regulate CD1d expression, which results in a reduction of iNKT cell anti-tumor functions. One approach to circumvent this critical barrier to iNKT cell activation is to develop an agonistic antibody that binds directly to the iTCR without the requirement for CD1d-mediated antigen presentation. Towards this end, we characterized the iNKT cell stimulatory properties of NKTT320, a novel recombinant humanized monoclonal antibody that binds selectively and with high affinity to the human iTCR. Strikingly, both soluble and immobilized NKTT320 induced a dose-dependent iNKT cell activation (upregulation of activation markers [CD25, CD69]), proliferation (by measuring CFSE dilution), degranulation (measured by exposure of CD107 on the cell surface) and activation of bystander immune cells. Additionally, iNKT cells stimulated by plate-bound NKTT320 also robustly secreted Th1 (IL-1β, IFN-γ, TNF-α, IL-2, IL-6) and Th2-type (IL-4, IL-5, IL-10) cytokines as well as chemokines. Our in vitro studies are consistent with in vivo data in Vα24 transgenic mice, which express the human iTCR alpha chain. Dosing of NKTT320 in these animals led to iNKT cell activation and IFN-γ production, as well as incorporation of BRDU indicating in vivo iNKT cell proliferation. Similarly, NKT14m (a murine iNKT-agonistic antibody) induced murine iNKT cell activation, cytokine production, and degranulation in vitro. Consistently, treatment of C57BL/6 (B6) mice with NKT14m led to rapid and robust iNKT cell activation and IFN-γ production. Furthermore, in vivo injection of NKT 14m in B6 mice induced robust T, B, and NK cell activation as well as DC maturation (as assessed by increased expression of MHC class II and co-stimulatory molecules [CD80 and CD86]). Importantly, treatment of T-lymphoma-bearing B6 mice with a single dose of NKT14m substantially repressed the growth of subcutaneous tumors. Taken together, these data suggest that iNKT-activating antibodies hold great potential for use in cell-based cancer immunotherapy. Studies are underway to examine whether treatment with NKT14m in combination with interleukin-12 (a potent iNKT and NK cell activating cytokine) can further augment iNKT cell activation in vivo. These studies are significant, as they provide a framework by which iNKT cell anti-cancer functions could be enhanced for therapeutic purposes. Citation Format: Rupali Das, Felix Scheuplein, Peng Guan, Robert Schaub, Kim E. Nichols. Cancer immunotherapeutic potential of NKTT320, a novel human invariant natural killer T-cell activating monoclonal antibody. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4294. doi:10.1158/1538-7445.AM2015-4294

Epigenetic modulation of CD1d-mediated antigen presentation by B cell lymphomas (TUM10P.1054)

Abstract Histone deacetylases (HDACs) are a family of enzymes that regulate diverse cellular events such as gene expression, cell proliferation, and immune pathways through deacetylation of their protein targets. We hypothesize that tumors use epigenetic mechanisms to dysregulate CD1d-mediated antigen processing and presentation, which leads to a functional impairment in the ability of natural killer T (NKT) cells to recognize and destroy cancerous cells. We examined CD1d-mediated antigen presentation to NKT cells following treatment with HDAC inhibitors (HDACi). Consistent with previous studies, we found that preventing deacetylation by treatment with Trichostatin A, a pan-HDACi, enhanced both CD1d and MHC class II-mediated antigen presentation. Similarly, treatment of B cell lymphomas with Panobinostat resulted in increased CD1d-dependent NKT cell responses, due at least in part to a dose-dependent increase in CD1d-cell surface expression. In addition, treatment with HDCAi results in a decrease in STAT3 expression as well as IL-10 secretion by B cell lymphomas. Overall, our studies demonstrate the efficacy of HDACi in restoring anti-tumor responses to B cell lymphomas through both cell-intrinsic and extrinsic factors. Collectively, these results suggest that HDACi may work to enhance the immune response and increases the immunogenicity of the tumor itself.

The inhibition of CD1d-mediated antigen presentation upon an HSV-1 infection is VP22-dependent (VIR1P.1134)

Abstract CD1d-restricted T (NKT) cells are important for controlling a herpes simplex virus (HSV) infection. One of the mechanisms of immune evasion by HSV is to down-regulate CD1d-mediated activation of NKT cells. VP22 is an HSV-1-encoded protein responsible for reorganizing the host cell’s cytoskeletal network and viral spreading. We have shown that modification of the cytoskeleton can alter CD1d-mediated antigen presentation. In this study, we found an HSV-1 lacking VP22 (ΔUL49) was impaired in its ability to inhibit CD1d-mediated antigen presentation compared to wild-type (WT) virus; this was reversed by a repair virus (UL49R) in CD1d-expressing cells. Of importance, the effects of VP22 were shown to be exerted on the T322/S323 dyad, critical residues for the virus to reduced antigen presentation. We further demonstrated that CD1d recycling was inhibited by infection with WT and UL49R, but not ΔUL49 virus. Rather than inhibiting antigen presentation by CD1d, ectopic expression of VP22 in CD1d-expressing cells complemented the VP22-deficient virus in inhibiting antigen presentation. Therefore, VP22 is required (but not sufficient) for the inhibition of CD1d-mediated antigen presentation by an HSV-1 infection.