• Defective Natural Killer T cell development and function in BRCA1 mutant mice

      Bates, Joshua Philip; Webb, Tonya J. (2016)
      NKT cells are significantly reduced in the blood of newly diagnosed breast cancer (BC) patients compared to healthy volunteers (HV). We hypothesize that one mechanism by which breast cancers escape immune detection is due to a loss in NKT cell number and function. Therefore, we used mice genetically predisposed to develop breast cancer (BRCA1-mut) to examine the phenotype and function of NKT cells throughout tumorigenesis. We found that BRCA1-mut mice have a 70-85% reduction of NKT cells in primary and secondary lymphoid organs, and that this reduction occurs in stage 3 of development. Moreover, the NKT cells of BRCA1-mut mice are functionally altered. Thus, our data suggest that NKT cells play a critical role in the development of breast tumorigenesis and further research is required to determine the mechanisms by which NKT cells are physically and functionally reduced in the absence of BRCA1.
    • Delineating the Role of NKT cell Activation in B cell Lymphoma

      Lee, Michael; Webb, Tonya J. (2020)
      Natural Killer T (NKT) cells play an important role in cancer surveillance and can reduce lymphoma burden in vivo; however, a hallmark of cancer is its ability to evade immune surveillance. Our goals were to elucidate novel mechanisms utilized by B cell lymphoma to evade NKT cell-mediated immune surveillance and determine the prognostic potential of assessing NKT cell function in lymphoma patients. We found that knockdown of sphingosine kinase 1 (SK1) in human lymphoma cells results in a significant increase in CD1d-mediated NKT cell activation. Lipidomic and co-culture studies identified cardiolipin as being upregulated in SK1 knockdown cells and implicated cardiolipin as an NKT cell-specific cancer neoantigen. We also sought to determine the efficacy of NKT cell-based therapy on survival and the induction of anti-tumor immune responses in a mouse model of B cell lymphoma. We found that activation of NKT cells via early administration of α-galactosylceramide (α-GalCer) only provided modest protection. Our data suggest that the lack of protection is due, at least in part, to the expansion of myeloid-derived suppressor cells in α-GalCer-treated tumor bearing mice. Lastly, we sought to identify novel immunological biomarkers in lymphoma patients. It was found that lymphoma patients have a reduction in NKT cell function compared to healthy donors. Furthermore, lymphoma patients have significantly higher levels of both pro- and anti-inflammatory cytokines in their sera compared to healthy donors. In addition, lymphoma patients who experience relapse have significantly reduced NKT cell function in the blood, compared to lymphoma patients who did not relapse. Collectively, our studies demonstrate the multifaceted role NKT cells play in immune responses to B cell lymphoma and will help inform the next generation of cancer immunotherapy.
    • Mechanisms of tumor evasion from NKT cell-mediated immunosurveillance

      Tiper, Irina; Webb, Tonya J. (2016)
      Natural killer T (NKT) cells are a unique subset of CD1d-restricted T cells that play an important role in mediating anti-tumor responses. NKT cells produce large amounts of cytokines and can directly mediate tumor cell lysis. However, NKT cells are numerically reduced and functionally impaired in cancer patients and the mechanisms by which tumor suppress NKT cell activation are poorly understood. Given that cancer cells possess epigenetic abnormalities, in addition to numerous genetic alterations, we hypothesize that tumors use epigenetic mechanisms to suppress anti-cancer immune responses by dysregulating antigen presentation and secreting soluble inhibitory factors. To test our hypothesis, lymphoma cells were pretreated with histone deacetylase inhibitors (HDACi) and then we assessed their ability to activate NKT cells. Treatment of B cell lymphomas with HDACi enhances anti-tumor immune responses by inducing CD1d-mediated antigen presentation, inhibiting STAT3, and subsequently, decreasing the production of STAT3-associated inflammatory cytokines. Moreover, we found that the enhanced immunogenicity observed following treatment with HDACi was HDAC2-dependent. Specifically, we performed chromatin immunoprecipitation (ChIP) to identify the HDAC that binds to the CD1D promoter. We found that HDAC2 binds to the CD1D promoter and negatively regulates CD1D transcription. In addition, we sought to investigate other mechanisms by which tumors suppress CD1d-mediated NKT cell activation. We found that, similar to lymphomas, ovarian cancers secrete molecules that can inhibit NKT cell activation. Additionally, we established a link between angiogenic factor secretion and suppression of antigen presentation, suggesting that existing anti-angiogenic therapies can positively impact anti-tumor immune responses. Thus, we identified cell- intrinsic and -extrinsic mechanisms by which tumors suppress CD1d-mediated antigen presentation, which can be targeted in an immunotherapeutic setting. Overall, our studies implicate roles of HDACi and anti-angiogenic therapies in immunotherapeutic approaches.
    • Natural Killer T Cell Development and Activation: Implications in Cancer Immunotherapy

      Shissler, Susannah; Webb, Tonya J.; 0000-0002-0660-9955 (2019)
      Natural killer T (NKT) cells are a population of innate-like lymphocytes that express both NK and T cell markers. Following activation by the recognition of glycolipid antigen presented in the context of CD1d, NKT cells have been shown to play important roles in infection, autoimmunity, and cancer. The research undertaken herein sought to determine factors important for NKT cell development and activation to further understand the potential of NKT cells in cancer immunotherapy. We characterized thymic NKT subsets in different strains of mice and demonstrated that both the size and the subset distribution of the NKT cell population is dependent on genetic factors. By developing sensitive methods to track cellular proliferation, we identified baseline differences in NKT cell subset activation parameters. Specifically, NK1.1+ NKT cells were less likely to enter cell cycle after stimulation, divided less when activated, and were more adversely affected by CD28 costimulatory blockade compared to NK1.1- NKT cells. In our lymphoma and breast cancer studies, we show that NKT cell recognition of cancer cells can be enhanced by the presence of an activating ligand, but not by PI3K inhibitors or CARP-1 functional mimetics. Additionally, NKT cell-mediated cytotoxicity is enhanced by, but not dependent on, CD1d engagement. Whereas NK1.1+ NKT cells are the optimal subset of NKT cells for use in cancer immunotherapy because of their potent cytolytic abilities, activation of NK1.1- NKT cells could pose a threat to anti-tumor immunity, attributable to their secretion of immunosuppressive or regulatory cytokines. Collectively, our data demonstrate a critical role for CD1d engagement and costimulation for optimal activation of NK1.1+ NKT cells. Due to the fact that cancers often downregulate CD1d and do not express costimulatory molecules, our data highlight the need for the development of strategies focused on inducing the activation of specific subsets of NKT cells for cancer immunotherapy.
    • The Role of Natural Killer T Cells in B Cell Lymphoma

      Balasubrahmanyam, Priyanka; Webb, Tonya J. (2014)
      Natural killer T (NKT) cells are a unique subset of T cells that recognize glycolipid antigens in the context of CD1d, a non-classical MHC class I-like molecule. NKT cells mount strong anti-tumor responses and are a major focus in developing effective cancer immunotherapy. However, little is known about the regulation of CD1d-mediated antigen presentation to NKT cells, particularly in the context of B cell lymphoma. Pro-survival factors of the Bcl-2 family, such as Bcl-xL are often upregulated in B cell lymphomas, and are associated with changes in the endocytic pathway, which is paramount for CD1d-mediated antigen presentation. We hypothesized that Bcl-xL can regulate this process, and found that over-expression or induction of Bcl-xL led to increased CD1d-mediated antigen presentation to NKT cells. Conversely, pharmacological inhibition or shRNA-mediated knockdown of Bcl-xL led to decreased antigen presentation. Surface CD1d expression was unchanged by the modulation of Bcl-xL, but its knockdown resulted in reduced CD1d trafficking to LAMP1+ compartments. Furthermore, Rab7, a late endosomal marker was upregulated following Bcl-xL knockdown, and CD1d molecules accumulated in the late endosomes. These results demonstrate that Bcl-xL modulates CD1d-mediated antigen presentation to NKT cells by altering the intracellular trafficking of CD1d. Thus, we have identified a potential tumor recognition mechanism that can impact current therapies targeting the Bcl-2 family, as well as emerging NKT cell based cancer immunotherapeutic strategies. We further studied the role of NKT cells in mantle cell lymphoma, a particularly aggressive form of non-Hodgkin's lymphoma, in vivo, using an IL-14α and c-Myc double-transgenic mouse model. We found that treatment with a single dose of the NKT cell agonist α-Galactosylceramide, increased survival and caused amelioration of disease. Ex vivo restimulation of splenocytes with α-GalCer showed increased IFN-γ responses, providing some insight into the mechanism underlying the enhanced anti-tumor response following α-GalCer administration. These studies indicate that NKT cells play an important role in mediating an effective immune response to lymphoma, warranting further investigation of the CD1d/NKT system. This small but powerful lymphocyte population bears high potential for translation into the next generation of cancer therapy.