Wenji Sun

NKT Cell Responses to B Cell Lymphoma

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.

Connecting the Dots: Artificial Antigen Presenting Cell-Mediated Modulation of Natural Killer T Cells

Natural killer T (NKT) cells constitute an important subset of T cells that can both directly and indirectly mediate antitumor immunity. However, we and others have reported that cancer patients have a reduction in both NKT cell number and function. NKT cells can be stimulated and expanded with α-GalCer and cytokines and these expanded NKT cells retain their phenotype, remain responsive to antigenic stimulation, and display cytotoxic function against tumor cell lines. These data strongly favor the use of ex vivo expanded NKT cells in adoptive immunotherapy. NKT cell based-immunotherapy has been limited by the use of autologous antigen-presenting cells, which can vary substantially in their quantity and quality. A standardized system that relies on artificial antigen-presenting cells (aAPCs) could produce the stimulating effects of dendritic cell (DC) without the pitfalls of allo- or xenogeneic cells. In this review, we discuss the progress that has been made using CD1d-based aAPC and how this acellular antigen presenting system can be used in the future to enhance our understanding of NKT cell biology and to develop NKT cell-specific adoptive immunotherapeutic strategies.

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCRα chain rearrangement: Vα14Jα18 in mice and Vα24Jα18 in humans, which is associated with Vβ chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with α-Galactosylceramide (α-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Invariant natural killer T cells generated from human adult hematopoietic stem-progenitor cells are poly-functional

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.

Human airway epithelia express catalytically active NEU3 sialidase

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 cell based Immunotherapy.

Natural killer T (NKT) cells play an important immunoregulatory role and are thought to bridge the innate and adaptive immune responses. Following activation through cognate interactions with lipid antigen presented in the context of CD1d molecules, NKT cells rapidly produce a plethora of cytokines and can also mediate cytotoxicity. Due to their potent effector functions, extensive research has been performed to increase our understanding on how to effectively modulate these cells. In fact, NKT cell agonists have been used as vaccine adjuvants to enhance antigen specific T and B cell responses to infections and malignancy. In this review, we will focus on recent advances in NKT cell-based vaccination strategies. Given the role that NKT cells play in autoimmune disease, infectious diseases, cancer, transplant immunology and dermatology, it is important to understand how to effectively guide their effector functions in order to develop novel immunotherapeutic strategies.

Development of a qPCR method to rapidly assess the function of NKT cells.

INTRODUCTION NKT cells comprise a rare, but important subset of T cells which account for ~0.2% of the total circulating T cell population. NKT cells are known to have anti-tumor functions and rapidly produce high levels of cytokines following activation. Several clinical trials have sought to exploit the effector functions of NKT cells. While some studies have shown promise, NKT cells are approximately 50% lower in cancer patients compared to healthy donors of the same age and gender, thus limiting their therapeutic efficacy. These studies indicate that baseline levels of activation should be assessed before initiating an NKT cell based immunotherapeutic strategy. AIM The goal of this study was to develop a sensitive method to rapidly assess NKT cell function. METHODS We utilized artificial antigen presenting cells in combination with qPCR in order to determine NKT cell function in peripheral blood mononuclear cells from healthy donors and breast cancer patients. RESULTS We found that NKT cell activation can be detected by qPCR, but not by ELISA, in healthy donors as well as in breast cancer patients following four hour stimulation. CONCLUSION This method utilizing CD1d-expressing aAPCs will enhance our knowledge of NKT cell biology and could potentially be used as a novel tool in adoptive immunotherapeutic strategies.