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The critical role of commensal microbiota in regulating the host immune response has been established. In addition, it is known that host–microbial interactions are bidirectional, and this interplay is tightly regulated to prevent chronic inflammatory disease. Although many studies have focused on the role of classic T cell subsets, unconventional lymphocytes such as NKT cells and innate lymphoid cells also contribute to the regulation of homeostasis at mucosal surfaces and influence the composition of the intestinal microbiota. In this review, we discuss the mechanisms involved in the cross-regulation between NKT cells, innate lymphoid cells, and the gut microbiota. Moreover, we highlight how disruptions in homeostasis can lead to immune-mediated disorders.

The incidence of lymphoma in the US is rising. While chemoimmunotherapy causes remissions in mantle cell lymphoma (MCL), responses are not durable and most patients relapse. New strategies that focus on eliminating tumor-associated immunosuppressive factors are needed to improve patient outcomes. One of the earliest pathways to be activated during an immune response is CD1d-mediated presentation of lipid antigens to natural killer T (NKT) cells. We have found that MCL patients have a significant reduction in circulating NKT cell number and function. We hypothesized that MCL-associated sphingosine-1 phosphate (S1P) can down regulate CD1d-dependent NKT cell responses to MCL. We found that inhibition of S1P signaling in MCL cell lines led to increased CD1d-mediated NKT cell activation, as assessed by cytokine production and cytotoxicity. Furthermore, knockdown of sphingosine kinase 1 in MCL cell lines resulted in the presentation of an activating endogenous antigen. These results demonstrate that S1P regulates CD1d-mediated antigen processing and presentation to NKT cells by altering the endogenous repertoire of lipid antigens and suggest that targeting this pathway could be an important immunotherapeutic strategy.

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.

Natural killer T (NKT) cells are a unique subset of T cells that recognize glycolipid antigens presented by CD1d molecules. Once activated, NKT cells can mount strong anti-tumor responses. However, little is known about the regulation of CD1d-mediated antigen processing and 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 regulates this process, and found that over-expression or induction of Bcl-xL led to increased antigen presentation to NKT cells. Conversely, inhibition or knockdown of Bcl-xL led to decreased NKT cell activation. Knockdown of Bcl-xL also resulted in the loss of CD1d trafficking to LAMP1+ compartments. Furthermore, Rab7, a late endosomal marker was upregulated following Bcl-xL knockdown, and CD1d molecules accumulated in the Rab7+ compartment. These results demonstrate that Bcl-xL regulates CD1d-mediated antigen presentation to NKT cells by altering the intracellular localization of CD1d. Thus, we have identified a new pathway involved in antigen presentation that can impact current therapies targeting the Bcl-2 family, as well as emerging NKT cell based cancer immunotherapeutic strategies.

Natural killer T (NKT) 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 NKT cell number and function, and these deficits limit the potential clinical application of NKT cells for cancer therapy. To overcome the problem of limited NKT cell numbers, we have investigated whether NKT cells can be generated in vitro from bone marrow-derived adult hematopoietic stem-progenitor cells (HSPC).Our data demonstrate that co-culture of human bone marrow HSPC with OP9 stromal cells transduced with Notch ligand delta-like 1 (OP9-DL1), generates a CD3+ T cell population. These CD3+ precursors can be further differentiated into CD8- NKT cells by secondary culture with CD1d-Ig based artificial antigen presenting cells (aAPC), expressing anti-CD28mAb and loaded with α-GalCer. Importantly, these in vitro-generated NKT cells are functional as demonstrated by their ability to proliferate and secrete IFN-γ following stimulation. These findings demonstrated for the first time that functional human NKT cells can be effectively generated from HSPC in the absence of genetic manipulation or viral transduction. These studies will serve to help understand and design NKT cell-based approaches to enhance current immunotherapeutic strategies for cancer.

Lymphomas are a heterogeneous group of malignancies, primarily divided into Hodgkin's and non-Hodgkin's lymphomas (NHL). Unlike other cancers, the incidence of NHL is steadily rising. Natural Killer T (NKT) cells are innate-like lymphocytes that recognize glycolipid antigens in the context of CD1d, an MHC class I-like molecule. They have strong anti-tumor activity and respond by massive cytokine production and/or cytotoxicity. It is known that pro-survival factors are upregulated in B cell lymphomas, but their role in antigen processing and presentation remains unknown. The effect of Bcl-xL on CD1d-mediated antigen presentation was examined utilizing the well-characterized WEHI-231 B cell lymphoma cell line. Bcl-xL expression was modulated via genetic, pharmacologic and biological approaches. We found a concomminant increase in CD1d-mediated NKT cell responses following the induction of Bcl-xL; however, cell surface expression of CD1d molecules remained unchanged. Induction of Bcl-xL in primary B cells led to increased CD1d-mediated activation of NKT cells in response to exogenous antigens, resulting in increased Th1 not Th2 responses. Taken together, these data suggest that aberrant expression of Bcl-xL may act as a sentry system to alert the CD1d/NKT system to neoplastic transformation.

Tumors often experience hypoxic microenvironments due to poor vascular access in the early stage of neoplastic growth and continue to experience oxygen deficits in late stage progression despite the increase in neovascularization to the primary tumor or metastasis. This persistent hypoxic state selects for tumors cells that are able to compensate for low oxygen levels by increasing use of the glycolytic cycle under aerobic conditions (The Warburg Effect), suppressing apoptosis and inducing decreased genomic stability. These effects have been shown to be mediated, at least in part, by the transcription factor hypoxia inducible factor (HIF)-1alpha. Natural Killer T (NKT) cells are a subset of T cells that recognize a wide range of lipid antigens presented by the MHC Class I-like CD1d molecule. In this study, we sought to investigate the effects of hypoxia induction on CD1d-mediated NKT cell responses. Pharmacological agents were used to induce HIF-1a in LCD1d, and then we assessed their ability to present endogenous antigen to NKT cells. It was found that the induction of HIF-1a resulted in a significant increase in CD1d-mediated NKT cell activation, as assessed by IL-2, GM-CSF, and IL-13 production. The increase in NKT function was not due to with an increase in cell surface expression of CD1d. These results suggest that hypoxia in tumor cells may alter the repertoire of endogenous lipids presented in the context of CD1d which permits their recognition by NKT cells.

Prostate cancer is the second leading cause of cancer mortality in men older than 40 years in the United States. African American (AA) men and men with a family history of prostate cancer are at a significantly increased risk for developing prostate cancer. A recent study found highly significant differences in the expression of immune regulatory genes and cytokines between tumors from AA and European-American (EA) patients. Therefore, we tested the hypothesis that genetic variations in immune regulatory genes including the CD1 locus may confer an increased disease risk and a poor outcome prostate cancer phenotype in African-American men. Natural Killer T (NKT) cells produce large amounts of cytokines following activation vis-à-vis recognition of lipid antigen presented in the context of CD1d molecules. Prostate tumors commonly express CD1 family members, thus polymorphisms in CD1 genes may be responsible for differences in tumor biology between AA and EA patients. We report that a polymorphism in CD1e occurs commonly in AA but not EA men. When the distribution of this polymorphism was examined in a large cohort (n=562), we found that it is ethnicity specific and occurs primarily in men of African ancestry (P

Natural killer T (NKT) cells are important in regulating immune responses against tumors. However, patients with mantle cell lymphoma (MCL), an aggressive subtype of Non-Hodgkins Lymphoma (NHL), have a reduction in both NKT cell number and function. We have found that NKT cells from MCL patients fail to be activated by autologous B cells, even in the presence of α-Galactosylceramide (α-GalCer), a potent activator of NKT cells. NKT cells from MCL patients had a significant reduction in their expansion potential and in cytokine production. Compared to serum from healthy donors, MCL serum induced chronic NKT cell stimulation and a rapid upregulation of PD-1 in cultured primary NKT cells (from healthy donors), resulting in an exhausted or anergic phenotype. Mass spectrometry demonstrated the presence of higher levels of sphingosine 1-phosphate (S1P) in patient sera compared to healthy controls sera. The functional involvement of S1P in NKT activation was supported by restoration of the anti-tumor function of NKT cells against MCL following S1P1 signaling blockade. These data suggest that tumor-related induction of S1P production and/or S1P signaling in MCL patients can inhibit NKT cell function, ultimately resulting in reduced anti-tumor responses.

Mantle cell lymphoma (MCL) is an aggressive subtype of Non-Hodgkin’s lymphoma that is characterized by the abnormal accumulation of CD20+CD5+ B cells in the lymph nodes, spleen, gastrointestinal tract, bone marrow and blood. Although treatment with combination chemotherapy can be effective, most patients relapse, and the outcome for MCL remains poor. Natural killer T (NKT) cells are important in regulating immune responses to tumors. The observation that lymphoma patients have a reduction in both NKT cell number and function suggests that the anti-tumor effects mediated by these cells may be compromised. In these studies, we have investigated NKT cell activation in response to MCL. We found that CD1d cell surface expression was highly variable in a panel of well-characterized MCL cell lines. Importantly, cell surface expression of CD1d did not directly correlate with the ability of the MCL lines to activate primary NKT cells. Circulating NKT cells in the peripheral blood of MCL patients were not stimulated by autologous B cells, even in the presence of the potent antigen, α-GalCer. However, when NKT cells from healthy donors were co-cultured with purified B cells from MCL patients, the NKT cells specifically recognized the malignant B cells and produced IFN-γ, but not inflammatory or regulatory cytokines. These data suggest that B cell lymphomas may condition autologous NKT cells not to recognize them in order to evade NKT cell mediated anti-tumor responses.

Natural killer T (NKT) cells have been shown to be important in regulating immune responses to tumors; however, the number and function of NKT cells are reduced in cancer patients. Genetic factors regulating NKT cell number and function have yet to be determined. The Breast Cancer Susceptibility gene 1 (BRCA-1) encodes a tumor suppressor gene that functions, in part, as a caretaker gene in preserving chromosomal stability. In this study we have investigated the role of BRCA-1 on NKT expression and function using a transgenic mouse model in which BRCA-1 is mutated. This mouse model has a direct translational link to a specific type of human breast cancer, because it develops the same genetic changes that are described in BRCA-1-related human cancer. BRCA-1 is highly expressed in the T cell lineages, and we have found that our NKT cell lines express BRCA-1 protein. When we examined the liver and spleens of BRCA-1 mutant mice, we found that the NKT cell population was significantly reduced, compared to wildtype control mice. The classical CD4 and CD8 T cell subpopulations were comparable between the BRCA-1 mutant mice and wildtype controls, which suggests that NKT cell population is specifically regulated BRCA-1. Collectively, our data suggest that determining whether genetic susceptibility alters the function of NKT cells, a key regulator of anti-tumor immune responses, could help in designing novel therapeutics targeting aggressive breast cancers.

Natural killer T (NKT) cells play a pivotal role in maintaining immune homeostasis. NKT cells rapidly secrete both proinflammatory and inflammatory cytokines following activation. However, the role costimulatory molecules during activation and their impact on effector functions have yet to be clearly defined. Our innovative system, which utilizes bead-based artificial Antigen Presenting Cells (aAPC), allows us to systematically evaluate the role of different costimulatory molecules on NKT cell activation and expansion. In this study, we have examined the effects of CD28, CD44, CD161, and OX-40 on NKT cell proliferation, phenotype and function. We found that the costimulatory molecules which increased NKT cell proliferation also enhanced cytokine production and cytotoxic function. These studies will enhance our knowledge of NKT cell biology and may aid in the development of novel NKT cell-based immunotherapeutic approaches for the treatment of infectious and autoimmune diseases, as well as cancer.