Natural killer T (NKT) cells recognize lipid antigens presented by CD1d molecules and can be classified into type I or type II based upon their usage of a semi-invariant TCR or diverse TCRs, respectively. Type II NKT cells are predominant in humans, whereas type I NKT cells are prevalent in the mouse. The biology and function of self-glycolipid-reactive CD1d-restricted type II NKT cells is poorly understood. We have identified a major subset of type II NKT cells that recognizes a myelin-derived glycolipid, sulfatide. Sulfatide-reactive type II but not type I NKT cells are enriched in the inflamed tissues during experimental autoimmune encephalomyelitis (EAE). Importantly, sulfatide-mediated activation of type II NKT cells induce a novel regulatory pathway accompanied by anergy induction in type I NKT cells and protection from autoimmune diseases. Here we propose to characterize in details the TCR V-gene repertoire of type II NKT cells with respect to the antigen fine specificity, tissue distribution and cytokine secretion pattern. Also we will study their activation and mechanism of immune regulation that involves interactions of NKT cells with other innate-like cells, including plasmacytoid dendritic cells (pDCs), myeloid-derived suppressor cells (MDSCs) which in turn control adaptive immunity. The TCR repertoire of sulfatide/CD1d-tetramer-sorted cells will be determined using flow cytometry, DNA sequencing and spectratyping of TCR V1 and V2 genes. Following determination of antigen specificity using retroviral vector-mediated transfection of dominant TCR 1 and 2 chain genes into a TCR-deficient cell line, most affine TCRs will be folded with sulfatide-loaded CD1d molecules for structural studies of the tri-molecular complex. The impact of the absence or presence of an excess amount of endogenous sulfatide on the TCR repertoire will be studied in ceramide galactosyl sulfotransferase CST-/- or saposin B-/- mice, respectively. The role of B cells in the activation of type II NKT cells and their regulatory ability will be investigated using B cell-deficient 5MT mice and their bone marrow chimeras. Since several autoimmune conditions are associated with the anti-sulfatide antibody response, the ability of type II NKT cells to provide help for the antibody response will be examined in CD1d-/-, J118-/- and type II NKT-depleted mice as well as in recipients of sulfatide/CD1d-tetramer+ cells. The role of pDCs vs. mDCs in activation of type II NKT cells and in regulation will be studied using CFSE-labeled CD1d-tetramer+ cells in CD1d-/- recipients. The role of type II NKT cells in the accumulation of MDSCs and their ability to suppress the encephalitogenic CD4+ T cells will be examined in adoptive transfer assays and following their impairment in mice treated with all-trans-retinoic acid. These studies are important not only for understanding the biology of type II NKT cells and the mechanisms of immune regulation, but also because of the highly conserved nature of CD1d molecules across species they will form the basis for manipulation of autoimmune diseases in humans and will have implications for augmenting anti-tumor immune responses.

Public Health Relevance

We have identified a distinct population of white blood lymphocytes that becomes activated by a self-glycolipid and is present in both mice and in humans. Upon their activation these cells are able to prevent and treat experimental autoimmune diseases mimicking multiple sclerosis (MS) in humans. This proposal seeks to understand the mechanism by which these lymphocytes control disease-causing cells in an effort to develop novel therapeutic approaches for the treatment of such diseases.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Innate Immunity and Inflammation Study Section (III)
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Howcroft, Thomas K
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Torrey Pines Institute for Molecular Studies
Port Saint Lucie
United States
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Dhodapkar, Madhav V; Kumar, Vipin (2017) Type II NKT Cells and Their Emerging Role in Health and Disease. J Immunol 198:1015-1021
Mathews, Stephanie; Feng, Dechun; Maricic, Igor et al. (2016) Invariant natural killer T cells contribute to chronic-plus-binge ethanol-mediated liver injury by promoting hepatic neutrophil infiltration. Cell Mol Immunol 13:206-16
Marrero, Idania; Aguilera, Carlos; Hamm, David E et al. (2016) High-throughput sequencing reveals restricted TCR V? usage and public TCR? clonotypes among pancreatic lymph node memory CD4(+) T cells and their involvement in autoimmune diabetes. Mol Immunol 74:82-95
Bandyopadhyay, Keya; Marrero, Idania; Kumar, Vipin (2016) NKT cell subsets as key participants in liver physiology and pathology. Cell Mol Immunol 13:337-46
Maricic, Igor; Sheng, Huiming; Marrero, Idania et al. (2015) Inhibition of type I natural killer T cells by retinoids or following sulfatide-mediated activation of type II natural killer T cells attenuates alcoholic liver disease in mice. Hepatology 61:1357-69
Maricic, Igor; Girardi, Enrico; Zajonc, Dirk M et al. (2014) Recognition of lysophosphatidylcholine by type II NKT cells and protection from an inflammatory liver disease. J Immunol 193:4580-9
Kumar, Vipin; Delovitch, Terry L (2014) Different subsets of natural killer T cells may vary in their roles in health and disease. Immunology 142:321-36
Maricic, Igor; Halder, Ramesh; Bischof, Felix et al. (2014) Dendritic cells and anergic type I NKT cells play a crucial role in sulfatide-mediated immune regulation in experimental autoimmune encephalomyelitis. J Immunol 193:1035-46
Kumar, Vipin (2013) NKT-cell subsets: promoters and protectors in inflammatory liver disease. J Hepatol 59:618-20
Girardi, Enrico; Maricic, Igor; Wang, Jing et al. (2012) Type II natural killer T cells use features of both innate-like and conventional T cells to recognize sulfatide self antigens. Nat Immunol 13:851-6

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