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Genetic Studies of Natural Glycosphingolipid Ligands for NKT Cells

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Invariant Natural Killer T-Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2388))

Abstract

Glycosphingolipids (GSL) are natural ligands of NKT cells. Several laboratories have reported the in vitro activity of isoglobotriosylceramide (iGb3) in stimulating NKT cells. However, the knockout mice of iGb3 synthase showed no deficiency in development and function of NKT cells. There is a lack of knowledge on the genetics of redundant natural glycosphingolipid ligands. We have identified additional glycosphingolipid with stimulatory activity to NKT cells, including fucosyl lactosylceramide (H antigen). Here we describe the procedures to generate mice with deficiencies in Fut1, Fut2, and Sec1 genes to deplete H antigen through BAC engineering for the generation of ES cell-targeting construct, as well as the mice with deficiency of both blood group H-GSL ligand and isoglobotriosylceramide.

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Abbreviations

Blood group A:

GalNAc α3 Fuc α2 Gal β

Blood group B:

Gal α3 Fuc α2 Gal β

Blood group H:

Fuc α2 Gal β

CFG :

Consortium of functional glycomics

Fut1:

α-2 fucosyltransferase I

Fut2 :

α-2 fucosyltransferase II

NKT:

natural killer T cells

Sec1:

α-2 fucosyltransferase III

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Acknowledgments

This work was supported by US NIH grant AI079232 (DZ), National Key Research and Development Plan grant 2021YFE0200500 and 2017YFA0505901 (DZ), Fundamental Research Funds for the Central Universities 22120210292 (DZ), National Natural Science Foundation of China grant 81570007 and 31870972 (DZ), Shanghai Science and Technology Committee grant 20410713500, and the Outstanding Clinical Discipline Project of Shanghai Pudong Grant Number: PWYgy2018–10 (DZ). All these sponsors have no roles in the study design or the collection, analysis, and interpretation of data.

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Authors and Affiliations

  1. Shanghai Pudong New Area Mental Health Center, School of Medicine, Tongji University, Shanghai, China

    Dapeng Zhou & Jiaxi Cheng

  2. Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, Yunnan University, Kunming, China

    Chengfeng Xia, Zhiyuan Li & Guanghui Ni

  3. School of Medicine, Southern University of Science and Technology, Shenzhen, China

    Peng G. Wang & Wenpeng Zhang

Authors
  1. Dapeng Zhou
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  2. Chengfeng Xia
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  3. Peng G. Wang
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  4. Zhiyuan Li
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  5. Wenpeng Zhang
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  6. Guanghui Ni
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  7. Jiaxi Cheng
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Corresponding authors

Correspondence to Dapeng Zhou , Chengfeng Xia or Peng G. Wang .

Editor information

Editors and Affiliations

  1. Department of Pathogen Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    Chaohong Liu

1 Electronic Supplementary Materials

Data S1

Primer sites for genotyping of Fut1/Fut2/Sec1 triple KO mice. Sites of PCR primers were marked for genotyping of △ Fut1/+Hyg allele and △Fut2DSec1 + (loxP-neo-loxP) allele (PPTX 38 kb)

Data S2

Stimulatory activities of blood group H GSLs to iNKT cells. iGb3 and blood group GSLs dissolved at 1 mg/mL in DMSO were diluted in cell culture medium at indicated concentrations and pulsed to mouse (C57BL/6) bone marrow-derived dendritic cells (105) for 8 h, after which the dendritic cells were washed with culture medium and mixed with DN32.D3 hybridoma cells (5 × 104). The resulting mixture was co-cultured for 24 h, and the released IL-2 was measured by bioassays using CTLL-2 cell line. Unit means international unit of IL2 (PPT 132 kb)

Data S3

Development of iNKT cells in Fut1/Fut2/Sec1−/− mice and Fut1/Fut2/Sec1/A3galt2−/− mice. Lymphocytes were purified from mouse thymus, spleen, and liver and stained by αGalCer/CD1d tetramer (APC-labelled, provided by NIAID tetramer facility at Emory University, Atlanta, GA) at cell surface, followed by staining with anti-CD3 antibody. % means the percentage of CD3+ PBS57/CD1d tetramer+ NKT cells among total CD3+ T cells. (A and B) Fut1/Fut2/Sec1−/− mice and wild-type littermates. (C and D) Fut1/Fut2/Sec1/A3galt2−/− mice and wild-type littermates (PPTX 550 kb)

Data S4

(DOCX 21 kb)

Data S5

(PPT 693 kb)

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Zhou, D. et al. (2021). Genetic Studies of Natural Glycosphingolipid Ligands for NKT Cells. In: Liu, C. (eds) Invariant Natural Killer T-Cells. Methods in Molecular Biology, vol 2388. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1775-5_2

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  • DOI: https://doi.org/10.1007/978-1-0716-1775-5_2

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1774-8

  • Online ISBN: 978-1-0716-1775-5

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