Abstract
Flow cytometry is an effective tool in immunology that uses laser as a light source to yield scattered and fluorescent light signals read by photomultiplier tubes or photodiodes for detection. Flow cytometry allows immunophenotyping using fluorescently conjugated antibodies for the identification of subgroups of immune cells at a single-cell level. Natural killer T (NKT) cells are CD1d-restricted T cells, which recognize lipid antigens, unlike conventional T lymphocytes that recognize peptide antigens presented by class I or class II MHC. The unique T cell receptor (TCR) of type I NKT or invariant natural killer T (iNKT) cells are comprised of an invariant α-chain that pairs with a limited repertoire of β-chains. Type I NKT cells play an essential role in the orchestration of the innate and adaptive immune responses against various diseases. Here, we will review the process of identifying mouse type I NKT cells by flow cytometry, which serves as a foundational technique for studying these cells.
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References
Godfrey DI, MacDonald HR, Kronenberg M, Smyth MJ, Van Kaer L (2004) NKT cells: what’s in a name? Nat Rev Immunol 4(3):231–237
Cameron G, Pellicci DG, Uldrich AP, Besra GS, Illarionov P, Williams SJ, La Gruta NL, Rossjohn J, Godfrey DI (2015) Antigen specificity of type I NKT cells is governed by TCR beta-chain diversity. J Immunol 195(10):4604–4614. https://doi.org/10.4049/jimmunol.1501222
Bendelac A, Savage PB, Teyton L (2007) The biology of NKT cells. Annu Rev Immunol 25:297–336. https://doi.org/10.1146/annurev.immunol.25.022106.141711
Tiwary S, Berzofsky JA, Terabe M (2019) Altered lipid tumor environment and its potential effects on NKT cell function in tumor immunity. Front Immunol 10:2187. https://doi.org/10.3389/fimmu.2019.02187
Terabe M, Berzofsky JA (2018) Tissue-specific roles of NKT cells in tumor immunity. Front Immunol 9:1838. https://doi.org/10.3389/fimmu.2018.01838
Godfrey DI, Stankovic S, Baxter AG (2010) Raising the NKT cell family. Nat Immunol 11(3):197–206. https://doi.org/10.1038/ni.1841
Baranek T, Lebrigand K, de Amat HC, Gonzalez L, Bogard G, Dietrich C, Magnone V, Boisseau C, Jouan Y, Trottein F, Si-Tahar M, Leite-de-Moraes M, Mallevaey T, Paget C (2020) High dimensional single-cell analysis reveals iNKT cell developmental trajectories and effector fate decision. Cell Rep 32(10):108116. https://doi.org/10.1016/j.celrep.2020.108116
Lee YJ, Wang H, Starrett GJ, Phuong V, Jameson SC, Hogquist KA (2015) Tissue-specific distribution of iNKT cells impacts their cytokine response. Immunity 43(3):566–578. https://doi.org/10.1016/j.immuni.2015.06.025
Lee YJ, Holzapfel KL, Zhu J, Jameson SC, Hogquist KA (2013) Steady-state production of IL-4 modulates immunity in mouse strains and is determined by lineage diversity of iNKT cells. Nat Immunol 14(11):1146–1154. https://doi.org/10.1038/ni.2731
Sag D, Krause P, Hedrick CC, Kronenberg M, Wingender G (2014) IL-10-producing NKT10 cells are a distinct regulatory invariant NKT cell subset. J Clin Invest 124(9):3725–3740. https://doi.org/10.1172/JCI72308
Lynch L, Michelet X, Zhang S, Brennan PJ, Moseman A, Lester C, Besra G, Vomhof-Dekrey EE, Tighe M, Koay HF, Godfrey DI, Leadbetter EA, Sant’Angelo DB, von Andrian U, Brenner MB (2015) Regulatory iNKT cells lack expression of the transcription factor PLZF and control the homeostasis of T(reg) cells and macrophages in adipose tissue. Nat Immunol 16(1):85–95. https://doi.org/10.1038/ni.3047
King IL, Fortier A, Tighe M, Dibble J, Watts GF, Veerapen N, Haberman AM, Besra GS, Mohrs M, Brenner MB, Leadbetter EA (2011) Invariant natural killer T cells direct B cell responses to cognate lipid antigen in an IL-21-dependent manner. Nat Immunol 13(1):44–50. https://doi.org/10.1038/ni.2172
Chang PP, Barral P, Fitch J, Pratama A, Ma CS, Kallies A, Hogan JJ, Cerundolo V, Tangye SG, Bittman R, Nutt SL, Brink R, Godfrey DI, Batista FD, Vinuesa CG (2011) Identification of Bcl-6-dependent follicular helper NKT cells that provide cognate help for B cell responses. Nat Immunol 13(1):35–43. https://doi.org/10.1038/ni.2166
Moreira-Teixeira L, Resende M, Devergne O, Herbeuval JP, Hermine O, Schneider E, Dy M, Cordeiro-da-Silva A, Leite-de-Moraes MC (2012) Rapamycin combined with TGF-beta converts human invariant NKT cells into suppressive Foxp3+ regulatory cells. J Immunol 188(2):624–631. https://doi.org/10.4049/jimmunol.1102281
Matsuda JL, Naidenko OV, Gapin L, Nakayama T, Taniguchi M, Wang CR, Koezuka Y, Kronenberg M (2000) Tracking the response of natural killer T cells to a glycolipid antigen using CD1d tetramers. J Exp Med 192(5):741–754
Benlagha K, Weiss A, Beavis A, Teyton L, Bendelac A (2000) In vivo identification of glycolipid antigen-specific T cells using fluorescent CD1d tetramers. J Exp Med 191(11):1895–1903
Van den Broeck W, Derore A, Simoens P (2006) Anatomy and nomenclature of murine lymph nodes: descriptive study and nomenclatory standardization in BALB/cAnNCrl mice. J Immunol Methods 312(1–2):12–19. https://doi.org/10.1016/j.jim.2006.01.022
Liu Y, Goff RD, Zhou D, Mattner J, Sullivan BA, Khurana A, Cantu C 3rd, Ravkov EV, Ibegbu CC, Altman JD, Teyton L, Bendelac A, Savage PB (2006) A modified alpha-galactosyl ceramide for staining and stimulating natural killer T cells. J Immunol Methods 312(1–2):34–39. https://doi.org/10.1016/j.jim.2006.02.009
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This work was supported by Intramural Research Program of the NIH, NCI.
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Joshi, V., Terabe, M. (2021). Detection of Mouse Type I NKT (iNKT) Cells by Flow Cytometry. 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_8
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DOI: https://doi.org/10.1007/978-1-0716-1775-5_8
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