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Mass Cytometry Assays for Antigen-Specific T Cells Using CyTOF

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1678)

Abstract

T Cells specific for a single antigen tend to be rare, even after expansion of memory cells. They are commonly detected by in vitro stimulation with peptides or protein, followed by staining for intracellular cytokines. In this protocol, we use CyTOF® mass cytometry to collect single-cell data on a large number of cytokines/chemokines, as well as cell-surface proteins that characterize T cells and other immune cells. We also include a method for magnetic bead enrichment of antigen-stimulated T cells, based on their expression of CD154 and CD69. Coupling magnetic enrichment with highly multi-parameter mass cytometry, this method enables the ability to dissect the frequency, phenotype, and function of antigen-specific T cells in greater detail than previously possible. Rare cell subsets can be examined, while minimizing run times on the CyTOF.

Key words

Antigen-specific Intracellular staining Cytokines T Cells CyTOF Magnetic enrichment 
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Notes

Acknowledgments

This work was supported by grants # 5U19AI057229 and S10RR027582 from the National Institutes of Health, and SFB 650 and the BCRT (FKZ 13GW0099).

References

  1. 1.
    Tanner SD, Bandura DR, Ornatsky O, Baranov VI, Nitz M, Winnik MA (2008) Flow cytometer with mass spectrometer detection for massively multiplexed single-cell biomarker assay. Pure Appl Chem 80(12):2627–2641. doi: 10.1351/pac200880122627
  2. 2.
    Bandura DR, Baranov VI, Ornatsky OI, Antonov A, Kinach R, Lou X, Pavlov S, Vorobiev S, Dick JE, Tanner SD (2009) Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry. Anal Chem 81(16):6813–6822. doi: 10.1021/ac901049w CrossRefGoogle Scholar
  3. 3.
    Ornatsky O, Bandura D, Baranov V, Nitz M, Winnik MA, Tanner S (2010) Highly multiparametric analysis by mass cytometry. J Immunol Methods 361(1–2):1–20. doi: 10.1016/j.jim.2010.07.002 CrossRefGoogle Scholar
  4. 4.
    Lovelace P, Maecker HT (2011) Multiparameter intracellular cytokine staining. Methods Mol Biol 699:165–178. doi: 10.1007/978-1-61737-950-5_8 CrossRefGoogle Scholar
  5. 5.
    Newell EW, Sigal N, Bendall SC, Nolan GP, Davis MM (2012) Cytometry by time-of-flight shows combinatorial cytokine expression and virus-specific cell niches within a continuum of CD8+ T cell phenotypes. Immunity 36(1):142–152. doi: 10.1016/j.immuni.2012.01.002 CrossRefGoogle Scholar
  6. 6.
    Lin DGS, Maecker HT (2015) Intracellular cytokine staining on PBMCs using CyTOF mass cytometry. Bioprotocol 5(1):1–7. doi: 10.21769/BioProtoc.1370 Google Scholar
  7. 7.
    Betts MR, Brenchley JM, Price DA, De Rosa SC, Douek DC, Roederer M, Koup RA (2003) Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods 281(1–2):65–78CrossRefGoogle Scholar
  8. 8.
    Betts MR, Koup RA (2004) Detection of T-cell degranulation: CD107a and b. Methods Cell Biol 75:497–512CrossRefGoogle Scholar
  9. 9.
    Chattopadhyay PK, Yu J, Roederer M (2005) A live-cell assay to detect antigen-specific CD4+ T cells with diverse cytokine profiles. Nat Med 11(10):1113–1117. doi: 10.1038/nm1293 CrossRefGoogle Scholar
  10. 10.
    Chattopadhyay PK, Yu J, Roederer M (2006) Live-cell assay to detect antigen-specific CD4+ T-cell responses by CD154 expression. Nat Protoc 1(1):1–6. doi: 10.1038/nprot.2006.1 CrossRefGoogle Scholar
  11. 11.
    Frentsch M, Arbach O, Kirchhoff D, Moewes B, Worm M, Rothe M, Scheffold A, Thiel A (2005) Direct access to CD4+ T cells specific for defined antigens according to CD154 expression. Nat Med 11(10):1118–1124. doi: 10.1038/nm1292 CrossRefGoogle Scholar
  12. 12.
    Bitmansour AD, Douek DC, Maino VC, Picker LJ (2002) Direct ex vivo analysis of human CD4(+) memory T cell activation requirements at the single clonotype level. J Immunol 169(3):1207–1218CrossRefGoogle Scholar
  13. 13.
    Miltenyi S, Muller W, Weichel W, Radbruch A (1990) High gradient magnetic cell separation with MACS. Cytometry 11(2):231–238. doi: 10.1002/cyto.990110203 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Fluidigm CorporationSouth San FranciscoUSA
  2. 2.Human Immune Monitoring Center, Institute for Immunity, Transplantation, and InfectionStanford University School of MedicineStanfordUSA

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