Simultaneous Analysis of HCV-Specific CD4+ and CD8+ T Cells by Multicolor Flow Cytometry

  • David Wolski
  • Georg M. LauerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1911)


CD4 T cell responses are key to effective control of HCV infection. However, their precise mechanisms of action and the molecular programs leading to effective T help versus CD4 T cell failure remain elusive. Direct visualization of HCV-specific CD4 T cells using HLA class II tetramers holds the promise to better define the function and phenotype of these cells and to isolate them for further molecular analysis. Here we describe how to utilize peptide-MHC (pMHC) class II tetramers in multicolor flow cytometry to define the expression of molecules on the surface and within HCV-specific CD4 T cells, how to analyze HCV-specific CD4 and CD8 T cells in the same tube, and how to sort live HCV-specific CD4 T cells as single cells or T cell populations for further analysis by RNAseq or other methods.

Key words

HCV-specific T cells CD4 CD8 Cell sorting Flow cytometry Tetramer Pentamer Peptide-MHC Intracellular staining Transcription factor 


  1. 1.
    Schulze zur Wiesch J, Lauer GM, Day CL et al (2005) Broad repertoire of the CD4+ Th cell response in spontaneously controlled hepatitis C virus infection includes dominant and highly promiscuous epitopes. J Immunol 175:3603–3613. Scholar
  2. 2.
    Schulze zur Wiesch J, Ciuffreda D, Lewis-Ximenez L et al (2012) Broadly directed virus-specific CD4+ T cell responses are primed during acute hepatitis C infection, but rapidly disappear from human blood with viral persistence. J Exp Med 209:61–75. Scholar
  3. 3.
    Altman JD, Davis MM (2003) MHC-peptide tetramers to visualize antigen-specific T cells. Curr Protoc Immunol Chapter 17:Unit 17:3. Scholar
  4. 4.
    Klenerman P, Cerundolo V, Dunbar PR (2002) Tracking T cells with tetramers: new tales from new tools. Nat Rev Immunol 2:263–272. Scholar
  5. 5.
    Vargas AL, Lechner F, Kantzanou M et al (2001) Ex vivo analysis of phenotype and TCR usage in relation to CD45 isoform expression on cytomegalovirus-specific CD8+ T lymphocytes. Clin Exp Immunol 125:432–439. Scholar
  6. 6.
    Bendall SC, Nolan GP, Roederer M, Chattopadhyay PK (2012) A deep profiler's guide to cytometry. Trends Immunol 33:323–332. Scholar
  7. 7.
    Perfetto SP, Chattopadhyay PK, Roederer M (2004) Seventeen-colour flow cytometry: unravelling the immune system. Nat Rev Immunol 4:648–655. Scholar
  8. 8.
    Schmid I, Ferbas J, Uittenbogaart CH, Giorgi JV (1999) Flow cytometric analysis of live cell proliferation and phenotype in populations with low viability. Cytometry A 35:64–74.<64::aid-cyto9>;2-pCrossRefGoogle Scholar
  9. 9.
    Shapiro HM (2005) Practical Flow Cytometry. Scholar
  10. 10.
    Hoffmann TK, Donnenberg VS, Friebe-Hoffmann U et al (2000) Competition of peptide-MHC class I tetrameric complexes with anti-CD3 provides evidence for specificity of peptide binding to the TCR complex. Cytometry A 41:321–328.<321::aid-cyto11>;2-0CrossRefGoogle Scholar
  11. 11.
    Wooldridge L, Scriba TJ, Milicic A et al (2006) Anti-coreceptor antibodies profoundly affect staining with peptide-MHC class I and class II tetramers. Eur J Immunol 36:1847–1855. Scholar
  12. 12.
    Clement M, Ladell K, Ekeruche-Makinde J et al (2011) Anti-CD8 Antibodies Can Trigger CD8+ T Cell Effector Function in the Absence of TCR Engagement and Improve Peptide–MHCI Tetramer Staining. J Immunol 187:654–663. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Gastrointestinal Unit and Liver Center, Massachusetts General Hospital andHarvard Medical SchoolBostonUSA

Personalised recommendations