Advertisement

Phenotypic and Functional Characterization of Cytotoxic T Lymphocytes by Flow Cytometry

  • Iulia PopescuEmail author
  • Matthew Pipeling
  • Jason Akulian
  • John McDyerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1186)

Abstract

Cytotoxic T lymphocytes (CTLs) are important constituents of the adaptive immune system. Development of CTLs are particularly important for bacterial and viral infections, in addition to tumor surveillance. Measuring T cell immune function is important in evaluating host defense, allergy, autoimmunity, transplant rejection, and tumor immunity. In these recent years it has become possible to measure multiple effector functions in a single cell such as cytokine, transcription factors, and cytolytic function. In addition these parameters can be evaluated in conjunction with cellular proliferation. In this chapter we detail these cellular based assays and the methods used to characterize and quantify both phenotype and function of CTL.

Key words

CD8+ T cells CTL Flow cytometry Cellular assay 

References

  1. 1.
    Williams MA, Bevan MJ (2007) Effector and memory CTL differentiation. Annu Rev Immunol 25:171–192, doi: 0.1146/annurev.immunol.25.022106.141548PubMedCrossRefGoogle Scholar
  2. 2.
    Kurtulus S, Tripathi P, Hildeman DA (2012) Protecting and rescuing the effectors: roles of differentiation and survival in the control of memory T cell development. Front Immunol 3:404. doi: 10.3389/fimmu. 2012. 00404, Epub 2013 Jan 23PubMedCentralPubMedGoogle Scholar
  3. 3.
    Kahan BD (2003) Individuality: the barrier to optimal immunosuppression. Nat Rev Immunol 3:831–838. doi: 10.1038/nri1204 PubMedGoogle Scholar
  4. 4.
    Kapsenberg ML (2003) Dendritic-cell control of pathogen-driven T-cell polarization. Nat Rev Immunol 3:984–993PubMedGoogle Scholar
  5. 5.
    McElroy DS, Badstibner AM, D’Orazio SEF (2007) Use of the CD107 mobilization assay reveals that cytotoxic T lymphocytes with novel MHC-Ib restriction are activated during Listeria monocytogenes infection. J Immunol Methods 328(1–2):45–52PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    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):65–78PubMedCrossRefGoogle Scholar
  7. 7.
    Popescu I, Macedo C, Abu-Elmagd K, Shapiro R, Hua Y, Thomson AW, Morelli AE, Storkus WJ, Metes D (2007) EBV-specific CD8+ T cell reactivation in transplant patients results in expansion of CD8+ type-1 regulatory T cells. Am J Transplant 7(5):1215–1223, Epub 2007 Feb 27PubMedCrossRefGoogle Scholar
  8. 8.
    Sarkar S, Kalia V, Haining WN, Konieczny BT, Subramaniam S, Ahmed R (2008) Functional and genomic profiling of effector CD8 T cell subsets with distinct memory fates. J Exp Med 205(3):625–640. doi: 10.1084/jem.20071641, Epub 2008 Mar 3PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Maecker HT, Frey T, Nomura L, Trotter J (2004) Selecting fluorochrome conjugates for maximum sensitivity. Cytometry A 62:169PubMedCrossRefGoogle Scholar
  10. 10.
    Maecker HT, Trotter J (2006) Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A 69:1037PubMedCrossRefGoogle Scholar
  11. 11.
    Roederer M (2008) How many events is enough? Are you positive? Cytometry A 73:384–385PubMedCrossRefGoogle Scholar
  12. 12.
    Crowther EJ, Stall A, Bishop JE, Sasaki D (2002) A setup system for compensation: BD CompBeads plus BD FACSDiva software Presented at ISAC XXI, San Diego, CA, 4–9 May Poster No. 57644Google Scholar
  13. 13.
    BD™CompBead and BD™CompBead Plus compensation particles, BD Biiosciences website: http://www.bdbiosciences.com/external_files/pm/doc/mkt_lit/salesSheets/live/web_enabled/23-13303-01.pdf
  14. 14.
    Molecular Probes Invitrogen web site: http://products.invitrogen.com/ivgn/product/L23105
  15. 15.
    Pipeling MR, Popescu ID, Orens JB, Pilewski JM, McDyer JF (2013) Dynamic changes in dominant antigen responses following primary CMV infection in lung transplant recipients. J Heart Lung Transplant 32(4):S298CrossRefGoogle Scholar
  16. 16.
    BD Biosciences web site tutorial protocol: http://www.bdbiosciences.com/resources/flowcytometry/index.jsp
  17. 17.
    Pipeling MR, West EE, Osborne CM, Whitlock AB, Dropulic LK, Willett MH, Forman M, Valsamakis A, Orens JB, Moller DR, Lechtzin N, Migueles SA, Connors M, McDyer JF (2008) Differential CMV-specific CD8+ effector T cell responses in the lung allograft predominate over the blood during human primary infection. J Immunol 181(1):546–556PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Gordy C et al (2011) Regulation of steady-state neutrophil homeostasis by macrophages. Blood 117:618PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Jia W et al (2011) Autophagy regulates endoplasmic reticulum homeostasis and calcium mobilization in T lymphocytes. J Immunol 186:5313PubMedCrossRefGoogle Scholar
  20. 20.
    Parish CR (1999) Cancer immunotherapy: the past, the present and the future. Immunol Cell Biol 77(6):499–508PubMedCrossRefGoogle Scholar
  21. 21.
    BioLegend website for Annexin V Pacific Blue protocol: http://www.biolegend.com/pacific-blue-annexin-v-5476.html
  22. 22.
    Hoegh-Petersen M, Roa L, Liu Y, Zhou F, Ugarte-Torres A, Louie P, Fonseca K, Khan F, Russell JA, Storek J (2012) Low cytomegalovirus-specific T-cell counts at reactivation are associated with progression to high-level viremia or disease in seropositive recipients of hematopoietic cell grafts from seropositive but not seronegative donors. Cytotherapy 14(2):194–204. doi: 10.3109/14653249.2011.634402, Epub 2011 Dec 7PubMedCrossRefGoogle Scholar
  23. 23.
    Wiesmayr S, Webber SA, Macedo C, Popescu I, Smith L, Luce J, Metes D (2012) Decreased NKp46 and NKG2D and elevated PD-1 are associated with altered NK-cell function in pediatric transplant patients with PTLD. Eur J Immunol 42(2):541–550. doi: 10.1002/eji.201141832. Epub 2011 Dec 16 PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Macedo C, Orkis EA, Popescu I, Elinoff BD, Zeevi A, Shapiro R, Lakkis FG, Metes D (2009) Contribution of naïve and memory T-cell populations to the human alloimmune response. Am J Transplant 9(9):2057–2066. doi: 10.1111/j.1600-6143.2009.02742.x, Epub 2009 Jul 16PubMedCrossRefGoogle Scholar
  25. 25.
    Akulian JA, Pipeling MR, John ER, Orens JB, Lechtzin N, McDyer JF (2013) High-quality CMV-specific CD4+ memory is enriched in the lung allograft and is associated with mucosal viral control. Am J Transplant 13:146–156PubMedCrossRefGoogle Scholar
  26. 26.
    Roederer M (2011) SPICE: exploration and analysis of post-cytometric complex multivariate datasets. Cytometry A 79(2011–01-01):167–174PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Parish CR, et al. (2009) Use of the intracellular fluorescent dye CFSE to monitor lymphocyte migration and proliferation. Curr Protoc Immunol Unit 4.9Google Scholar
  28. 28.
    Miller MJ et al (2002) Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science 296:1869PubMedCrossRefGoogle Scholar
  29. 29.
    Jung T, Schauer U, Heusser C, Neumann C, Rieger C (1993) Detection of intracellular cytokines by flow cytometry. J Immunol Methods 159(1–2):197–207PubMedCrossRefGoogle Scholar
  30. 30.
    Foster B, Prussin C (2012) Current protocols in immunology. Wiley, New York, NY), Unit 6.24; (NIAID, NIH Bethesda MD)Google Scholar
  31. 31.
    Alberts B, Johnson A, Lewis J et al (2002) Molecular biology of the cell, 4th edn. Garland Science, New YorkGoogle Scholar
  32. 32.
  33. 33.
  34. 34.
    Ramaswami B, Popescu I, Macedo C, Luo C, Shapiro R, Metes D, Chalasani G, Randhawa PS (2011) The Polyomavirus BK large T-antigen-derived peptide elicits an HLA-DR promiscuous and polyfunctional CD4+ T-cell response. Clin Vaccine Immunol 18(5):815–824. doi: 10.1128/CVI.00487-10, Epub 2011 Mar 2PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Ramaswami B, Popescu I, Macedo C, Metes D, Bueno M, Zeevi A, Shapiro R, Viscidi R, Randhawa PS (2009) HLA-A01-, -A03-, and -A024-binding nanomeric epitopes in polyomavirus BK large T antigen. Hum Immunol 70(9):722–728. doi: 10.1016/j.humimm.2009.05.003, Epub 2009 May 14PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Zhu J, Yamane H, Paul WE (2010) Differentiation of effector CD4 T cell populations. Annu Rev Immunol 28:445–489PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Zhou L, Chong MM, Littman DR (2009) Plasticity of CD4+ T cell lineage differentiation. Immunity 30:646–655PubMedCrossRefGoogle Scholar
  38. 38.
    Chatenoud L, Bach JF (2006) Adaptive human regulatory T cells: myth or reality? J Clin Invest 116:2325–2327PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Mitchell P, Afzali B, Lombardi G, Lechler RI (2009) The T helper 17-regulatory T cell axis in transplant rejection and tolerance. Curr Opin Organ Transplant 14:326–331PubMedCrossRefGoogle Scholar
  40. 40.
    Chang SH, Dong C (2009) IL-17 F: regulation, signaling and function in inflammation. Cytokine 46:7–11PubMedCentralPubMedCrossRefGoogle Scholar
  41. 41.
    Pearce EL, Mullen AC, Martins GA, Krawczyk CM, Hutchins AS, Zediak VP, Banica M, DiCioccio CB, Gross DA, Mao CA, Shen H, Cereb N, Yang SY, Lindsten T, Rossant J, Hunter CA, Reiner SL (2003) Control of effector CD8+ T cell function by the transcription factor Eomesodermin. Science 302(5647):1041–1043PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Division of Pulmonary, Allergy and Critical Care Medicine, Department of MedicineUniversity of Pittsburgh School of MedicinePittsburghUSA
  2. 2.Interventional Pulmonary, Division of Pulmonary and Critical CareUniversity of North Carolina School of MedicineChaper HillUSA

Personalised recommendations