Measurement of T Cell Activation After 16‐hr In Vitro Stimulation with Concanavalin A

Borros M. Arneth1

1 Institute of Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg University Mainz, Mainz, Germany
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.28
DOI:  10.1002/0471142956.cy0628s51
Online Posting Date:  January, 2010
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Abstract

A flow cytometry assay that can be used to directly determine the proportion of activated T lymphocytes in human whole blood samples after stimulation with concanavalin A is presented here. Human whole blood is incubated with fluorescently labeled antibodies (against CD3, CD4, CD8, and CD69), erythrocytes are then lysed, and the samples are analyzed using a flow cytometer. The assay presented is able to differentiate between CD4+ and CD8+ T lymphocytes. Thus, it is possible to quantify both lymphocyte populations in parallel, as well as the respective proportions of activated T lymphocytes, all from one sample. An additional advantage of this assay is that it was developed to assay whole blood, thereby reducing the likelihood of introducing pre‐analytic error and facilitating the calculation of quantitative ratios. Thus, the method can be used for both in vitro and ex vivo experiments. Curr. Protoc. Cytom. 51:6.28.1‐6.28.10. © 2010 by John Wiley & Sons, Inc.

Keywords: activated T cells; lymphocytes; lymphocyte proliferation assay; activation marker; CD4 T helper cells; CD8 cytotoxic T cells; CD69 activation marker; T lymphocytes; CD69; CD4 T cells

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Immunophenotypic Analysis of the Relative Distribution of Activated and Non‐Activated CD4 and CD8 T Lymphocyte Subsets in Unwashed Erythrocyte‐Lysed Whole Peripheral Blood Samples
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Immunophenotypic Analysis of the Relative Distribution of Activated and Non‐Activated CD4 and CD8 T Lymphocyte Subsets in Unwashed Erythrocyte‐Lysed Whole Peripheral Blood Samples

  Materials
  • Patients
  • Anticoagulant, e.g., heparin, Sarstedt; Fraxiparine (nadroparin), Glaxo Smith Kline; or Fragmin (dalteparin), Pfizer
  • Concanavalin A solution (1µg/µl) (e.g., ConA, Sigma Aldrich)
  • Fluorochrome‐conjugated monoclonal antibodies that recognize cell surface antigens CD3, CD4, CD8, and CD69 (Table 6.28.1):
    • CD8 FITC, CD69 PE, and CD3 PerCP antibodies (e.g., FastImmune CD8 FITC, CD69 PE and CD3 PerCP antibody mix, BD Bioscience, Becton Dickinson)
  • CD 4 APC antibody (e.g., CD4 antibody, BD Bioscience, Becton Dickinson)
  • Isotype antibody controls corresponding to the antibodies used (e.g., FITC/PE/ PerCP/APC Mouse Ig Fluorescence Controls, BD Bioscience, Becton Dickinson; FastImmune Control γFITC/γPE/CD3 PerCP, BD Bioscience, Becton Dickinson)
  • Ammonium chloride erythrocyte lysing solution (e.g., 10× BD FACS lysing solution, BD Bioscience, Becton Dickinson)
  • Blood withdrawal tubes (e.g., blood withdrawal monovettes, Sarstedt)
  • 12 × 75–mm polypropylene tubes
  • 37°C incubator (e.g., Binder KB 53 incubator)
  • Flow cytometer equipped with four fluorescence detectors (e.g., FACS‐Calibur, Becton Dickinson)
    Table 6.8.1   MaterialsPanel of Monoclonal Antibody Reagents Used to Measure the Number of Activated T Lymphocytes as well as the Percentage of Activated (CD69+) CD4+ and CD8+ T‐Cell Subsets

    Antigen Fluorescent label a Purpose of admixture
    CD3 PerCP CD3 versus side scatter, gate on lymphocytes, count 30,000
    CD4 APC CD69 versus CD4, count CD69+ and CD69 CD4 cells
    CD8 FITC CD69 versus CD8, count CD69+ and CD69 CD8 cells
    CD69 PE Marker for activated T cells

     aFluorescent labels shown above yield the highest sensitivity.
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Figures

  •   FigureFigure 6.28.1 Antibody staining of a ConA‐stimulated sample. This experiment shows CD4‐ and CD8‐positive populations, including those positive for CD69 and CD4 and those positive for CD69 and CD8. These double‐positive populations represent the activated CD4 and CD8 T lymphocytes, respectively.
  •   FigureFigure 6.28.2 Antibody staining of an unstimulated sample. This sample shows CD4‐positive and CD8‐positive populations, but very few CD69‐positive T cells.
  •   FigureFigure 6.28.3 Isotype control staining of an unstimulated whole blood sample. CD4‐ and CD8‐positive populations are absent. This experiment demonstrates the lack of nonspecific binding.
  •   FigureFigure 6.28.4 Isotype control staining of a ConA‐stimulated sample. Although the sample was activated by a mitogen, it does not show CD4 and CD8 populations because it was incubated with the isotype control and not with the staining antibodies. Thus, no nonspecific binding is observed.

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Literature Cited

Literature Cited
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   Gimenez‐Camarasa, J.M., Garcia‐Calderon, P., Asensio, J., and De Moragas, J.M. 1975a. Lymphocyte transformation test in allergic contact nickel dermatitis. Br. J. Dermatol. 92:9‐15.
   Gimenez‐Camarasa, J.M., Garcia‐Calderon, P., and de Moragas, J.M. 1975b. Lymphocyte transformation test in fixed drug eruption. N. Engl. J. Med. 292:819‐821.
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   Lindsey, W.B., Lowdell, M.W., Marti, G.E., Abbasi, F., Zenger, V., King, K.M., and Lamb, L.S. Jr. 2007. CD69 expression as an index of T‐cell function: Assay standardization, validation and use in monitoring immune recovery. Cytotherapy 9:123‐132.
   Maino, V.C., Suni, M.A., and Ruitenberg, J.J. 1995. Rapid flow cytometric method for measuring lymphocyte subset activation. Cytometry 20:127‐133.
   Mardiney, M., Brown, M.R., and Fleisher, T.A. 1996. Measurement of T‐cell CD69 expression: A rapid and efficient means to assess mitogen‐ or antigen‐induced proliferative capacity in normals. Cytometry 26:305‐310.
   Rutella, S., Rumi, C., Lucia, M.B., Barberi, T., Puggioni, P.L., Lai, M., Romano, A., Cauda, R., and Leone, G. 1999. Induction of CD69 antigen on normal CD4+ and CD8+ lymphocyte subsets and its relationship with the phenotype of responding T‐cells. Cytometry 38:95‐101.
   Simms, P.E. and Ellis, T.M. 1996. Utility of flow cytometric detection of CD69 expression as a rapid method for determining poly‐ and oligoclonal lymphocyte activation. Clin. Diagn. Lab. Immunol. 3:1071‐1412.
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