The Purification and Functional Analysis of Human CD4+CD25high Regulatory T Cells

Clare M. Baecher‐Allan1, David A. Hafler1

1 Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.4B
DOI:  10.1002/0471142735.im0704bs72
Online Posting Date:  May, 2006
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Abstract

Regulatory T cells were initially identified and isolated in the mouse, by virtue of their endogenous expression of CD25 (IL‐2R αchain) and shown to inhibit both the in vivo development of autoimmunity and the in vitro proliferation of nonregulatory, CD4+CD25 T cells. In contrast to mouse cells, human regulatory T cells are not purified by isolating all CD25‐expressing CD4 T cells ex vivo. Such cells can be isolated by targeting only the small percentage of human CD4 T cells that express high levels of CD25. This is best achieved by FACS sorting using the level of CD25 expressed on CD4 T cells to place the gate for discriminating high expression of CD25. This unit provides two widely used methods to isolate (FACS) or to enrich (magnetic beads) human CD4+CD25+ regulatory T cells from blood, along with an in vitro coculture assay to measure the anergic and suppressive features of human CD4+CD25+ regulatory T cells.

Keywords: T cell subpopulation; CD25; inhibition; regulation; suppression

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of CD4+CD25high Regulatory T and CD4+CD25− Nonregulatory Target T Cells by FACS
  • Alternate Protocol 1: Enrichment of CD4+CD25+ Regulatory T Cells by Magnetic Separation
  • Basic Protocol 2: IN Vitro Analysis of the Anergic and Antiproliferative Properties of Isolated CD4+CD25high T Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of CD4+CD25high Regulatory T and CD4+CD25− Nonregulatory Target T Cells by FACS

  Materials
  • Freshly obtained heparinized peripheral blood
  • Complete RPMI medium (see recipe), serum free (ice cold)
  • Complete RPMI medium (see recipe) containing 5% heat‐inactivated pooled human AB serum (RPMI‐HuS), ice cold
  • Fluorescently labeled mAbs (all mouse, all from BD Pharmingen):
    • PE‐labeled anti‐CD4: mAb RPA‐T4 (mIgG1)
    • PE‐Cy5 labeled anti‐CD25: mAb M‐A251 (mIgG1)
    • FITC‐labeled anti‐monocyte surface antigens: anti‐CD14 mAb M5E2 (mIgG2a), anti‐CD32 mAb 3D3 (mIgG1), and anti‐CD116 mAb M5D12 (mIgG1)
  • Fluorescently labeled isotype controls (BD Pharmingen):
    • PE‐labeled mIgG1
    • PE‐Cy5‐labeled mIgG1
    • Mixture of FITC‐labeled mIgG1 and mIgG2a to control for the monocyte‐specific mAbs
  • Fluorescence‐activated cell sorter (FACS; Chapter 5), capable of detecting three colors
  • Additional reagents and equipment for preparing PBMC by Ficoll‐Hypaque centrifugation (unit 7.1), flow cytometry (Chapter 5), and counting viable cells ( appendix 3B)

Alternate Protocol 1: Enrichment of CD4+CD25+ Regulatory T Cells by Magnetic Separation

  • CD4 negative‐selection magnetic beads (Miltenyi Biotech or Dynal)
  • Phosphate‐buffered saline (PBS; appendix 2A) containing 0.5% fetal bovine serum (FBS)
  • Anti‐CD25‐coupled small magnetic beads for positive selection (Miltenyi Biotec)
  • Magnetic separator and separation columns (Miltenyi Biotech)
  • Additional reagents and equipment for magnetic separations (unit 3.5)

Basic Protocol 2: IN Vitro Analysis of the Anergic and Antiproliferative Properties of Isolated CD4+CD25high T Cells

  • Isolated CD4+CD25+ regulatory T cells (see protocol 1 or protocol 2)
  • Isolated CD4+CD25 responder T cells (see protocol 1 or protocol 2)
  • Complete RPMI medium (see recipe) containing 5% heat‐inactivated pooled human AB serum (RPMI‐HuS)
  • PBMC to be used as accessory cells (see protocol 1, step )
  • Anti‐CD2 or anti‐CD3 magnetic beads (Dynal Biotech)
  • Anti‐CD3 mAb (clone UCHT1 from BD Pharmingen)
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • Cesium irradiator
  • 96‐well polystyrene, U‐bottom tissue culture plates
  • Centrifuge with microtiter plate carrier
  • Additional reagents and equipment for magnetic purification of peripheral blood T cells (unit 7.47.4A), cytokine ELISAs (Chapter 6), and measuring cell proliferation ( appendix 3D)
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Figures

Videos

Literature Cited

Literature Cited
   Asano, M., Toda, M., Sakaguchi, N., and Sakaguchi, S. 1996. Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J. Exp. Med. 184:387‐396.
   Baecher‐Allan, C., Brown, J.A., Freeman, G.J., and Hafler, D.A. 2001. CD4+CD25 high regulatory cells in human peripheral blood. J. Immunol. 167:1245‐1253.
   Baecher‐Allan, C., Viglietta, V., and Hafler, D.A. 2002. Inhibition of human CD4(+)CD25(+high) regulatory T cell function. J. Immunol. 169: 6210‐6217.
   Dieckmann, D.,  Plottner, H.,  Berchtold, S.,  Berger, T., and Schuler, G. 2001. Ex vivo isolation and characterization of CD4(+)CD25(+) T cells with regulatory properties from human blood. J. Exp. Med. 193:1303‐1310.
   Jonuleit, H., Schmitt, E., Stassen, M., Tuettenberg, A., Knop, J., and Enk, A.H. 2001. Identification and functional characterization of human CD4(+)CD25(+) T cells with regulatory properties isolated from peripheral blood. J. Exp. Med. 193:1285‐1294.
   Levings, M.K., Sangregorio, R., and Roncarolo, M.G. 2001. Human cd25(+)cd4(+) t regulatory cells suppress naive and memory T cell proliferation and can be expanded in vitro without loss of function. J. Exp. Med. 193:1295‐1302.
   Salomon, B., Lenschow, D.J., Rhee, L., Ashourian, N., Singh, B., Sharpe, A., and Bluestone, J.A. 2000. B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity 12:431‐440.
   Shevach, E.M., McHugh, R.S., Piccirillo, C.A., and Thornton, A.M. 2001. Control of T‐cell activation by CD4+ CD25+ suppressor T cells. Immunol. Rev. 182:58‐67.
   Thornton, A.M. and Shevach, E.M. 1998. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188:287‐296.
   Wing, K., Ekmark, A., Karlsson, H., Rudin, A., and Suri‐Payer, E. 2002. Characterization of human CD25+CD4+ T cells in thymus, cord, and adult blood. Immunology 106:190–199.
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