UNIT 7.4B The Purification and Functional Analysis of Human CD4+CD25high Regulatory T Cells

  1. Clare M. Baecher-Allan,
  2. David A. Hafler

Published Online: 1 MAY 2006

DOI: 10.1002/0471142735.im0704bs72

Current Protocols in Immunology

Current Protocols in Immunology

How to Cite

Baecher-Allan, C. M. and Hafler, D. A. 2006. The Purification and Functional Analysis of Human CD4+CD25high Regulatory T Cells. Current Protocols in Immunology. 72:I:7.4B:7.4B.1–7.4B.12.

Author Information

  1. Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts

Publication History

  1. Published Online: 1 MAY 2006
  2. Published Print: APR 2006


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.


  • T cell subpopulation;
  • CD25;
  • inhibition;
  • regulation;
  • suppression