A live-cell assay to detect antigen-specific CD4+ T cells with diverse cytokine profiles

Nat Med. 2005 Oct;11(10):1113-7. doi: 10.1038/nm1293. Epub 2005 Sep 25.


Recently activated, but not resting, CD4(+) T cells express CD154, providing costimulatory signals to B cells and antigen-presenting cells (APCs). Therefore, de novo CD154 expression after stimulation identifies antigen-specific CD4(+) T cells. Previous assays were limited by the transient nature of surface CD154 expression; we overcame this by including fluorescently conjugated CD154-specific antibody during stimulation. Our assay is fully compatible with intracellular cytokine staining, and can be used for stimulations as long as 24 h. Notably, it is nonlethal, providing a means to purify viable antigen-specific CD4(+) T cells for further analysis. Using this assay, we found that stimulated cells expressing tumor necrosis factor (TNF)-alpha, interleukin (IL)-2 or interferon (IFN)-gamma were predominantly CD154(+). Furthermore, some cells expressing none of these cytokines also expressed CD154, suggesting that CD154 marks cells with other effector functions. For vaccine- or pathogen-specific responses, we found substantial heterogeneity in expression of CD154 and cytokines, suggesting previously unrecognized diversity in abilities of responding cells to stimulate APCs through CD40.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • CD4 Antigens / analysis
  • CD4 Antigens / metabolism
  • CD4-Positive T-Lymphocytes / cytology
  • CD4-Positive T-Lymphocytes / immunology*
  • CD4-Positive T-Lymphocytes / metabolism*
  • CD40 Ligand / analysis
  • CD40 Ligand / metabolism
  • Cell Separation / methods*
  • Cell Survival
  • Cells, Cultured
  • Cytokines / analysis
  • Cytokines / metabolism*
  • Gene Expression
  • Humans
  • Immunoassay
  • Infections / immunology
  • Vaccination


  • CD4 Antigens
  • Cytokines
  • CD40 Ligand