Table 1.

Methods of characterization of allergen-specific CD4+ T cells.

Method	Advantages	Limitations	Time	T cell assay compatibility
MHCII-peptide tetramer	Sensitive, epitope-specific, cells are viabl	Availability of recombinant MHCII molecule, knowledge of epitopes	Ex vivo: 5 h in vitro: 5–14 days	ICS, CFSE, CCA, CD154
Intracellular cytokine staining (ICS)	Allows functional detection of whole antigen-specific CD4+ T cell response.	Cells are non-viable, dependent on specific T-cell function	4–12 h	CFSE, CD154, MHCII tetramer
Cytokine capture assay (CCA)	Allows functional detection of whole antigen-specific CD4+ T cell response, cells are viable.	Limited to a few cytokines, carry-over of irrelevant cells	4–12 h	MHCII tetramer, CD154, CFSE
CD154 assay	Allows detection of whole antigen-specific CD4+ T cell response.	Dependent on specific T-cell activation	3–8 h	ICS, CFSE, CCA, MHCII tetramer
Elispot	Sensitive, provides both qualitative and quantitative information.	Limited to a few cytokines, dependent on specific T-cell function	Ex vivo: 2–3 days in vitro: 5–14 days
CFSE-dilution assay	Allows detection of whole antigen-specific CD4+ T cell response	Limited to dividing cells, by-stander activation	3–10 days	ICS, CCA, MHCII tetramer, CD154