Antigen-specific T cell analyse

CD8: MHC multimer analyses (combinatorial coding)
CD4: CD40L assays

CD8: MHC multimer analyses (combinatorial coding)

The detection and characterization of antigen-specific CD8+ T cells is crucial for the understanding of their role in for instance vaccination or anti-cancer therapies. The detection of antigen-specific CD8+ T cells recognizing an HLA binding peptide is possible by staining these cells with fluorescently labelled multimers.

To date, a limited number of antigen-specific T cell populations per sample can be detected. This limits the ability to perform a comprehensive analysis of disease-associated or therapy-induced T cell responses. In particular for clinical samples this can be a considerable disadvantage. Combinatorial coding is a new high-tech method to circumvent the limitation of sample size. With this technique monitoring of T cell recognition of the MHC-peptide complexes can be performed quickly in small sample volumes.

Illustratie 8

Also see:

CD4: CD40L assays

The characterization of CD4+ T cells is crucial for understanding their role in immunotherapeutic approaches such as vaccinations or tolerizing therapies. Measuring CD4+ T cell responses after different antigen stimulations will help defining the proteins/peptides responsible for certain immune responses.

Detecting the antigen specificity of T cells can be executed by tetramer binding. Using class I tetramers for CD8+ T cell antigen specificity is a very powerful and robust technique (CD8: MHC multimer analyses (combinatorial coding)). Using class II tetramers to determine CD4+ T cell specificities is less robust than class I tetramer detection, due to larger HLA variability and less stable multimer: T cell complexes. Therefore, a different approach has been developed to determine antigen specific CD4+ T cells. After antigen specific TCR stimulation CD4+ T cells up regulate CD40L (CD154), this can be achieved by culturing PBMCs with antigen or peptide of choice. Detection of CD40L by flow cytometry can be combined with functional analysis such as cytokine production.

With this assay we can detect antigen specific CD4+ T cells, whilst simultaneously characterizing the cytokine profile of the antigen specific cells.


PBMCs unstimulated (left graph) or with CMV (rigth graph) overnight. Stained with CD40L and measured by fow cytometry to determine the percentage of CMV specific CD4+ T cells

Also see:


Van Buuren MM, Dijkgraaf FE, Linnemann C, Toebes M, Chang CX, Mok JY, Nguyen M, van Esch WJ, Kvistborg P, Grotenbreg GM, Schumacher TN. HLA micropolymorphisms strongly affect peptide-MHC multimer-based monitoring of antigen-specific CD8+ T cell responses. J Immunol 2014; 192(2):641-8.

Bakker AH, Hoppes R, Linnemann C, Toebes M, Rodenko B, Berkers CR, Hadrup SR, van Esch WJ, Heemskerk MH, Ovaa H, Schumacher TN. Conditional MHC class I ligands and peptide exchange technology for the human MHC gene products HLA-A1, -A3, -A11, and -B7. Proc Natl Acad Sci U S A 2008; 105(10):3825-30.

Rodenko B, Toebes M, Hadrup SR, van Esch WJ, Molenaar AM, Schumacher TN, Ovaa H. Generation of peptide-MHC class I complexes through UV-mediated ligand exchange. Nat Protoc 2006; 1(3):1120-32.

Andersen RS, Kvistborg P, Frøsig TM, Pedersen NW, Lyngaa R, Bakker AH, Shu CJ, Straten Pt, Schumacher TN, Hadrup SR. Parallel detection of antigen-specific T cell responses by combinatorial encoding of MHC multimers. Nat Protoc 2012; 7(5):891-902.

Last edited on: 29 October 2015