Using the CD40L assay, we can measure the percentage of antigen-specific T helper cell subsets in peripheral blood mononuclear cells (PBMCs).
CD40L (CD154) is upregulated on the surface of CD4+ T cells, after T cell receptor (TCR) stimulation by its specific ligand/peptide. This CD40L expression can be detected by flow cytometry. This analysis can thereby identify activated antigen-specific T cells.
The advantage of flow cytometry is that it allows determination of additional characteristics on these CD40L-expressing T cells using additional markers, such as membrane markers or intracellular cytokine profiling.
PBMCs are stimulated with peptides from the antigen of choice, followed by flow cytometric analyses of CD40L expression.
- Cytokine production (flow cytometry)
- Transcription factor expression (flow cytometry)
Acquired thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder that results from the development of auto-antibodies against ADAMTS13. The patient analyzed suffered from relapsing TTP that was resolved following splenectomy. The The ADAMTS13 peptide FINVAPHAR was recognized predominantly in this patient. When altering the anchor residues of the peptide the T cells failed to recognize the epitope (negative control).
CD4+ T cells from patients with acquired thrombotic thrombocytopenic purpura recognize CUB2 domain-derived peptides.
Verbij FC, Turksma AW, de Heij F, Kaijen P, Lardy N, Fijnheer R, Sorvillo N, ten Brinke A and Voorberg J. Blood 2016; 127(12):1606-1609