TetramerStaining

TetramerStaining is a flow cytometry-based technique to detect antigen-specific T cells using peptide–MHC tetramers labeled with a fluorochrome. Each tetramer comprises four peptide–MHC complexes bound to a streptavidin backbone, enabling multivalent TCR engagement. Class I tetramers detect CD8+ T cells; class II tetramers detect CD4+ T cells. Successful staining requires the correct HLA type and a qualifying peptide.

Preparation includes producing biotinylated peptide–MHC monomers, assembling them into tetramers with streptavidin-fluorochrome, and validating specificity. In practice, cells are stained at 4°C, often after Fc receptor blocking. Tetramers are used with surface markers and a viability dye, followed by flow cytometry. Gating starts with singlets, lymphocytes, live cells, then CD3+ T cells, and finally CD4+ or CD8+ subsets, with tetramer+ cells quantified within those gates.

Controls and interpretation: Negative controls include tetramers with irrelevant peptides or unloaded tetramers; positive controls use known antigen-specific tetramers. Non-specific binding can occur via CD8 interactions or binding to B cells; thus background should be assessed. Limitations include the need for known peptide–HLA combinations, potential underrepresentation of low-affinity TCRs, and variability due to activation state or reagent quality. Tetramer staining is often complemented by functional assays or single-cell sequencing for confirmation.

Variants and applications: Dextramers or pentamers provide higher avidity. The method is used to study immune responses to infections, cancer, vaccines, and autoimmunity, to quantify and characterize antigen-specific T cells and to isolate them for downstream analyses.