How MHC Tetramer can detect T cells, and how to make it?

Class I and class II major histocompatibility complex (MHC) tetramer reagents allow rapid and simple detection of antigen-specific T cells. MHC tetramer technology is based on the ability of MHC-peptide complexes to recognize the antigen-specific T cells at a single cell level. This breakthrough technology enables researchers to precisely measure targeted T-cell responses in infectious diseases, cancer, and autoimmune diseases.
T-Select MHC Tetramers are available as ready-to-use reagents conjugated to a fluorochrome such as fluorescein isothiocyanate (FITC), phycoerythrin (PE), or allophycocyanin (APC). Whole blood, isolated peripheral blood mononuclear cells (PBMCs) , and expanded cell lines are appropriate sample types for tetramer analysis. MHC tetramers are used for the analyses of T cell immunity, as these reagents allow accurate enumeration and efficient immunomagnetic sorting of antigen-specific T cells, regardless of the T cell's functional capacity. MHC tetramers allow combined phenotypic and functional analyses of antigen-specific T cells using antibodies against surface and intracellular proteins. Monitoring of antigen-specific T-cell immune responses is thought to be the most important and relevant outcome of anti-tumor or anti-viral responses for the development of vaccines and therapies. These antigen-specific T cells can be detected using MBL T-Select MHC Tetramers.

Most T lymphocytes express a clonal and highly specific antigen receptor (TCR) on the cell surface. TCR (T cell receptor) binds to the MHC-peptide complex and recognizes the antigenic peptide presented in the context of MHC molecules. Such interaction initiates the generation of adaptive cellular immunity such as cell-mediated and humoral immunity. Because TCR has a low avidity and fast off-rates for MHC-peptide complexes, recombinant soluble monomeric MHC-peptide complexes have not been used to detect antigen-specific T cells. Altman et al.¹⁾ have introduced MHC-peptide tetrameric complexes (so-called MHC tetramers) for detection of antigen-specific T cells. MHC tetramers have increased avidity for their cognate TCRs and are successfully used to directly visualize antigen-specific T cells ex vivo by flow cytometry.

Reference:
1) Altman JD et al. Science 274, 94-96 (1996)

   ■ Steps for custom MHC class I tetramer

MHC Tetramers are complexes of four MHC molecules, associated with a specific peptide and bound to a fluorochrome. The T cell surface CD8 enhances T cell antigen recognition by binding to HLA class I molecules. Therefore, we produced T-Select HLA class I Tetramers with one point mutation (Ala245Val) at the HLA α3 domain known to reduce the CD8-HLA interaction. These mutated tetramers showed a greatly diminished nonspecific binding but retained specific binding²⁾. MHC Tetramers are generated essentially as described by Altman et al.¹⁾. The manufacture process is outlined in the figure on the right side.
Purified recombinant MHC (heavy chain) and human β2-microglobulin (β2m) are refolded in molar excess of the appropriate 8- to 10-mer peptide for several days. High-performance liquid chromatography (HPLC) has been extensively used to monitor the generation of monomeric MHC-peptide complexes (monomers). The refolded monomer is biotinylated with a single biotin by the BirA enzyme at the C-terminal end of the heavy chain. The biotinylated monomers are purified by streptavidin–agarose affinity column chromatography. Subsequently, purified monomers are linked by the addition of fluorochrome-labeled streptavidin.

Reference:
2) Bodinier M et al. Nat. Med. 6, 707-710 (2000)