TCR Tetramers

MBL has developed and manufactured various major histocompatibility complex (MHC) Tetramer products as tools for detecting antigen-specific T cells. These products have been used to monitor T cell immune responses in research studies and clinical trials.

MBL has efficiently manufactured a TCR Tetramer reagent, which is recognized by MHC, and has successfully commercialized it. Moreover, our cancer antigen-specific (survivin-2B, PBF) TCR Tetramer reagents* have also been assessed for their staining properties by flow cytometry.

*These products are licensed under JP patent No. P2017-169566A.

What is a TCR Tetramer?

A TCR Tetramer is a reagent prepared by tetramerizing biotinylated antigen-specific TCRs with the help of phycobiliprotein-labeled streptavidin.

Product Highlights

Product Code	PRODUCT NAME
TS-TCR01-1	Human TCR Tetramer-PE (HLA-A*24:02 survivin-2B80-88-AYACNTSTL)
TS-TCR02-1	Human TCR Tetramer-PE (HLA-A*24:02 PBF145-153-AYRPVSRNI)

Preparation of TCR Tetramers

The manufacturing process is outlined in the figure below. Purified recombinant TCR α and β chains are refolded for several days to obtain soluble monomeric TCRs (monomers). The refolded monomer is conjugated with a single biotin molecule as the expressed recombinant β chain is already biotinylated at the C-terminal end. The biotinylated monomers were purified and subsequently linked by adding fluorochrome-labeled streptavidin.

Comparison between MHC and TCR Tetramers

	MHC TETRAMER	TCR TETRAMER
Structure	MHC/peptide	TCR (α chain/β chain)
Recognition	Specific TCR	Specific MHC/peptide complex
Detection	Antigen-specific T cells	Antigen-specific tumors and antigen presenting cells
Application	・Quantification ・Monitoring of antigen-specific T cells	・Quantification ・Evaluation of the efficacy of cancer immunotherapies ・Quality control of cancer vaccine

Staining using TCR Tetramers

Example #1: Cell Staining (Survivin-2B TCR Tetramer)

C1R cells (HLA-A-deficient) that stably expressed HLA-A*24:02 (called C1R-A24), were prepared; these cells were pulsed with survivin-2B peptide (AYACNTSTL) for 4 hours. Thereafter, the cells were stained with PE-labeled survivin-2B TCR Tetramer and Anti-HLA-A24 (Human) mAb-FITC (Code No. K0209-4) and further analyzed by flow cytometry. 96.42% of HLA-A24+/Tetramer+ cells were detected while staining peptide-pulsed C1R-A24 cells.

Numbers in the upper right quandrants represent the percentages of TCR Tetramer+ cells relative to the total HLA-A24+ cells

Example #2: Staining of MHC Tetramer-conjugated beads (Survivin-2B TCR Tetramer)

Streptavidin-coated beads were loaded with biotinylated peptide/HLA complex (HLA-A*24:02 presenting survivin-2B peptide, AYACNTSTL) at dilution ratios ranging from 1 to 10−3. This mimics the difference in the pMHC expression in cells.

The following data reveal the staining of the beads using Survivin-2B TCR Tetramer. A representative histogram indicates TCR Tetramer-positive populations in the normalized bead count. The noncoated beads were used as a negative control (R=0).

Reactivity of TCR Tetramer depends on the number of peptide/HLA complexes coated on the beads.