A robust method for production of MHC tetramers with small molecule fluorophores

Abstract

Tetramers of major histocompatibility complex molecules (MHC) are now well-established reagents for the detection of antigen-specific T cells by flow cytometry. MHC tetramers are prepared by mixing enzymatically biotinylated MHC molecules with commercial preparations of streptavidin, usually conjugated to a fluorescent phycobiliprotein such as phycoerythrin (PE) or allophycocyanin (APC). While data obtained with MHC tetramers prepared with small molecule fluorophores has been reported, considerable lot-to-lot variation among conventional streptavidin conjugates to small molecules prevents routine preparation of such reagents. We now report robust preparation of MHC tetramers with small molecule fluorophores, using a recombinant mutant of streptavidin incorporating a carboxy-terminal cysteine in each of the four identical subunits that is conjugated to maleimide derivatives of any of several small molecule fluorophores. These reagents significantly expand the versatility of the MHC tetramer methodology.

Figure 1. The carboxylate of biotin bound to subunit D (yellow) is 7.25 Å from the ε-amino group of lysine-121 (green) of the neighboring subunit A (otherwise magenta)

The carboxy-termini of all subunits, shown in red space-filling models, are very much further away from the biotin carboxylates.

Figure 2. Purified, recombinant StvC behaves like commercial preparations of stretpavidin on SDS-PAGE

Non-boiled StvC and commercial streptavidin migrate as intact tetramers, while the boiled proteins migrate as monomeric subunits.

Figure 3. Normalized emission spectra of fluorophores conjugated to streptavidin

The emission spectra of the maleimide-modified StvC derivatives are shown as dotted lines, while the commercial preparations of NHS-modified streptavidins are shown as solid lines.

Figure 4. StvC-based tetramers are predominantly tetramers, and do not contain high concentrations of higher-molecular weight aggregates

StvC modified with Alexa647-maleimide was mixed with a slight excess of HLA-A*0201 complexed with the HCMV.pp65 peptide NLVPMVATV an analyzed by size exclusion chromatography.

Figure 5. Flow cytometric analysis of tetramer staining from whole blood samples

Whole blood samples from an HLA-A*0201⁺, HCMV⁺ donor were stained for 20 minutes at room temperature with A2/CMV.pp65.495–503 tetramers (peptide sequence NLVPMVATV) prepared with StvC labeled with fluorophores as indicated in the figure, together with anti-CD3 and anti-CD8 antibodies. Fluorophore labels on the anti-CD3 and anti-CD8 reagents vary from panel-to-panel and were chosen to minimize spillover from their fluorescence into the tetramer channel.