MHC-Peptide Tetramers - (Apr/14/2006 )
Could anyone please tell me how MHC-Peptide tetramers work in the detection of antigen-specific T cells?
hi,
briefly, you charge the tetramer with your peptide, incubate them w/ your cells and pass the mix to cytometer
Seb
The principle is that a specific MHC molecule has been expressed which will bind the antigenic peptide you're interested in (i.e. a specific 8mer/9mer peptide from a viral protein). So you need to know/have predicted what MHC molecule binds your peptide.
As Sebastien said, this peptide is loaded onto the MHC molecule, which is made tetrameric and fluorescent by using biotinylated MHC molecules and steptavidin-conjugated to a fluorochrome (FITC, PE etc.).
T-cells expressing a T-cell receptor (TCR) capable of recognising this peptide i.e specific for the peptide of interest (in the context of MHC of course) will bind the tetramer and will be fluorescently labelled on the flow cytometer.
So it's like using a fluoresently tagged ab but the specificity is due to the specific nature of the MHC/peptide and TCR interaction.
Hope that helps,
Ceri
As Sebastien said, this peptide is loaded onto the MHC molecule, which is made tetrameric and fluorescent by using biotinylated MHC molecules and steptavidin-conjugated to a fluorochrome (FITC, PE etc.).
T-cells expressing a T-cell receptor (TCR) capable of recognising this peptide i.e specific for the peptide of interest (in the context of MHC of course) will bind the tetramer and will be fluorescently labelled on the flow cytometer.
So it's like using a fluoresently tagged ab but the specificity is due to the specific nature of the MHC/peptide and TCR interaction.
Hope that helps,
Ceri
OK, so maybe I'm about to ask a particularly naive question, but is this something you can do in vitro? If I know the MHC molecule, can I just add my peptide, incubate, remove excess peptide etc and hey preto, there's my laoded MHC molecule, ready for T cell detection/capture? WHere do you get unloaded MHC molcules? HLA-R-Us? And what strength binding do you get?
Thanks! Helps a Lot
As Sebastien said, this peptide is loaded onto the MHC molecule, which is made tetrameric and fluorescent by using biotinylated MHC molecules and steptavidin-conjugated to a fluorochrome (FITC, PE etc.).
T-cells expressing a T-cell receptor (TCR) capable of recognising this peptide i.e specific for the peptide of interest (in the context of MHC of course) will bind the tetramer and will be fluorescently labelled on the flow cytometer.
So it's like using a fluoresently tagged ab but the specificity is due to the specific nature of the MHC/peptide and TCR interaction.
Hope that helps,
Ceri
Swanny,
In theory I guess yes if you have purified MHC. I'm assuming these are expressed recombinantly to be secreted rather that purifying specific MHC from the different MHC molecules expressed on a cell. Although my reply was based on my theoretical knowledge not on practical experience. There are programs to predict peptide binding to different MHC molecules. People who look at CTL use them for predicting peptide binding from their antigen of interest.
Ceri