I want to investigate antigen specific CD8 T cells using tetramers. Since these specific cells are rare in PBMCs, I am using in vitro stimulation. I want to ask if it is possible to isolate CD8+ T cells (positive magnetic selection) and use the negative fraction irradiated as APCs. Does anyone know if the positive selection of CD8+ might affect tetramer staining after the in vitro culture?
I don't see an issue and a number of papers have used this technique http://www.jimmunol.org/content/166/9/5712.full.pdf is an example but its involving mouse model research. As for affecting tetrameter staining, I would not imagine it causing a problem, and if you are culturing with the peptide prior to tetrameter staining. If you are exposing the cells to epitope over a relatively short period, then culturing to increase the CD8+ memory T cells reactive to the epitope, id imagine an increase in tetramer response in the cultured cells vs the "day 0" or unexposed cells. In my experience in tetramer staining and in culturing CD8+ memory T cells, I find a culture medium with IL-7 (approx 50U/ml) 1uM Sodium Pyruvate and R10 (with AB serum in preference to FCS) works well if you add IL-15 (approx 50u/ml) to the culture as well at day 3 works well. Using IL-7/IL-15 produces less death than IL-2. Also keep the peptide exposure as short as possible, possibly running multiple experiments with different exposure periods (i.e. 30min, 1hr, 24hrs and length of culture) sometimes extended exposure to the reactive peptide can cause other memory T cell responses which can then overwhelm the response you are targeting as these T cells may proliferate in preference or in line with your target memory T cells. which would reduce your response to tetramers..........I hope some of this helps, and I hope it makes sense. I am writing this late at night after a loooong day in the lab. Good luck
I recommend Prof. Dr. Dirk Busch, Microbiology, Technical University Munich. He is world-expert.
We do this in vitro stimulation routinely and there is no need for positive isolation of the CD8s or irradiation of the feeders. We recently also published on this http://www.ncbi.nlm.nih.gov/pubmed/25134947 Beware that T cells down regulate their TCR following peptide stimulation so detection with tetramers can not be used after short term peptide stimulation (24h). alternative markers such as CD137 can help in this case. Please feel free to contact me directly should you have any more questions!
Hi, I have experience in monitoring T cell resposes to self peptides in type 1 diabetes. CD8 is critical for the binding of CD3 to the MHC-peptide, therefore covering CD8 with an antibody (for the positive selection) can impair the binding of tetramer. Same is for the antibody you use to select the CD8+ population for FACS. Have a look to the proimmune website. they recommend anti CD8 antibodies for FACS that do not interfere with the binding to tetramers. With respect to the positive selection: why dont you just stimulate PBMC with the peptide plus IL-7 without sorting?
As alluded to by Cazaly and Monti above- remember tetrame biningis not a null event- the T cell will be activated by binding. If you have polyclonal T cells the different clones will bind tetramer differently due to affinity of the TCR, amount of the TCR and the amount of CD8 expressed. PE tetramer are espcailly sticky for mouse B cells. For very rare population (e.g. naive cells) we use two colors of tetramers and look for double positives.
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