These CD8+ T cells would be isolated from the spleen of vaccinated mice and enriched to ~95% purity. I would like to find out if it is one or more clones of CD8+ T cells that are expanding after vaccination.

Dear Felicity, you should remember that only about 0.1% of clones responded to common antigens. You have few chances to obtain informative data working with ex vivo isolated CD8 cells without enrichment of antigenspecific clones. You can enrich antigen-specific T cells by sorting of CD8 T cells after staining with MHC-tetramers. If MHC-tetramers are unavailable, you can use standart limiting dilution assay to obtain and identify antigenspecific clones of CTLs before performing molecular analysis. Also, you can obtain T cell hybridomas by the fusion of activated CD8 T cells with BWZ.36CD8alpha (from Dr. Nilabh Shastri). Then, you will have sufficient time to analyse panel and verify specificity of obtained clones.

Hi Dmitry, Thank you. Since there is no tetramer (no identified CD8 specific epitopes yet) I'm thinking I'll try out limiting dilutions then. I had figured that the majority of the rapidly proliferating CD8 T cells (7 days after vaccination) would be antigen specific for a handful of clones (less than 10 I guessed), but from the complicated protocols I've found, I'm thinking this is more difficult than it appears. Thanks for the fusion with BWZ.36CD8alpha idea, I'll look into that too.

I think, 7 days after vaccination you will see no more than 2% (at the best, 5%) of antigenspecific T cells between ex vivo isolated CD8 cells. Certainly, this estimation is conditional.

Hi Felicity,

If you do not know what epitope to expect, a common way how to proceed is to use libraries of overlapping 15-mer peptides, which overlap each other by 10 aa and span the entire length of your antigen target (usually a protein, but it may be an entire viral genome as well).

Typically you stimulate the splenocytes at day 7 post infection with the pooled peptide library for 6h in the presence of Brefeldin A and do an ICCS for IFNg.

The peptide library will tell you if the protein is an (immunodominant) antigen or not. To zoom on the specific epitope, you will need to order the peptide library of as a set of individual peptides, and test them individually.

You can make pools of about 7-8 peptides per pool, which allows you to proceed in a two-step deconvolution strategy, where in the first step you define the pool of 7 peptides that gives you a positive ICCS signal, and in the second one the peptide itself (thus you do not need to perform 350 reactions to identify the 15-mer out of a 1500aa protein, but rather 50 reactions in the first round of testing and seven in the second one).

Here a reference that may serve you for starters:

http://www.ncbi.nlm.nih.gov/pubmed/23129156

Good luck

BTW. We used the strategy I described above to identify the West-Nile Virus immunodominant peptides in BALB/c mice. It is in this paper:

http://www.ncbi.nlm.nih.gov/pubmed/22916012

Credits go to James D. Brien and Jen Uhrlaub, who did the actual screening on 500+ splenocyte probes.

You may be interested in this method (PMID: 24020931) if you have access to next gen sequencing