I want to activate CD8 T cells of 2C mice in vivo. CD8 T cells will be purified from 2C mice and then will be transferred IV to B6 mice. Next, splenocytes of Balb/C mice will be injected IV in to the B6 as Ag for 2C CD8 T cells.
My question is that how many CD8 T cells and how many Balb/C cells should be transferred and how long after injection CD8 T cells of 2C acquire the activated phenotype (CD44 hi/CD62L lo)?
Did you need to separate your 2C T cells from B6 mice? You could stain your 2C T cells with CFSE and you should see both proliferation (reduction in CFSE staining) and activation markers within 3 days. I would inject 5-10x 10e6 2C T cells, while you might do different doses of Balb/c splenocytes (Might start with 1 x 10e7 splenocytes and go up from there)
Yes David. I need to purify them after activation. I will transfer 2C CD8 T LY5.2 cells IV to B6 LY5.2 and after activation I will purify them by CD45.1 biotin+SA magnetic beads by AutoMACS. I will write my results here next week. Thank you very much for your help.
The number of T cells you decide to adoptively transfer really does depend on the goals of your study. IF you want to assess development of memory cells your best bet is to look at endogenous T cells using tetramers. Since you are looking at CD8 cells, that makes your job easier, since Class I tetramers are much easier to make/use. If you want to look at initial activation events, you should still limit the number transferred. As Marzo & Lefrançois demonstrated, the initial frequency of the Ag-specific T cell effects development of memory T cells. And they do a good job to show that it is due to competition for MHC-peptide interactions. We do not transfer over 10e5 CD8 T cells (seeding ~10%, initial frequency is still a whopping 10e4 Ag-specific cells). With this number, you may or may not be able to assess CFSE dilutions in CD8 T cells (they proliferate much "faster" and more often than the CD4 T cells, in our hands. You could use re-stimulation and IFNg+ or IFNg/IL2+ cells as a means to quantify responses. As for how long after Ag/adjuvant treatment, we find that it is very difficult to remove the T cells from the spleens (single cells, at least) for the first 48h. Vella et al showed that it is probably due to strong interactions w/APC and losing the T cells to the tissue architecture. Therefore, we typically begin "counting" Ag-specific cells ~60h after treatment. The fewer cells given, the longer it takes to get to "peak" clonal expansion (might sound obvious, but sometimes obvious is overlooked). BTW, in looking closely at my data and similar studies in the literature, no matter the number transferred, there appears to be a ceiling to the number of peptide-specific T cells (again probably correlated to APC-MHC-peptide availability/competition) that approaches 2x10e6. Since epigenetic changes occur during cellular division, the more divisions it takes to reach that ceiling (lower initial frequency), the more opportunity the resultant population has had to differentiate). Our "mid-level transfer" peaks approximately 72h post-Ag txt. Transfering ~200 OTI cells leaves us to approximately 7 days to peak expansion. Our 2007 Science paper (Mata-Haro et al) used the 10e5 mid-level transfer, if you would like to look at the expansion kinetics. Which brings me to one last point...if you are using purified peptides or proteins that you know are endotoxin-free or other contaminant-free, you will need to include some sort of adjuvant to see the sort of kinetics we get with addition of LPS. Without an adjuvant of some sort, there is limited clonal expansion, and then tolerance. Good luck with your studies. I hope some of this will be helpful to you.
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