Hi everyone,
I am working on human Primary CD8+ T-cells and on their behaviour under various conditions.
once activated in a OKT-3 coated plate and anti-CD28 for one hour, i put my cells in culture under various conditions such as IL-2, IL-15, IL-12, IL-10 (all in different well, at concentration of 30 U/ml for IL-2, 50 ng/ml for the others).
I aim to analyze various reactions to these conditions on day1, 4 and 7.
However, on day7, most of my cells are dead (up to 95% death). I tried to switch fresh medium on day 4. This helped for some cultures (IL-15 culture up to 35-40% survival, which can be considered acceptable for this specific interleukin), however, for the others it didn't change much and they desperatly stood almost all dead on day7 (up to 95% of the cells dead).
I know that CD8+ T cells are not supposed to stay alive a Long time post-activation. However, the survival of only 10 to 15% for IL-2 stays pretty astonishing, since various labs work on IL-2 cultures of CD8+ T-cells for more than a week and don't appear to have this Kind of Problem, or at least not on such scale.
do you have any advice for this Kind of Problem, and have you ever encounter such death rate for human CD8+ T cells culture?
thanks a lot for your help and have anice day!
Did you add feeders (irradiated self PBMCs)? Purified hCD8 recently stimulated are cytotoxic and can became exhausted. Feeders and low cell concentration of the purified activated CD8s may extend their life, but at day 8 you're reaching their limit for re-estimulation.
thank you for your answer,
I didn't go with Feeders for now, since I wanted to keep my culture as free as possible from any other Kind of interaction than the one with the specific Interleukin. adding feeders might bring complexity to the model. that is anyway an alternative i will study, thank you!
low cell concentration might help, however, my concentrations are not particularly high... i will control this alternative.
also, I just checked for any possible difference of survival in a non-activated Group: the death rate stood high, even with IL-2, which is also surprising.
- CD8s are "terminal" cells. If you do nothing they die, if you do too much they die after having completed their mission. Unless you're studying memory, the ideal time point for you to measure any activity differences are in the first 72 hours post-activation, when everyone does their cytotoxic assays. 7 days is stretching too much.
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