Hi, guys
I`m quite fresh in the field of culturing CD8 T-cells in vitro and I need an advice. I want to test if a population of CD8 T-cells may give protection against LCMV-Armstrong virus infection in mice. So, we want to isolate the cells from LCMV-immune mice, expand, then transfer into naive hosts and challenge the mice with LCMV. As a measure of protection we want to use viral titer. We rather doubt if the cells are protective.
The point is that antigen specificity of the overall cell population is not known. The cells of interest contain some antiviral effectors, but relatively few and it`s not known whether these effectors play any role in the response.
TCR-transgenic mice are unavailable as far as induction of the marker I`m studying is not possible to achieve.
So, I`d avoid antigen-mediated CD8 T-cell expansion as far as it can promote some cell while depleting the rest from the population of interest. Initially I planned to use IL15-mediated expansion. Preliminary tests with that cells from naive mice were promising for 50 and 100 ng/ml of IL15. Cells grew very fast and within 1 week I was able to get 5-6 millions cells starting from 0.1 million of sorted cells.
I am making a preliminary trial with total memory CD8 T-cells from LCMV-immune mice. The cells grew quite fast on IL15 and expanded more than 20 times within 1 week. They were viable, proliferated in response to LCMV-peptides and secreted IFNg.
I wanted to expand them more to make some additional tests, but after 1 week of culture in vitro the cells stoped growing. Right now 1 week passed, but they hasn`t expanded even a little. They are just alive, neither die, nor proliferate.
Unfortunately, after stimulation with plate-bound aCD3e (1 mcg/ml overnight) that IL15-expanded CD8 T-cells from LCMV-immune mice died very fast. So I`m not sure if I can use this method.
Any ideas how to expand them? Should I try weak aCD3 stimulation with plate-bound antibodies and cytokines? Or I`d rather try some stimulator cells with soluble aCD3? Or some cytokines? IL15/IL15Ra?
Hi Dmytro,
How many cells do you need for adoptive transfer?
Either way, I recommend the use of CD3/CD28 Dynabeads + recombinant IL-2
These will kill some of your cells, but will result in robust expansion. Wash the beads off on day 2 and keep the cells in culture with IL-2 for 2 weeks.
Good luck.
Link:
https://www.thermofisher.com/us/en/home/references/protocols/proteins-expression-isolation-and-analysis/t-cell-activation-and-expansion/dynabeads-human-t-activator-cd3-cd28.html
@Mohamed!
thanks for your advice!
according to the published data I'd expect that 0.1 million good aLCMV cells must be enough to make a significant effect. after 45 days aLCMV cells should constitute ~10% of all memory Tcells in immune mice. thus, I'd expect that I need at least ~1 million cells / mouse. totally 5-6 millions, but I wished to make a titration trial.
So, you suggest to use dynabeads+IL2/IL15, right? May be reasonable, but when I tried to stimulate the IL15-cultured cells with plate-bound aCD3 (1 mcg/ml overnight) + soluble aCD28 (0.4 mcg/ml overnight) alone or with IL2 (15 U/ml) or IL15 (25 ng/ml) the cells died after 3 day culture.
is it reasonable to stimulate cells with dynabeads and IL15? I've read that it may stimulate generation of long-term cells.
Dmytro
To obtain 5-6 millions, start with 50,000 to 100000 cells, and culture with Dynabeads + IL-2 [and IL-15]. The important step is to wash the beads off on day 1 [or after 24 hours] and keep IL-2 + IL-15 in culture throughout. This should provide robust expansion.
Also, reactivation of cells with beads/plate-bound anti-CD3 will cause Activation-induced Cell Death (AICD) or more appropriately called Restimulation-induced cell Death (RICD) (PMID: 28462523)
Hi, thanks alot!
I'll try. I'm fresh with this method.
so, to keep cells healthy I need to stimulate for a short period of time and remove the stimulator from the cells and leave the cytokines, right?
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