Hello, I am currently using a lentiguide CRISPR system to knockout a gene that may be important in initial Th17 T cell differentiation, but not important in the maintenance of the cells once they have differentiated. Our sgRNA works, as I have seen reductions in the protein level with transduced cell lines and T cells, though it takes a number of days for the protein level to dropout fully. What I am wondering is, is it possible to plate naive CD4+ T cells (purified with a bead system)--- wait until they are 'activated' after 24-48 hours, then add viral supernatant (leave on for 18 hours before replacing media), and then keep them proliferating in culture for a few days without having them commit to a certain T helper lineage? I plate the T cells on anti-CD3/anti-CD28 antibody coated plates, which should 'activate' them, though I have no idea in which direction they will commit if I don't include any other cytokines or antibodies in the media. I would want to skew them to Th17 at around 3 or 4 days following transduction, but am worried that the lentivirus may also cause some form of inflammation before that point and would cause them to differentiate. I have heard IL-7 or IL-2 can be used to culture T cells in a more naive-like state, though I know IL-2 inhibits Th17 differentiation, so I'm more hesitant to use that.
Hello
kindly look at link here
Article Plasticity of CD4(+) T Cell Lineage Differentiation
Good Luck
Dear Nikolas,
This is indeed a tricky question. In our lab, we used to do lentivirus after 40+/-2 hours of TCR activation (plate bound like you), and then add polarizing cytokines for Th17. No worry for IL2, whether you put it or not, activated T cells will produce it, so you can not avoid it. I would suggest to do first a simple technical solution like this one / the one you propose to check whether there is an effect at all. If you see a difference you don't need to think further.
Also, redifferentiating cells in Th17 usually quite works because Th17 is dominant, provided you do it early (
Interesting.
IL-2 knockout mice have no observable T cell defect with IL-7 taking on much of the proliferation signalling role of IL-2. Try to find a reference where the skewing of Th cells towards defined patterns of cytokine synthesis is examined in these mice. As for IL-2 you could block endogenous IL-2 activity with a neutralizing antibody.
I personally don't find activation of cells using anti-CD3/anti-CD28 to be at all physiological but in a "model" system such as yours it is likely required so you will have to live with the limitations of your system.
In my opinion you should do that experiment, i.e., not adding anything but the lentivirus, or a sham version of it, plus minus neutralizing anti-IL2 mAb to give you an idea of the effects of each without having to worry about variables whose activity can be determined.
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